AAC Accepted Manuscript Posted Online 4 January 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.01844-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
Klebsiella pneumoniae from a New York City Hospital Belonging to ST258 and Carrying blaKPC-2- and
2
blaVIM-4-Encoding Genes
3 4 5
Short running title: KPC-/VIM-producer in the USA
6 7 8
Mariana Castanheira1,
9
Lalitagauri M. Deshpande1,
10
Janet C. Mills1,
11
Ronald N. Jones1,2,
12
Rosemary Soave3
13
Stephen G. Jenkins3,
14
and
15
Audrey N. Schuetz3
16 17 1
18
JMI Laboratories, North Liberty, Iowa, USA 52317;
2
19 20
Tufts University School of Medicine, Boston, Massachusetts, USA 02111; and
3
Weill Cornell Medical College/NewYork-Presbyterian Hospital, New York, New York, USA
21 22 23 24 25 26 27 28 29 30
*Corresponding Author:
Mariana Castanheira, PhD JMI Laboratories 345 Beaver Kreek Ctr, Ste A North Liberty, Iowa, 52317, USA Phone: 319-665-3370 Fax: 319-665-3371 [email protected]
1
31
Abstract
32
Among 69/139 (49.6%) carbapenem non-susceptible Enterobacteriaceae carrying blaKPC, one K.
33
pneumoniae was also positive for blaVIM. The isolate belonged to ST258 and carried blaKPC-2 on a copy of
34
Tn4401a and blaVIM-4 on a class 1 integron. Genes were located on distinct plasmids belonging to Inc
35
types A/C and FII. Elevated expression of the efflux pump AcrAB-TolC (acrA, 15.3X) and reduced
36
expression of outer membrane proteins ompK35 and ompK37 (0.16X and 0.081X, respectively)
37
associated with various amino acid alterations on ompK37 were observed. The presence of two
38
carbapenemases in ST258 K. pneumoniae is of great concern due to the ability of this organism to widely
39
disseminate.
40 41
Keywords: KPC, VIM, carbapenem-resistant Enterobacteriaceae, ST258
42
2
43
The first reported KPC-producing isolate was a Klebsiella pneumoniae collected in 1996 from a
44
patient hospitalized in North Carolina (1). Over the past decade, KPC variants became the most clinically
45
significant β-lactamase among Class A derivatives, and KPC-producing Enterobacteriaceae have been
46
reported worldwide. Within the United States, there are differences in the prevalence of KPC-producing
47
isolates which have been reported in all states except Alaska, Maine, and Idaho; however, these strains
48
seem to be endemic in the New York City area (2).
49
Overall, the dissemination of blaKPC has been associated with clonally-related strains, and the
50
majority of blaKPC-carrying K. pneumoniae belong to multilocus sequence type (MLST) 258 and double-
51
locus variants, which may have contributed to the worldwide dissemination of KPC-producing strains (3).
52
Moreover, there is mounting evidence that blaKPC genes are also consistently associated with a specific
53
genetic element (i.e. transposon Tn4401) (3). Tn4401 is a Tn3-like transposon that has been identified in
54
distinct plasmids carried by KPC-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates
55
from various geographic areas (4).
56
During 2013, a total of 139 Enterobacteriaceae isolates with elevated imipenem or meropenem
57
MIC values (≥2 µg/ml) were collected from U.S. medical centers as part of the SENTRY Antimicrobial
58
Surveillance Program. Susceptibilities were performed on the isolates using the reference broth
59
microdilution method as described by the Clinical and Laboratory Standards Institute (CLSI) (5).
60
Categorical interpretations from M100-S24 (6) were used for all antimicrobials, and quality control (QC)
61
was performed using Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. All QC
62
results were within specified ranges as published by CLSI (6).
63
Carbapenem non-susceptible isolates were screened for the presence of blaKPC, blaNDM, blaVIM,
64
blaIMP, blaSME, blaIMI, blaNMC-A and blaOXA-48 in a multiplex polymerase chain reaction (PCR). Amplicons
65
generated were sequenced on both strands; nucleotide and deduced amino acid sequences were
66
analysed using the Lasergene software package (DNASTAR, Madison, Wisconsin, USA). Amino acid
67
sequences were compared with those available via internet sources (http://www.ncbi.nlm.nih.gov/blast/).
68
Among 139 carbapenem-resistant Enterobacteriaceae isolates detected in the USA in 2013 as
69
part of the program, 123 were K. pneumoniae. A total of 69/139 (49.6%) Enterobacteriaceae isolates
70
carried blaKPC and, among those, 34 (49.3%) were collected in hospitals located in New York state. Ten
3
71
KPC-producing K. pneumoniae were recovered in one hospital, and one strain from this hospital was also
72
positive for blaVIM.
73
This isolate was recovered from a nephrostomy urine specimen of a 23 year-old female who 10
74
months prior to presentation had received a matched unrelated donor allogeneic stem cell transplant for
75
T-cell acute lymphoblastic leukemia. Her post-transplant course was complicated by severe BK virus-
76
associated hemorrhagic cystitis refractory to cidofovir treatment, chronic graft-versus-host-disease,
77
pancytopenia, and multiple blood and urine cultures positive for Gram-negative and Gram-positive
78
bacteria.
79
Shortly after transplant, the patient had three episodes of bacteremia over the course of eight
80
months due to a variety of organisms, including initially a pan-susceptible K. pneumoniae, and later E. coli
81
and K. pneumoniae, both with an extended-spectrum β-lactamase (ESBL) resistance profile. The ESBL
82
bacteremia episodes were each treated with meropenem for two weeks, and bacteremia cleared. During
83
this time, BK nephropathy and hemorrhagic cystitis developed, with urine cultures positive repeatedly for
84
50,000 CFU/mL of vancomycin-resistant Enterococcus faecium and >100,000 CFU/mL of K. pneumoniae.
85
Urinary BK levels measured by PCR were consistently high, peaking at 7.0 x 108 copies/mL Engraftment
86
was achieved, but neutropenia and pancytopenia generally continued throughout her course.
87
Two months prior to this presentation, urinary symptoms worsened, and bilateral nephrostomy
88
tubes were placed after urine cultures were positive for >100,000 CFU/mL of ESBL E. coli (resistant to
89
gentamicin). Meropenem was initiated, and a hematoma developed around the bladder. Cultures of the
90
hematoma, the nephrostomy tube clots, and the urine were positive for a variety of organisms including
91
carbapenem-resistant [CR] K. pneumoniae (susceptible to gentamicin), ESBL E. coli (resistant to
92
gentamicin), and VRE. Gentamicin was added to meropenem therapy to treat the CR K. pneumoniae.
93
Despite therapeutic measures to abate the refractory bladder hemorrhage including intravenous
94
and intravesicular cidofovir, bilateral nephrostomy tube placement, bilateral iliac artery gelfoam
95
embolization, and intravesicular balloon placement, the patient’s urinary symptoms and bleeding
96
worsened still. The bladder was subsequently formalinized (instilled with formalin) twice to stop the
97
bleeding. Urine cultures obtained from the nephrostomy tubes at the time of formalinization were positive
98
for >100,000 CFU/mL of carbapenem-resistant K. pneumoniae, also resistant to gentamicin. Gentamicin
4
99
was discontinued, the nephrostomy tubes were removed, and meropenem was discontinued two weeks
100
later. The patient’s urinary symptoms and bleeding improved after bladder formalinization. During this
101
time, her central Broviac line which had been placed at the time of transplant was removed, and was
102
negative on culture. At the time of isolation of the bacterium presented in this report, the patient had been
103
on an almost continuous course of meropenem for six weeks due to the presence of bilateral
104
nephrostomy tubes, immunosuppressed status, and neutropenia with ongoing BK virus-induced infection
105
with concern for sepsis.
106
The patient currently has a formalinized bladder with no instrumentation, and this isolate has not
107
since been recovered from her urine cultures. This isolate which produced two carbapenemases (KPC
108
and VIM) was resistant to all β-lactams, including aztreonam, aminoglycosides (gentamicin and
109
tobramycin), quinolones and trimethoprim-sulfamethoxazole, but showed low MICs to polymyxin B,
110
colistin and tigecycline (Table 1).
111
MLST performed for the K. pneumoniae isolate according to instructions on the website
112
http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html revealed that the isolate belonged
113
to ST258, and sequencing of amplicons confirmed that the isolate carried blaKPC-2 and blaVIM-4. Additional
114
screening for β-lactamase-encoding genes, performed as previously described (7), demonstrated that this
115
K. pneumoniae also carried blaCMY-4, blaTEM-1 and blaSHV-11.
116
The Tn4401 blaKPC-carrying element was amplified with primers targeting the surrounding
117
structures and the carbapenemase-encoding gene. Amplicons were digested with EagI, and restriction
118
fragment length polymorphism patterns were compared to reference Tn4401 blaKPC-carrying elements
119
previously sequenced. The region directly upstream of blaKPC was sequenced and blaKPC-2 was located on
120
Tn4401a element with a 99-bp deletion upstream of this carbapenemase gene.
121
Primers annealing to the 5’ and 3’ conserved sequence (CS) regions of class 1 integron were
122
used in combination with the blaVIM primers to determine the size and structure of the integron. Additional
123
primers targeting the genes detected in the integron were used to complete sequencing. Sequencing was
124
analyzed as described above. blaVIM-4 was embedded in the first position of a 4.5-Kb class 1 integron
125
followed by aacA7, dhfrA1, aadA1-like gene and qacEΔ1/sul1. The integron sequence has been
126
deposited in GenBank under accession number KJ551510.
5
127
Genetic location of the carbapenemase-encoding genes was assessed by ICeuI and with S1
128
nuclease digestion of chromosomal DNA resolved by electrophoresis on CHEF DRII (BioRad, Richmond,
129
California) and followed by Southern blotting and hybridization with digoxigenin labelled (Roche
130
Diagnostics GmbH, Mannheim, Germany) blaKPC- and blaVIM-specific probes. Plasmid sizes were
131
estimated using concatenated Lambda DNA ladder (8). Plasmid incompatibility was determined by
132
multiplex PCR tests described by Carattoli et al. (9). Genes encoding KPC-2 and VIM-4 were located on
133
distinct plasmids of approximately 120-Kb and 190-Kb, respectively. Incompatibility (Inc) types for these
134
plasmids were A/C and FII, respectively.
135
The expression of acrAB-TolC, ompK35, ompK36 and ompK37 was determined by quantitative
136
real-time PCR (qRT-PCR) using DNA-free RNA preparations. Total RNA was extracted from mid-log-
137
phase bacterial cultures (cell density at OD600 of 0.3-0.5) using RNA Protect Reagent and RNeasy Mini
138
Kit (Qiagen, Hilden, Germany) in the Qiacube workstation (Qiagen), and residual DNA was eliminated
139
with RNase-free DNase (Promega, Wisconsin). Quantification of mRNA and sample quality was assessed
140
using the RNA 6000 Pico kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara,
141
California) according to manufacturer instructions. Only preparations with RNA integrity number (RIN) ≥7
142
that showed no visual degradation were used for experiments. Relative quantification of target genes was
143
performed in triplicate by normalization to an endogenous reference gene (rpsL) on the StepOne Plus
144
instrument (Life Technologies, Carlsbad, California) using custom designed primers showing >93.0%
145
efficiency. Transcription levels were considered significantly different if at least a five-fold difference was
146
noted compared with K. pneumoniae ATCC 13383. This isolate had elevated expression of the efflux
147
pump AcrAB-TolC (15.3X) and reduced expression of ompK35 and ompK37 (0.16X and 0.081X,
148
respectively) when compared to control K. pneumoniae strain (Table 1).
149
Outer membrane proteins of K. pneumoniae were sequenced in full using specific primers.
150
Amplicons were sequenced on both strands; nucleotide and deduced amino acid sequences were
151
analysed as described above. Although the role of OmpK37 in carbapenems resistance is not clear,
152
multiple mutations and insertions have likely disrupted the function of this protein.
153 154
Klebsiella pneumoniae isolates co-harboring KPC and VIM have been described from Greece (10, 11), Germany (12) and recently in Colombia (13) and Italy (14), but have not been described from the
6
155
U.S. Interestingly, K. pneumoniae isolates carrying blaKPC-2, blaVIM-4 and blaCMY-4 have been reported from
156
Athens, Greece in 2010, but in a strain with different sequence type (ST383) compared to the isolate
157
reported in this study (10).
158
The KPC-2- and VIM-4-producing K. pneumoniae detected in this study harbored multiple β-
159
lactam resistance mechanisms, including a transferable cephalosporinase, elevated expression of a
160
tripartite efflux pump system and reduced expression and/or disruptions of at least two outer membrane
161
proteins. Additionally, this isolate was resistant to many other antimicrobial classes, limiting the treatment
162
options for the infection cause by it in a patient with a severe underlying condition and compromised
163
immunological system.
164
The occurrence of VIM-producing isolates in the USA is rare and the presence of two
165
carbapenemases in K. pneumoniae ST258 is of great concern due to the ability of this organism to
166
disseminate. This patient had had no travel history and had not traveled outside of New York City.
167
Furthermore, these results suggest that K. pneumoniae isolates are able to accumulate various
168
resistance mechanisms contributing to the success of this pathogen.
169
Surveillance initiatives are of great importance to create awareness to these resistance
170
mechanisms that are infrequent and to highlight the need for more therapeutic options to treat these
171
multidrug resistant organisms.
172 173
7
174
Acknowledgements
175
This study was presented at the 114th General Meeting American Society for Microbiology (Boston,
176
2014).
177 178
JMI Laboratories, Inc. has received research and educational grants in 2012-2014 from – Achaogen,
179
Actelion, Affinium, American Proficiency Institute (API), AmpliPhi Bio, Anacor, Astellas, AstraZeneca,
180
Basilea, BioVersys, Cardeas, Cempra, Cerexa, Cubist, Daiichi, Dipexium, Durata, Fedora, Forest
181
Research Institute, Furiex, Genentech, GlaxoSmithKline, Janssen, Johnson & Johnson, Medpace, Meiji
182
Seika Kaisha, Melinta, Merck, Methylgene, Nabriva, Nanosphere, Novartis, Pfizer, Polyphor, Rempex,
183
Roche, Seachaid, Shionogi, Synthes, The Medicines Co., Theravance, ThermoFisher, Venatorx, Vertex,
184
Waterloo and some other corporations. Some JMI employees are advisors/consultants for Astellas,
185
Cubist, Pfizer, Cempra, Cerexa-Forest, and Theravance. In regards to speakers bureaus and stock
186
options-none to declare.
187
8
188
References
189
1.
Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, Alberti
190
S, Bush K, Tenover FC. 2001. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a
191
carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45: 1151-
192
1161.
193
2.
Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, Cornaglia
194
G, Garau J, Gniadkowski M, Hayden MK, Kumarasamy K, Livermore DM, Maya JJ,
195
Nordmann P, Patel JB, Paterson DL, Pitout J, Villegas MV, Wang H, Woodford N, Quinn JP.
196
2013. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases.
197
Lancet. Infect. Dis. 13: 785-796.
198
3.
199 200
Nordmann P, Naas T, Poirel L. 2011. Global spread of Carbapenemase-producing Enterobacteriaceae. Emerg. Infect. Dis. 17: 1791-1798.
4.
Naas T, Cuzon G, Villegas MV, Lartigue MF, Quinn JP, Nordmann P. 2008. Genetic structures
201
at the origin of acquisition of the beta-lactamase bla KPC gene. Antimicrob. Agents Chemother. 52:
202
1257-1263.
203
5.
Clinical and Laboratory Standards Institute. 2012. M07-A9. Methods for dilution antimicrobial
204
susceptibility tests for bacteria that grow aerobically; approved standard: ninth edition. Clinical
205
and Laboratory Standards Institute, Wayne, PA.
206
6.
Clinical and Laboratory Standards Institute. 2014. M100-S24. Performance standards for
207
antimicrobial susceptibility testing: 24th informational supplement. Clinical and Laboratory
208
Standards Institute, Wayne, PA.
209
7.
Castanheira M, Farrell SE, Deshpande LM, Mendes RE, Jones RN. 2013. Prevalence of β-
210
lactamase encoding genes among Enterobacteriaceae bacteremia isolates collected in 26 USA
211
hospitals: Report from the SENTRY Antimicrobial Surveillance Program (2010). Antimicrob.
212
Agents Chemother. 57: 3012-3020.
213 214
8.
Barton BM, Harding GP, Zuccarelli AJ. 1995. A general method for detecting and sizing large plasmids. Anal. Biochem. 226: 235-240.
9
215
9.
216 217
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63: 219-228.
10.
Papagiannitsis CC, Giakkoupi P, Vatopoulos AC, Tryfinopoulou K, Miriagou V, Tzouvelekis
218
LS. 2010. Emergence of Klebsiella pneumoniae of a novel sequence type (ST383) producing
219
VIM-4, KPC-2 and CMY-4 beta-lactamases. Int. J. Antimicrob. Agents 36: 573-574.
220
11.
Giakkoupi P, Papagiannitsis CC, Miriagou V, Pappa O, Polemis M, Tryfinopoulou K,
221
Tzouvelekis LS, Vatopoulos AC. 2011. An update of the evolving epidemic of blaKPC-2-
222
carrying Klebsiella pneumoniae in Greece (2009-10). J. Antimicrob. Chemother. 66: 1510-1513.
223
12.
Steinmann J, Kaase M, Gatermann S, Popp W, Steinmann E, Damman M, Paul A, Saner F,
224
Buer J, Rath P. 2011. Outbreak due to a Klebsiella pneumoniae strain harbouring KPC-2 and
225
VIM-1 in a German university hospital, July 2010 to January 2011. Euro. Surveill. 16.
226
13.
Rojas LJ, Mojica MF, Blanco VM, Correa A, Montealegre MC, De La Cadena E, Maya JJ,
227
Camargo RD, Quinn JP, Villegas MV. 2013. Emergence of Klebsiella pneumoniae Coharboring
228
KPC and VIM Carbapenemases in Colombia. Antimicrob. Agents Chemother. 57: 1101-1102.
229
14.
Perilli M, Bottoni C, Grimaldi A, Segatore B, Celenza G, Mariani M, Bellio P, Frascaria P,
230
Amicosante G. 2013. Carbapenem-resistant Klebsiella pneumoniae harbouring blaKPC-3 and
231
blaVIM-2 from central Italy. Diagn. Microbiol. Infect. Dis. 75: 218-221.
232
15.
Tygacil. 2010.Tygacil Package Insert. Available at www.tygacil.com. Accessed October, 2010.
233 234 235
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236 237 238
Table 1. Antimicrobial susceptibility and molecular characterization of KPC-2- and VIM-4-producing K. pneumoniae recovered in a New York City hospital. Test/Characteristic Antimicrobial susceptibility testing Imipenem Meropenem Ceftazidime Cefepime Aztreonam Piperacillin-tazobactam Ciprofloxacin Amikacin Gentamicin Tobramycin Trimethoprim-sulfamethoxazole Tigecycline Colistin Polymyxin B Genetic location of carbapenemase genes blaKPC-2 plasmid size blaVIM-4 plasmid size Plasmid Inc types Tn4401 isoform (blaKPC-2) Gene expression acrAB ompK35 ompK36 ompK37
Results for K. pneumoniae isolate MIC in (µg/mL); (categorical interpretation when availablea): >8 (R) >8 (R) >32 (R) >16 (R) >16 (R) >64 (R) >4 (R) 32 (I) >8 (R) >16 (R) >4 (R) 1 (S) 0.5 (S) 0.5 Molecular size/Inc type/Tn4401 type: 120-Kb 190-Kb IncA/C and IncFIIS Tn4401a (99-base pair deletion upstream of blaKPC) Relative expressionb,c: 15.3 0.16 6.6 0.08
Amino acid alterationsd,e: G211S, V241I R345H R239K, 237-TERY-238 insertion, E244D, N274S, OmpK37 D275T, 275-SSTNGG-276 insertion, V277I, V295G, D350G a. S= susceptible; I= intermediate; R= resistant. Interpretive criteria as published by the Clinical and Laboratory Standards Institute (CLSI) (6) except when otherwise noted; European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria utilized for colistin (http://www.eucast.org/); US Food and Drug Administration (FDA) criteria utilized for tigecycline (15). b. K. pneumoniae ATCC13883 was used as baseline for the analysis of the expression results. c. Underlined values are considered significant. d. Amino acid sequences were compared to K. pneumoniae 342 and ATCC 13883 sequences available over the internet.
OMP mutations OmpK35 OmpK36
239 240 241 242 243 244 245 246
11
247 248
e. Alterations that might be significant are underlined.
12
249 250 251
Figure 1. Schematic representation of the structure carrying blaVIM-2. Genes are represented as open arrows and the direction of the arrows indicate the transcription orientation. Filed circles represent the 59be recombination sites.
59’-be
attI1 5´CS
3´CS
intI1
blaVIM-4
aacA7
dhfrA1 aadA1-like qacEΔ1/sul1
13
59’-be
attI1 5´CS
3´CS
intI1
blaVIM-4
aacA7
dhfrA1 aadA1-like qacEΔ1/sul1
1
Klebsiella pneumoniae from a New York City Hospital Belonging to ST258 and Carrying blaKPC-2- and
2
blaVIM-4-Encoding Genes
3 4 5
Short running title: KPC-/VIM-producer in the USA
6 7 8
Mariana Castanheira1,
9
Lalitagauri M. Deshpande1,
10
Janet C. Mills1,
11
Ronald N. Jones1,2,
12
Rosemary Soave3
13
Stephen G. Jenkins3,
14
and
15
Audrey N. Schuetz3
16 17 1
18
JMI Laboratories, North Liberty, Iowa, USA 52317;
2
19 20
Tufts University School of Medicine, Boston, Massachusetts, USA 02111; and
3
Weill Cornell Medical College/NewYork-Presbyterian Hospital, New York, New York, USA
21 22 23 24 25 26 27 28 29 30
*Corresponding Author:
Mariana Castanheira, PhD JMI Laboratories 345 Beaver Kreek Ctr, Ste A North Liberty, Iowa, 52317, USA Phone: 319-665-3370 Fax: 319-665-3371 [email protected]
1
31
Abstract
32
Among 69/139 (49.6%) carbapenem non-susceptible Enterobacteriaceae carrying blaKPC, one K.
33
pneumoniae was also positive for blaVIM. The isolate belonged to ST258 and carried blaKPC-2 on a copy of
34
Tn4401a and blaVIM-4 on a class 1 integron. Genes were located on distinct plasmids belonging to Inc
35
types A/C and FII. Elevated expression of the efflux pump AcrAB-TolC (acrA, 15.3X) and reduced
36
expression of outer membrane proteins ompK35 and ompK37 (0.16X and 0.081X, respectively)
37
associated with various amino acid alterations on ompK37 were observed. The presence of two
38
carbapenemases in ST258 K. pneumoniae is of great concern due to the ability of this organism to widely
39
disseminate.
40 41
Keywords: KPC, VIM, carbapenem-resistant Enterobacteriaceae, ST258
42
2
43
The first reported KPC-producing isolate was a Klebsiella pneumoniae collected in 1996 from a
44
patient hospitalized in North Carolina (1). Over the past decade, KPC variants became the most clinically
45
significant β-lactamase among Class A derivatives, and KPC-producing Enterobacteriaceae have been
46
reported worldwide. Within the United States, there are differences in the prevalence of KPC-producing
47
isolates which have been reported in all states except Alaska, Maine, and Idaho; however, these strains
48
seem to be endemic in the New York City area (2).
49
Overall, the dissemination of blaKPC has been associated with clonally-related strains, and the
50
majority of blaKPC-carrying K. pneumoniae belong to multilocus sequence type (MLST) 258 and double-
51
locus variants, which may have contributed to the worldwide dissemination of KPC-producing strains (3).
52
Moreover, there is mounting evidence that blaKPC genes are also consistently associated with a specific
53
genetic element (i.e. transposon Tn4401) (3). Tn4401 is a Tn3-like transposon that has been identified in
54
distinct plasmids carried by KPC-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates
55
from various geographic areas (4).
56
During 2013, a total of 139 Enterobacteriaceae isolates with elevated imipenem or meropenem
57
MIC values (≥2 µg/ml) were collected from U.S. medical centers as part of the SENTRY Antimicrobial
58
Surveillance Program. Susceptibilities were performed on the isolates using the reference broth
59
microdilution method as described by the Clinical and Laboratory Standards Institute (CLSI) (5).
60
Categorical interpretations from M100-S24 (6) were used for all antimicrobials, and quality control (QC)
61
was performed using Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. All QC
62
results were within specified ranges as published by CLSI (6).
63
Carbapenem non-susceptible isolates were screened for the presence of blaKPC, blaNDM, blaVIM,
64
blaIMP, blaSME, blaIMI, blaNMC-A and blaOXA-48 in a multiplex polymerase chain reaction (PCR). Amplicons
65
generated were sequenced on both strands; nucleotide and deduced amino acid sequences were
66
analysed using the Lasergene software package (DNASTAR, Madison, Wisconsin, USA). Amino acid
67
sequences were compared with those available via internet sources (http://www.ncbi.nlm.nih.gov/blast/).
68
Among 139 carbapenem-resistant Enterobacteriaceae isolates detected in the USA in 2013 as
69
part of the program, 123 were K. pneumoniae. A total of 69/139 (49.6%) Enterobacteriaceae isolates
70
carried blaKPC and, among those, 34 (49.3%) were collected in hospitals located in New York state. Ten
3
71
KPC-producing K. pneumoniae were recovered in one hospital, and one strain from this hospital was also
72
positive for blaVIM.
73
This isolate was recovered from a nephrostomy urine specimen of a 23 year-old female who 10
74
months prior to presentation had received a matched unrelated donor allogeneic stem cell transplant for
75
T-cell acute lymphoblastic leukemia. Her post-transplant course was complicated by severe BK virus-
76
associated hemorrhagic cystitis refractory to cidofovir treatment, chronic graft-versus-host-disease,
77
pancytopenia, and multiple blood and urine cultures positive for Gram-negative and Gram-positive
78
bacteria.
79
Shortly after transplant, the patient had three episodes of bacteremia over the course of eight
80
months due to a variety of organisms, including initially a pan-susceptible K. pneumoniae, and later E. coli
81
and K. pneumoniae, both with an extended-spectrum β-lactamase (ESBL) resistance profile. The ESBL
82
bacteremia episodes were each treated with meropenem for two weeks, and bacteremia cleared. During
83
this time, BK nephropathy and hemorrhagic cystitis developed, with urine cultures positive repeatedly for
84
50,000 CFU/mL of vancomycin-resistant Enterococcus faecium and >100,000 CFU/mL of K. pneumoniae.
85
Urinary BK levels measured by PCR were consistently high, peaking at 7.0 x 108 copies/mL Engraftment
86
was achieved, but neutropenia and pancytopenia generally continued throughout her course.
87
Two months prior to this presentation, urinary symptoms worsened, and bilateral nephrostomy
88
tubes were placed after urine cultures were positive for >100,000 CFU/mL of ESBL E. coli (resistant to
89
gentamicin). Meropenem was initiated, and a hematoma developed around the bladder. Cultures of the
90
hematoma, the nephrostomy tube clots, and the urine were positive for a variety of organisms including
91
carbapenem-resistant [CR] K. pneumoniae (susceptible to gentamicin), ESBL E. coli (resistant to
92
gentamicin), and VRE. Gentamicin was added to meropenem therapy to treat the CR K. pneumoniae.
93
Despite therapeutic measures to abate the refractory bladder hemorrhage including intravenous
94
and intravesicular cidofovir, bilateral nephrostomy tube placement, bilateral iliac artery gelfoam
95
embolization, and intravesicular balloon placement, the patient’s urinary symptoms and bleeding
96
worsened still. The bladder was subsequently formalinized (instilled with formalin) twice to stop the
97
bleeding. Urine cultures obtained from the nephrostomy tubes at the time of formalinization were positive
98
for >100,000 CFU/mL of carbapenem-resistant K. pneumoniae, also resistant to gentamicin. Gentamicin
4
99
was discontinued, the nephrostomy tubes were removed, and meropenem was discontinued two weeks
100
later. The patient’s urinary symptoms and bleeding improved after bladder formalinization. During this
101
time, her central Broviac line which had been placed at the time of transplant was removed, and was
102
negative on culture. At the time of isolation of the bacterium presented in this report, the patient had been
103
on an almost continuous course of meropenem for six weeks due to the presence of bilateral
104
nephrostomy tubes, immunosuppressed status, and neutropenia with ongoing BK virus-induced infection
105
with concern for sepsis.
106
The patient currently has a formalinized bladder with no instrumentation, and this isolate has not
107
since been recovered from her urine cultures. This isolate which produced two carbapenemases (KPC
108
and VIM) was resistant to all β-lactams, including aztreonam, aminoglycosides (gentamicin and
109
tobramycin), quinolones and trimethoprim-sulfamethoxazole, but showed low MICs to polymyxin B,
110
colistin and tigecycline (Table 1).
111
MLST performed for the K. pneumoniae isolate according to instructions on the website
112
http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html revealed that the isolate belonged
113
to ST258, and sequencing of amplicons confirmed that the isolate carried blaKPC-2 and blaVIM-4. Additional
114
screening for β-lactamase-encoding genes, performed as previously described (7), demonstrated that this
115
K. pneumoniae also carried blaCMY-4, blaTEM-1 and blaSHV-11.
116
The Tn4401 blaKPC-carrying element was amplified with primers targeting the surrounding
117
structures and the carbapenemase-encoding gene. Amplicons were digested with EagI, and restriction
118
fragment length polymorphism patterns were compared to reference Tn4401 blaKPC-carrying elements
119
previously sequenced. The region directly upstream of blaKPC was sequenced and blaKPC-2 was located on
120
Tn4401a element with a 99-bp deletion upstream of this carbapenemase gene.
121
Primers annealing to the 5’ and 3’ conserved sequence (CS) regions of class 1 integron were
122
used in combination with the blaVIM primers to determine the size and structure of the integron. Additional
123
primers targeting the genes detected in the integron were used to complete sequencing. Sequencing was
124
analyzed as described above. blaVIM-4 was embedded in the first position of a 4.5-Kb class 1 integron
125
followed by aacA7, dhfrA1, aadA1-like gene and qacEΔ1/sul1. The integron sequence has been
126
deposited in GenBank under accession number KJ551510.
5
127
Genetic location of the carbapenemase-encoding genes was assessed by ICeuI and with S1
128
nuclease digestion of chromosomal DNA resolved by electrophoresis on CHEF DRII (BioRad, Richmond,
129
California) and followed by Southern blotting and hybridization with digoxigenin labelled (Roche
130
Diagnostics GmbH, Mannheim, Germany) blaKPC- and blaVIM-specific probes. Plasmid sizes were
131
estimated using concatenated Lambda DNA ladder (8). Plasmid incompatibility was determined by
132
multiplex PCR tests described by Carattoli et al. (9). Genes encoding KPC-2 and VIM-4 were located on
133
distinct plasmids of approximately 120-Kb and 190-Kb, respectively. Incompatibility (Inc) types for these
134
plasmids were A/C and FII, respectively.
135
The expression of acrAB-TolC, ompK35, ompK36 and ompK37 was determined by quantitative
136
real-time PCR (qRT-PCR) using DNA-free RNA preparations. Total RNA was extracted from mid-log-
137
phase bacterial cultures (cell density at OD600 of 0.3-0.5) using RNA Protect Reagent and RNeasy Mini
138
Kit (Qiagen, Hilden, Germany) in the Qiacube workstation (Qiagen), and residual DNA was eliminated
139
with RNase-free DNase (Promega, Wisconsin). Quantification of mRNA and sample quality was assessed
140
using the RNA 6000 Pico kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara,
141
California) according to manufacturer instructions. Only preparations with RNA integrity number (RIN) ≥7
142
that showed no visual degradation were used for experiments. Relative quantification of target genes was
143
performed in triplicate by normalization to an endogenous reference gene (rpsL) on the StepOne Plus
144
instrument (Life Technologies, Carlsbad, California) using custom designed primers showing >93.0%
145
efficiency. Transcription levels were considered significantly different if at least a five-fold difference was
146
noted compared with K. pneumoniae ATCC 13383. This isolate had elevated expression of the efflux
147
pump AcrAB-TolC (15.3X) and reduced expression of ompK35 and ompK37 (0.16X and 0.081X,
148
respectively) when compared to control K. pneumoniae strain (Table 1).
149
Outer membrane proteins of K. pneumoniae were sequenced in full using specific primers.
150
Amplicons were sequenced on both strands; nucleotide and deduced amino acid sequences were
151
analysed as described above. Although the role of OmpK37 in carbapenems resistance is not clear,
152
multiple mutations and insertions have likely disrupted the function of this protein.
153 154
Klebsiella pneumoniae isolates co-harboring KPC and VIM have been described from Greece (10, 11), Germany (12) and recently in Colombia (13) and Italy (14), but have not been described from the
6
155
U.S. Interestingly, K. pneumoniae isolates carrying blaKPC-2, blaVIM-4 and blaCMY-4 have been reported from
156
Athens, Greece in 2010, but in a strain with different sequence type (ST383) compared to the isolate
157
reported in this study (10).
158
The KPC-2- and VIM-4-producing K. pneumoniae detected in this study harbored multiple β-
159
lactam resistance mechanisms, including a transferable cephalosporinase, elevated expression of a
160
tripartite efflux pump system and reduced expression and/or disruptions of at least two outer membrane
161
proteins. Additionally, this isolate was resistant to many other antimicrobial classes, limiting the treatment
162
options for the infection cause by it in a patient with a severe underlying condition and compromised
163
immunological system.
164
The occurrence of VIM-producing isolates in the USA is rare and the presence of two
165
carbapenemases in K. pneumoniae ST258 is of great concern due to the ability of this organism to
166
disseminate. This patient had had no travel history and had not traveled outside of New York City.
167
Furthermore, these results suggest that K. pneumoniae isolates are able to accumulate various
168
resistance mechanisms contributing to the success of this pathogen.
169
Surveillance initiatives are of great importance to create awareness to these resistance
170
mechanisms that are infrequent and to highlight the need for more therapeutic options to treat these
171
multidrug resistant organisms.
172 173
7
174
Acknowledgements
175
This study was presented at the 114th General Meeting American Society for Microbiology (Boston,
176
2014).
177 178
JMI Laboratories, Inc. has received research and educational grants in 2012-2014 from – Achaogen,
179
Actelion, Affinium, American Proficiency Institute (API), AmpliPhi Bio, Anacor, Astellas, AstraZeneca,
180
Basilea, BioVersys, Cardeas, Cempra, Cerexa, Cubist, Daiichi, Dipexium, Durata, Fedora, Forest
181
Research Institute, Furiex, Genentech, GlaxoSmithKline, Janssen, Johnson & Johnson, Medpace, Meiji
182
Seika Kaisha, Melinta, Merck, Methylgene, Nabriva, Nanosphere, Novartis, Pfizer, Polyphor, Rempex,
183
Roche, Seachaid, Shionogi, Synthes, The Medicines Co., Theravance, ThermoFisher, Venatorx, Vertex,
184
Waterloo and some other corporations. Some JMI employees are advisors/consultants for Astellas,
185
Cubist, Pfizer, Cempra, Cerexa-Forest, and Theravance. In regards to speakers bureaus and stock
186
options-none to declare.
187
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References
189
1.
Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, Alberti
190
S, Bush K, Tenover FC. 2001. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a
191
carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45: 1151-
192
1161.
193
2.
Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, Cornaglia
194
G, Garau J, Gniadkowski M, Hayden MK, Kumarasamy K, Livermore DM, Maya JJ,
195
Nordmann P, Patel JB, Paterson DL, Pitout J, Villegas MV, Wang H, Woodford N, Quinn JP.
196
2013. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases.
197
Lancet. Infect. Dis. 13: 785-796.
198
3.
199 200
Nordmann P, Naas T, Poirel L. 2011. Global spread of Carbapenemase-producing Enterobacteriaceae. Emerg. Infect. Dis. 17: 1791-1798.
4.
Naas T, Cuzon G, Villegas MV, Lartigue MF, Quinn JP, Nordmann P. 2008. Genetic structures
201
at the origin of acquisition of the beta-lactamase bla KPC gene. Antimicrob. Agents Chemother. 52:
202
1257-1263.
203
5.
Clinical and Laboratory Standards Institute. 2012. M07-A9. Methods for dilution antimicrobial
204
susceptibility tests for bacteria that grow aerobically; approved standard: ninth edition. Clinical
205
and Laboratory Standards Institute, Wayne, PA.
206
6.
Clinical and Laboratory Standards Institute. 2014. M100-S24. Performance standards for
207
antimicrobial susceptibility testing: 24th informational supplement. Clinical and Laboratory
208
Standards Institute, Wayne, PA.
209
7.
Castanheira M, Farrell SE, Deshpande LM, Mendes RE, Jones RN. 2013. Prevalence of β-
210
lactamase encoding genes among Enterobacteriaceae bacteremia isolates collected in 26 USA
211
hospitals: Report from the SENTRY Antimicrobial Surveillance Program (2010). Antimicrob.
212
Agents Chemother. 57: 3012-3020.
213 214
8.
Barton BM, Harding GP, Zuccarelli AJ. 1995. A general method for detecting and sizing large plasmids. Anal. Biochem. 226: 235-240.
9
215
9.
216 217
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63: 219-228.
10.
Papagiannitsis CC, Giakkoupi P, Vatopoulos AC, Tryfinopoulou K, Miriagou V, Tzouvelekis
218
LS. 2010. Emergence of Klebsiella pneumoniae of a novel sequence type (ST383) producing
219
VIM-4, KPC-2 and CMY-4 beta-lactamases. Int. J. Antimicrob. Agents 36: 573-574.
220
11.
Giakkoupi P, Papagiannitsis CC, Miriagou V, Pappa O, Polemis M, Tryfinopoulou K,
221
Tzouvelekis LS, Vatopoulos AC. 2011. An update of the evolving epidemic of blaKPC-2-
222
carrying Klebsiella pneumoniae in Greece (2009-10). J. Antimicrob. Chemother. 66: 1510-1513.
223
12.
Steinmann J, Kaase M, Gatermann S, Popp W, Steinmann E, Damman M, Paul A, Saner F,
224
Buer J, Rath P. 2011. Outbreak due to a Klebsiella pneumoniae strain harbouring KPC-2 and
225
VIM-1 in a German university hospital, July 2010 to January 2011. Euro. Surveill. 16.
226
13.
Rojas LJ, Mojica MF, Blanco VM, Correa A, Montealegre MC, De La Cadena E, Maya JJ,
227
Camargo RD, Quinn JP, Villegas MV. 2013. Emergence of Klebsiella pneumoniae Coharboring
228
KPC and VIM Carbapenemases in Colombia. Antimicrob. Agents Chemother. 57: 1101-1102.
229
14.
Perilli M, Bottoni C, Grimaldi A, Segatore B, Celenza G, Mariani M, Bellio P, Frascaria P,
230
Amicosante G. 2013. Carbapenem-resistant Klebsiella pneumoniae harbouring blaKPC-3 and
231
blaVIM-2 from central Italy. Diagn. Microbiol. Infect. Dis. 75: 218-221.
232
15.
Tygacil. 2010.Tygacil Package Insert. Available at www.tygacil.com. Accessed October, 2010.
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Table 1. Antimicrobial susceptibility and molecular characterization of KPC-2- and VIM-4-producing K. pneumoniae recovered in a New York City hospital. Test/Characteristic Antimicrobial susceptibility testing Imipenem Meropenem Ceftazidime Cefepime Aztreonam Piperacillin-tazobactam Ciprofloxacin Amikacin Gentamicin Tobramycin Trimethoprim-sulfamethoxazole Tigecycline Colistin Polymyxin B Genetic location of carbapenemase genes blaKPC-2 plasmid size blaVIM-4 plasmid size Plasmid Inc types Tn4401 isoform (blaKPC-2) Gene expression acrAB ompK35 ompK36 ompK37
Results for K. pneumoniae isolate MIC in (µg/mL); (categorical interpretation when availablea): >8 (R) >8 (R) >32 (R) >16 (R) >16 (R) >64 (R) >4 (R) 32 (I) >8 (R) >16 (R) >4 (R) 1 (S) 0.5 (S) 0.5 Molecular size/Inc type/Tn4401 type: 120-Kb 190-Kb IncA/C and IncFIIS Tn4401a (99-base pair deletion upstream of blaKPC) Relative expressionb,c: 15.3 0.16 6.6 0.08
Amino acid alterationsd,e: G211S, V241I R345H R239K, 237-TERY-238 insertion, E244D, N274S, OmpK37 D275T, 275-SSTNGG-276 insertion, V277I, V295G, D350G a. S= susceptible; I= intermediate; R= resistant. Interpretive criteria as published by the Clinical and Laboratory Standards Institute (CLSI) (6) except when otherwise noted; European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria utilized for colistin (http://www.eucast.org/); US Food and Drug Administration (FDA) criteria utilized for tigecycline (15). b. K. pneumoniae ATCC13883 was used as baseline for the analysis of the expression results. c. Underlined values are considered significant. d. Amino acid sequences were compared to K. pneumoniae 342 and ATCC 13883 sequences available over the internet.
OMP mutations OmpK35 OmpK36
239 240 241 242 243 244 245 246
11
247 248
e. Alterations that might be significant are underlined.
12
249 250 251
Figure 1. Schematic representation of the structure carrying blaVIM-2. Genes are represented as open arrows and the direction of the arrows indicate the transcription orientation. Filed circles represent the 59be recombination sites.
59’-be
attI1 5´CS
3´CS
intI1
blaVIM-4
aacA7
dhfrA1 aadA1-like qacEΔ1/sul1
13
59’-be
attI1 5´CS
3´CS
intI1
blaVIM-4
aacA7
dhfrA1 aadA1-like qacEΔ1/sul1
