Original Article
Characterization of carbapenem resistance mechanisms and in vitro synergistic activity of colistin plus meropenem in carbapenem resistant Klebsiella pneumoniae
Sort Running Title: CRKP and colistin-meropenem synergy
Mohammed ALI M. MARIE1 $*; Lakshmana GOWDA KRISHNAPPA1 $; Yazeed A. AL SHEIKH1, 2; Khaled HOMOUD M. DABWAN1;
1
Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University,
Riyadh, Kingdom of Saudi Arabia 2
Chair of Medical and Molecular Genetics Research, Clinical Laboratory Sciences Department, College of
Applied Medical Sciences, King Saud University, Po Box 10219, Riyadh 11433, Saudi Arabia. $ Equally contributed *
Corresponding Author
Clinical Laboratory Sciences Department., College of Applied Medical Sciences., King Saud University., Riyadh., Kingdom of Saudi Arabia., P. O. Box 10219., Riyadh 11433., Kingdom of Saudi Arabia. Tel: +9661-4696022., Fax: +966-1-4693736., Mobile: +966-505 477409 E-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, Typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12199. This article is protected by copyright. All rights reserved.
1
Abstract Unreasonable antibiotics consumption is rooted from the rapid emergence of carbapenem resistant Klebsiella pneumoniae (CRKP) with increasing treatment failure being noted. With this sound knowledge we collected consecutive isolates of K. pneumoniae and tested for the presence of different resistant mechanisms of carbapenem using various tests such as the modified Hodge test (MHT) and Metallo-β-Lactamase Confirmative Identification by Rosco Neo-SensitabsTM-Carbapenemases (RNS). Isolates, in which resistant mechanism was not identified by the RNS, were tested for the presence of different mechanisms of carbapenem resistance using Phenylalanine arginine β naphthylamide (PABN) based E-test. A single target polymerase chain reaction for blaNDM-1 gene was performed. Randomly selected 135 isolates were tested for in vitro meropenem-colistin synergy by an E-test method. Among 235 isolates collected, 98 % showed carbapenemase production by MHT. RNS were positive in 97 % of isolates which showed inhibition with dipicolinic acid signifying the production of MBL. None produced K. pneumoniae carbapenamases (KPC) or AmpC β-Lactamase (AmpC BL). Eight isolates did not show inhibition with any of the inhibitor containing disks. 220 isolates (94%) were found to possess the blaNDM-1. Among the 230 isolates positive by the MHT, 223 were MBLs positive by the RNS. Colistin-meropenem Synergy was observed only in small proportion (37%) of the study isolates. This needs further investigation with more accurate tests such as the time-kill assay. Colistin-meropenem combinations may have a role in the treatment of carbapenem resistant K. pneumoniae and may possibly slow the selection of heteroresistant subpopulations during colistin therapy.
Key words: K. pneumoniae; Modified Hodge test (MHT), Metallo-β-Lactamase; Rosco Neo-SensitabsTMCarbapenemases.
2
Introduction Klebsiella pneumoniae, a member of the Enterobacteriaceae is a causative agent for nosocomial infections
such as pneumonia, bacteremia, urinary tract infections and intra-abdominal abscesses (1). This organism is notorious for rapid evolution of drug resistance which is accomplished by vertical (intra species) and horizontal (inter species) dissemination (2). More over irrational use of antimicrobials has raised the rapid emergence of drug resistance and in past few years it is become a major problem being faced in health care settings across the world (3). Until recently, Gram-negative nosocomial infections were effectively treated using carbapenems. However with the emergence of carbapenem resistant Enterobacteriaceae (CRE) in the past decade, increasing treatment failure is being noted (4). Carbapenemases are β-lactamases considered to have potential capability to hydrolyze well protected β-lactam ring. Among the different classes and members of carbapenemases, New Delhi metallo β-lactamase - 1 (NDM-1) is reported recently and of greater concern in Saudi region (5). On the other hand, noncarbapenemase mediated mechanisms include hyper-functioning efflux pumps or loss of porin channels,coupled with increased secretion of lower β-lactamases such as extended spectrum β-lactamases (ESBL) or AmpC β-lactamases (AmpC BL) (2). Apart from resistance to carbapenems, these organisms also possess intrinsic resistant determinants to most of the available antimicrobials. With rapid worldwide dissemination of these pan drug resistant organisms and very few treatment options in hand; we are pushed to face a therapeutic dead end (5). Colistin, a neglected antibiotic is now drawing the attention as it is found to be efficient in treatment of carbapenem resistant organisms. Polymyxins are among the sole antibiotics used as part of a combination treatment for carbapenem resistant K.pneumoniae infection. Colistin resistant strains are increasing and account for an alarming proportion of isolates and they are associated with higher attributable mortality (6). Combining colistin with meropenem or imipenem is found to exert a synergistic action and is superior to colistin mono-therapy (6). In this work, we intended to describe the mechanisms of carbapenem resistance in K. pneumoniae and to check synergistic activity of colistin in combination with meropenem against the carbapenem resistant K. 3
pneumoniae. Materials and methods Bacterial strain. This descriptive study was conducted at Al Iman Hospital of King Saud University Hospital, Riyadh. Successive and unrepeated isolates of K. pneumoniae were collected over a period of 24 months, from January 2010 to December 2013. All laboratory tests were carried out as per the standard protocol of our microbiology laboratory. Inclusion and exclusion criteria: K. pneumoniae isolates included for study were isolated only from blood, respiratory and urine samples. Strains must be resistant to imipenem and meropenem by Kirby-Bauer disc diffusion test. Only those isolates with Minimum inhibitory concentration (MIC) of ≥ 2 µg/ml for both the imipenem and meropenem (resistant) were included in the study. Those isolates which had MIC in susceptible range like less than 1 μg/ml to any of carbapenem by agar dilution method were excluded from study. Antimicrobial susceptibility profile and MIC of imipenem and meropenem The antimicrobial susceptibility profile of the isolates was determined for the first line antimicrobial agent by the disc diffusion technique. Susceptibility of the isolates to antimicrobials such as colistin and tigecycline were also determined using the disk diffusion method and MIC of imipenem and meropenem was determined for all the isolates by agar dilution method using the EUCAST recommended criteria. Characterization of resistance mechanisms The modified Hodge test (MHT) and Metallo-β-Lactamase Confirmative Identification by Rosco NeoSensitabsTM- Carbapenemases kit (RNS) were performed on all the study isolates to define different mechanisms of carbapenem resistance. The MIC of meropenem was determined using the E-test both in the presence and absence of Phenylalanine arginine β naphthylamide assay (PABN) only on isolates in which the mechanism of resistance was not identifiable by the RNS. Randomly selected 135 isolates were tested for in vitro meropenem-colistin synergy by an E-test method with the most active drug incorporated in the medium. These entire tests were done according to manufacturer instruction. A single target polymerase chain reaction for blaNDM-1 gene was performed as described earlier (5). Five isolates positive for the blaNDM-1 gene were randomly selected for sequencing and BLAST matching.
4
Results A total of 235 isolates were included which passed the selection criteria for the study. This comprised of 31 isolates from respiratory samples, 79 from urine and 125 from blood. 145 out of 235 isolates (62%) were obtained from hospitalized patients. Samples from hospitalized patients were obtained from any of the wards or intensive care units of the hospital. 17% (40 out of 235) were from intensive care units and 45% (105 out of 235) were from different wards. The sex distribution was found to be slightly on the higher side for males. 148 isolates (63%) were from males and 87 (37%) were from females. The age of the patients ranged from one day to 81 years. Maximum number of isolates was seen to be from the age group of 40 to 60 years (Figure. 1). Antimicrobial susceptibility profile and MIC of imipenem and meropenem According to EUCAST 2012 criteria, all the 235 isolates were resistant to all β-lactams and β-lactamase inhibitor combinations, gentamicin and ciprofloxacin. Very few isolates showed susceptibility to aztreonam (6%), amikacin (11%) and co-trimoxazole (10%). 97% of the isolates were found to be susceptible to colistin (228 out of 235) while 69% (162 out of 235) were susceptible to tigecycline (Figure. 2). The MICs to imipenem and meropenem of the study isolates are provided in figure 3. Characterization of resistance mechanisms Among 235 isolates, 98% (230) were positive for carbapenemase production by Modified Hodge test. 97 % (227) isolates were positive by RNS and showed inhibition with dipicolinic acid signifying the production of MBL. None produced KPC or AmpC BL. Eight isolates did not show inhibition with any of the inhibitor containing disks. 223 were found to produce MBLs by the RNS among 230 isolates positive for carbapenemases by the MHT. The remaining seven MHT positive isolates did not produce MBLs or KPC. A total of five isolates which were negative by the MHT, in that four were found to produce MBLs by the RNS. Only one MHT negative isolate was negative for MBLs and KPC (Table 1). The eight isolates with unknown mechanism of resistance (seven MHT positive with MBL negative and one MHT negative with MBL negative) were checked for the presence of efflux pumps using the PABN assay and all were found to be negative for efflux. Test was considered to be positive for drug efflux pumps when there was a≥ 3 dilution decrease in the MIC of the meropenem in combination to PABN when compared to the MIC of meropenem alone. 5
Polymerase chain reaction for blaNDM-1 gene, sequencing and BLAST matching 220 isolates (94%) were found to possess the blaNDM-1. Five positive isolates were randomly selected, sequenced, BLAST matched. The sequences were identical to each other and also to those identified elsewhere. Meropenem - colistin in vitro synergy testing using E-test 135 isolates were tested randomly for the synergistic activity of colistin in combination with meropenem. Colistin was known 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. As the interpretive MIC breakpoints of colistin for the Enterobacteriaceae are not provided by the CLSI, the breakpoints of EUCAST were used to interpret results for the study isolates. 130 of the 135 isolates were susceptible to colistin (MIC ≤2 µg/ml) and remaining five had a colistin MIC of ≥ 6 µg/ml and was considered resistant (Figure. 4). The drug combination showed synergy in 49 of the 130 (38 %) colistin susceptible isolates and one of the five (20%) colistin resistant isolates. Overall, synergy was observed in 37% (50 out of 135) of the isolates. In the remaining isolates the combination was found to be either additive or indifferent. Antagonism was not observed.
Discussion In this study K. pneumoniae was chosen as a representative organism to elucidate the different mechanisms of carbapenem resistance. This was based on the consensus derived from a preliminary literature search by which K. pneumoniae was established to be the generally notorious multidrug resistant nosocomial organism with regard to the emergence and spread of carbapenem resistance (2, 7). During the sample collection phase, CRE belonging to other genera were noted. Since the study included only K. pneumoniae, the prevalence of CRE was not able to be precisely identified. Retrospective search of data from the annual reports of 2009 and 2012 revealed the prevalence of CRE to be around 8 to 10% (Data not shown). This data correlates well with that of another study from an Indian tertiary care hospital where there was 8% prevalence of CRE in the blood cultures derived from the intensive care units (8). During the study period of 24 months, 235 isolates of carbapenem resistant K. pneumoniae were collected mainly from blood, urine and respiratory tract samples. The majority of the isolates were obtained from the 6
hospitalized patients (81%) while few isolates (19%) were from community acquired infections suggesting the presence of community strains. Most studies done on CRE across the world have almost exclusively obtained their samples from the critically ill hospitalized patients (9). The most probable reason for this in a tertiary care setup would be due to the patients being referred from different hospitals after unsuccessful therapy with multiple antimicrobials. But one must also be aware that the possibility of community acquired CRE infection is not remote in our country due to the presence of these organisms in the common water sources (10). Community acquired CRE infections have been reported to occur in other countries (11). Isolates were more susceptible to colistin and tigecyclin, while resistant to most of the first line antimicrobial agents. Susceptibility to aztreonam, amikacin and co-trimoxazole was noted but reduced compared to previous years (data not provided). Colistin was found to have higher susceptibility rates than tigecycline for the study isolates. However CLSI does not provide breakpoint for these drugs and as these drugs are widely used in the treatment of CRE infection, accurate interpretive breakpoints are immediately needed. We conclude the results with presence of high carbapenemase mediated resistant mechanisms than the noncarbapenemase mediated mechanisms, as we see 98% of the study isolates gave a positive MHT. The blaNDM-1 gene was detected by PCR in 94% (224 out of 235) of the study isolates suggesting that NDM-1 was the most common mechanism of carbapenem resistance. This correlates to available literature and suggests the increasing prevalence of NDM-1 in the Saudi and Asian subcontinent (9). The number of isolates which were positive for blaNDM-1 gene were found to be a subset of these RNS positive isolates, suggesting that the blaNDM1
gene when present was expressed (Table 1). The remaining seven isolates produced MBLs other than NDM-
1 which was observed to be the second most common mechanism of carbapenem resistance characterized in the study. The results of a tertiary hospital based study conducted in Asia by Azim et al. suggests IMP and VIM to be the predominant MBLs in CRE just before the advent of the NDM-1 era (13). KPC production was not observed in any of our study isolates. Kitchel et al. while describing the epidemiology of KPC, mentioned its presence in Indian continent based on unpublished CDC data (14). The resistance mechanisms of eight of the study isolates could not be identified using the Neo sensitabs. Of these eight isolates with unknown mechanisms, seven were MHT positive and one was MHT negative. These isolates were tested for the presence of efflux pumps by an inhibitor based assay using PABN. None of the 7
isolates had efflux pumps as a possible mechanism of resistance. Usually the organisms with efflux mediated resistance show a low level susceptibility to imipenem and complete resistance to meropenem (15). Three possible mechanisms could exist behind the MHT positive isolates. The first and the most probable mechanism would be the production of OXA carbapenemases which could not be detected by inhibitor based tests such as the Rosco NeoSensitabsTM. This was predicted based on the report on CRE by Castanheira et al. which states OXA-181 as an important carbapenemase in Asia apart from NDM-1 (16, 17). The second probability would be a co-existence of more than one resistance mechanism, most likely being an OXA with a MBL (not NDM-1 in this case, as it would have been detected by the PCR). This assumption is supported by the existing reports which have identified the co-production of different classes of carbapenemases (9, 13). The third possibility would be a false positive MHT result due to excess production of ESBLs (Here, AmpC BLs have been ruled out by the Neo sensitabs). The single MHT negative isolate was the only one with a non-carbapenemase mediated mechanism. 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 over-activity were excluded by the Rosco NeoSensitabsTM and PABN inhibitor E-test respectively. The mechanisms of carbapenem resistance of all the isolates detected in the study are summarized in table 1. In view of evaluating the efficacy of the drug combination as an option for therapy randomly selected 135 isolates were tested for meropenem plus colistin synergistic effect testing using E-test with the most active drug incorporated in the medium. The colistin-carbapenem combination was found to perform consistently well in only 37% of the study isolates. However, confirmation with the time kill assay would be appropriate before deeming this combination ineffective. Results of the modified Hodge test suggest that carbapenemases contributed to the majority of the resistance (98%). Among the carbapenemases, NDM-1 was the most prevalent (94%). Mechanisms such as K. pneumoniae carbapenemase, AmpC β-lactamases and hyperfunctioning efflux pumps were not observed. The synergy between the colistin-meropenem combination was tested in vitro using an E-test method. Synergy was observed only in small proportion (34%) of the study isolates, this needs further investigation with more accurate tests such as the time-kill assay. The exact prevalence of CRE in our hospital setting was not able to be identified since the study included only 8
K. pneumoniae isolates. The prevalence of carbapenem resistant K. pneumoniae was also not calculated because, the study included only the isolates from blood, respiratory samples and urine, while those isolated from other sources were not included. 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 false positive and false negative results (18). Other MBLs, OXA carbapenemases and co-existence of more than one mechanism of resistance were not detected. Hence the resistance mechanism in eight of the isolates was not able to be characterized. In our study, the MICs of the study isolates were interpreted using the guidelines of EUCAST. Isolates with MIC value of 2 µg/ml would have been considered resistant to imipenem or meropenem if the CLSI 2011 guidelines were followed and would have been included from the study (12). The guidelines issued by the CLSI in 2011 do not recommend the detection of resistance mechanisms. But this is controversial, as it is necessary to identify the predominant mechanism of resistance in a particular geographical region in order to compare with other studies on the same mechanism and to formulate effective control measures. ACKNOWLEDGMENTS The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project no. RGP-VPP-314. Disclosure The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
9
References 1. Nordmann P, Cuzon G, Naas T. (2009) The real threat of K. pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis 9:228-236. 2. Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi S. (1983) Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of K. pneumoniae and Serratia marcescens. Infection 11:315-317. 3. Zhang R, Cai JC, Zhou HW, Nasu M, Chen GX. (2011) Genotypic characterization and in vitro activities of tigecycline and polymyxin B for members of the Enterobacteriaceae with decreased susceptibility to carbapenems. J Med Microbiol 60:1813-9. 4. Guh AY, Limbago BM, Kallen AJ. (2014) Epidemiology and prevention of carbapenem-resistant Enterobacteriaceae in the United States. Expert Rev Anti Infect Ther May;12(5):565-80. 5. Dortet L, Poirel L, Nordmann P. (2014) Worldwide Dissemination of the NDM-Type Carbapenemases in Gram-Negative Bacteria. Biomed Res Int 2014;2014:249856 6. Landman D, Georgescu C, Martin DA, Quale J. (2008) Polymyxins revisited. Clin. Microbiol. Rev 21:449465. 7. MacKenzie FM, Forbes KJ, Dorai-John T, Amyes SG, Gould IM. (1997) Emergence of a carbapenemresistant K. pneumoniae. Lancet 350:783. 8. Deshpande P, Rodrigues C, Shetty A, Kapadia F, Hedge A, Soman R. (2010) New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae: treatment options with carbapenems compromised. J Assoc Physicians India 58:147-149. 9. Lascols C, Hackel M, Marshall SH, Hujer AM, Bouchillon S, Badal R, Hoban D, Bonomo RA. (2011) Increasing prevalence and dissemination of NDM-1 metallo-{beta}-lactamase in India: data from the SMART study (2009). J Antimicrob. Chemother 66:1992-1997. 10. Walsh TR, Weeks J, Livermore DM, Toleman MA. (2011) Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 11:355-362. 10
11. Athanassios Tsakris, Aggeliki Poulou, Fani Markou, Vassiliki Pitiriga, Evangelia-Theophano Piperaki, Ioulia Kristo,3 Spyros Pournaras. (2010) Recurrent healthcare-associated community-onset infections due to K. pneumoniaeproducing VIM-1 metallo-beta-lactamase. J Antimicrob Chemother 65:2538-2542 12. Clinical and Laboratory Standards Institute. (2011) Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational supplement. M100-S21 ed. Wayne, PA, USA. 13. Azim A, Dwivedi M, Rao PB, Baronia AK, Singh RK, Prasad KN, Poddar B, Mishra A, Gurjar M, Dhole TN. (2010) Epidemiology of bacterial colonization at intensive care unit admission with emphasis on extended-spectrum beta-lactamase-and metallo-beta-lactamase-producing Gram-negative bacteria--an Indian experience. J Med Microbiol 59:955-960 14. Kitchel B, Rasheed JK, Patel JB, Srinivasan A, Navon-Venezia S, Carmeli Y, Brolund A, Giske CG. (2009) Molecular epidemiology of KPC-producing K. pneumoniae isolates in the United States: clonal expansion of multilocus sequence type 258. Antimicrob Agents Chemother 53:3365-3370. 15. Quale J, Bratu S, Gupta J, Landman D. (2006) Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 50:16331641. 16. Castanheira M, Deshpande LM, Mathai D, Bell JM, Jones RN, Mendes RE. (2011) Early dissemination of NDM-1- and OXA-181-producing Enterobacteriaceae in Indian hospitals: report from the SENTRY Antimicrobial Surveillance Program, 2006-2007. Antimicrob Agents Chemother 55:1274-1278. 17. Colorado Epson EE, Pisney LM, Wendt JM, MacCannell DR, Janelle SJ, Kitchel B, Rasheed JK, Limbago BM, Gould CV, Kallen AJ, Barron MA, Bamberg WM. (2014) Carbapenem-resistant K. pneumoniae producing New Delhi metallo-β-lactamase at an acute care hospital. Infect Control Hosp Epidemiol 35:390-7. 18. Carvalhaes CG, Picão RC, Nicoletti AG, Xavier DE, Gales AC. (2010) Cloverleaf test (modified Hodge test) for detecting carbapenemase production in K. pneumoniae: be aware of false positive results. Journal of Antimicrobial Chemotherapy 65:249 -251.
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Figure 1. Age distribution of patients from which K. pneumoniae strains were isolated (Total number of patients, n= 235) Figure 2. AST profile of study isolates for common antimicrobials. Figure 3. MICs of imepenem and meropenem for the study isolates. Figure 4. Comparative MICs of colistin for the study isolates showing synergy. Table 1. Summary of mechanism of carbapenem resistance in study isolates.
Abbreviation Carbapenem resistant Enterobacteriaceae (CRE) New Delhi metallo β-lactamase - 1 (NDM-1) Extended spectrum β-lactamases (ESBL) Amp C β-lactamases (AmpC BL) Minimum inhibitory concentration (MIC) Modified Hodge test (MHT) Carbapenem resistance K. pneumoniae (CRKP) Metallo-β-Lactamase (MBL) Rosco Neo-SensitabsTM-Carbapenemases (RNS). Phenylalanine arginine β naphthylamide (PABN)
12
Table 1. Summary of the mechanism of carbapenem resistance in the study isolates MHT MBL NDM‐1 No. of isolates Reason Concordant in results for all three tests 218 + + + Concordant in results for MBL other than NDM‐1 5 + + ‐
+ ‐
‐
7
Concordant in results for all three tests for carbapenemases other than MBL or KPC, most probably OXA (or) Co‐existence of more than one mechanism (Non NDM‐1) (or) Due to ESBLs coupled with porin loss MHT ends in false positive
‐ +
+
2
May be low level production of NDM‐1 results in false negative MHT
‐ +
‐
2
May be low level production of MBL other than NDM‐1 cause false negative MHT due to
‐
‐
1
Concordant in results for all three tests most probably ESBL with porin loss
‐
13
Figure 1
14
Figure 2
15
Figure 3
16
Figure 4
17
Characterization of carbapenem resistance mechanisms and in vitro synergistic activity of colistin plus meropenem in carbapenem resistant Klebsiella pneumoniae
Sort Running Title: CRKP and colistin-meropenem synergy
Mohammed ALI M. MARIE1 $*; Lakshmana GOWDA KRISHNAPPA1 $; Yazeed A. AL SHEIKH1, 2; Khaled HOMOUD M. DABWAN1;
1
Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University,
Riyadh, Kingdom of Saudi Arabia 2
Chair of Medical and Molecular Genetics Research, Clinical Laboratory Sciences Department, College of
Applied Medical Sciences, King Saud University, Po Box 10219, Riyadh 11433, Saudi Arabia. $ Equally contributed *
Corresponding Author
Clinical Laboratory Sciences Department., College of Applied Medical Sciences., King Saud University., Riyadh., Kingdom of Saudi Arabia., P. O. Box 10219., Riyadh 11433., Kingdom of Saudi Arabia. Tel: +9661-4696022., Fax: +966-1-4693736., Mobile: +966-505 477409 E-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, Typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12199. This article is protected by copyright. All rights reserved.
1
Abstract Unreasonable antibiotics consumption is rooted from the rapid emergence of carbapenem resistant Klebsiella pneumoniae (CRKP) with increasing treatment failure being noted. With this sound knowledge we collected consecutive isolates of K. pneumoniae and tested for the presence of different resistant mechanisms of carbapenem using various tests such as the modified Hodge test (MHT) and Metallo-β-Lactamase Confirmative Identification by Rosco Neo-SensitabsTM-Carbapenemases (RNS). Isolates, in which resistant mechanism was not identified by the RNS, were tested for the presence of different mechanisms of carbapenem resistance using Phenylalanine arginine β naphthylamide (PABN) based E-test. A single target polymerase chain reaction for blaNDM-1 gene was performed. Randomly selected 135 isolates were tested for in vitro meropenem-colistin synergy by an E-test method. Among 235 isolates collected, 98 % showed carbapenemase production by MHT. RNS were positive in 97 % of isolates which showed inhibition with dipicolinic acid signifying the production of MBL. None produced K. pneumoniae carbapenamases (KPC) or AmpC β-Lactamase (AmpC BL). Eight isolates did not show inhibition with any of the inhibitor containing disks. 220 isolates (94%) were found to possess the blaNDM-1. Among the 230 isolates positive by the MHT, 223 were MBLs positive by the RNS. Colistin-meropenem Synergy was observed only in small proportion (37%) of the study isolates. This needs further investigation with more accurate tests such as the time-kill assay. Colistin-meropenem combinations may have a role in the treatment of carbapenem resistant K. pneumoniae and may possibly slow the selection of heteroresistant subpopulations during colistin therapy.
Key words: K. pneumoniae; Modified Hodge test (MHT), Metallo-β-Lactamase; Rosco Neo-SensitabsTMCarbapenemases.
2
Introduction Klebsiella pneumoniae, a member of the Enterobacteriaceae is a causative agent for nosocomial infections
such as pneumonia, bacteremia, urinary tract infections and intra-abdominal abscesses (1). This organism is notorious for rapid evolution of drug resistance which is accomplished by vertical (intra species) and horizontal (inter species) dissemination (2). More over irrational use of antimicrobials has raised the rapid emergence of drug resistance and in past few years it is become a major problem being faced in health care settings across the world (3). Until recently, Gram-negative nosocomial infections were effectively treated using carbapenems. However with the emergence of carbapenem resistant Enterobacteriaceae (CRE) in the past decade, increasing treatment failure is being noted (4). Carbapenemases are β-lactamases considered to have potential capability to hydrolyze well protected β-lactam ring. Among the different classes and members of carbapenemases, New Delhi metallo β-lactamase - 1 (NDM-1) is reported recently and of greater concern in Saudi region (5). On the other hand, noncarbapenemase mediated mechanisms include hyper-functioning efflux pumps or loss of porin channels,coupled with increased secretion of lower β-lactamases such as extended spectrum β-lactamases (ESBL) or AmpC β-lactamases (AmpC BL) (2). Apart from resistance to carbapenems, these organisms also possess intrinsic resistant determinants to most of the available antimicrobials. With rapid worldwide dissemination of these pan drug resistant organisms and very few treatment options in hand; we are pushed to face a therapeutic dead end (5). Colistin, a neglected antibiotic is now drawing the attention as it is found to be efficient in treatment of carbapenem resistant organisms. Polymyxins are among the sole antibiotics used as part of a combination treatment for carbapenem resistant K.pneumoniae infection. Colistin resistant strains are increasing and account for an alarming proportion of isolates and they are associated with higher attributable mortality (6). Combining colistin with meropenem or imipenem is found to exert a synergistic action and is superior to colistin mono-therapy (6). In this work, we intended to describe the mechanisms of carbapenem resistance in K. pneumoniae and to check synergistic activity of colistin in combination with meropenem against the carbapenem resistant K. 3
pneumoniae. Materials and methods Bacterial strain. This descriptive study was conducted at Al Iman Hospital of King Saud University Hospital, Riyadh. Successive and unrepeated isolates of K. pneumoniae were collected over a period of 24 months, from January 2010 to December 2013. All laboratory tests were carried out as per the standard protocol of our microbiology laboratory. Inclusion and exclusion criteria: K. pneumoniae isolates included for study were isolated only from blood, respiratory and urine samples. Strains must be resistant to imipenem and meropenem by Kirby-Bauer disc diffusion test. Only those isolates with Minimum inhibitory concentration (MIC) of ≥ 2 µg/ml for both the imipenem and meropenem (resistant) were included in the study. Those isolates which had MIC in susceptible range like less than 1 μg/ml to any of carbapenem by agar dilution method were excluded from study. Antimicrobial susceptibility profile and MIC of imipenem and meropenem The antimicrobial susceptibility profile of the isolates was determined for the first line antimicrobial agent by the disc diffusion technique. Susceptibility of the isolates to antimicrobials such as colistin and tigecycline were also determined using the disk diffusion method and MIC of imipenem and meropenem was determined for all the isolates by agar dilution method using the EUCAST recommended criteria. Characterization of resistance mechanisms The modified Hodge test (MHT) and Metallo-β-Lactamase Confirmative Identification by Rosco NeoSensitabsTM- Carbapenemases kit (RNS) were performed on all the study isolates to define different mechanisms of carbapenem resistance. The MIC of meropenem was determined using the E-test both in the presence and absence of Phenylalanine arginine β naphthylamide assay (PABN) only on isolates in which the mechanism of resistance was not identifiable by the RNS. Randomly selected 135 isolates were tested for in vitro meropenem-colistin synergy by an E-test method with the most active drug incorporated in the medium. These entire tests were done according to manufacturer instruction. A single target polymerase chain reaction for blaNDM-1 gene was performed as described earlier (5). Five isolates positive for the blaNDM-1 gene were randomly selected for sequencing and BLAST matching.
4
Results A total of 235 isolates were included which passed the selection criteria for the study. This comprised of 31 isolates from respiratory samples, 79 from urine and 125 from blood. 145 out of 235 isolates (62%) were obtained from hospitalized patients. Samples from hospitalized patients were obtained from any of the wards or intensive care units of the hospital. 17% (40 out of 235) were from intensive care units and 45% (105 out of 235) were from different wards. The sex distribution was found to be slightly on the higher side for males. 148 isolates (63%) were from males and 87 (37%) were from females. The age of the patients ranged from one day to 81 years. Maximum number of isolates was seen to be from the age group of 40 to 60 years (Figure. 1). Antimicrobial susceptibility profile and MIC of imipenem and meropenem According to EUCAST 2012 criteria, all the 235 isolates were resistant to all β-lactams and β-lactamase inhibitor combinations, gentamicin and ciprofloxacin. Very few isolates showed susceptibility to aztreonam (6%), amikacin (11%) and co-trimoxazole (10%). 97% of the isolates were found to be susceptible to colistin (228 out of 235) while 69% (162 out of 235) were susceptible to tigecycline (Figure. 2). The MICs to imipenem and meropenem of the study isolates are provided in figure 3. Characterization of resistance mechanisms Among 235 isolates, 98% (230) were positive for carbapenemase production by Modified Hodge test. 97 % (227) isolates were positive by RNS and showed inhibition with dipicolinic acid signifying the production of MBL. None produced KPC or AmpC BL. Eight isolates did not show inhibition with any of the inhibitor containing disks. 223 were found to produce MBLs by the RNS among 230 isolates positive for carbapenemases by the MHT. The remaining seven MHT positive isolates did not produce MBLs or KPC. A total of five isolates which were negative by the MHT, in that four were found to produce MBLs by the RNS. Only one MHT negative isolate was negative for MBLs and KPC (Table 1). The eight isolates with unknown mechanism of resistance (seven MHT positive with MBL negative and one MHT negative with MBL negative) were checked for the presence of efflux pumps using the PABN assay and all were found to be negative for efflux. Test was considered to be positive for drug efflux pumps when there was a≥ 3 dilution decrease in the MIC of the meropenem in combination to PABN when compared to the MIC of meropenem alone. 5
Polymerase chain reaction for blaNDM-1 gene, sequencing and BLAST matching 220 isolates (94%) were found to possess the blaNDM-1. Five positive isolates were randomly selected, sequenced, BLAST matched. The sequences were identical to each other and also to those identified elsewhere. Meropenem - colistin in vitro synergy testing using E-test 135 isolates were tested randomly for the synergistic activity of colistin in combination with meropenem. Colistin was known 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. As the interpretive MIC breakpoints of colistin for the Enterobacteriaceae are not provided by the CLSI, the breakpoints of EUCAST were used to interpret results for the study isolates. 130 of the 135 isolates were susceptible to colistin (MIC ≤2 µg/ml) and remaining five had a colistin MIC of ≥ 6 µg/ml and was considered resistant (Figure. 4). The drug combination showed synergy in 49 of the 130 (38 %) colistin susceptible isolates and one of the five (20%) colistin resistant isolates. Overall, synergy was observed in 37% (50 out of 135) of the isolates. In the remaining isolates the combination was found to be either additive or indifferent. Antagonism was not observed.
Discussion In this study K. pneumoniae was chosen as a representative organism to elucidate the different mechanisms of carbapenem resistance. This was based on the consensus derived from a preliminary literature search by which K. pneumoniae was established to be the generally notorious multidrug resistant nosocomial organism with regard to the emergence and spread of carbapenem resistance (2, 7). During the sample collection phase, CRE belonging to other genera were noted. Since the study included only K. pneumoniae, the prevalence of CRE was not able to be precisely identified. Retrospective search of data from the annual reports of 2009 and 2012 revealed the prevalence of CRE to be around 8 to 10% (Data not shown). This data correlates well with that of another study from an Indian tertiary care hospital where there was 8% prevalence of CRE in the blood cultures derived from the intensive care units (8). During the study period of 24 months, 235 isolates of carbapenem resistant K. pneumoniae were collected mainly from blood, urine and respiratory tract samples. The majority of the isolates were obtained from the 6
hospitalized patients (81%) while few isolates (19%) were from community acquired infections suggesting the presence of community strains. Most studies done on CRE across the world have almost exclusively obtained their samples from the critically ill hospitalized patients (9). The most probable reason for this in a tertiary care setup would be due to the patients being referred from different hospitals after unsuccessful therapy with multiple antimicrobials. But one must also be aware that the possibility of community acquired CRE infection is not remote in our country due to the presence of these organisms in the common water sources (10). Community acquired CRE infections have been reported to occur in other countries (11). Isolates were more susceptible to colistin and tigecyclin, while resistant to most of the first line antimicrobial agents. Susceptibility to aztreonam, amikacin and co-trimoxazole was noted but reduced compared to previous years (data not provided). Colistin was found to have higher susceptibility rates than tigecycline for the study isolates. However CLSI does not provide breakpoint for these drugs and as these drugs are widely used in the treatment of CRE infection, accurate interpretive breakpoints are immediately needed. We conclude the results with presence of high carbapenemase mediated resistant mechanisms than the noncarbapenemase mediated mechanisms, as we see 98% of the study isolates gave a positive MHT. The blaNDM-1 gene was detected by PCR in 94% (224 out of 235) of the study isolates suggesting that NDM-1 was the most common mechanism of carbapenem resistance. This correlates to available literature and suggests the increasing prevalence of NDM-1 in the Saudi and Asian subcontinent (9). The number of isolates which were positive for blaNDM-1 gene were found to be a subset of these RNS positive isolates, suggesting that the blaNDM1
gene when present was expressed (Table 1). The remaining seven isolates produced MBLs other than NDM-
1 which was observed to be the second most common mechanism of carbapenem resistance characterized in the study. The results of a tertiary hospital based study conducted in Asia by Azim et al. suggests IMP and VIM to be the predominant MBLs in CRE just before the advent of the NDM-1 era (13). KPC production was not observed in any of our study isolates. Kitchel et al. while describing the epidemiology of KPC, mentioned its presence in Indian continent based on unpublished CDC data (14). The resistance mechanisms of eight of the study isolates could not be identified using the Neo sensitabs. Of these eight isolates with unknown mechanisms, seven were MHT positive and one was MHT negative. These isolates were tested for the presence of efflux pumps by an inhibitor based assay using PABN. None of the 7
isolates had efflux pumps as a possible mechanism of resistance. Usually the organisms with efflux mediated resistance show a low level susceptibility to imipenem and complete resistance to meropenem (15). Three possible mechanisms could exist behind the MHT positive isolates. The first and the most probable mechanism would be the production of OXA carbapenemases which could not be detected by inhibitor based tests such as the Rosco NeoSensitabsTM. This was predicted based on the report on CRE by Castanheira et al. which states OXA-181 as an important carbapenemase in Asia apart from NDM-1 (16, 17). The second probability would be a co-existence of more than one resistance mechanism, most likely being an OXA with a MBL (not NDM-1 in this case, as it would have been detected by the PCR). This assumption is supported by the existing reports which have identified the co-production of different classes of carbapenemases (9, 13). The third possibility would be a false positive MHT result due to excess production of ESBLs (Here, AmpC BLs have been ruled out by the Neo sensitabs). The single MHT negative isolate was the only one with a non-carbapenemase mediated mechanism. 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 over-activity were excluded by the Rosco NeoSensitabsTM and PABN inhibitor E-test respectively. The mechanisms of carbapenem resistance of all the isolates detected in the study are summarized in table 1. In view of evaluating the efficacy of the drug combination as an option for therapy randomly selected 135 isolates were tested for meropenem plus colistin synergistic effect testing using E-test with the most active drug incorporated in the medium. The colistin-carbapenem combination was found to perform consistently well in only 37% of the study isolates. However, confirmation with the time kill assay would be appropriate before deeming this combination ineffective. Results of the modified Hodge test suggest that carbapenemases contributed to the majority of the resistance (98%). Among the carbapenemases, NDM-1 was the most prevalent (94%). Mechanisms such as K. pneumoniae carbapenemase, AmpC β-lactamases and hyperfunctioning efflux pumps were not observed. The synergy between the colistin-meropenem combination was tested in vitro using an E-test method. Synergy was observed only in small proportion (34%) of the study isolates, this needs further investigation with more accurate tests such as the time-kill assay. The exact prevalence of CRE in our hospital setting was not able to be identified since the study included only 8
K. pneumoniae isolates. The prevalence of carbapenem resistant K. pneumoniae was also not calculated because, the study included only the isolates from blood, respiratory samples and urine, while those isolated from other sources were not included. 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 false positive and false negative results (18). Other MBLs, OXA carbapenemases and co-existence of more than one mechanism of resistance were not detected. Hence the resistance mechanism in eight of the isolates was not able to be characterized. In our study, the MICs of the study isolates were interpreted using the guidelines of EUCAST. Isolates with MIC value of 2 µg/ml would have been considered resistant to imipenem or meropenem if the CLSI 2011 guidelines were followed and would have been included from the study (12). The guidelines issued by the CLSI in 2011 do not recommend the detection of resistance mechanisms. But this is controversial, as it is necessary to identify the predominant mechanism of resistance in a particular geographical region in order to compare with other studies on the same mechanism and to formulate effective control measures. ACKNOWLEDGMENTS The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project no. RGP-VPP-314. Disclosure The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
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References 1. Nordmann P, Cuzon G, Naas T. (2009) The real threat of K. pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis 9:228-236. 2. Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi S. (1983) Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of K. pneumoniae and Serratia marcescens. Infection 11:315-317. 3. Zhang R, Cai JC, Zhou HW, Nasu M, Chen GX. (2011) Genotypic characterization and in vitro activities of tigecycline and polymyxin B for members of the Enterobacteriaceae with decreased susceptibility to carbapenems. J Med Microbiol 60:1813-9. 4. Guh AY, Limbago BM, Kallen AJ. (2014) Epidemiology and prevention of carbapenem-resistant Enterobacteriaceae in the United States. Expert Rev Anti Infect Ther May;12(5):565-80. 5. Dortet L, Poirel L, Nordmann P. (2014) Worldwide Dissemination of the NDM-Type Carbapenemases in Gram-Negative Bacteria. Biomed Res Int 2014;2014:249856 6. Landman D, Georgescu C, Martin DA, Quale J. (2008) Polymyxins revisited. Clin. Microbiol. Rev 21:449465. 7. MacKenzie FM, Forbes KJ, Dorai-John T, Amyes SG, Gould IM. (1997) Emergence of a carbapenemresistant K. pneumoniae. Lancet 350:783. 8. Deshpande P, Rodrigues C, Shetty A, Kapadia F, Hedge A, Soman R. (2010) New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae: treatment options with carbapenems compromised. J Assoc Physicians India 58:147-149. 9. Lascols C, Hackel M, Marshall SH, Hujer AM, Bouchillon S, Badal R, Hoban D, Bonomo RA. (2011) Increasing prevalence and dissemination of NDM-1 metallo-{beta}-lactamase in India: data from the SMART study (2009). J Antimicrob. Chemother 66:1992-1997. 10. Walsh TR, Weeks J, Livermore DM, Toleman MA. (2011) Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 11:355-362. 10
11. Athanassios Tsakris, Aggeliki Poulou, Fani Markou, Vassiliki Pitiriga, Evangelia-Theophano Piperaki, Ioulia Kristo,3 Spyros Pournaras. (2010) Recurrent healthcare-associated community-onset infections due to K. pneumoniaeproducing VIM-1 metallo-beta-lactamase. J Antimicrob Chemother 65:2538-2542 12. Clinical and Laboratory Standards Institute. (2011) Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational supplement. M100-S21 ed. Wayne, PA, USA. 13. Azim A, Dwivedi M, Rao PB, Baronia AK, Singh RK, Prasad KN, Poddar B, Mishra A, Gurjar M, Dhole TN. (2010) Epidemiology of bacterial colonization at intensive care unit admission with emphasis on extended-spectrum beta-lactamase-and metallo-beta-lactamase-producing Gram-negative bacteria--an Indian experience. J Med Microbiol 59:955-960 14. Kitchel B, Rasheed JK, Patel JB, Srinivasan A, Navon-Venezia S, Carmeli Y, Brolund A, Giske CG. (2009) Molecular epidemiology of KPC-producing K. pneumoniae isolates in the United States: clonal expansion of multilocus sequence type 258. Antimicrob Agents Chemother 53:3365-3370. 15. Quale J, Bratu S, Gupta J, Landman D. (2006) Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 50:16331641. 16. Castanheira M, Deshpande LM, Mathai D, Bell JM, Jones RN, Mendes RE. (2011) Early dissemination of NDM-1- and OXA-181-producing Enterobacteriaceae in Indian hospitals: report from the SENTRY Antimicrobial Surveillance Program, 2006-2007. Antimicrob Agents Chemother 55:1274-1278. 17. Colorado Epson EE, Pisney LM, Wendt JM, MacCannell DR, Janelle SJ, Kitchel B, Rasheed JK, Limbago BM, Gould CV, Kallen AJ, Barron MA, Bamberg WM. (2014) Carbapenem-resistant K. pneumoniae producing New Delhi metallo-β-lactamase at an acute care hospital. Infect Control Hosp Epidemiol 35:390-7. 18. Carvalhaes CG, Picão RC, Nicoletti AG, Xavier DE, Gales AC. (2010) Cloverleaf test (modified Hodge test) for detecting carbapenemase production in K. pneumoniae: be aware of false positive results. Journal of Antimicrobial Chemotherapy 65:249 -251.
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Figure 1. Age distribution of patients from which K. pneumoniae strains were isolated (Total number of patients, n= 235) Figure 2. AST profile of study isolates for common antimicrobials. Figure 3. MICs of imepenem and meropenem for the study isolates. Figure 4. Comparative MICs of colistin for the study isolates showing synergy. Table 1. Summary of mechanism of carbapenem resistance in study isolates.
Abbreviation Carbapenem resistant Enterobacteriaceae (CRE) New Delhi metallo β-lactamase - 1 (NDM-1) Extended spectrum β-lactamases (ESBL) Amp C β-lactamases (AmpC BL) Minimum inhibitory concentration (MIC) Modified Hodge test (MHT) Carbapenem resistance K. pneumoniae (CRKP) Metallo-β-Lactamase (MBL) Rosco Neo-SensitabsTM-Carbapenemases (RNS). Phenylalanine arginine β naphthylamide (PABN)
12
Table 1. Summary of the mechanism of carbapenem resistance in the study isolates MHT MBL NDM‐1 No. of isolates Reason Concordant in results for all three tests 218 + + + Concordant in results for MBL other than NDM‐1 5 + + ‐
+ ‐
‐
7
Concordant in results for all three tests for carbapenemases other than MBL or KPC, most probably OXA (or) Co‐existence of more than one mechanism (Non NDM‐1) (or) Due to ESBLs coupled with porin loss MHT ends in false positive
‐ +
+
2
May be low level production of NDM‐1 results in false negative MHT
‐ +
‐
2
May be low level production of MBL other than NDM‐1 cause false negative MHT due to
‐
‐
1
Concordant in results for all three tests most probably ESBL with porin loss
‐
13
Figure 1
14
Figure 2
15
Figure 3
16
Figure 4
17
