Antimicrobial Original Research Paper
Characterization of carbapenem resistance mechanisms in Klebsiella pneumoniae and in vitro synergy of the colistin–meropenem combination Lakshmana Gowda Krishnappa1, Mohammed Ali M. Marie1, Yazeed A. Al Sheikh1,2 1
Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia, 2Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia In this prospective study, consecutive isolates of Klebsiella pneumoniae were tested for different mechanisms of carbapenem resistance using the modified Hodge test (MHT), Rosco Neo-Sensitabs (ROSCO). Phenylalanine arginine beta-naphthylamide assay (PABN) inhibitor-based test was done on isolates in which the mechanism of resistance was not identifiable by the ROSCO. Among 105 selected isolates, carbapenemase production was noted in 100 (95%) by MHT and ROSCO showed 97 (92.4%) inhibition with dipicolinic acid signifying the production of MBL. PCR amplification was positive in 90 (86%) isolates for blaNDM-1 and 46 (44%) isolates for blaOXA-48. 54 (51%) isolates were positive for blaCTX-M and all belonged to blaCTX-M group 1. Isolates co produced blaOXA-48 (31/105, 30%) and blaCTX-M (40/105, 38%) in combination with the carbapenemase (blaNDM-1) gene. Five colistin-resistant isolates were positive for blaOXA-48. Eight isolates did not show inhibition with any of the inhibitor containing disks and found to be positive for blaOXA-48. Isolates were tested for colistin-meropenem synergy and detection rate was higher by the checkerboard (48%) than E-test method (35%). Our study necessitates continuous surveillance to recognize the predominant machinery of resistance in a particular geographical region to formulate effective control measures. Keywords: Carbapenem-resistant Klebsiella pneumoniae (CRKP), Colistin-meropenem, In vitro synergy, Modified Hodge test (MHT)
Introduction The widespread irrational use of antimicrobials together with speedy appearance of multidrug resistance bacteria is a chief hitch faced in health-care settings around the world.1,2 Multidrug and pan drug-resistant Gramnegative bacilli of the Enterobacteriaceae family are among the most common causes of nosocomial and systemic infections.2 Klebsiella pneumoniae, a member of the Enterobacteriaceae, plays a causative role in nosocomial infections such as pneumonia, bacteremia, urinary tract infections, and intra-abdominal abscesses.3,4 Most of the bacteria in Enterobacteriaceae family including K. pneumoniae are notorious for quick evolution of antibiotic resistance, which is accomplished of both vertical (intra species) and horizontal (inter species) dissemination.4,5 Correspondence to: M. A. M. Maire, Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, PO Box 10219, Riyadh 11433, Saudi Arabia. Email: dr.mmarieali@gmail. com
ß 2014 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000197
The commonest drug resistance mechanism identified in Enterobacteriaceae in K. pneumoniae is production of beta-lactamases such as MBL and extended spectrum beta-lactamases (ESBLs).2,4 Until recently, Gram-negative nosocomial infections were effectively treated using carbapenems. Though with the appearance of carbapenem-resistant Enterobacteriaceae (CRE) in the past decade, rising treatment failure is being noted.4,6 Carbapenem resistance in Gram-negative bacteria may be either due to carbapenemases or mechanisms other than carbapenemases. Carbapenemases are superior beta-lactamases capable of hydrolysing the wellprotected beta-lactam ring of carbapenems. Among the different classes and members of carbapenemases, New Delhi metallo-beta-lactamase-1 (NDM-1) is of greater concern in our country.4,7 Another carbapenem-hydrolysing class D beta-lactamase OXA-48 was first described in a K. pneumoniae isolate from Turkey in 2004 and has now been reported from other
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country.4,8,9 On the other hand, non-carbapenemasemediated mechanisms include hyper-functioning efflux pumps or loss of porin channels, coupled with increased secretion of other beta-lactamases, such as ESBL or Amp C beta-lactamases (AmpC BL).3 With speedy worldwide distribution of these pandrug-resistant organisms and few treatment options in hand, we are pushed to face a therapeutic dead end.5 Colistin, an old abandoned antibiotic, is now of renewed interest as it is found to be effective in treatment of carbapenem-resistant organisms. Combining colistin with meropenem is found to exert a synergistic action and is superior to colistin monotherapy.10 The drug combination could be tested in vitro for synergy using various methods.11 The present work is a descriptive study carried out on this background in a tertiary medical care centre in Saudi Arabia, intending to characterize the mechanisms of carbapenem resistance in Klebsiella pneumoniae and to check for synergy of the colistin–meropenem combination in vitro.
Materials and Methods In a prospective and descriptive study conducted in the microbiology laboratory of King Saud University Hospital (Riyadh, Saudi Arabia), consecutive isolates of Klebsiella pneumoniae was collected over a period of 15 months, from December 2010 to February 2012.
Inclusion criteria Biochemically confirmed that Klebsiella pneumoniae must have been isolated from either blood, respiratory, or urine samples and should be resistant to imipenem and meropenem by disc diffusion test. Only those isolates with minimum inhibitory concentration (MIC) of §2 mg/ml for both the carbapenems (intermediate and resistant) were included in the study.
Exclusion criteria Isolates with MIC of susceptible range (#1 mg/ml) to either imipenem or meropenem by agar dilution method was excluded from study. Isolation, identification, and disc diffusion testing were carried out as per the standard operating procedure of microbiology laboratory described earlier.12,13 The antimicrobial susceptibility profile of the isolates was determined for the first-line antimicrobial agents by the disc diffusion technique. MICs of imipenem and meropenem were determined for all the isolates as per the CLSIrecommended agar dilution method.13 Susceptibility of the isolates to antimicrobials colistin and tigecycline was also determined using the agar dilution method and results were interpreted using EUCAST MIC criteria for Enterobacteriaceae against tigecycline and colistin.14 All the study isolates were tested for the presence of different mechanisms of carbapenem resistance using the modified Hodge test (Microbiologics, Inc., USA),
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Rosco Neo-SensitabsTM Carbapenemases/Metallo-betaLactamase Confirmative Identification Pack (ROSCO Diagnostica A/S, Denmark) as described earlier in previous literature.15 Phenylalanine arginine betanaphthylamide assay (PABN) was done as described earlier16 to detect efflux pumps on isolates in which the mechanism of resistance was not identifiable by the Rosco Neo-SensitabsTM Carbapenemases/Metallobeta-Lactamase Confirmative Identification Pack. All the tests procedure was followed according to manufactures instruction.
Meropenem–colistin in vitro synergy testing using E-test and checkerboard assay All the isolates were tested for the presence synergy between meropenem and colistin in view of evaluating the efficacy of the drug combination as an option for therapy. Colistin was identified to act synergistically to meropenem when there was a §3 dilution reduction in the MIC of the combination compared to the MIC of meropenem alone. MIC breakpoints to the isolates for antimicrobials tigecycline and colistin were determined according to criteria recommended by EUCAST.14 Four different sets of MHA plates were prepared, depending upon the colistin MICs of the organisms to be tested. The colistin susceptible isolates with MICs of 0.38, 0.5, and 0.75 mg/ml were tested on MHA plates incorporated with 0.2, 0.25, and 0.4 mg/ml of colistin sulphate, respectively. MHA plates containing 4 mg/ml colistin sulphate were used for the four colistin-resistant isolates. According to accepted criteria, results were recorded as follows: #0.5, synergy; 0.5–1.0, additivity; 1.0–4.0, indifference; and .4, antagonism. Also chequerboard synergy testing was performed in duplicate with all isolates. Test was performed and interpretation of the results was determined as described earlier in the literature.17,18 In brief, Fractional Inhibitory Concentration Index was used to assess synergistic activity, which was determined by the addition of fractional inhibitory concentrations of the meropenem–colistin used in the combination. Fractional inhibitory concentration of each agent was calculated as a ratio of MIC when used in combination and MIC when used alone.
Polymerase chain reaction for blaNDM-1, blaCTX-M, and blaOXA-48 PCR amplification was carried out for the detection of blaNDM-1, blaCTX-M, and blaOXA-48 using previously described primers and methodology.7,19,20 The subgroups of blaCTX-M were determined by PCR using the procedures of Woodford et al.21 Five representative isolates positive for the any of gene were randomly selected for sequencing and BLAST matching.
Results After passing the inclusion and exclusion criteria, 105 isolates were included in the study. This was composed
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combinations. Very few isolates showed susceptibility to aztreonam (7%), amikacin (9.5%), and co-trimoxazole (10%). According to EUCAST 2011 criteria, 95% of the isolates were found to be susceptible to colistin (100 out of 105), while 59% (62 out of 105) were susceptible to tigecycline (Fig. 2). The MICs of the study isolates are provided in Fig. 3.
Results of modified Hodge test (MHO) and Rosco Neo-Sensitabs Carbapenemases/Metallo-betaLactamase Confirmative Identification Pack Figure 1 Age distribution.
of seven isolates from respiratory samples, 44 from urine and 54 from blood. Eighty-five out of 105 isolates (81%) were obtained from hospitalized patients. These inpatient samples were obtained from any of the 28 wards or 8 intensive care units of the hospital. Twentyseven per cent (28 out of 105) were from intensive care units and 54% (57 out of 105) were from different wards. Medical intensive care unit along with the high dependency unit was the single source which contributed to the maximum number of inpatient samples (12 out of 85). Also a considerable number of isolates were from outpatient samples (19%). The sex distribution was found to be slightly on the higher side for males: 58 isolates (55%) were from males and 47 (45%) were from females. The age of the patients ranged from 1 day to 80 years. When observed for age distribution, the maximum number of isolates was from adults of 20–60 years (Fig. 1).
Antimicrobial susceptibility profiles and MICs of imipenem and meropenem All isolates were resistant to all gentamicin, ciprofloxacin, beta-lactams, and beta-lactamase inhibitor
Carbapenemase production was noted in 95% (100 out of 105) of the isolates By MHT. Results of ROSCO showed that 97 out of 105 isolates (92.4%) showed inhibition with dipicolinic acid signifying the production of MBL. Among the 100 positive isolates by the MHT, 93 were found to produce MBLs by the ROSCO. The remaining seven MHT-positive isolates did not produce MBLs or KPC. Among the five isolates which were negative for carbapenemases by the MHT, four were found to produce MBLs by the ROSCO. Only one MHT-negative isolate was negative for MBLs and KPC (Table 1).None of them produced KPC or AmpC BL. Eight isolates did not show inhibition with any of the inhibitor containing disks. In addition, no difference was observed among NDM1 and OXA-48 co-producers for synergy testing with DPA in the ROSCO kit. All NDM-1 and OXA-48 coproducers always gave synergy.
Phenylalanine arginine beta-naphthylamide assay The MIC of meropenem (E-test; Biomeurix, France) was determined in the presence and absence of PABN. Drug efflux pumps were said to present when there was a § 3 dilution decrease in the MIC of the meropenem along with PABN when compared to the MIC of meropenem alone. The eight isolates in which
Figure 2 AST profile of study isolates for common antimicrobials.
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Figure 3 MICs of imepenem and meropenem for the study isolates.
the mechanism of resistance could not be identified (seven MHT-positive with MBL-negative and one MHT-negative with MBL-negative) were checked for the presence of efflux pumps using the PABN assy. All the eight isolates were negative for drug efflux.
Meropenem–colistin in vitro synergy testing Overall, synergy was observed in 34% (36 out of 105) of the isolates. The drug combination showed synergy in 35 of the 100 (35%) colistin susceptible isolates and one of the five (20%) colistin-resistant isolates (Fig. 4). In the remaining isolates, the combination was found to be either additive or indifferent. The detection rate of synergy was seen in 50 (48%) isolates by the checkerboard slightly higher than E-test method, and the detection rate of indifference was significantly higher by the E-test than by the checkerboard method. In addition, no statistically significant difference was detected between the checkerboard and E-test methods for the detection rates of interaction types. Antagonism was not observed.
Polymerase chain reaction for blaNDM-1, blaCTX-M, and blaOXA-48 Randomly selected isolates sequence were identical to those identified elsewhere and in NCBI database. Ninety of 105 isolates (86%) were found to possess the blaNDM-1. The carbapenemase gene blaOXA-48 was
detected in 46 (44%) isolates and blaCTX-M in 54 (51%), and all belonged to blaCTX-M group 1. The isolates often carried two and occasionally all three different genes. In this study, we detected the presence of blaOXA-48 (31/105, 30%) and blaCTX-M (40/105, 38%) in combination with the carbapenemase (blaNDM-1) gene in the isolates. Five isolates with resistance to colistin were positive for blaOXA-48 alone. All eight isolates which showed no inhibition with any of the inhibitor containing disks in ROSCO kit were positive for blaOXA-48.
Discussion Klebsiella pneumoniae is found to be the most notorious member of the family Enterobacteriaceae with regard to the emergence and spread of carbapenem resistance.22,23 During the study period, 105 isolates of carbapenemresistant Klebsiella pneumoniae were collected from blood, urine and respiratory tract samples. The majority of the isolates were obtained from the inpatients (81%), which correlates to the fact that the organism is a nosocomial pathogen, while few isolates (19%) were from community acquired infections suggesting the presence of community strains. The study isolates were resistant to most of the first line antimicrobial agents. Susceptibility to aztreonam, amikacin, and co-trimoxazole was infrequently noted.
Table 1 Summary of the mechanism of carbapenem resistance in the study isolates No. of MHT MBL NDM-1 isolates
4
z z
z z
z 2
88 5
z
2
2
7
2 2 2
z z 2
z 2 2
2 2 1
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Reason All three tests concordant for NDM-1 All three tests concordant for MBL other than NDM-1 All three tests concordant for carbapenemases other than MBL or KPC, most probably OXA (or) Co-existence of more than one mechanism (Non NDM-1) (or) False-positive MHT due to ESBLs coupled with porin loss False-negative MHT due to low level production of NDM-1 False-negative MHT due to low level production of MBL other than NDM-1 All three tests concordant for non-carbapenemase mechanism most probably ESBL with porin loss
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Figure 4 Comparative MICs of colistin for the study isolates showing synergy.
Colistin was found to have higher susceptibility rates than tigecycline for the study isolates. Ninety-five per cent of the isolates were susceptible to colistin, while only 59% susceptibility was noted for tigecycline. As these drugs are widely used in the treatment of CRE infection, accurate interpretive breakpoints are urgently needed. In this study, the carbapenem MICs of the study isolates were interpreted using the revised guidelines issued by the CLSI in 2011.13 Carbapenem MIC for five isolates (three for imipenem and two for meropenem) was 2 mg/ml. These five isolates would have been considered susceptible to imipenem or meropenem if the CLSI 2010 guidelines or the EUCAST guidelines were followed and would have been excluded from the study. Ninety-five per cent of the study isolates gave a positive MHT signifying that carbapenemase-mediated mechanisms operated at a greater frequency than the non-carbapenemase-mediated mechanisms. The Rosco Neo-Sensitabs identified 97 isolates as MBL producers. The 90 isolates which were positive for blaNDM-1 gene were found to be a subset of these 97 isolates, suggesting that the blaNDM-1 gene when present was expressed. The resistance mechanisms of eight of the study isolates could not be identified using the Rosco Neo-Sensitabs. These isolates were tested for the presence of efflux pumps by an inhibitor-based assay using PABN. None of the isolates had efflux pumps as a possible mechanism of resistance. The reason for not detecting efflux pump over-activity in our may lies in our inclusion criteria. Usually the organisms with efflux-mediated resistance show a low-level susceptibility to imipenem and complete resistance to meropenem. This phenomenon is often observed with the efflux pumps of Pseudomonas aeruginosa and a similar behaviour of AcrAB-TolC pumps of the Enterobacteriaceae is
questionable.24 Although only the isolates resistant to both imipenem and meropenem were included in the study, none of sample recruitment was found to be resistant to one carbapenem and susceptible to the other. This ruled out the possibility of the selection bias which would have eliminated the isolate resistance by efflux mechanisms. Of the eight isolates with unknown mechanisms, seven were MHT-positive and one was MHT-negative. Other possible mechanisms could exist behind the MHT-positive isolates. The probable mechanism would be the one which could not be detected by inhibitorbased tests such as the Rosco Neo-Sensitabs. The other possibility would be a false-positive MHT result due to excess production of ESBLs (where AmpC BLs have been ruled out by the Neo-Sensitabs). Resistant mechanism could not be ruled out in single MHT-negative isolate and we assume that porin loss coupled with excessive ESBL secretion could be the reason for carbapenem resistance in this isolate, since the production of AmpC BLs and efflux pump overactivity were excluded by the Rosco Neo-Sensitabs and PABN inhibitor E-test, respectively. The MHT used in the study to identify carbapenemase production had certain drawbacks. The qualitative nature of this test makes it prone to observer bias, which was attempted to be reduced in the study by interpreting with two different persons. Also the test was not free of falsepositive and false negative-results.15 Among the carbapenemases, NDM-1 was the most prevalent seen in 90 (86%) followed by blaOXA-48 in 46 (44%) and blaCTX-M in 54 (51%) isolates. This result is quite different from the study by Shibl et al. which report that blaOXA-48 (78%) was more prevalent than blaNDM-1 (20%) in Riyadh, Saudi Arabia.9 The isolates often carried two and occasionally all three tested genes in PCR. This corresponds to most of the currently
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available literature, which suggests the increasing prevalence of multidrug-resisatant organism specially harbouring NDM-1 in the Asian subcontinent.2,4,25 The colistin–carbapenem combination was found to perform consistently well in 50 (48%) and 36 (34%) isolates by checkerboard assay and E-test, respectively. However, confirmation with the time kill assay would be appropriate before deeming this combination ineffective. As for now colistin last choice of drugs of choice for the treatment for CRE, report of widespread occurrence of these isolates in our setting is of major concern. A revision in drug policies urgently needs further studies to clearly map out the epidemiology. A progression of improved inspection for these multidrug-resistant pathogens is immediately advocated so that appropriate infection control measures are instituted to stem further dissemination.
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Disclaimer Statements Contributors LGK and MAM designed the study, collected the data, and wrote the manuscript. MAM supervised the study and related work in Vellore. YAAS participated in the design of the study and assisted with writing the manuscript. LGK performed the lab work. All authors read and approved the final manuscript. Funding Research Center at the College of Applied Medical Sciences and the Deanship of Scientific Research at King Saud University. Conflicts of interest The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Ethics approval Study was approved by institutional ethical board.
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References 1 Zhang R, Cai JC, Zhou HW, Nasu M, Chen GX. Genotypic characterization and in vitro activities of tigecycline and polymyxin B for members of the Enterobacteriaceae with decreased susceptibility to carbapenems. J Med Microbiol. 2011;60:1813–9. 2 Arunagiri K, Sekar B, Sangeetha G, John J. Detection and characterization of metallo-beta-lactamases in Pseudomonas aeruginosa by phenotypic and molecular methods from clinical samples in a tertiary care hospital. West Indian Med J. 2012;61:778–83. 3 Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis. 2009;9:228–36. 4 Marie MA, John J, Krishnappa LG, Sangeetha G. Molecular characterization of the b-lactamases in Escherichia coli and Klebsiella pneumoniae from a tertiary care hospital in Riyadh, Saudi Arabia. Microbiol Immunol. 2013;57:805–10. 5 Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi S. Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection. 1983;11:315–7. 6 Schwaber MJ, Klarfeld-Lidji S, Navon-Venezia S, Schwartz D, Leavitt A, Carmeli Y. Predictors of carbapenem-resistant Klebsiella pneumoniae acquisition among hospitalized adults
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and effect of acquisition on mortality. Antimicrob Agents Chemother. 2008;52:1028–33. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 2010;10:597–602. Poirel L, He´ritier C, Tolu¨n V, Nordmann P. Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae. Antimicrob Agents Chemother. 2004;48:15–22. Shibl A, Al-Agamy M, Memish Z, Senok A, Khader SA, Assiri A. The emergence of OXA-48- and NDM-1-positive Klebsiella pneumoniae in Riyadh, Saudi Arabia. Int J Infect Dis. 2013; 17:e1130–3. Pongpech P, Amornnopparattanakul S, Panapakdee S, Fungwithaya S, Nannha P, Dhiraputra C, et al. Antibacterial activity of carbapenem-based combinations against multidrugresistant Acinetobacter baumannii. J Med Assoc Thai. 2010;93: 161–71. Landman D, Georgescu C, Martin DA, Quale J. Polymyxins revisited. Clin Microbiol Rev. 2008;21:449–65. Siegel JD, Rhinehart EM, Jackson M, Chiarello L; HICPAC. Guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings 2007. Atlanta, GA: CDC; 2007. Available from: www.cdc.gov/ncidod/dhqp/pdf/ isolation2007.pdf (accessed 19 Jan 2014). Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 21st Informational Supplement. M100-S21 ed. Wayne, PA: CLSI; 2011. European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters, Version 1.3. Va¨xjo¨: EUCAST; 2011. Carvalhaes CG, Pica˜o RC, Nicoletti AG, Xavier DE, Gales AC. Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results. J Antimicrob Chemother. 2010;65:249– 51. Lamers RP, Cavallari JF, Burrows LL. The efflux inhibitor phenylalanine-arginine beta-naphthylamide (PAbN) permeabilizes the outer membrane of Gram-negative bacteria. PLoS One. 2013;8:e60666. Cetin ES, Tekeli A, Ozseven AG, Us E, Aridogan BC. Determination of in vitro activities of polymyxin B and rifampin in combination with ampicillin/sulbactam or cefoperazone/ sulbactam against multidrug-resistant Acinetobacter baumannii by the E-test and checkerboard methods. Jpn J Infect Dis. 2013;66:463–8. Berc¸ot B, Poirel L, Dortet L, Nordmann P. In vitro evaluation of antibiotic synergy for NDM-1-producing Enterobacteriaceae. J Antimicrob Chemother. 2011;66:2295–7. Dallenne C, Da Costa A, Decre´ D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important betalactamases in Enterobacteriaceae. J Antimicrob Chemother 2010;65:49 Gu¨lmez D, Woodford N, Palepou MF, Mushtaq S, Metan G, Yakupogullari Y, et al. Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48like carbapenemases and outer membrane protein loss. Int J Antimicrob Agents. 2008;31:523–6. Woodford N, Fagan EJ, Ellington MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. J Antimicrob Chemother. 2006;57:154–5. MacKenzie FM, Forbes KJ, Dorai-John T, Amyes SG, Gould IM. Emergence of a carbapenem-resistant Klebsiella pneumoniae. Lancet. 1997;350:783. Deshpande P, Rodrigues C, Shetty A, Kapadia F, Hedge A, Soman R. New Delhi metallo-beta lactamase (NDM-1) in Enterobacteriaceae: treatment options with carbapenems compromised. J Assoc Physicians India. 2010;58:147–9. Quale J, Bratu S, Gupta J, Landman D. Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother. 2006;50:1633–41. Lascols C, Hackel M, Marshall SH, Hujer AM, Bouchillon S, Badal R, et al. Increasing prevalence and dissemination of NDM-1 metallo-b-lactamase in India: data from the SMART study (2009). J Antimicrob Chemother. 2011;66:1992–7.
Characterization of carbapenem resistance mechanisms in Klebsiella pneumoniae and in vitro synergy of the colistin–meropenem combination Lakshmana Gowda Krishnappa1, Mohammed Ali M. Marie1, Yazeed A. Al Sheikh1,2 1
Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia, 2Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia In this prospective study, consecutive isolates of Klebsiella pneumoniae were tested for different mechanisms of carbapenem resistance using the modified Hodge test (MHT), Rosco Neo-Sensitabs (ROSCO). Phenylalanine arginine beta-naphthylamide assay (PABN) inhibitor-based test was done on isolates in which the mechanism of resistance was not identifiable by the ROSCO. Among 105 selected isolates, carbapenemase production was noted in 100 (95%) by MHT and ROSCO showed 97 (92.4%) inhibition with dipicolinic acid signifying the production of MBL. PCR amplification was positive in 90 (86%) isolates for blaNDM-1 and 46 (44%) isolates for blaOXA-48. 54 (51%) isolates were positive for blaCTX-M and all belonged to blaCTX-M group 1. Isolates co produced blaOXA-48 (31/105, 30%) and blaCTX-M (40/105, 38%) in combination with the carbapenemase (blaNDM-1) gene. Five colistin-resistant isolates were positive for blaOXA-48. Eight isolates did not show inhibition with any of the inhibitor containing disks and found to be positive for blaOXA-48. Isolates were tested for colistin-meropenem synergy and detection rate was higher by the checkerboard (48%) than E-test method (35%). Our study necessitates continuous surveillance to recognize the predominant machinery of resistance in a particular geographical region to formulate effective control measures. Keywords: Carbapenem-resistant Klebsiella pneumoniae (CRKP), Colistin-meropenem, In vitro synergy, Modified Hodge test (MHT)
Introduction The widespread irrational use of antimicrobials together with speedy appearance of multidrug resistance bacteria is a chief hitch faced in health-care settings around the world.1,2 Multidrug and pan drug-resistant Gramnegative bacilli of the Enterobacteriaceae family are among the most common causes of nosocomial and systemic infections.2 Klebsiella pneumoniae, a member of the Enterobacteriaceae, plays a causative role in nosocomial infections such as pneumonia, bacteremia, urinary tract infections, and intra-abdominal abscesses.3,4 Most of the bacteria in Enterobacteriaceae family including K. pneumoniae are notorious for quick evolution of antibiotic resistance, which is accomplished of both vertical (intra species) and horizontal (inter species) dissemination.4,5 Correspondence to: M. A. M. Maire, Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, PO Box 10219, Riyadh 11433, Saudi Arabia. Email: dr.mmarieali@gmail. com
ß 2014 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000197
The commonest drug resistance mechanism identified in Enterobacteriaceae in K. pneumoniae is production of beta-lactamases such as MBL and extended spectrum beta-lactamases (ESBLs).2,4 Until recently, Gram-negative nosocomial infections were effectively treated using carbapenems. Though with the appearance of carbapenem-resistant Enterobacteriaceae (CRE) in the past decade, rising treatment failure is being noted.4,6 Carbapenem resistance in Gram-negative bacteria may be either due to carbapenemases or mechanisms other than carbapenemases. Carbapenemases are superior beta-lactamases capable of hydrolysing the wellprotected beta-lactam ring of carbapenems. Among the different classes and members of carbapenemases, New Delhi metallo-beta-lactamase-1 (NDM-1) is of greater concern in our country.4,7 Another carbapenem-hydrolysing class D beta-lactamase OXA-48 was first described in a K. pneumoniae isolate from Turkey in 2004 and has now been reported from other
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country.4,8,9 On the other hand, non-carbapenemasemediated mechanisms include hyper-functioning efflux pumps or loss of porin channels, coupled with increased secretion of other beta-lactamases, such as ESBL or Amp C beta-lactamases (AmpC BL).3 With speedy worldwide distribution of these pandrug-resistant organisms and few treatment options in hand, we are pushed to face a therapeutic dead end.5 Colistin, an old abandoned antibiotic, is now of renewed interest as it is found to be effective in treatment of carbapenem-resistant organisms. Combining colistin with meropenem is found to exert a synergistic action and is superior to colistin monotherapy.10 The drug combination could be tested in vitro for synergy using various methods.11 The present work is a descriptive study carried out on this background in a tertiary medical care centre in Saudi Arabia, intending to characterize the mechanisms of carbapenem resistance in Klebsiella pneumoniae and to check for synergy of the colistin–meropenem combination in vitro.
Materials and Methods In a prospective and descriptive study conducted in the microbiology laboratory of King Saud University Hospital (Riyadh, Saudi Arabia), consecutive isolates of Klebsiella pneumoniae was collected over a period of 15 months, from December 2010 to February 2012.
Inclusion criteria Biochemically confirmed that Klebsiella pneumoniae must have been isolated from either blood, respiratory, or urine samples and should be resistant to imipenem and meropenem by disc diffusion test. Only those isolates with minimum inhibitory concentration (MIC) of §2 mg/ml for both the carbapenems (intermediate and resistant) were included in the study.
Exclusion criteria Isolates with MIC of susceptible range (#1 mg/ml) to either imipenem or meropenem by agar dilution method was excluded from study. Isolation, identification, and disc diffusion testing were carried out as per the standard operating procedure of microbiology laboratory described earlier.12,13 The antimicrobial susceptibility profile of the isolates was determined for the first-line antimicrobial agents by the disc diffusion technique. MICs of imipenem and meropenem were determined for all the isolates as per the CLSIrecommended agar dilution method.13 Susceptibility of the isolates to antimicrobials colistin and tigecycline was also determined using the agar dilution method and results were interpreted using EUCAST MIC criteria for Enterobacteriaceae against tigecycline and colistin.14 All the study isolates were tested for the presence of different mechanisms of carbapenem resistance using the modified Hodge test (Microbiologics, Inc., USA),
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Rosco Neo-SensitabsTM Carbapenemases/Metallo-betaLactamase Confirmative Identification Pack (ROSCO Diagnostica A/S, Denmark) as described earlier in previous literature.15 Phenylalanine arginine betanaphthylamide assay (PABN) was done as described earlier16 to detect efflux pumps on isolates in which the mechanism of resistance was not identifiable by the Rosco Neo-SensitabsTM Carbapenemases/Metallobeta-Lactamase Confirmative Identification Pack. All the tests procedure was followed according to manufactures instruction.
Meropenem–colistin in vitro synergy testing using E-test and checkerboard assay All the isolates were tested for the presence synergy between meropenem and colistin in view of evaluating the efficacy of the drug combination as an option for therapy. Colistin was identified to act synergistically to meropenem when there was a §3 dilution reduction in the MIC of the combination compared to the MIC of meropenem alone. MIC breakpoints to the isolates for antimicrobials tigecycline and colistin were determined according to criteria recommended by EUCAST.14 Four different sets of MHA plates were prepared, depending upon the colistin MICs of the organisms to be tested. The colistin susceptible isolates with MICs of 0.38, 0.5, and 0.75 mg/ml were tested on MHA plates incorporated with 0.2, 0.25, and 0.4 mg/ml of colistin sulphate, respectively. MHA plates containing 4 mg/ml colistin sulphate were used for the four colistin-resistant isolates. According to accepted criteria, results were recorded as follows: #0.5, synergy; 0.5–1.0, additivity; 1.0–4.0, indifference; and .4, antagonism. Also chequerboard synergy testing was performed in duplicate with all isolates. Test was performed and interpretation of the results was determined as described earlier in the literature.17,18 In brief, Fractional Inhibitory Concentration Index was used to assess synergistic activity, which was determined by the addition of fractional inhibitory concentrations of the meropenem–colistin used in the combination. Fractional inhibitory concentration of each agent was calculated as a ratio of MIC when used in combination and MIC when used alone.
Polymerase chain reaction for blaNDM-1, blaCTX-M, and blaOXA-48 PCR amplification was carried out for the detection of blaNDM-1, blaCTX-M, and blaOXA-48 using previously described primers and methodology.7,19,20 The subgroups of blaCTX-M were determined by PCR using the procedures of Woodford et al.21 Five representative isolates positive for the any of gene were randomly selected for sequencing and BLAST matching.
Results After passing the inclusion and exclusion criteria, 105 isolates were included in the study. This was composed
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combinations. Very few isolates showed susceptibility to aztreonam (7%), amikacin (9.5%), and co-trimoxazole (10%). According to EUCAST 2011 criteria, 95% of the isolates were found to be susceptible to colistin (100 out of 105), while 59% (62 out of 105) were susceptible to tigecycline (Fig. 2). The MICs of the study isolates are provided in Fig. 3.
Results of modified Hodge test (MHO) and Rosco Neo-Sensitabs Carbapenemases/Metallo-betaLactamase Confirmative Identification Pack Figure 1 Age distribution.
of seven isolates from respiratory samples, 44 from urine and 54 from blood. Eighty-five out of 105 isolates (81%) were obtained from hospitalized patients. These inpatient samples were obtained from any of the 28 wards or 8 intensive care units of the hospital. Twentyseven per cent (28 out of 105) were from intensive care units and 54% (57 out of 105) were from different wards. Medical intensive care unit along with the high dependency unit was the single source which contributed to the maximum number of inpatient samples (12 out of 85). Also a considerable number of isolates were from outpatient samples (19%). The sex distribution was found to be slightly on the higher side for males: 58 isolates (55%) were from males and 47 (45%) were from females. The age of the patients ranged from 1 day to 80 years. When observed for age distribution, the maximum number of isolates was from adults of 20–60 years (Fig. 1).
Antimicrobial susceptibility profiles and MICs of imipenem and meropenem All isolates were resistant to all gentamicin, ciprofloxacin, beta-lactams, and beta-lactamase inhibitor
Carbapenemase production was noted in 95% (100 out of 105) of the isolates By MHT. Results of ROSCO showed that 97 out of 105 isolates (92.4%) showed inhibition with dipicolinic acid signifying the production of MBL. Among the 100 positive isolates by the MHT, 93 were found to produce MBLs by the ROSCO. The remaining seven MHT-positive isolates did not produce MBLs or KPC. Among the five isolates which were negative for carbapenemases by the MHT, four were found to produce MBLs by the ROSCO. Only one MHT-negative isolate was negative for MBLs and KPC (Table 1).None of them produced KPC or AmpC BL. Eight isolates did not show inhibition with any of the inhibitor containing disks. In addition, no difference was observed among NDM1 and OXA-48 co-producers for synergy testing with DPA in the ROSCO kit. All NDM-1 and OXA-48 coproducers always gave synergy.
Phenylalanine arginine beta-naphthylamide assay The MIC of meropenem (E-test; Biomeurix, France) was determined in the presence and absence of PABN. Drug efflux pumps were said to present when there was a § 3 dilution decrease in the MIC of the meropenem along with PABN when compared to the MIC of meropenem alone. The eight isolates in which
Figure 2 AST profile of study isolates for common antimicrobials.
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Figure 3 MICs of imepenem and meropenem for the study isolates.
the mechanism of resistance could not be identified (seven MHT-positive with MBL-negative and one MHT-negative with MBL-negative) were checked for the presence of efflux pumps using the PABN assy. All the eight isolates were negative for drug efflux.
Meropenem–colistin in vitro synergy testing Overall, synergy was observed in 34% (36 out of 105) of the isolates. The drug combination showed synergy in 35 of the 100 (35%) colistin susceptible isolates and one of the five (20%) colistin-resistant isolates (Fig. 4). In the remaining isolates, the combination was found to be either additive or indifferent. The detection rate of synergy was seen in 50 (48%) isolates by the checkerboard slightly higher than E-test method, and the detection rate of indifference was significantly higher by the E-test than by the checkerboard method. In addition, no statistically significant difference was detected between the checkerboard and E-test methods for the detection rates of interaction types. Antagonism was not observed.
Polymerase chain reaction for blaNDM-1, blaCTX-M, and blaOXA-48 Randomly selected isolates sequence were identical to those identified elsewhere and in NCBI database. Ninety of 105 isolates (86%) were found to possess the blaNDM-1. The carbapenemase gene blaOXA-48 was
detected in 46 (44%) isolates and blaCTX-M in 54 (51%), and all belonged to blaCTX-M group 1. The isolates often carried two and occasionally all three different genes. In this study, we detected the presence of blaOXA-48 (31/105, 30%) and blaCTX-M (40/105, 38%) in combination with the carbapenemase (blaNDM-1) gene in the isolates. Five isolates with resistance to colistin were positive for blaOXA-48 alone. All eight isolates which showed no inhibition with any of the inhibitor containing disks in ROSCO kit were positive for blaOXA-48.
Discussion Klebsiella pneumoniae is found to be the most notorious member of the family Enterobacteriaceae with regard to the emergence and spread of carbapenem resistance.22,23 During the study period, 105 isolates of carbapenemresistant Klebsiella pneumoniae were collected from blood, urine and respiratory tract samples. The majority of the isolates were obtained from the inpatients (81%), which correlates to the fact that the organism is a nosocomial pathogen, while few isolates (19%) were from community acquired infections suggesting the presence of community strains. The study isolates were resistant to most of the first line antimicrobial agents. Susceptibility to aztreonam, amikacin, and co-trimoxazole was infrequently noted.
Table 1 Summary of the mechanism of carbapenem resistance in the study isolates No. of MHT MBL NDM-1 isolates
4
z z
z z
z 2
88 5
z
2
2
7
2 2 2
z z 2
z 2 2
2 2 1
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Reason All three tests concordant for NDM-1 All three tests concordant for MBL other than NDM-1 All three tests concordant for carbapenemases other than MBL or KPC, most probably OXA (or) Co-existence of more than one mechanism (Non NDM-1) (or) False-positive MHT due to ESBLs coupled with porin loss False-negative MHT due to low level production of NDM-1 False-negative MHT due to low level production of MBL other than NDM-1 All three tests concordant for non-carbapenemase mechanism most probably ESBL with porin loss
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Figure 4 Comparative MICs of colistin for the study isolates showing synergy.
Colistin was found to have higher susceptibility rates than tigecycline for the study isolates. Ninety-five per cent of the isolates were susceptible to colistin, while only 59% susceptibility was noted for tigecycline. As these drugs are widely used in the treatment of CRE infection, accurate interpretive breakpoints are urgently needed. In this study, the carbapenem MICs of the study isolates were interpreted using the revised guidelines issued by the CLSI in 2011.13 Carbapenem MIC for five isolates (three for imipenem and two for meropenem) was 2 mg/ml. These five isolates would have been considered susceptible to imipenem or meropenem if the CLSI 2010 guidelines or the EUCAST guidelines were followed and would have been excluded from the study. Ninety-five per cent of the study isolates gave a positive MHT signifying that carbapenemase-mediated mechanisms operated at a greater frequency than the non-carbapenemase-mediated mechanisms. The Rosco Neo-Sensitabs identified 97 isolates as MBL producers. The 90 isolates which were positive for blaNDM-1 gene were found to be a subset of these 97 isolates, suggesting that the blaNDM-1 gene when present was expressed. The resistance mechanisms of eight of the study isolates could not be identified using the Rosco Neo-Sensitabs. These isolates were tested for the presence of efflux pumps by an inhibitor-based assay using PABN. None of the isolates had efflux pumps as a possible mechanism of resistance. The reason for not detecting efflux pump over-activity in our may lies in our inclusion criteria. Usually the organisms with efflux-mediated resistance show a low-level susceptibility to imipenem and complete resistance to meropenem. This phenomenon is often observed with the efflux pumps of Pseudomonas aeruginosa and a similar behaviour of AcrAB-TolC pumps of the Enterobacteriaceae is
questionable.24 Although only the isolates resistant to both imipenem and meropenem were included in the study, none of sample recruitment was found to be resistant to one carbapenem and susceptible to the other. This ruled out the possibility of the selection bias which would have eliminated the isolate resistance by efflux mechanisms. Of the eight isolates with unknown mechanisms, seven were MHT-positive and one was MHT-negative. Other possible mechanisms could exist behind the MHT-positive isolates. The probable mechanism would be the one which could not be detected by inhibitorbased tests such as the Rosco Neo-Sensitabs. The other possibility would be a false-positive MHT result due to excess production of ESBLs (where AmpC BLs have been ruled out by the Neo-Sensitabs). Resistant mechanism could not be ruled out in single MHT-negative isolate and we assume that porin loss coupled with excessive ESBL secretion could be the reason for carbapenem resistance in this isolate, since the production of AmpC BLs and efflux pump overactivity were excluded by the Rosco Neo-Sensitabs and PABN inhibitor E-test, respectively. The MHT used in the study to identify carbapenemase production had certain drawbacks. The qualitative nature of this test makes it prone to observer bias, which was attempted to be reduced in the study by interpreting with two different persons. Also the test was not free of falsepositive and false negative-results.15 Among the carbapenemases, NDM-1 was the most prevalent seen in 90 (86%) followed by blaOXA-48 in 46 (44%) and blaCTX-M in 54 (51%) isolates. This result is quite different from the study by Shibl et al. which report that blaOXA-48 (78%) was more prevalent than blaNDM-1 (20%) in Riyadh, Saudi Arabia.9 The isolates often carried two and occasionally all three tested genes in PCR. This corresponds to most of the currently
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available literature, which suggests the increasing prevalence of multidrug-resisatant organism specially harbouring NDM-1 in the Asian subcontinent.2,4,25 The colistin–carbapenem combination was found to perform consistently well in 50 (48%) and 36 (34%) isolates by checkerboard assay and E-test, respectively. However, confirmation with the time kill assay would be appropriate before deeming this combination ineffective. As for now colistin last choice of drugs of choice for the treatment for CRE, report of widespread occurrence of these isolates in our setting is of major concern. A revision in drug policies urgently needs further studies to clearly map out the epidemiology. A progression of improved inspection for these multidrug-resistant pathogens is immediately advocated so that appropriate infection control measures are instituted to stem further dissemination.
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Disclaimer Statements Contributors LGK and MAM designed the study, collected the data, and wrote the manuscript. MAM supervised the study and related work in Vellore. YAAS participated in the design of the study and assisted with writing the manuscript. LGK performed the lab work. All authors read and approved the final manuscript. Funding Research Center at the College of Applied Medical Sciences and the Deanship of Scientific Research at King Saud University. Conflicts of interest The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Ethics approval Study was approved by institutional ethical board.
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