How to detect carbapenemase producers? A literature review of phenotypic and molecular methods.

MIMET-04491; No of Pages 13 Journal of Microbiological Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

1

Review

4

D. Hammoudi a,b,⁎, C. Ayoub Moubareck a,b,c, D. Karam Sarkis a,b a

8

a r t i c l e

9 10 11 12 13

Article history: Received 9 July 2014 Received in revised form 25 September 2014 Accepted 26 September 2014 Available online xxxx

14 15 16 17 18 19

Keywords: Carbapenemases Detection Screening Phenotypic methods Molecular methods

i n f o

R O

Microbiology Laboratory, School of Pharmacy, Saint-Joseph University, Beirut, Lebanon Rodolph Merieux Laboratory, Beirut, Lebanon Department of National Science and Public Health, College of Sustainability Sciences and Humanities, Zayed University, Dubai, United Arab Emirates

a b s t r a c t

E

D

P

The production of carbapenemases by Gram negative bacterial pathogens has become a worldwide threat to successful antibiotic therapy. Carbapenem resistance has been increasingly reported in recent years, and given the paucity of reliable antimicrobials, focus has shifted towards early surveillance of carbapenemases in microbiology laboratories. Detection of carbapenemases is primarily based upon careful recognition of decreased in vitro susceptibility to carbapenems by measurement of their MIC values or inhibition zone diameters. This is followed by a set of conventional phenotypic methods of variable efficiencies, such as the modified Hodge test and culturebased tests utilizing carbapenemase inhibitors. Among these, boronic acid compounds are used to inhibit Ambler class A carbapenemases, and EDTA and dipicolinic acid are used to inhibit Ambler class B carbapenemases. While the detection of carbapenemase producers is possible using screening culture media, the identification of carbapenemase genes relies on molecular techniques. Polymerase chain reaction experiments allow the detection of well-known carbapenemase genes, and sequencing is essential to the identification of new genes. Innovative biochemical and spectrometric detection are being developed to complement the molecular methods and shorten processing times needed for detection of carbapenemase activity. These are promising options to become routinely applied for rapid detection of carbapenemase-producing organisms with high precision and are most useful for epidemiologic purposes. Molecular techniques are nevertheless expensive, time consuming, and require well-trained personnel. This review is a summary of the current state-of-art of carbapenemase detection methods, with a description of the advantages and limitations of each. © 2014 Published by Elsevier B.V.

E

C

c

T

b

O

5 6 7

F

3

How to detect carbapenemase producers? A literature review of phenotypic and molecular methods

2

R

37

Q2

1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Screening of carbapenemase-producers based on antibiotic susceptibility tests Phenotypic detection methods . . . . . . . . . . . . . . . . . . . . . 3.1. Modified Hodge test . . . . . . . . . . . . . . . . . . . . . . . 3.2. Inhibitor-based tests . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Inhibitors of Ambler class A carbapenemases . . . . . . . 3.2.2. Inhibitors of Ambler class B carbapenemases (MBLs) . . . . 3.2.3. Inhibitors of Ambler class C carbapenemases . . . . . . . 3.2.4. Inhibitors of Ambler class D carbapenemases . . . . . . . 3.3. Detection using carbapenem-including culture media . . . . . . . . Analytical and biochemical detection methods . . . . . . . . . . . . . . 4.1. Isoelectric focusing . . . . . . . . . . . . . . . . . . . . . . . 4.2. Spectrophotometric detection . . . . . . . . . . . . . . . . . . 4.2.1. UV-spectrophotometry . . . . . . . . . . . . . . . . . 4.2.2. Mass spectrometry . . . . . . . . . . . . . . . . . . . 4.3. Carba NP test . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Immunochromatography . . . . . . . . . . . . . . . . . . . .

N C O

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Contents

U

40 43 42

R

41 39 38

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

⁎ Corresponding author at: Saint-Joseph University, Campus of Medical Sciences, Damascus Road, PO BOX: 11-5076 Riad El solh, Beirut 1107-2180, Lebanon. Tel.: +961 76 549030; fax: +961 1 421022. E-mail addresses: [email protected] (D. Hammoudi), [email protected] (C. Ayoub Moubareck), [email protected] (D. Karam Sarkis).

http://dx.doi.org/10.1016/j.mimet.2014.09.009 0167-7012/© 2014 Published by Elsevier B.V.

Please cite this article as: Hammoudi, D., et al., How to detect carbapenemase producers? A literature review of phenotypic and molecular methods, J. Microbiol. Methods (2014), http://dx.doi.org/10.1016/j.mimet.2014.09.009

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Q3 36

2

61 62 63 64 65 66 67

D. Hammoudi et al. / Journal of Microbiological Methods xxx (2014) xxx–xxx

5.

Molecular detection of carbapenemase genes . . . 5.1. Carbapenemase detection by PCR-sequencing 5.2. Cloning and sequencing of new genes . . . 5.3. Molecular analysis of clonal relatedness . . 6. Conclusion . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

0 0 0 0 0 0 0

68

86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123

C

84 85

E

83

R

F

O

R O

81 82

R

79 80

O

77 78

C

75 76

N

73 74

U

71 72

P

Carbapenemases are emerging resistance determinants in Gram negative pathogens, including Enterobacteriaceae, Pseudomonas and Acinetobacter. These carbapenem-hydrolyzing enzymes confer resistance to a broad variety of β-lactams, and are located on self-conjugative plasmids carrying other resistance determinants, capable of disseminating among bacteria and resulting in spread of resistance to multiple classes of antibiotics like fluoroquinolones, aminoglycosides and cotrimoxazole (Falagas et al., 2011; Nordmann and Poirel, 2013). The first carbapenemase was described in France in the year 1993 and its gene blaNmcA was localized on the chromosome of an Enterobacter cloacae (Naas and Nordmann, 1994). In 1995, a report from Japan described a plasmid-borne gene blaIMP-1 capable of hydrolyzing carbapenems in Serratia (Ito et al., 1995). In 2001, a similar report from North Carolina documented another plasmid-borne, carbapenem-hydrolyzing gene blaKPC-1 that was recovered from an isolate of Klebsiella pneumoniae (Yigit et al., 2001). Since then, a number of newly recognized carbapenemases has proliferated and disseminated generating a major therapeutic and epidemiological concern, due to restriction in patient treatment options and infection control strategies (El-Herte et al., 2012). Carbapenems are indeed broad spectrum antibiotics that represent key agents in life-threatening nosocomial infections, transplantations, hospitalizations in intensive care units, and surgeries (Nordmann et al., 2012a). Their use has increased in clinical practice as a result of expanding resistance to other β-lactam antibiotics, being the sole antibiotics of this class with proven efficacy against extended-spectrum β-lactamase (ESBL) producing Gram negative bacteria (Hawkey and Livermore, 2012). Resistance to carbapenems has been largely reported as a consequence of acquisition of carbapenemase-encoding genes, even though other resistance mechanisms, such as reduced permeability of the outer membrane due to porin alterations, or high efflux pump activity, may be responsible of carbapenem resistance (Nordmann et al., 2011a). The clinically significant carbapenemases belong to Ambler class A (KPC, GES) with serine active sites and low hydrolysis of all β-lactams except cephamycins; or Ambler class B (NDM, VIM, and IMP) which are zinc-dependent metallo-β-lactamases (MBLs) strongly hydrolyzing all β-lactams except aztreonam; or Ambler class D (OXA-type) with weak hydrolysis of carbapenems, and no effect on broad spectrum cephalosporins and aztreonam (Queenan and Bush, 2007; Poirel et al., 2012). The Ambler class C cephalosporinases (AmpC cephalosporinases) have very little hydrolytic activity, if any, against carbapenems (Jacoby, 2009). However, the production of plasmid-encoded AmpC cephalosporinases has emerged as an important resistance determinant to carbapenems in isolates with impermeability mechanisms or efflux pump overactivity (Gutierrez et al., 2007; Dahmen et al., 2012). A comparison of the clinically significant carbapenemases is shown in Table 1. Because carbapenemases represent a versatile family of βlactamases of increasing incidence, the optimization of their detection techniques is necessary, an action that may be challenging for the microbiology laboratory (Queenan and Bush, 2007). The effective and timely detection of carbapenemase-producing organisms is an urgent issue, not only for the selection of appropriate therapeutic schemes but also for the implementation of infection control measures. However, this detection includes a number of difficulties, because it cannot be

124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140

D

70

simply based on resistance profile, and its accurate methodology has not been yet adequately standardized (Miriagou et al., 2010). In general, a preliminary screening of carbapenemase producers relies on recognition of decreased susceptibility to carbapenems in antibiotic susceptibility tests, followed by other phenotypic and biochemical tests (Cohen Stuart and Leverstein-Van Hall, 2010). However, and although not available in many laboratories, carbapenemase gene recognition by molecular methods remains the gold standard of detection (Nordmann and Poirel, 2013). Other newer, alternative techniques based on analytical methods or innovative technologies are also being developed and show potential for use due to high efficiency. The purpose of this review is to summarize available methods that have been proposed for laboratory identification of carbapenemase-producing Gram negative bacteria. A scheme for possible utilization of the different methods is presented in Fig. 1. The article constitutes a relevant laboratory tool for the detection of carbapenemases and opens wide perspectives in clinical and experimental microbiology.

2. Screening of carbapenemase-producers based on antibiotic 141 susceptibility tests 142

E

1. Introduction

The first cause of suspicion of carbapenemase production in a clinical isolate is an increase in carbapenem minimum inhibitory concentration (MIC) or a decreased in inhibition zone diameter. This result renders a bacterial isolate eligible for further analysis for carbapenemase production using more specific methods (Miriagou et al., 2010). The carbapenem susceptibility ranges for Enterobacteriaceae, Pseudomonas, and Acinetobacter are shown in Table 2. According to the 2014 recommendations of the European Community on Antimicrobial Susceptibility testing (EUCAST), the MIC breakpoints of imipenem and meropenem for Enterobacteriaceae are greater than 8 mg/L, while the MIC breakpoint of ertapenem is greater than 1 mg/L (EUCAST, 2014). According to the 2014 US guidelines of the Clinical Laboratory and Standards Institute (CLSI), these breakpoints are greater than or equal to 4 mg/L for imipenem and meropenem and greater than or equal to 2 mg/L for ertapenem (CLSI, 2014). Ertapenem seems to be a good candidate for detecting most carbapenemase producers among Enterobacteriaceae because MIC values of ertapenem are usually higher than MICs of other carbapenems (Nordmann et al., 2012c). However, detection of carbapenemase producers based only on MIC values may lack sensitivity. Many carbapenemase producing Enterobacteriaceae show broad range of MICs with sometimes values within the susceptibility range. Indeed, intermediate susceptibility, or even sensitivity to carbapenems has been observed for producers of all types of carbapenemases especially the OXA-48/OXA-181 producing Enterobacteriaceae that do not co-harbor an ESBL (Table 3) (Nordmann, 2010; Nordmann et al., 2011a). Carbapenem MICs are expected to substantially rise only in the presence of an additional resistance mechanism, like permeability lesions due to outer membrane protein mutation, or simultaneous production of AmpC cephalosporinases or ESBLs (Livermore and Woodford, 2006). To avoid false negative results, or to maximize detection sensitivity, it has been proposed to screen enterobacterial isolates for carbapenemase activity if they exhibit MICs of ertapenem greater than or equal to 0.5 mg/L or MICs of imipenem or meropenem greater than or equal to 1 mg/L, or to screen any enterobacterial isolate displaying a slight decrease in susceptibility to

T

69


143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177

Dortet et al. (2012)

carbapenems compared with the wild-type organism (Nordmann and Poirel, 2013). Concerning Pseudomonas and Acinetobacter, EUCAST MIC breakpoints of imipenem and meropenem are greater than 8 mg/L while MIC breakpoint of doripenem is greater than 2 mg/L (EUCAST, 2014). Ertapenem is not considered for treatment of infections caused by the latter two organisms: (i) It lacks sufficient antipseudomonal activity to be clinically useful due to a combination of reduced membrane permeability and possibly an increased affinity for efflux pumps (Zhanel et al., 2007); (ii) Acinetobacter baumannii strains have intrinsically a smaller number and size of porins compared with other Gram negative organisms, contributing to natural outer membrane impermeability to ertapenem (Abbott et al., 2013). In Pseudomonas, where IMP, VIM and SPM MBLs are increasingly scattered, a more consistent reduction in susceptibility to carbapenems is observed in comparison to Enterobacteriaceae with the same enzymes. This may be explained by the existence of parallel impermeability of the outer membrane as well as the activity of multidrug efflux pumps (Livermore and Woodford, 2006). In A. baumannii, the most frequently encountered carbapenemases are of the OXA-type, and possess only frail enzymatic activity (Livermore and Woodford, 2006). The high carbapenem MIC levels in this genus are, in part, caused by co-determinants of resistance like porin protein mutations (Poirel and Nordmann, 2006). Apart from MICs of carbapenems, a concomitant examination of MICs of additional β-lactam antibiotics may be helpful to detect carbapenemase-producing organisms. In fact, the acquisition of carbapenemases diminishes susceptibility to a wide variety of β-lactam antibiotics regardless of the actual level of carbapenem resistance. More specifically, a carbapenemase-producing isolate exhibits at least resistance to penicillins and narrow-spectrum cephalosporins. If the isolate harbors either class A or class B carbapenemase, it is expected to be resistant to broader-spectrum cephalosporins like ceftazidime, ceftriaxone, and cefotaxime. These latter antibiotics, however, are not affected by the production of class D carbapenemases (Queenan and Bush, 2007). Despite the fact that MIC determination is suitable and appropriate for the clinical laboratory, its routine use may be associated with reproducibility issues for most conventional methods (Miriagou et al., 2010). In a study comparing various susceptibility testing methods, significant discrepancies were observed in determination of susceptibility of five VIM-1 producing K. pneumoniae isolates to imipenem and meropenem using broth microdilution, Etest, disc diffusion, and the automated systems Vitek 2, Phoenix, and MicroScan. The isolates displayed major changes from susceptible to resistant especially when the automated systems were used (Giakkoupi et al., 2005). Also, in another study on K. pneumoniae, 15% of KPC-producing isolates were susceptible to imipenem in automated broth microdilution methods, while only 4% were susceptible by the Etest method (Bratu et al., 2005b). It is suggested that such results are at least partially attributed to the effect of inoculum density, where low inocula made many strains appear susceptible or intermediate to imipenem. A less prominent inoculum effect was observed for meropenem, while not observed at all for ertapenem, suggesting that this compound may be preferable for detection of KPC-producing isolates (Bratu et al., 2005b). A similar inoculum effect was observed for KPC-producing Enterobacter species (Bratu et al., 2005a). Hence inoculation should be done adequately for susceptibility testing to be reliable; if the proper inoculum cannot be assured using an inoculation wand (loop) method, inocula should be prepared using standardized CLSI procedures (CLSI, 2014). The failure of automated systems to regularly detect KPC-expressing isolates was demonstrated by Tenover et al., where among 15 KPC-producing K. pneumonia, the number of imipenem susceptible isolates varied between 1, 2 and 13 for MicroScan, Phoenix, and Sensititre respectively. The authors recommend that if treatment failure with carbapenems is observed for isolates previously reported as susceptible, testing should be repeated with a nonautomated method like disc diffusion (Tenover et al., 2006). Other

O

R O

T

E

D

P

Positive

C

Negative

t1:14

Positive Positive, but not for GES-type enzymes

R

Modified Hodge test Common inhibitors Detection by selective media (CHROMagar, SUPERCARBA) Detection by Carba NP test t1:9 t1:10 t1:11 t1:12 t1:13

EDTA = ethylene diamine tetra-acetic acid; ESBLs = extended spectrum ß-lactamases.

E

Positive None Positive Variable EDTA, dipicolinic acid Positive

R

Affected β-lactam substrates t1:8

Positive Boronic acid, low inhibition by clavulanate Positive

Most commonly affected species t1:7

All β-lactams including monobactams, except cephamycins (cefoxitine, cefotetan) GES does not hydrolyze aztreonam, and shows low hydrolysis of carbapenems

N C O

U

3

F

Oxacillin and cloxacillin No hydrolysis of aztreonam and extended-spectrum cephalosporins weak hydrolysis of carbapenems

Penicillins, oxyimino cephalosporins, and aztreonam Almost no effect on cefepime and carbapenems except if coupled to porin loss or ESBLs Variable Boronic acid, cloxacillin Negative

Thomson (2010) Thomson (2010) Nordmann et al. (2012b)

Queenan and Bush (2007), Kanj and Kanafani (2011)

Enterobacteriaceae, A. baumannii K. pneumoniae, Salmonella, E. coli

Reference Ambler class D

Serine Turkey, Middle East OXA-23, OXA-24, OXA-58, OXA-48, OXA-181

Plasmid-mediated Ambler class C

Serine South Korea, USA CMY, FOX, MOX, ACC, DHA

Ambler class B

Zinc Japan, India VIM-2, IMP-1, IMP-2, NDM, SPM-1 Enterobacteriaceae, A. baumannii, Pseudomonas All β-lactams except aztreonam. Strong hydrolysis of carbapenems Serine France, England, USA Chromosomal: NmcA, SME, IMI-1, SFC-1 Plasmid-borne: KPC, GES, IMI-2 Enterobacteriaceae, Acinetobacter baumannii, Pseudomonas Active enzyme site Geographic epicenter Important enzymes t1:4 t1:5 t1:6

Ambler class A Ambler class

t1:3

Table 1 Classification, comparison of properties, and detection methods of carbapenemases. t1:1 t1:2

Queenan and Bush (2007) El-Herte et al. (2012) Queenan and Bush (2007), Patel and Bonomo (2013) Livermore and Woodford (2006)



178 179 180 181 182 183 184 185 186 187 Q4 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243


U

N

C

O

R

R

E

C

T

E

D

P

R O

O

F

4

Fig. 1. Simplified scheme for detection of carbapenemases. MIC, minimum inhibitory concentration; DDST, double disc synergy test; APBP; aminophenyl boronic acid; EDTA, ethylene diamine tetra-acetic acid; IEF, isoelectric focusing; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; PCR, polymerase chain reaction; PFGE, pulsed field gel electrophoresis; MLST, multilocus sequence typing.

244 245 246 247 248 249

studies that employed gradient diffusion methodologies such as the Etest did also demonstrate inconsistency in the results due to colonies present in the zone of inhibition (Tenover et al., 2006; Samuelsen et al., 2008). Accordingly, the broth microdilution and disc diffusion methods are considered to be more reliable for detection of all types of carbapenem-mediated resistance (Miriagou et al., 2010).

3. Phenotypic detection methods

250

Given the variability of carbapenemase screening results by antibiotic susceptibility tests, detection of carbapenemase-producing organisms by phenotypic, culture-based techniques is an optional step that avoids delayed reporting of such strains to the clinic, in case genotypic tests

251 252


253 254


CLSI

Doripenem Ertapenem Imipenem Meropenem Doripenem Ertapenem Imipenem Meropenem Doripenem Ertapenem Imipenem Meropenem

Pseudomonas

Acinetobacter

MIC breakpoint (mg/L)

S≤

RN

S≥

Rb

S≤

R≥

S≥

R≤

1 0.5 2 2 1 – 4 2 1 – 2 2

2 1 8 8 2 – 8 8 2 – 8 8

24 25 22 22 25 – 20 24 23 – 23 21

21 22 16 16 22 – 17 18 20 – 17 15

1 0.5 1 1 2 – 2 2 2 – 2 2

4 2 4 4 8 – 8 8 8 – 8 8

23 22 23 23 19 – 19 19 18 – 22 18

19 18 19 19 15 – 15 15 14 – 18 14

MIC, minimum inhibitory concentration; S, sensitive; R, resistant.

255 256

are not readily available. They include the modified Hogde test, inhibitorbased tests, and the use of specific culture media.

257

3.1. Modified Hodge test

258

The clover leaf technique, or modified Hodge test, is available in routine clinical settings and has been extensively used as a phenotypic tool to detect carbapenemase activity. This test is a modification of an original simple test designed by Hodge et al. to detect penicillinaseproducing Neisseria gonorrhea (Hodge et al., 1978). The modified Hodge test is performed on Mueller–Hinton agar, and is based upon inactivation of a carbapenem by a carbapenemase-producing test strain. An indicator organism, usually Escherichia coli ATCC 25922 at a turbidity of 0.5 McFarland standards, is used to inoculate the plate surface, and a carbapenem disc is placed at the center. The test strain is heavily streaked from the disc to the plate periphery. After overnight incubation, the cloverleaf-shaped indentation of growth of the test strain versus the susceptible indicator strain is interpreted as a positive result for carbapenemase production by the tested strain (Lee et al., 2001). One advantage of the modified Hodge test is that it assumes excellent sensitivity for detection of Ambler class A and class D carbapenemases, allowing therefore detection of enzymes with weak carbapenemase activity such as OXA-23, GES-5 and GES-6 (Hornstein et al., 1997; Vourli et al., 2004). While evaluating different methods, the modified Hodge test used in combination to carbapenem

270 271 272 273 274 275 276 277

t3:1 t3:2 t3:3 t3:4 t3:5

C

E

268 269

R

267

R

265 266

N C O

263 264

Table 3 Ranges of MICs of carbapenems for clinical Gram negative bacteria expressing the main carbapenemases (Nordmann, 2010; Nordmann et al., 2012c). MIC (mg/L)

U

261 262

T

t2:19

259 260

Imipenem Meropenem Ertapenem

t3:6 t3:7 t3:8

Enterobacteriaceae KPC (Ambler class A) IMP/VIM/NDM (Ambler class B)

0.5 to N32 0.5 to N32 0.5 to N32 0.5 to N64

t3:9

OXA-48/OXA-181 (Ambler class D)

0.25 to 64

t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20

Pseudomonas KPC (Ambler class A) IMP/VIM/NDM (Ambler class B) Acinetobacter KPC (Ambler class A) IMP/VIM/NDM (Ambler class B) OXA-23/OXA-40/OXA-58/OXA-143 (Ambler class D) MIC, minimum inhibitory concentration.

Zone diameter breakpoint (mm) for 10 μg disc

0.5 to 64

0.5 to N32 0.38 to N32 0.38 to N32

2 to N64 N64

2 to N64 N64

– –

2 to N64 1 to N64 N32

2 to N64 1 to N64 N32

– – –

F

Enterobacteriaceae

Zone diameter breakpoint (mm) for 10 μg disc

O

t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18

EUCAST MIC breakpoint (mg/L)

R O

Carbapenem

susceptibility testing was selected to confirm KPC-producers among isolates of Enterobacteriaceae (Anderson et al., 2007). In this study, ertapenem resistance coupled with positive modified Hodge test was considered sensitive enough to detect KPC carbapenemases. The detection of MBL-producers by the modified Hodge test lacks sensitivity (Thomson, 2010). Girlich et al. addressed this issue in a study on carbapenem nonsusceptible Enterobacteriaceae, where despite the correct detection of class A and class D carbapenemase-producers by the modified Hodge test, its sensitivity to detect NDM-producers was only 50%. This was justified by a possible secretion of a substance such as colicin, a bacteriocin-peptide released by certain bacteria, which may inhibit the growth of the indicator strain and interfere on results of the test (Girlich et al., 2012). To improve the detection limits for NDM-producers, the authors investigated the addition of zinc sulfate (100 μg/mL) to the Muller–Hinton agar media. Since MBL activity is zinc-dependent, this modification increased sensitivity of the test to 85.7%. Similarly, and when detecting MBLs of Pseudomonas aeruginosa and Acinetobacter by the modified Hodge test, Lee et al. also demonstrated that the test can be improved by using an imipenem disc to which 10 μL of 50 mM zinc sulfate (140 μg/disc) has been added or by using Mueller–Hinton agar to which zinc sulfate has been added to a final concentration of 70 μg/mL (Lee et al., 2003). In parallel, even though contested, it has been proposed that the performance of the modified Hodge test was better with MacConkey's agar due to enhanced release of β-lactamases from the cells in the presence of bile compounds (Lee et al., 2010; Girlich et al., 2012). The modified Hodge test has also other limitations apart its sensitivity to detect class B carbapenemases (Miriagou et al., 2010). These limitations include the type of carbapenem optimal for confirmation of carbapenemase production, where imipenem appears to be the least specific agent although it is the most sensitive to detect OXA-type enzymes. Moreover, the modified Hodge test may lack sensitivity to detect carbapenemase activity in Enterobacter species (Nordmann and Poirel, 2013). In addition, false positive results are expected with AmpC producers, more likely with imipenem than with other carbapenems (Thomson, 2010). A study by Pasteran et al. indicated high sensitivity of the modified Hodge test to detect class A carbapenemases using 3 carbapenem discs (ertapenem, meropenem, and imipenem). However, false-positive results were obtained for CTX-M producing strains with reduced outer membrane permeability, and to a lesser extent for those hyperproducing AmpC cephalosporinases. To circumvent this, authors did recommend other confirmatory tests such as inhibitor-based methods to be applied for strains co-expressing CTX-M enzymes (Pasteran et al., 2009). In short, the modified Hodge test is an easy and inexpensive tool that can be used while screening for carbapenemases following to

P

Microorganism

t2:4 t2:5

D

t2:3

Table 2 Breakpoints of carbapenem susceptibility for Enterobacteriaceae, Pseudomonas, and Acinetobacter according to European (EUCAST, 2014) and US (CLSI, 2014) guidelines.

E

t2:1 t2:2 Q1

5


278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323

In the presence of specific carbapenemase inhibitors, the activity of certain carbapenemases decreases and carbapenemase producers become more sensitive to ß-lactams. Phenotypic tests involving these inhibitors are based upon in vitro observation of an increase in inhibition zone diameter (or reduction of the MIC) of the tested isolate in the presence of a carbapenem combined with a carbapenemase inhibitor compared to the same carbapenem alone (Cohen Stuart and Leverstein-Van Hall, 2010). The phenomenon of synergy between the carbapenem and the inhibitor can be practically verified by several techniques:

348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385

3.2.2. Inhibitors of Ambler class B carbapenemases (MBLs) All MBLs require divalent cations, usually zinc, as metal cofactors for enzyme activity, and accordingly are affected by the removal of zinc from the active site (Walsh et al., 2005). Studies addressing MBL detection have relied upon this principle, and a variety of inhibitors that take advantage of metalloenzyme inhibition has been considered. These inhibitors include EDTA, EDTA plus 1,10-phenanthroline, thiol compounds like 2-mercaptopropionic acid or sodium mercaptoacetic acid, and dipicolinic acid. Detection is based upon synergy between such inhibitors and a carbapenem (imipenem and/or meropenem) and/or an oximiminocephalosporin (ceftazidime) (Miriagou et al., 2010). However, the level of inhibition of the above-mentioned compounds is variable, and MBLs are inconstant in their ability to confer resistance to the commonly used screening compounds (imipenem and ceftazidime). Therefore, there is no perfect inhibitor that can detect all MBLs, and some of these carbapenemases can be missed especially in Enterobacteriaceae which are carbapenem intermediate or susceptible (Walsh et al., 2005). Tests utilizing MBL inhibitors were applied using various techniques. Lee et al. applied the double disc synergy test to investigate synergy between imipenem and a set of MBL inhibitors including EDTA and thiol compounds like mercaptopropionic acid and sodium mercaptoacetic acid, in IMP-1 and VIM-2 producing Pseudomonas and Acinetobacter strains. EDTA discs were the most accurate for detecting MBLproducing strains among Pseudomonas, while mercaptopropionic acid and sodium mercaptoacetic acid discs were preferable for Acinetobacter, suggesting that thiol compounds may be more promising to reduce false positive MBL detection in the latter organism. Combined discs of EDTA and sodium mercaptoacetic acid showed higher sensitivity compared to single inhibitor discs with both organisms (Lee et al., 2003). In another study, a combined disc with 10 μg imipenem plus 750 μg of EDTA differentiated all MBL-producing Pseudomonas, and the sensitivity and specificity for Acinetobacter were 95.7 and 91.0% respectively. The criterion for MBL production was an increase in inhibition zone diameter produced by discs with EDTA by at least 7 mm. Because the addition of EDTA to imipenem discs is a time-consuming process, the authors investigated stability of dried imipenem/EDTA discs at 4 and − 20 °C; the combined discs were stable without significant loss of activity

T

346 347

C

344 345

E

342 343

3.2.1. Inhibitors of Ambler class A carbapenemases Phenotypic tests were developed to detect class A carbapenemaseproducing strains in the presence of boronic acid compounds, usually the 3-aminophenylboronic acid (APBA). These compounds are activesite-directed serine-type β-lactamase inhibitors that are not based on a β-lactam structure like clavulanate and tazobactam. In fact, class A carbapenemases are not reliably detected by the inhibitors clavulanate and tazobactam, and boronic acid compounds represent a more sensitive method of inhibition (Thomson, 2010). For example, in a KPC-2-producing K. pneumoniae, the recommended CLSI confirmatory test utilizing clavulanate for the detection of Ambler class A carbapenemases yielded a positive result with ceftazidime only, while the use of clavulanate and boronic acid gave a positive result with cefotaxime, cefepime and carbapenems (Tsakris et al., 2008). In addition, the combined disc test using APBA has been applied very often, and evaluated as being better than the double-disc synergy test (Miriagou et al., 2010). It is proposed that an increase in inhibition zone diameter by 4–7 mm with meropenem discs with or without 400 μg of APBA is a cut-off value for the production of class A carbapenemases (Tsakris et al., 2009). In tests utilizing MIC, a three-fold or greater reduction of MIC of the carbapenem in the presence of 0.3 g/L of APBA was proposed as another cut-off value (Pasteran et al., 2009). The synergy between boronic acid and carbapenems in combined disc tests was used to detect the first KPC-producing K. pneumoniae in a Greek hospital (Tsakris et al., 2008). Later, Tsakris et al. evaluated boronic acid synergy with imipenem or meropenem using combined disc tests for a large panel of KPC-producing K. pneumoniae isolates with variable carbapenem MICs. A total of 57 KPC-producers yielded positive results, while 106 non-KPC producers were negative, indicating

R

340 341

- In the combined disc test, the inhibitor is added to a commercially available disc of carbapenem; the combined disc and a disc of the same carbapenem are placed on Muller–Hinton agar streaked with the test strain (Pasteran et al., 2009). The combination disc can be either prepared in-house and dried in air or purchased as a ready combination tablet (Rosco Diagnostica, Denmark). - In the double-disc synergy test (disc approximation method), discs of carbapenems are placed at variable distances from sterile discs impregnated with the inhibitor; the observation of synergy between the carbapenem and the inhibitor is considered a positive result (Pasteran et al., 2009). - Etest strips employing imipenem and imipenem combined with EDTA are available to test for metallo-β-lactamase activity (Queenan and Bush, 2007). - Observation of the reduction in carbapenem MIC, or an increase in carbapenem inhibition zone diameter, in the presence of an inhibitor in the culture medium is also a positive test for carbapenemases in agar dilution assays (Pasteran et al., 2009).

R

339

O

337 338

C

335 336

N

333 334

U

331 332

F

329 330

O

3.2. Inhibitor-based tests

R O

328

respectively a sensitivity and specificity of 100% with both imipenem and meropenem. When ertapenem was used, the differentiation was still correct; however, 5 Amp. producers gave false positive results decreasing specificity to 95.3% (Tsakris et al., 2009). Similarly, Giske et al. reported 100% sensitivity and 98% specificity of APBA in detecting KPC-producing K. pneumoniae; AmpC producers were sensitive to APBA, but were differentiated by synergy with cloxacillin, a commonly used AmpC inhibitor (Giske et al., 2011). In short, boronic acid testing is reported to be specific for KPC if performed with imipenem or meropenem, but not if performed with ertapenem in an isolate co-producing a plasmid-mediated AmpC β-lactamase (Tsakris et al., 2009). Other authors revealed that boronic acid compounds exhibit potent activity against a wide range of β-lactamase classes and may complicate the detection of carbapenemases (Pournaras et al., 2010). AmpC producers are sensitive to APBA, but are differentiated by inactivation with cloxacillin, a commonly used AmpC inhibitor (Giske et al., 2011). Unfortunately, boronic acid compounds inhibit also several class A β-lactamases, such as the chromosomal penicillinase of Bacillus cereus, and some of the CTX-M-type ESBLs leading to false positive results. In addition, it is worth mentioning that APBA-based assays failed to detect KPC-producing K. pneumoniae isolates in the case of coproduction of VIM enzyme (Giakkoupi et al., 2009). Therefore, following to the susceptibility testing, APBA inhibition assays using combined discs with imipenem and meropenem may be useful for the detection of class A carbapenemase-producers even though their specificity needs to be further evaluated (Miriagou et al., 2010).

P

326 327

antimicrobial susceptibility tests. It may be useful to incorporate this test within the infection control process as an add-on strategy which facilitates checking carbapenemase activity in outbreaks caused by suspected carbapenemase producers.

D

324 325


E

6


386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450


472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500

501 502 503 504 505 506 507 508 509 510 511 512 513 514

3.2.3. Inhibitors of Ambler class C carbapenemases The recognition of plasmid-mediated AmpC enzymes is considered necessary for therapeutic and infection control considerations (Jacoby, 2009). These enzymes affect a broad category of β-lactams including penicillins, oxyiminocephalosporins, cephamycins, and variably aztreonam, with the exception of ACC that does not confer resistance to cephamycins and is cefoxitin inhibited (Girlich et al., 2000). Plasmid mediation of AmpC carries, furthermore, the threat of spread to other organisms within a hospital or a geographic region (Jacoby, 2009). Therefore, because the plasmid-encoded AmpC cephalosporinases like DHA, ACC, and CMY may exist in carbapenem-nonsusceptible isolates with other resistance mechanisms, and because they may contribute to a greater risk of poor therapeutic outcome, their investigation is needed (Thomson, 2010). This is especially important in organisms

537

3.3. Detection using carbapenem-including culture media

552

The widespread dissemination of carbapenemase-producing Enterobacteriaceae in healthcare settings has emphasized the use of rapid surveillance methods (Adler et al., 2011). A commonly used, cheap and available screening technique is to culture clinical specimens, especially stool, on MacConkey's agar supplemented with 1 μg/mL of imipenem, rendering it selective for carbapenem-nonsusceptible strains (Samra et al., 2008; Adler et al., 2011). Several commercial chromogenic media are available for detection of carbapenemase-producing isolates; they contain chromogenic molecules that contribute to the recognition of enterobacterial species. A medium initially designed to screen for ESBL producers that contains cefpodoxime, the ChromID ESBL (BioMérieux, La Balme-les-Grottes, France), may be used to detect carbapenemase producers. This medium has good sensitivity for detection of IMP, VIM and KPC-type carbapenemases with high level of resistance to both cephalosporins and carbapenems. Its main disadvantage is its lack of detection of OXA-48-like producers that are susceptible to cefpodoxime in the absence of coproduction of an ESBL (Carrer et al., 2010). In addition, this medium is not specific for carbapenem-resistant bacteria, since widespread ESBL-producing isolates will be also selected (Nordmann et al., 2012b). To circumvent this issue, a disc of carbapenem may be applied on this medium to select carbapenemase producers. Another option is the use of a more selective medium developed for rapid detection of carbapenem-resistant Gram negative bacilli: the CHROMagar KPC (CHROMagar, Paris, France). This medium is supplemented with

553 554

O

F

3.2.4. Inhibitors of Ambler class D carbapenemases Class D carbapenemases are usually not inhibited by clavulanic acid, tazobactam, sulbactam, cloxacillin, boronic acid compounds or zinc chelators. It has been reported that the activities of class D β-lactamases maybe inhibited in vitro by sodium chloride at a concentration of 100 mM (Poirel et al., 2010). This feature is not shared by β-lactamases of other classes, thus defining it as a useful characteristic for in vitro identification (Poirel et al., 2004, 2005). However, this property has not been exploited in carbapenemase detection as a routine phenotypic test. The large extent of variability in amino acid sequences of the carbapenem-hydrolyzing class D enzymes like the OXA-48/OXA-181 Amp. cephalosporinases group has limited the development of specific inhibitor tests for their characterization, and molecular techniques remain the gold standard for their correct laboratory identification (Miriagou et al., 2010; Poirel et al., 2010; Nordmann et al., 2012c).

R O

470 471

P

468 469

515 516

D

466 467

that do not produce chromosomal AmpC cephalosporinases like Klebsiella, Salmonella, and Proteus mirabilis. Boronic acid and cloxacillin are the most commonly used AmpC inhibitors, and upon inhibition of AmpC cephalosporinases, they potentiate the activity of cephalosporins (Jacoby, 2009). In a study by Yagi et al., all AmpC varieties in K. pneumoniae and E. coli were reliably detected based upon at least 5 mm increase in the inhibition zone diameter when 300 μg of APBA was added to ceftazidime or cefotaxime discs (Yagi et al., 2005). Boronic acid inhibition tests were also useful to detect inducible and noninducible AmpC cephalosporinases in P. aeruginosa (Upadhyay et al., 2011). However, these inhibition tests need careful interpretation because boronic acid inhibits KPC enzymes, some ESBLs and OXA-12, and may inhibit the growth of certain bacterial strains (Mammeri et al., 2008; Thomson, 2010). The inhibition of AmpC cephalosporinases using cloxacillin can be accomplished either by incorporating cloxacillin powder in the growth medium to a final concentration of 200 μg/mL, or by the use of a cloxacillin disc. Combined Etest strips with a gradient of cefotetan or cefoxitin on one half and the same combined with fixed concentration of cloxacillin on the other half have also been used (Jacoby, 2009). Compared to APBAbased tests, cloxacillin inhibitor tests do not exhibit problems of false positive results with strains harboring KPC or ESBL (Ruppe et al., 2006).

E

464 465

T

462 463

C

460 461

E

458 459

R

457

R

455 456

N C O

453 454

after 12 and 16 weeks respectively (Yong et al., 2002). In a recent investigation comparing various phenotypic methods for detection of NDM in 27 NDM-positive enterobacterial isolates, 25 isolates were positive in the imipenem/imipenem-EDTA Etest strip, while all 27 isolates were positive in a combined disc of EDTA and imipenem. The authors suggested that the combined disc method is to be applied for routine detection of MBL producers, while the combined Etest is more suitable for laboratories which do not screen MBLs on a daily basis, or which perform susceptibility testing in liquid media and rarely apply disc diffusion techniques (Nordmann et al., 2011b). Moreover, EDTA synergy tests do have few limitations and are inappropriate for detection of MBLs in Enterobacteriaceae with low imipenem resistance (MIC ≤ 4 mg/L) (Yan et al., 2004). In A. baumannii, some studies have reported failure of EDTA inhibition tests to detect MBLs (Ikonomidis et al., 2008; Loli et al., 2008), while others have reported possibility of false positive results in strains that produce OXA-23, due to possibility of conversion of this enzyme into a less active monomeric form in presence of EDTA (Segal and Elisha, 2005). Furthermore, it has been observed that EDTA itself can inhibit some bacteria due to increased permeability of the outer membrane, leading to false positive results (Chu et al., 2005). In general, it is necessary to test the intrinsic activity of the chelator alone to avoid false-positive results in case it inhibits the tested isolate (Ratkai et al., 2009). The addition of zinc to the culture media to test MBL production in VIM-2 producing P. aeruginosa was addressed in a study by Giakkoupi et al. (2008). Zinc sulfate, in a final concentration of 70 mg/L, was incorporated into the Muller–Hinton agar. This improved sensitivity of phenotypic MBL detection methods using Etest, double-disc synergy test and combined disc test with imipenem and EDTA to 100, 96 and 67% respectively. The result was explained by possible enhanced formation of functional MBL molecules in the periplasmic space of bacteria, as these enzymes are dependent on zinc for their activity. Also, the relatively high Zn2 + concentrations during growth reduce expression of P. aeruginosa porins like OprD, and consequently carbapenem diffusion rates, further enhancing the effects of carbapenemase activity. This study warranted more intensive investigation of the role of zinc as an adjunct for MBL detection tests in P. aeruginosa. The early detection of MBL-producers is essential for infection control and prevention of dissemination. However, the inhibitor-based detection tests are not yet standardized, and there is no single inhibitor or method that proved appropriate for all circumstances and for different bacteria. While EDTA may prove a promising inhibitor for MBL detection in Enterobactereiaceae and Pseudomonas species, thiol derivatives appear more effective in A. baumannii. Moreover, while the disc synergy methods are sensitive to detect MBLs, the Etest is more practical but not cost-effective in health care institutions where the prevalence of MBL producers is uncommon. The selection of the appropriate inhibitor and methodology of detection of MBLs, therefore, should be done depending upon the characteristics of MBL producers in a particular setting.

U

451 452

7


517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536

538 539 540 541 542 543 544 545 546 547 548 549 550 551

555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577

601 602 603 604 605 606 607 608 Q5 609 610 611 612 613 614 615 616 617 618 619 620 621 Q6 622 623 624 625 Q7 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643

672

4.1. Isoelectric focusing

673

This is a type of electrophoresis where proteins are separated into bands based on their isoelectric point. The electrophoresis is performed on an immobilized gel, composed of polyacrylamide, agarose, or starch, where a pH gradient has been established. A high degree of resolution is obtained by the method, because focusing of the proteins is caused by forces that act against diffusion and proteins are therefore concentrated during their separation. This technique has been previously used for the separation of β-lactamases (Mathew et al., 1975). The localization of β-lactamases on the gel is accomplished after electrophoresis, by overlaying the gel with filter papers soaked in 0.7 mg/mL of the chromogenic cephalosporin nitrocephin, which undergoes a color change upon hydrolysis by a β-lactamase. An overlay of the gel with clavulanic acid or EDTA can detect sensitivity of the enzymes to these potential inhibitors, indicating classes Ambler A or B respectively (Queenan and Bush, 2007). Another method to measure carbapenemase activity by isoelectric focusing is the use of two consecutive overlays: an overlay of Mueller– Hinton agar with imipenem and another with a susceptible indicator organism like E. coli ATCC 25922. After overnight incubation, the growth of colonies of this organism over an enzyme band localizes a potential carbapenemase (Schneider et al., 2006) (Lee et al., 2002). Such procedure was previously utilized upon characterization of the MBL AsbM1 from Aeromonas sobria, a carbapenemase having weak hydrolysis rates with nitrocefin (Yang and Bush, 1996). Isoelectric focusing is a possible method to detect variants of KPC with different hydrolytic properties (Wolter et al., 2009). In a previous study by Wolter et al., isoelectric focusing was used to characterize a new KPC enzyme in P. aeruginosa, KPC-5. The isoelectric point of KPC5 was similar to that of KPC-4, an evolutionary variant of KPC-5, while another variant, KPC-2, had a different isoelectric point. By further analysis, these data were correlated with a higher resistance to imipenem, a lower hydrolysis of ceftazidime, and a less potent inhibition by clavulanic acid in the KPC-2 producing isolates.

674

O

F

4. Analytical and biochemical detection methods

R O

599 600

P

597 598

644 645

D

595 596

A selective and chromogenic agar medium, CHROMagar Acinetobacter (CHROMagar, Paris, France), has been developed for the rapid identification of multi-drug resistant A. baumannii. It contains agents that inhibit the growth of Gram-positive organisms, yeasts, and carbapenemsusceptible Gram negative bacilli, and incorporates substrates enabling color-based identification of colonies (Acinetobacter appears in red color in opposition to non-inhibited Gram negative strains). In a study investigating A. baumannii in fecal and perianal swabs, this medium was about 90% sensitive and specific in detecting A. baumannii with OXA-type carbapenemases (Gordon and Wareham, 2009). More recently, modification of the CHROMagar Acinetobacter via the addition of liquid “K. pneumoniae carbapenemase supplement” (KPC: CHROMagar, Paris, France), also called CR102, improved the medium for specifically selecting only carbapenem-resistant A. baumannii instead of all MDR isolates (Wareham and Gordon, 2011). Commercially available carbapenem-containing media are imperfect to differentiate between carbapenem resistance due to inactivation by enzymes or due to other resistance mechanisms (Moran Gilad et al., 2011). SUPERCARBA medium has a slight advantage over other media since theoretically, it inhibits carbapenem-resistant but noncarbapenemase-producing organisms (Nordmann et al., 2012b). In fact, the phenotypic tests described above are not satisfactorily sharp to allow clear differentiation between carbapenemase classes or between the plasmid-encoded and chromosomal AmpC enzymes. Therefore, the current “gold standard” for such discrimination is through molecular methods, as explained below in Section 5. Nevertheless, biochemical and analytical diagnostics have found their application in microbiological detection of carbapenemases, as explained below.

T

593 594

C

591 592

E

589 590

R

587 588

R

585 586

O

584

C

582 583

N

580 581

carbapenems to inhibit the growth of Gram positive/Gram negative carbapenem-sensitive bacteria (Samra et al., 2008). The cultures present different colors that allow differentiation of various organisms, for instance, metallic blue for K. pneumoniae, pink to red for E. coli, and transparent or yellow to green for P. aeruginosa (Merlino et al., 1996). In one study, the performance of CHROMagar in detection of KPC in K. pneumoniae was compared to PCR for blaKPC genes and to MacConkey's agar supplemented with carbapenem discs (Samra et al., 2008). The sensitivity and specificity relative to PCR were 100% and 98.4%, respectively, for CHROMagar KPC and 92.7% and 95.9%, respectively, for MacConkey's agar. CHROMagar KPC allows recovery of highlevel-resistant carbapenemase-producing Enterobacteriaceae harboring KPC, IMP, or VIM, but its sensitivity is limited for isolates with MICs less than 4 μg/mL (Carrer et al., 2010). A suboptimal performance of CHROMagar KPC was reported in a study by Gilad et al., where it showed 100% sensitivity in detection of KPC-producing K. pneumoniae and E. cloacae. However, it exhibited only 71.4% sensitivity in detection of KPC-producing E. coli, and 72.7% sensitivity in recovery of KPC-negative K. pneumoniae which were resistant to ertapenem due to porin loss combined with ESBL production. The Brilliance CRE medium (Oxoid, Thermofisher Scientific, Illkick, France) is another commercially available solid medium supplemented with a carbapenem and capable of inhibiting the growth of carbapenem-sensitive bacteria (Bracco et al., 2013). The presumptive identification of carbapenem resistant colonies is dependent on variable colony pigmentation. In a recent study, the sensitivity of this medium in recovery of carbapenem resistant Enterobacteriaceae was 94%, but differed per carbapenemase type (100% for KPC, NDM, and GIM, 90% for VIM, and 84% for OXA-48-producing isolates). The specificity of the medium was 71%, because it did allow growth of AmpC- and/or ESBLproducing isolates (Cohen Stuart et al., 2013). Hence, this medium is efficient in the detection of KPC and MBL-producing Enterobacteriaceae, but demonstrates low specificity by allowing growth of non-carbapenemase producers. Another prototype medium designated for detection of carbapenemase-producing Enterobacteriaceae is chromID Carba (bioMérieux, La Balme-les-Grottes, France). This medium inhibits growth of Gram-positive organisms and carbapenem sensitive bacteria, and yields specific colony colors allowing differentiation of carbapenem non-susceptible E. coli, Klebsiella spp., Enterobacter spp., Serratia spp., and Citrobacter spp. In a rectal swab surveillance study, chromID Carba exhibited 92% sensitivity and 97% specificity in detecting KPCpositive and VIM-positive K. pneumoniae, and KPC-positive Enterobacter aerogenes strains (Vrioni et al., 2012). The recently developed SUPERCARBA medium consists of Drigalski agar supplemented with ertapenem, zinc sulfate to enhance expression of MBL producers, and cloxacillin to inhibit AmpC natural producers such as E. cloacae, E. aerogenes, Morganella morganii, and Serratia marcescens (Nordmann et al., 2012b). The species that naturally produce AmpC enzymes are clinically significant sources of carbapenem resistance associated with an outer membrane permeability defect, and their inhibition increases specificity of the medium in detecting carbapenemase producers. Plus, this medium is sensitive for detection of carbapenemase producers with low-level resistance, including KPC, VIM, IMP, as well as OXA-48 producers. In a study by Girlich et al., comparing SUPERCARBA medium to CHROMagar KPC and Brillance CRE agar, although the SUPERCARBA medium exhibited the highest sensitivity (96.5%), its specificity was lower than that of CHROMagar KPC (60.7 versus 67.8%) due to detection of non-carbapenemase producing isolates (Girlich et al., 2013). Nevertheless, the SUPERCARBA medium provides a significant improvement for detection of the most common types of carbapenemase producers, is easy to implement, and is cost-effective. It is also warranted that this medium could be further improved by the addition of chromogenic molecules for presumptive identification of enterobacterial species.

U

578 579


E

8


646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671

675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706


717 718

4.2.1. UV-spectrophotometry Measurement of carbapenem hydrolysis, indicating carbapenemase activity, can be reliably detected using a UV spectrophotometer, and this method is available in many reference laboratories (Miriagou et al., 2010). An 18 hour culture of the tested strain is centrifuged, subject to protein extraction, and either the crude cell extracts or the purified β-lactamases are mixed with imipenem at a final concentration of 150 μmol/L. Hydrolysis of imipenem is then quantified using a UV spectrophotometer at a wavelength of 297 nm (Bernabeu et al., 2012). This method exhibited 100% sensitivity and 98.5% specificity for detecting carbapenemase activity in a study by Bernabeu et al. KPC producers gave higher rates of imipenem hydrolysis than OXA-48 and MBL producers (Bernabeu et al., 2012). The technique is cheap and can detect any type of carbapenemase; it can also differentiate carbapenemase producers from non-carbapenemase producers having combined mechanisms of resistance (e.g., outermembrane permeability defect, overproduction of cephalosporinases, ESBLs) and from strains expressing broad spectrum ß-lactamases without carbapenemase activity (ESBLs, plasmid and chromosome-encoded cephalosporinases) (Bernabeu et al., 2012; Nordmann and Poirel, 2013). The technique, however, is time-consuming, technically-demanding and laborious, so should be performed only in reference laboratories (Miriagou et al., 2010). Also, it cannot indicate types of carbapenemases or detect carbapenemases with very weak activity unless large amounts of cell extracts are used (Queenan and Bush, 2007).

731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768

C

729 730

E

727 728

R

725 726

4.2.2. Mass spectrometry Matrix-assisted laser desorption ionization-time of flight (MALDITOF) mass spectrometry (MS) has been recently introduced into the diagnostic microbiology laboratory (Nordmann and Poirel, 2013). It has become routinely used for identification of bacteria and fungi, and is being validated for detection of antibiotic resistance, focusing on the group of β-lactam antibiotics, especially carbapenems (Alvarez-Buylla et al., 2013). The basic MALDI-TOF MS principle relies upon the detection of antibiotic degradation products by bacterial hydrolyzing enzymes, mixed with a suitable matrix which has different mass spectra than both the antibiotic and the degradation product (Hrabak et al., 2013). The detection of carbapenemase activity is performed using a fresh overnight bacterial culture, suspended in a buffer, and centrifuged, and the pellet is resuspended in a reaction buffer containing the carbapenem molecule. After incubation for 1 to 3 h at 35 °C, the mixture is centrifuged and the supernatant is mixed with a proper matrix and measured by MALDI-TOF MS. Spectra corresponding to the intact carbapenem molecule and/or its degradation products are then analyzed (Hrabak et al., 2013). MALDI-TOF MS was able to detect meropenem and its hydrolysis products in a study involving 124 Gram-negative carbapenemnonsusceptible isolates of which 30 were carbapenemase-producers. The sensitivity and specificity of the technique were above 95%, and it did detect VIM-2 and IMP-7 in P. aeruginosa, and VIM-1, KPC-2 and NDM-1 in Enterobacteriaceae (Hrabak et al., 2011). Also, significant peaks for imipenem were obtained in another study involving IMPand OXA-producing Acinetobacter species (Alvarez-Buylla et al., 2013).

R

723 724

N C O

721 722

U

719 720

4.3. Carba NP test

793

F

4.2. Spectrophotometric detection

T

716

O

714 715

R O

713

769 770

P

711 712

Although the initial costs of MALDI-TOF MS are relatively high, the cost of identifying one carbapenemase-producing isolate remains low compared to the cost of systematic biochemical or molecular genetic techniques (Wieser et al., 2012). Another advantage of the MALDI-TOF MS is reduction of the turnover time needed to obtain results till an average of about 4 h if a fresh bacterial culture is available (Hrabak et al., 2013). Despite such advantages, the MALDI-TOF MS exhibits some limitations which confine its use in routine laboratories; the interpretation of the spectra needs special skills, and the identification of the carbapenemase type is not possible. Given this, it should be accompanied with genetic techniques for epidemiological purposes (Hrabak et al., 2013). MALDI-TOF MS may not be suitable to detect carbapenemase activity in certain samples that require an enrichment step, like surveillance swabs, urine or wound specimens. Recently, MS was combined with liquid chromatography to detect carbapenem hydrolysis upon incubation with carbapenemase-producing bacteria in enrichment broth. A set of 402 Gram negative isolates, including both Enterobacteriaceae and non-Enterobacteriaceae, and expressing carbapenemases of class A, B, or D, were analyzed by this method. It was possible to detect intact and hydrolyzed carbapenems, with a high performance for imipenem, and reducing the time needed for conventional phenotypic methods by about 18 h (Kulkarni et al., 2014).

D

709 710

Although isoelectric focusing cannot specifically identify a carbapenemase, it can give sufficient data on isoelectric point and inhibition characteristics, and is especially valuable for separation and detection of multiple β-lactamases present in the same isolate (Queenan and Bush, 2007). This technique concentrates carbapenemases at their isoelectric points and allows them to be separated on the basis of very small charge differences. It is a sensitive method that can allow comparison of patterns of carbapenemase production by various organisms without a need for purification of crude cellular extracts.

This is a recent biochemical detection method of carbapenemases in Enterobacteriaceae, and Pseudomonas. The principle is based upon hydrolysis of the β-lactam ring of imipenem by the tested strain, followed by color change of a pH indicator, usually phenol red from red to yellow/orange (Nordmann et al., 2012d). The test differentiates carbapenemase producers from isolates resistant via other mechanisms, such as outer membrane permeability and/or production of cephalosporinases and/or ESBLs as well as from carbapenemsusceptible strains with non-carbapenem hydrolyzing ESBL, plasmidencoded or chromosome-encoded cephalosporinases (Nordmann et al., 2012d). The specificity and sensitivity of the test are reported to be 100 and 94.4% respectively in a survey of Pseudomonas, while it was 100% specific and sensitive in a survey of Enterobacteriaceae (Dortet et al., 2012; Nordmann et al., 2012d). The test requires less than 2 h, and eliminates the need of other in vitro phenotypic tests like the modified Hodge test or the inhibitor-based techniques. Not only the test detects all known carbapenemases of Ambler classes A, B, and D, but also identifies virtually any newly emerging carbapenemase, in contrast to molecular methods. It is also cheap and has no requirement for special equipment (Nordmann and Poirel, 2013). However, the test cannot differentiate among carbapenemase classes and does not detect GES-type carbapenemases which have a rather weak carbapenemase activity; this should be considered in areas where these enzymes have a high prevalence like Brazil and South Africa (Dortet et al., 2012). Furthermore, in a recent Canadian study on carbapenemase-producing Enterobacteriaceae and Pseudomonas, and although the Carba NP test was 100% sensitive, it was only 80% specific; false-negative results correlated with enzymes displaying low carbapenemase activity, particularly OXA-48-like, which has emerged globally in Enterobacteriaceae (Tijet et al., 2013). A recent study described an updated version of the Carba NP test, called the CarbAcineto NP test, intended to detect carbapenemases in Acinetobacter spp. Applying the same principle as the original test, the CarbAcinetobacter NP test utilizes modified lysis conditions and an increased inoculum size. In contrast to the Carba NP test which detects only MBLs among Acinetobacter spp., the newly designated test can detect all carbapenemase types in this organism and its sensitivity and specificity are very high (Dortet et al., 2014).

E

707 708

9


771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792

794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831


874

5.1. Carbapenemase detection by PCR-sequencing

875 876

PCR is nowadays becoming a routine method in many clinical laboratories to circumvent the difficulties associated with phenotypic detection (Miriagou et al., 2010). A PCR technique performed on genomic DNA can give results within 4–6 h, and may be followed by sequencing if needed for precise identification of a carbapenemase variant, rather than just its group (e.g. VIM-type, KPC-type, NDM-type, and OXA-type) (Poirel et al., 2011b). PCR can be single, multiplex, or real-time and a set of recently published primers and standardized cycling conditions are available to allow easy and relevant recognition of clinically relevant carbapenemase genes (Dallenne et al., 2010; Poirel et al., 2011b). Multiplex PCR for plasmid mediated AmpC detection has been developed with six primer pairs to detect genes for MOX, CMY, LAT, BIL, DHA, ACC, MIR, ACT, and FOX (Perez-Perez and Hanson, 2002). A seventh pair has been as well added to allow detection of CFE-1 (Nakano et al., 2004). Apart from such “in-house” assays, commercially available PCR kits are also available. Some of these, like the Check-MDR CT102 DNA microarray is based on DNA amplification followed by amplicon detection in a tube microarray; this offers the advantage of detection of multiple genes in a single test. In a study by

854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870

877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893

C

852 853

E

850 851

R

848 849

R

846 847

O

844 845

C

842 843

N

840 841

U

838 839

F

5. Molecular detection of carbapenemase genes

837

O

873

835 836

R O

871 872

Immunodiagnostic assays including ELISA, immunofluorescence, and immunochromatography rely on specific antigen–antibody reactions to obtain accurate results, and are often used in infectious disease diagnosis. Among these, immunochromatography finds common application due to promptness and ease of handling (Kitao et al., 2011). Immunochromatography is based on an immunological reaction carried out on chromatographic paper by capillary action, and two types of specific antibodies against an antigen (in this case the carbapenemase molecule) are used. One of the antibodies is immobilized on the chromatographic paper, and the other is labeled with colloidal gold and infiltrated into a sample pad. An immunochromatographic unit is completed by attaching the sample pad at the end of the membrane. When a specimen is dropped on the sample pad, the antigen in the specimen forms an immunocomplex with the gold-labeled antibody. This complex moves along with the liquid sample, contacts the antibody immobilized on the membrane, and forms an immuno-complex that results in color change (Koivunen, 2006). Two independent research groups in Japan have recently utilized an immnuchromatographic commercially available diagnostic kit to detect carbapenemases. Using this kit, Kitao et al. detected IMP-type MBLs in P. aeruginosa, and the results were fully consistent with molecular data (Kitao et al., 2011). Notake et al. detected different varieties of IMP enzymes in Enterobacteriaceae and non-glucose-fermenting Gram negative rods with a specificity and sensitivity of 100% (Notake et al., 2013). It is suggested that this newly developed assay can substitute molecular detection of carbapenemases, and is reliable, sensitive, and easy to use, warranting the likelihood of its crucial role in infection-control measures and epidemiological research. Nevertheless, it is specific for type of carbapenemase but not for subtype. For example, it can differentiate IMP from VIM or NDM but cannot differentiate IMP-1 from IMP-2 or IMP-7, nor can identify novel mutations of target genes (Kitao et al., 2011). Despite their applicability, the analytical and biochemical detection methods demonstrate a number of limitations. The most important limitation is the specific identification and differentiation of carbapenemases, only possible by molecular techniques (Nordmann et al., 2012c). Although this precise identification of carbapenemases is not crucial for selection of patient therapy or containment of outbreaks, it may be interesting for research and epidemiological purposes (Nordmann and Poirel, 2013).

P

833 834

Stuart et al., this system had 100% specificity and 97% sensitivity in detecting KPC, NDM, VIM, OXA-48 and VIM plus KPC producing enterobacterial isolates whose MICs of meropenem were greater or equal to 0.5 mg/L (Stuart et al., 2012). Another study reported sensitivity and specificity values for carbapenemase detection by this array to be 100% and 95.7% respectively for 41 carbapenemase-producing enterobacterial strains with a similar set of enzymes (Woodford et al., 2011). In another type of commercial amplification kits, enzyme-linked immunosorbent assay (ELISA) follows carbapenemase gene amplification (Gazin et al., 2012). In the hyplex®-MBL ID Multiplex PCR-ELISA, bacterial DNA is amplified by multiplex PCR, immobilized on polystyrene-ELISA plates, hybridized to specific oligonucleotide probes, and the hybridized complexes are detected by a peroxidase conjugated antibody resulting in color change. In a Greek study, this method showed 98% sensitivity and 98.6% specificity in detection of VIMproducers among P. aeruginosa, A. baumannii, and Enterobacteriaceae (Avlami et al., 2010). The required time for the utilization of the hyplex®-MBL ID Multiplex PCR-ELISA method is 4.5–6 h (Ambretti et al., 2013). A loop-mediated isothermal amplification (LAMP) method can be used for the rapid and sensitive detection of NDM gene (Qi et al., 2012). This method facilitates genetic testing for rapid identification of bacteria and viruses, and has been recently evaluated for detection of carbapenemase genes as well. LAMP consists of an auto-cycling strand displacement DNA synthesis performed by the Bst DNA polymerase, with 4 to 6 primers recognizing 6 to 8 distinct regions of the target gene and generating the loop-mediated amplification under isothermal conditions ranging from 60 °C to 65 °C for about 60 min, resulting in large amounts of amplification products with many types of structures. Compared to PCR, LAMP is more specific and quicker to perform. Furthermore, gel electrophoresis is not required, because the LAMP method synthesizes large amounts of DNA where products can easily be detected by turbidity or fluorescence (Notomi et al., 2000; Chen and Cui, 2009). In a recent Chinese study, LAMP assays were used for NDM-1 gene detection and exhibited good specificity and higher sensitivity than the conventional PCR, with a detection limit of 1 pg genomic DNA per tube of NDM-1-positive reference strain. The detection result for 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. LAMP method demonstrated a potential and valuable means for detection of NDM-1 gene (Qi et al., 2012). Cunningham et al. reported the use of a real time PCR assay that applies fluorescence resonance energy transfer (FRET) hybridization probe based detection of KPC and NDM-encoding genes simultaneously. In 134 carbapenemase producing Gram negative bacilli, the assay was 100% sensitive and specific and it had a turnaround time of 90 min from colony to results (Cunningham et al., 2013). Also, another realtime PCR with SYBR Green detection was developed for screening of KPC-producing organisms in stool specimens and nasal swabs. To avoid inhibition of PCR in these specimens, the assay included an internal control formed of oligonucleotides that can be amplified by the KPC primers, but displays a lower melting temperature than the amplified KPC gene. This assay was sensitive and specific, can be performed in less than 2 h, and is at least two-times less expensive than real time PCR with probes, due to the low cost of the fluorescent molecule SYBR Green (Wang et al., 2012). The main disadvantages of the PCR-based methods are their cost, the requirement for high degree of expertise, and the inability to detect novel unidentified genes (Nordmann et al., 2012c). The high diversity of carbapenemase-encoding genes and the escalating number of new variants imply that a negative PCR result concerning a carbapenem resistant strain in a local laboratory setting requires re-evaluation by a reference laboratory for further genotypic analysis (Cohen Stuart and Leverstein-Van Hall, 2010).

D

4.4. Immunochromatography

T

832

E

10


894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 Q8 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959


Epidemiological information on the clonality of carbapenemproducing isolates is needed for surveillance programs and helps in identification of high-risk clones that have potential of dissemination. Molecular typing should be done not only in outbreak conditions, but also on sporadic isolates which should be compared with epidemic strains (Miriagou et al., 2010). In low resource settings, analysis of clonal relatedness of strains may be performed using Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR. In this technique, consensus primers are utilized in PCRs to amplify ERIC sequences then fingerprints are produced for different bacterial genomes (Versalovic et al., 1991; Ferreira et al., 2011). Pulsed field gel electrophoresis (PFGE) is a more accurate tool to establish clonality. The principles of this technique depend upon cutting bacterial genomes by specific restriction enzymes, and separating the resulting large DNA fragments using an electric field switched periodically between different directions. The resulting patterns can then be interpreted using well-accepted criteria to establish the degree of relatedness (Tenover et al., 1995). PFGE revealed spread of highly related strains of A. baumannii in Turkey and Enterobacteriaceae in New York while variable clonal lineages in OXA-producing A. baumannii in Sweden and Iran were described (Deshpande et al., 2006; Meric et al., 2008; Karah et al., 2011; Shahcheraghi et al., 2011). Despite using PFGE, multilocus sequence typing (MLST) method appears nowadays to be more appropriate. The MLST procedure characterizes isolates of a given microbial species using DNA sequences of multiple housekeeping genes. Approximately 450–500 bp internal fragments of each of these genes are used, as these can be accurately sequenced using an automated DNA sequencer. For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles. For each isolate, the alleles at all loci define the allelic profile or sequence type (ST), obtained by importing sequences into MLST database. In areas with long-term persistence of carbapenemase-producing isolates, MLST offers the opportunity of tracking clones and the exchange of allelic information among different geographic areas. In areas with low prevalence, MLST helps to identify emergence of highly epidemic clones associated with specific carbapenemases (Miriagou et al., 2010). An example of such clones is P. aeruginosa ST235, known to produce VIM-1 enzyme although previously recognized to produce PER-1 ESBL (Empel et al., 2007; Libisch et al., 2008). MLST was also used to type NDM-producing enterobacterial isolates from Switzerland, and revealed a link with isolates detected in India or the Balkans (Poirel et al., 2011a). In another study, carbapenem-resistant A. baumannii isolated from Korean hospitals, were split by MLST into 2 main clones designated by ST22 and ST28, which were clearly distinct in types of OXA and ADC carbapenemases and in antimicrobial susceptibility profiles (Park et al., 2009). Data on ST are available via certain public databases like the Institut Pasteur MLST database and the PubMLST developed by University of Oxford.

985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022

C

983 984

E

981 982

R

979 980

R

977 978

N C O

970 971

U

968 969

1027

Because bacterial resistance to carbapenems is increasingly reported, the diagnostic microbiology laboratory has a pivotal role in optimal detection of carbapenemase-producing isolates in order to aid in therapy selection and improve infection control. The techniques used to detect carbapenemase-producing Gram-negative pathogens are diverse and abundant. While regular screening techniques and inhibitor-based tests are easy and affordable to implement in routine settings, their sensitivity and specificity remain inconclusive. On the other hand, novel culture media and biochemical tests have demonstrated the ability to detect carbapenemase-producers with high efficiency. Genotypic techniques are the reference methods to precisely confirm carbapenemase genes, and are mostly used in reference laboratories. The current knowledge on carbapenemase detection should be updated regularly due to ongoing technical developments and the emergence of new carbapenemase variants.

1028

F

975 976

966 967

6. Conclusion

O

5.3. Molecular analysis of clonal relatedness

965

R O

974

963 964

Funding

P

972 973

Because gene amplification with PCR can only identify carbapenemase genes with predetermined sequences, identification of new carbapenemase genes requires initial gene cloning and sequencing. In cloning experiments, genomic DNA from bacterial strains is cut by restriction enzymes and then allowed to hybridize with a plasmid that has been cut via the same enzyme to create “sticky ends”. When the fragment of DNA is joined with the cloning vector, the resulting recombinant DNA molecule harboring the carbapenemase gene can be used to transform competent bacterial cells, in which the new gene can be reproduced along with the recipient cell's DNA and purified before sequencing. Several studies have utilized cloning experiments to identify new carbapenemase genes (Mugnier et al., 2008; Moubareck et al., 2009; Won et al., 2011; Bogaerts et al., 2012).

1023 1024 1025 1026

1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043

This work was supported by the Saint-Joseph University Research 1044 Council (project No. FPH37) and by the Lebanese National Council for 1045 Research and Development (project No. 02-09-12). 1046

D

961 962

These databases tend to provide a common language on microbial strain typing, epidemiology and evolution. MLST designates a highly discriminative typing method for purposes of regional and global epidemiologic analysis and investigation of clonal spread.

E

5.2. Cloning and sequencing of new genes

T

960

11

References

1047

Abbott, I., Cerqueira, G.M., et al., 2013. Carbapenem resistance in Acinetobacter baumannii: laboratory challenges, mechanistic insights and therapeutic strategies. Expert Rev. Anti-Infect. Ther. 11 (4), 395–409. Adler, A., Navon-Venezia, S., et al., 2011. Laboratory and clinical evaluation of screening agar plates for detection of carbapenem-resistant Enterobacteriaceae from surveillance rectal swabs. J. Clin. Microbiol. 49 (6), 2239–2242. Alvarez-Buylla, A., Picazo, J.J., et al., 2013. Optimized method for Acinetobacter species carbapenemase detection and identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 51 (5), 1589–1592. Ambretti, S., Gaibani, P., Berlingeri, A., Cordovana, M., Tamburini, M.V., Bau, G., Landini, M.P., Sambri, V., 2013. Evaluation of phenotypic and genotypic approaches for the detection of class A and class B carbapenemasescin Enterobacteriaceae. Microb. Drug Resist. Anderson, K.F., Lonsway, D.R., et al., 2007. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J. Clin. Microbiol. 45 (8), 2723–2725. Avlami, A., Bekris, S., et al., 2010. Detection of metallo-beta-lactamase genes in clinical specimens by a commercial multiplex PCR system. J. Microbiol. Methods 83 (2), 185–187. Bernabeu, S., Poirel, L., et al., 2012. Spectrophotometry-based detection of carbapenemase producers among Enterobacteriaceae. Diagn. Microbiol. Infect. Dis. 74 (1), 88–90. Bogaerts, P., Bebrone, C., et al., 2012. Detection and characterization of VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in a clinical isolate of Enterobacter cloacae. Antimicrob. Agents Chemother. 56 (6), 3283–3287. Bracco, S., Migliavacca, R., et al., 2013. Evaluation of brilliance CRE agar for the detection of carbapenem-resistant Gram-negative bacteria. New Microbiol. 36 (2), 181–186. Bratu, S., Landman, D., et al., 2005a. Detection of KPC carbapenem-hydrolyzing enzymes in Enterobacter spp. from Brooklyn, New York. Antimicrob. Agents Chemother. 49 (2), 776–778. Bratu, S., Mooty, M., et al., 2005b. Emergence of KPC-possessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection. Antimicrob. Agents Chemother. 49 (7), 3018–3020. Carrer, A., Fortineau, N., et al., 2010. Use of ChromID extended-spectrum beta-lactamase medium for detecting carbapenemase-producing Enterobacteriaceae. J. Clin. Microbiol. 48 (5), 1913–1914. Chen, C.M., Cui, S.J., 2009. Detection of porcine parvovirus by loop-mediated isothermal amplification. J. Virol. Methods 155 (2), 122–125. Chu, Y.W., Cheung, T.K., et al., 2005. EDTA susceptibility leading to false detection of metallo-beta-lactamase in Pseudomonas aeruginosa by Etest and an imipenemEDTA disk method. Int. J. Antimicrob. Agents 26 (4), 340–341. CLSI, 2014. Performance Standards for Antimicrobial Suceptibility Testing: Twenty-first Informational Supplement, (Wayne, PA, USA). Cohen Stuart, J., Leverstein-Van Hall, M.A., 2010. Guideline for phenotypic screening and confirmation of carbapenemases in Enterobacteriaceae. Int. J. Antimicrob. Agents 36 (3), 205–210.

1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 Q9 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092


D

P

R O

O

F

Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and multidrugresistant Pseudomonas aeruginosa. Mayo Clin. Proc. 86 (3), 250–259. Karah, N., Giske, C.G., et al., 2011. A diversity of OXA-carbapenemases and class 1 integrons among carbapenem-resistant Acinetobacter baumannii clinical isolates from Sweden belonging to different international clonal lineages. Microb. Drug Resist. 17 (4), 545–549. Kitao, T., Miyoshi-Akiyama, T., et al., 2011. Development of an immunochromatographic assay for diagnosing the production of IMP-type metallo-beta-lactamases that mediate carbapenem resistance in Pseudomonas. J. Microbiol. Methods 87 (3), 330–337. Koivunen, M.E.K.R., 2006. Principles of immunochemical techniques used in clinical laboratories. Lab. Med. 37 (8), 8. Kulkarni, M.V., Zurita, A.N., et al., 2014. Use of Imipenem to detect KPC, NDM, OXA, IMP, and VIM carbapenemase activity from Gram-negative rods in 75 minutes using liquid chromatography–tandem mass spectrometry. J. Clin. Microbiol. 52 (7), 2500–2505. Lee, K., Chong, Y., et al., 2001. Modified Hodge and EDTA-disk synergy tests to screen metallo-beta-lactamase-producing strains of Pseudomonas and Acinetobacter species. Clin. Microbiol. Infect. 7 (2), 88–91. Lee, K., Lim, J.B., et al., 2002. bla(VIM-2) cassette-containing novel integrons in metallobeta-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital. Antimicrob. Agents Chemother. 46 (4), 1053–1058. Lee, K., Lim, Y.S., et al., 2003. Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-beta-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp.. J. Clin. Microbiol. 41 (10), 4623–4629. Lee, K., Kim, C.K., et al., 2010. Improved performance of the modified Hodge test with MacConkey agar for screening carbapenemase-producing Gram-negative bacilli. J. Microbiol. Methods 83 (2), 149–152. Libisch, B., Watine, J., et al., 2008. Molecular typing indicates an important role for two international clonal complexes in dissemination of VIM-producing Pseudomonas aeruginosa clinical isolates in Hungary. Res. Microbiol. 159 (3), 162–168. Livermore, D.M., Woodford, N., 2006. The beta-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol. 14 (9), 413–420. Loli, A., Tzouvelekis, L.S., et al., 2008. Outbreak of Acinetobacter baumannii with chromosomally encoded VIM-1 undetectable by imipenem-EDTA synergy tests. Antimicrob. Agents Chemother. 52 (5), 1894–1896. Mammeri, H., Eb, F., et al., 2008. Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered in a French hospital. J. Antimicrob. Chemother. 61 (3), 498–503. Mathew, A., Harris, A.M., et al., 1975. The use of analytical isoelectric focusing for detection and identification of beta-lactamases. J. Gen. Microbiol. 88 (1), 169–178. Meric, M., Kasap, M., et al., 2008. Emergence and spread of carbapenem-resistant Acinetobacter baumannii in a tertiary care hospital in Turkey. FEMS Microbiol. Lett. 282 (2), 214–218. Merlino, J., Siarakas, S., et al., 1996. Evaluation of CHROMagar Orientation for differentiation and presumptive identification of Gram-negative bacilli and Enterococcus species. J. Clin. Microbiol. 34 (7), 1788–1793. Miriagou, V., Cornaglia, G., et al., 2010. Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues. Clin. Microbiol. Infect. 16 (2), 112–122. Moran Gilad, J., Carmeli, Y., et al., 2011. Laboratory evaluation of the CHROMagar KPC medium for identification of carbapenem-nonsusceptible Enterobacteriaceae. Diagn. Microbiol. Infect. Dis. 70 (4), 565–567. Moubareck, C., Bremont, S., et al., 2009. GES-11, a novel integron-associated GES variant in Acinetobacter baumannii. Antimicrob. Agents Chemother. 53 (8), 3579–3581. Mugnier, P., Poirel, L., et al., 2008. Carbapenem-resistant and OXA-23-producing Acinetobacter baumannii isolates in the United Arab Emirates. Clin. Microbiol. Infect. 14 (9), 879–882. Naas, T., Nordmann, P., 1994. Analysis of a carbapenem-hydrolyzing class A betalactamase from Enterobacter cloacae and of its LysR-type regulatory protein. Proc. Natl. Acad. Sci. U. S. A. 91 (16), 7693–7697. Nakano, R., Okamoto, R., et al., 2004. CFE-1, a novel plasmid-encoded AmpC betalactamase with an ampR gene originating from Citrobacter freundii. Antimicrob. Agents Chemother. 48 (4), 1151–1158. Nordmann, P., 2010. Résistance aux carbapénèmes chez les bacilles à Gram négatif. Med./Sci. 26, 950–959. Nordmann, P., Poirel, L., 2013. Strategies for identification of carbapenemase-producing Enterobacteriaceae. J. Antimicrob. Chemother. 68 (3), 487–489. Nordmann, P., Naas, T., et al., 2011a. Global spread of Carbapenemase-producing Enterobacteriaceae. Emerg. Infect. Dis. 17 (10), 1791–1798. Nordmann, P., Poirel, L., et al., 2011b. How to detect NDM-1 producers. J. Clin. Microbiol. 49 (2), 718–721. Nordmann, P., Dortet, L., et al., 2012a. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol. Med. 18 (5), 263–272. Nordmann, P., Girlich, D., et al., 2012b. Detection of carbapenemase producers in Enterobacteriaceae by use of a novel screening medium. J. Clin. Microbiol. 50 (8), 2761–2766. Nordmann, P., Gniadkowski, M., et al., 2012c. Identification and screening of carbapenemase-producing Enterobacteriaceae. Clin. Microbiol. Infect. 18 (5), 432–438. Nordmann, P., Poirel, L., et al., 2012d. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg. Infect. Dis. 18 (9), 1503–1507. Notake, S., Matsuda, M., et al., 2013. Detection of IMP metallo-beta-lactamase in carbapenem-nonsusceptible Enterobacteriaceae and non-glucose-fermenting Gram-negative rods by immunochromatography assay. J. Clin. Microbiol. 51 (6), 1762–1768.

N

C

O

R

R

E

C

T

Cohen Stuart, J., Voets, G., Rottier, W., Voskuil, S., Scharringa, J., Van Dijk, K., Fluit, A.C., Leverstein-Van Hall, M., 2013. Evaluation of the Oxoid Brillance™ CRE agar for the detection of carbapenemase-producing Enterobacteriaceae. Eur. J. Clin. Microbiol. Infect. Dis. Cunningham, S.A., Noorie, T., et al., 2013. Rapid and simultaneous detection of genes encoding Klebsiella pneumoniae carbapenemase (blaKPC) and New Delhi metallo-beta-lactamase (blaNDM) in Gram-negative bacilli. J. Clin. Microbiol. 51 (4), 1269–1271. Dahmen, S., Mansour, W., et al., 2012. Imipenem resistance in Klebsiella pneumoniae is associated to the combination of plasmid-mediated CMY-4 AmpC beta-lactamase and loss of an outer membrane protein. Microb. Drug Resist. 18 (5), 479–483. Dallenne, C., Da Costa, A., et al., 2010. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother. 65 (3), 490–495. Deshpande, L.M., Jones, R.N., et al., 2006. Occurrence and characterization of carbapenemase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program (2000–2004). Microb. Drug Resist. 12 (4), 223–230. Dortet, L., Poirel, L., et al., 2012. Rapid detection of carbapenemase-producing Pseudomonas spp. J. Clin. Microbiol. 50 (11), 3773–3776. Dortet, L., Poirel, L., et al., 2014. CarbAcineto NP Test for rapid detection of carbapenemase-producing Acinetobacter spp.. J. Clin. Microbiol. 52 (7), 2359–2364. El-Herte, R.I., Kanj, S.S., et al., 2012. The threat of carbapenem-resistant Enterobacteriaceae in Lebanon: an update on the regional and local epidemiology. J. Infect. Public Health 5 (3), 233–243. Empel, J., Filczak, K., et al., 2007. Outbreak of Pseudomonas aeruginosa infections with PER-1 extended-spectrum beta-lactamase in Warsaw, Poland: further evidence for an international clonal complex. J. Clin. Microbiol. 45 (9), 2829–2834. EUCAST, E. c. o. a. s. t., 2014. Breakpoint Tables for Interpretation of MICs and Zone Diameters. Falagas, M.E., Karageorgopoulos, D.E., et al., 2011. Therapeutic options for infections with Enterobacteriaceae producing carbapenem-hydrolyzing enzymes. Future Microbiol 6 (6), 653–666. Ferreira, A.E., Marchetti, D.P., et al., 2011. Molecular characterization of clinical multiresistant isolates of Acinetobacter sp. from hospitals in Porto Alegre, State of Rio Grande do Sul, Brazil. Rev. Soc. Bras. Med. Trop. 44 (6), 725–730. Gazin, M., Paasch, F., et al., 2012. Current trends in culture-based and molecular detection of extended-spectrum-beta-lactamase-harboring and carbapenem-resistant Enterobacteriaceae. J. Clin. Microbiol. 50 (4), 1140–1146. Giakkoupi, P., Tzouvelekis, L.S., et al., 2005. Discrepancies and interpretation problems in susceptibility testing of VIM-1-producing Klebsiella pneumoniae isolates. J. Clin. Microbiol. 43 (1), 494–496. Giakkoupi, P., Vourli, S., et al., 2008. Supplementation of growth media with Zn2+ facilitates detection of VIM-2-producing Pseudomonas aeruginosa. J. Clin. Microbiol. 46 (4), 1568–1569. Giakkoupi, P., Pappa, O., et al., 2009. Emerging Klebsiella pneumoniae isolates coproducing KPC-2 and VIM-1 carbapenemases. Antimicrob. Agents Chemother. 53 (9), 4048–4050. Girlich, D., Naas, T., et al., 2000. Biochemical-genetic characterization and regulation of expression of an ACC-1-like chromosome-borne cephalosporinase from Hafnia alvei. Antimicrob. Agents Chemother. 44 (6), 1470–1478. Girlich, D., Poirel, L., et al., 2012. Value of the modified Hodge test for detection of emerging carbapenemases in Enterobacteriaceae. J. Clin. Microbiol. 50 (2), 477–479. Girlich, D., Poirel, L., et al., 2013. Comparison of the SUPERCARBA, CHROMagar KPC, and Brilliance CRE screening media for detection of Enterobacteriaceae with reduced susceptibility to carbapenems. Diagn. Microbiol. Infect. Dis. 75 (2), 214–217. Giske, C.G., Gezelius, L., et al., 2011. A sensitive and specific phenotypic assay for detection of metallo-beta-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin. Microbiol. Infect. 17 (4), 552–556. Gordon, N.C., Wareham, D.W., 2009. Evaluation of CHROMagar Acinetobacter for detection of enteric carriage of multidrug-resistant Acinetobacter baumannii in samples from critically ill patients. J. Clin. Microbiol. 47 (7), 2249–2251. Gutierrez, O., Juan, C., et al., 2007. Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from Spanish hospitals. Antimicrob. Agents Chemother. 51 (12), 4329–4335. Hawkey, P.M., Livermore, D.M., 2012. Carbapenem antibiotics for serious infections. BMJ 344, e3236. Hodge, W., Ciak, J., et al., 1978. Simple method for detection of penicillinase-producing Neisseria gonorrhoeae. J. Clin. Microbiol. 7 (1), 102–103. Hornstein, M., Sautjeau-Rostoker, C., et al., 1997. Oxacillin-hydrolyzing beta-lactamase involved in resistance to imipenem in Acinetobacter baumannii. FEMS Microbiol. Lett. 153 (2), 333–339. Hrabak, J., Walkova, R., et al., 2011. Carbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 49 (9), 3222–3227. Hrabak, J., Chudackova, E., et al., 2013. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for detection of antibiotic resistance mechanisms: from research to routine diagnosis. Clin. Microbiol. Rev. 26 (1), 103–114. Ikonomidis, A., Ntokou, E., et al., 2008. Hidden VIM-1 metallo-beta-lactamase phenotypes among Acinetobacter baumannii clinical isolates. J. Clin. Microbiol. 46 (1), 346–349. Ito, H., Arakawa, Y., et al., 1995. Plasmid-mediated dissemination of the metallo-betalactamase gene blaIMP among clinically isolated strains of Serratia marcescens. Antimicrob. Agents Chemother. 39 (4), 824–829. Jacoby, G.A., 2009. AmpC beta-lactamases. Clin. Microbiol. Rev. 22 (1), 161–182 (Table of Contents). Kanj, S.S., Kanafani, Z.A., 2011. Current concepts in antimicrobial therapy against resistant Gram-negative organisms: extended-spectrum beta-lactamase-producing

U

1093 1094 1095 1096 Q10 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178


E

12


1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 Q11 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264


N C O

R

R

E

C

D

P

R O

O

F

Thomson, K.S., 2010. Extended-spectrum-beta-lactamase, AmpC, and Carbapenemase issues. J. Clin. Microbiol. 48 (4), 1019–1025. Tijet, N., Boyd, D., et al., 2013. Evaluation of the Carba NP test for rapid detection of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 57 (9), 4578–4580. Tsakris, A., Kristo, I., et al., 2008. First occurrence of KPC-2-possessing Klebsiella pneumoniae in a Greek hospital and recommendation for detection with boronic acid disc tests. J. Antimicrob. Chemother. 62 (6), 1257–1260. Tsakris, A., Kristo, I., et al., 2009. Evaluation of boronic acid disk tests for differentiating KPC-possessing Klebsiella pneumoniae isolates in the clinical laboratory. J. Clin. Microbiol. 47 (2), 362–367. Upadhyay, S., Sen, M.R., et al., 2011. Diagnostic utility of combination of inducer and inhibitor based assay in detection of Pseudomonas aeruginosa producing AmpC beta-lactamase. J. Microbiol. Methods 87 (1), 116–118. Versalovic, J., Koeuth, T., et al., 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19 (24), 6823–6831. Vourli, S., Giakkoupi, P., et al., 2004. Novel GES/IBC extended-spectrum beta-lactamase variants with carbapenemase activity in clinical enterobacteria. FEMS Microbiol. Lett. 234 (2), 209–213. Vrioni, G., Daniil, I., et al., 2012. Comparative evaluation of a prototype chromogenic medium (ChromID CARBA) for detecting carbapenemase-producing Enterobacteriaceae in surveillance rectal swabs. J. Clin. Microbiol. 50 (6), 1841–1846. Walsh, T.R., Toleman, M.A., et al., 2005. Metallo-beta-lactamases: the quiet before the storm? Clin. Microbiol. Rev. 18 (2), 306–325. Wang, L., Gu, H., et al., 2012. Rapid low-cost detection of Klebsiella pneumoniae carbapenemase genes by internally controlled real-time PCR. J. Microbiol. Methods 91 (3), 361–363. Wareham, D.W., Gordon, N.C., 2011. Modifications to CHROMagar Acinetobacter for improved selective growth of multi-drug resistant Acinetobacter baumannii. J. Clin. Pathol. 64 (2), 164–167. Wieser, A., Schneider, L., et al., 2012. MALDI-TOF MS in microbiological diagnosticsidentification of microorganisms and beyond (mini review). Appl. Microbiol. Biotechnol. 93 (3), 965–974. Wolter, D.J., Kurpiel, P.M., et al., 2009. Phenotypic and enzymatic comparative analysis of the novel KPC variant KPC-5 and its evolutionary variants, KPC-2 and KPC-4. Antimicrob. Agents Chemother. 53 (2), 557–562. Won, S.Y., Munoz-Price, L.S., et al., 2011. Emergence and rapid regional spread of Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae. Clin. Infect. Dis. 53 (6), 532–540. Woodford, N., Warner, M., et al., 2011. Evaluation of a commercial microarray to detect carbapenemase-producing Enterobacteriaceae. J. Antimicrob. Chemother. 66 (12), 2887–2888. Yagi, T., Wachino, J., et al., 2005. Practical methods using boronic acid compounds for identification of class C beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. J. Clin. Microbiol. 43 (6), 2551–2558. Yan, J.J., Wu, J.J., et al., 2004. Comparison of the double-disk, combined disk, and Etest methods for detecting metallo-beta-lactamases in Gram-negative bacilli. Diagn. Microbiol. Infect. Dis. 49 (1), 5–11. Yang, Y., Bush, K., 1996. Biochemical characterization of the carbapenem-hydrolyzing beta-lactamase AsbM1 from Aeromonas sobria AER 14 M: a member of a novel subgroup of metallo-beta-lactamases. FEMS Microbiol. Lett. 137 (2–3), 193–200. Yigit, H., Queenan, A.M., et al., 2001. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45 (4), 1151–1161. Yong, D., Lee, K., et al., 2002. Imipenem-EDTA disk method for differentiation of metallobeta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp.. J. Clin. Microbiol. 40 (10), 3798–3801. Zhanel, G.G., Wiebe, R., et al., 2007. Comparative review of the carbapenems. Drugs 67 (7), 1027–1052.

E

T

Notomi, T., Okayama, H., et al., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28 (12), E63. Park, Y.K., Choi, J.Y., et al., 2009. Two distinct clones of carbapenem-resistant Acinetobacter baumannii isolates from Korean hospitals. Diagn. Microbiol. Infect. Dis. 64 (4), 389–395. Pasteran, F., Mendez, T., et al., 2009. Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae. J. Clin. Microbiol. 47 (6), 1631–1639. Patel, G., Bonomo, R.A., 2013. “Stormy waters ahead”: global emergence of carbapenemases. Front. Microbiol. 4, 48. Perez-Perez, F.J., Hanson, N.D., 2002. Detection of plasmid-mediated AmpC betalactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol. 40 (6), 2153–2162. Poirel, L., Nordmann, P., 2006. Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin. Microbiol. Infect. 12 (9), 826–836. Poirel, L., Heritier, C., et al., 2004. Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae. Antimicrob. Agents Chemother. 48 (1), 15–22. Poirel, L., Marque, S., et al., 2005. OXA-58, a novel class D {beta}-lactamase involved in resistance to carbapenems in Acinetobacter baumannii. Antimicrob. Agents Chemother. 49 (1), 202–208. Poirel, L., Naas, T., et al., 2010. Diversity, epidemiology, and genetics of class D betalactamases. Antimicrob. Agents Chemother. 54 (1), 24–38. Poirel, L., Schrenzel, J., et al., 2011a. Molecular analysis of NDM-1-producing enterobacterial isolates from Geneva, Switzerland. J. Antimicrob. Chemother. 66 (8), 1730–1733. Poirel, L., Walsh, T.R., et al., 2011b. Multiplex PCR for detection of acquired carbapenemase genes. Diagn. Microbiol. Infect. Dis. 70 (1), 119–123. Poirel, L., Potron, A., et al., 2012. OXA-48-like carbapenemases: the phantom menace. J. Antimicrob. Chemother. 67 (7), 1597–1606. Pournaras, S., Poulou, A., et al., 2010. Inhibitor-based methods for the detection of KPC carbapenemase-producing Enterobacteriaceae in clinical practice by using boronic acid compounds. J. Antimicrob. Chemother. 65 (7), 1319–1321. Qi, J., Du, Y., et al., 2012. A loop-mediated isothermal amplification method for rapid detection of NDM-1 gene. Microb. Drug Resist. 18 (4), 359–363. Queenan, A.M., Bush, K., 2007. Carbapenemases: the versatile beta-lactamases. Clin. Microbiol. Rev. 20 (3), 440–458 (table of contents). Ratkai, C., Quinteira, S., et al., 2009. Controlling for false positives: interpreting MBL Etest and MBL combined disc test for the detection of metallo-beta-lactamases. J. Antimicrob. Chemother. 64 (3), 657–658. Ruppe, E., Bidet, P., et al., 2006. First detection of the Ambler class C 1 Amp. betalactamase in Citrobacter freundii by a new, simple double-disk synergy test. J. Clin. Microbiol. 44 (11), 4204–4207. Samra, Z., Bahar, J., et al., 2008. Evaluation of CHROMagar KPC for rapid detection of carbapenem-resistant Enterobacteriaceae. J. Clin. Microbiol. 46 (9), 3110–3111. Samuelsen, O., Buaro, L., et al., 2008. Evaluation of phenotypic tests for the detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa in a low prevalence country. J. Antimicrob. Chemother. 61 (4), 827–830. Schneider, I., Queenan, A.M., et al., 2006. Novel carbapenem-hydrolyzing oxacillinase OXA-62 from Pandoraea pnomenusa. Antimicrob. Agents Chemother. 50 (4), 1330–1335. Segal, H., Elisha, B.G., 2005. Use of Etest MBL strips for the detection of carbapenemases in Acinetobacter baumannii. J. Antimicrob. Chemother. 56 (3), 598. Shahcheraghi, F., Abbasalipour, M., et al., 2011. Isolation and genetic characterization of metallo-beta-lactamase and carbapenamase producing strains of Acinetobacter baumannii from patients at Tehran hospitals. Iran. J. Microbiol. 3 (2), 68–74. Stuart, J.C., Voets, G., et al., 2012. Detection of carbapenemase-producing Enterobacteriaceae with a commercial DNA microarray. J. Med. Microbiol. 61 (Pt 6), 809–812. Tenover, F.C., Arbeit, R.D., et al., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33 (9), 2233–2239. Tenover, F.C., Kalsi, R.K., et al., 2006. Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing. Emerg. Infect. Dis. 12 (8), 1209–1213.

U

1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324

13


1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384

Bloodstream infections caused by Pseudomonas spp.: how to detect carbapenemase producers directly from blood cultures.

Reply to: differentiation between KPC and IMP carbapenemase need phenotypic and genotypic methods.

Differentiation between KPC and IMP carbapenemase needs phenotypic and genotypic methods.

Imipenem heteroresistance: high prevalence among Enterobacteriaceae Klebsiella pneumoniae carbapenemase producers.

How to contribute occupationally to ecological sustainability: a literature review.

Evaluation of several phenotypic methods for the detection of carbapenemase-producing Pseudomonas aeruginosa.

The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide.

An evaluation of multiple phenotypic screening methods for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae.

How to detect atrial fibrosis.

Methods to detect protein glutathionylation.

Carbapenem-Resistant Enterobacteriaceae: A Major Prevalence Difference due to the High Performance of Carbapenemase Producers when compared to the Nonproducers.

First report of Klebsiella pneumonia carbapenemase-producing Pseudomonas aeruginosa isolated from burn patients in Iran: phenotypic and genotypic methods.

Double- and multi-carbapenemase-producers: the excessively armored bacilli of the current decade.

Endoscopic techniques to detect small-bowel neuroendocrine tumors: A literature review.

Evaluation of four molecular methods to detect Leishmania infection in dogs.

Simplified Protocol for Carba NP Test for Enhanced Detection of Carbapenemase Producers Directly from Bacterial Cultures.

Laboratory methods to detect antiphospholipid antibodies.

N-acetylglutamate synthase deficiency: Novel mutation associated with neonatal presentation and literature review of molecular and phenotypic spectra.

Improved Phenotype-Based Definition for Identifying Carbapenemase Producers among Carbapenem-Resistant Enterobacteriaceae.

Identification of geosmin and 2-methylisoborneol in cyanobacteria and molecular detection methods for the producers of these compounds.

How much Literature Review is enough for a Case Report?

Modern acupuncture-like stimulation methods: a literature review.

New screening method to detect carriage of carbapenemase-producing Enterobacteriaceae in patients within 24 hours.

How reliable are case formulations? A systematic literature review.