crossmark
Frequency of BKC-1-Producing Klebsiella Species Isolates Willames M. B. S. Martins,a Adriana G. Nicoletti,a Silvia R. Santos,b Jorge L. M. Sampaio,b,c Ana C. Galesa Laboratório Alerta, Division of Infectious Diseases, Department of Internal Medicine, Escola Paulista de Medicina, Federal University of São Paulo, São Paulo, Brazila; School of Pharmacy, University of São Paulo, São Paulo, Brazilb; Fleury Diagnostic Medicine, Microbiology Section, São Paulo, Brazilc
BKC-1 is a new class A serine carbapenemase that was recently identified in Klebsiella pneumoniae clinical isolates. The principal objective of this study was to evaluate the frequency of blaBKC-1 by testing a collection of Klebsiella isolates. Only 2 of 635 Klebsiella isolates (0.3%) carried blaBKC-1. The two BKC-1-producing isolates belonged to clonal complex 442 and possessed identical pulsed-field gel electrophoresis patterns. The blaBKC-1 gene was inserted into a 10-kb plasmid that was identical to the previously reported plasmid, p60136. The BKC-producing K. pneumoniae isolates presented also possessed other mechanisms for beta-lactam resistance, such as genes encoding extended-spectrum beta-lactamases and mutations in the genes ompK35 and ompK36, encoding the major porins.
I
n the past decade, carbapenemase-encoding genes such as blaKPC-like, blaOXA-48, and blaNDM-like have emerged as the main mechanisms of carbapenem resistance in Enterobacteriaceae. These carbapenem resistance genes are frequently harbored on mobile genetic elements that are highly transmissible, contributing to the increasing frequency of carbapenem-resistant Enterobacteriaceae (1). BKC-1 (Brazilian Klebsiella carbapenemase) is a class A serine carbapenemase that was recently identified in Klebsiella pneumoniae isolates from Brazil that belonged to sequence type 1781 (ST1781) (clonal complex 442 [CC442]) (2). The carbapenemase was encoded by blaBKC-1, which was located on a small nonconjugative plasmid (2). Here we investigated the frequency of blaBKC-1 among Brazilian clinical isolates of Klebsiella spp., and we performed full characterization of BKC-1-producing isolates. A total of 635 Klebsiella strains collected from previous surveillance studies were randomly selected for this study, in order to represent distinct Brazilian geographic regions. Since BKC-1 is a weak carbapenemase and other mechanisms could be associated with carbapenemase production, we selected Klebsiella strains independent of the resistance phenotype. The Klebsiella strains were isolated from different clinical specimens obtained between 2008 and 2014 (Fig. 1A). Among the 635 Klebsiella strains, 523 (82.3%) and 22 (3.4%) were previously identified as K. pneumoniae and Klebsiella oxytoca, respectively. The search for resistance genes was carried out by PCR using specific primers (2), followed by DNA sequencing (ABI 3500 Genetic Analyzer; Applied Biosystems, Foster City, CA). Only 2 BKC-1-producing K. pneumoniae isolates (0.3%), i.e., Kpn252 and KpnJ, were detected. These strains were isolated from blood and rectal swab cultures from two patients admitted to different hospitals located in the same region (the city of São Paulo and the surrounding area) in 2010 and 2014 (2 and 6 years, respectively, after the first isolation of BKC-1 [2]). The clonal relationships among the BKC-1-producing isolates were determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) (3). PFGE was performed using SpeI as the restriction enzyme. Fingerprint patterns were analyzed using BioNumerics software (Applied Maths, Austin, TX), and a dendrogram was generated by unweighted pair group method with arithmetic mean (UPGMA) cluster analyses, using a 1.5% Dice coefficient and 1% tolerance. For comparison purposes, the first reported BKC-producing strains, i.e., KP60134,
5044
aac.asm.org
KP60135, and KP6013, were included in the PFGE experiment (2). The Kpn252 and KpnJ strains showed more than 80% similarity to the KP60134, KP60135, and KP60136 strains and were grouped in the same cluster (Fig. 1B). Despite the clonal relationship, Kpn252 and KpnJ belonged to distinct STs, namely, ST1781 and ST442, respectively. ST1781 is a single-locus variant of ST442, and both are grouped in CC442. To date, all isolates identified as producers of BKC-1 have belonged to CC442. Antimicrobial susceptibility testing was performed by the broth microdilution method, according to CLSI guidelines (4, 5). The Kpn252 and KpnJ strains showed resistance to all beta-lactams and fluoroquinolones. Kpn252 showed susceptibility to tigecycline (MIC, 0.25 g/ml) and polymyxin B (MIC, 0.5 g/ml), while KpnJ was susceptible only to gentamicin (MIC, 0.25 g/ml) (Table 1). KpnJ was resistant to polymyxin B (MIC, 16 g/ml), and molecular investigation revealed disruption of mgrB by IS903. To date, inactivation of mgrB has been the main mechanism of polymyxin resistance observed in K. pneumoniae isolates (6). The genes blaBKC-1, blaSHV-110-like, and aph3A-VI were detected in both strains; in addition, qnrS1 and blaCTX-M-2 were present only in Kpn252 (Table 1). Plasmid extraction of K. pneumoniae isolates was performed using the method described by Kieser (7). The Kpn252 and KpnJ strains showed different plasmid profiles. However, Southern blot analysis revealed that the blaBKC-1 gene was located on the same 10-kb plasmid in both isolates. To determine whether this plasmid was identical to p60136, the first plasmid identified as carrying blaBKC-1, we performed whole-plasmid sequencing of the plasmid recovered from KpnJ. Initially, we performed transformation by electroporation into Escherichia coli Top10. Transformant cells
Received 29 February 2016 Returned for modification 16 March 2016 Accepted 18 May 2016 Accepted manuscript posted online 23 May 2016 Citation Martins WMBS, Nicoletti AG, Santos SR, Sampaio JLM, Gales AC. 2016. Frequency of BKC-1-producing Klebsiella species isolates. Antimicrob Agents Chemother 60:5044 –5046. doi:10.1128/AAC.00470-16. Address correspondence to Willames M. B. S. Martins, [email protected]. W.M.B.S.M. and A.G.N. contributed equally to this work. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Antimicrobial Agents and Chemotherapy
August 2016 Volume 60 Number 8
blaBKC-1 in Klebsiella Isolates
FIG 1 (A) Map of Brazil, showing the geographic distribution of Klebsiella isolates that were investigated for the blaBKC-1 gene. PA, Pará; MA, Maranhão; CE, Ceará; RN, Rio Grande do Norte; BA, Bahia; MG, Minas Gerais; GO, Goiás; SP, São Paulo; RJ, Rio de Janeiro; PR, Paraná; SC, Santa Catarina; RS, Rio Grande do Sul. (B) Dendrogram and computer-generated image of PFGE results, analyzed by BioNumerics, for the BKC-1-producing K. pneumoniae isolates.
were selected on LB agar plates supplemented with 0.5 g/ml imipenem. The sequencing of the plasmid obtained from the transformant strain was carried out using an Ion Torrent PGM system (Thermo Fisher Scientific, Waltham, MA). Newbler v. 2.8 software (Roche Diagnostics GmbH, Penzberg, Germany) was employed for plasmid sequence assembly. The whole-plasmid se-
TABLE 1 Antimicrobial susceptibility profiles of BKC-1-producing K. pneumoniae clinical isolates MIC (g/ml) (susceptibility category)a Antimicrobial
Kpn252
KpnJ
Gentamicin Amikacin Ertapenem Imipenem Meropenem Aztreonam Piperacillin-tazobactam Ceftazidime Ceftriaxone Levofloxacin Ciprofloxacin Tigecycline Polymyxin B
256 (R) 256 (R) 32 (R) 32 (R) 8 (R) ⬎128 (R) ⬎128 (R) ⬎128 (R) ⬎64 (R) 64 (R) ⬎8 (R) 0.25 (S) 0.5 (S)
0.25 (S) 256 (R) 64 (R) 32 (R) 32 (R) ⬎128 (R) ⬎128 (R) 128 (R) ⬎64 (R) 256 (R) ⬎8 (R) 2 (R) 16 (R)
a The resistance genes in Kpn252 were blaCTX-M-2, blaSHV-110-like, blaBKC-1, aph3A-VI, and qnrS1, and the resistance genes in KpnJ were blaSHV-110-like, blaBKC-1, and aph3AVI. S, sensitive; R, resistant.
August 2016 Volume 60 Number 8
quence of the 10-kb KpnJ plasmid was identical to that of the blaBKC-1-carrying plasmid p60136 (GenBank accession number KP689347.1). During the experiments, it was observed that blaBKC-1 was easily lost in the absence of selective pressure. To evaluate the stability of p60136 harbored by the Kpn252 isolate, we performed serial passages of the Kpn252 isolate on Mueller-Hinton agar plates without the addition of antibiotic. PCR was used to search for blaBKC-1 in K. pneumoniae colonies obtained from daily cultures (approximately 50 colonies per day). We verified that, in the absence of selective pressure, the plasmid harboring the blaBKC-1 gene, p60136, was unstable. A total of 20%, 32%, and 34% of colonies grown on the plates lost blaBKC-1 after 3, 4, and 5 passages, respectively. SDS-PAGE of outer membrane proteins (OMPs) and DNA sequencing of the genes coding for porins (ompK35 and ompk36) were performed. By SDS-PAGE, both isolates showed only one of the main OMPs. DNA sequencing revealed mutations in the ompK36 gene of Kpn252 and nucleotide insertion in the ompK35 gene of KpnJ. These data might suggest that, although genetically related, the isolates were probably subjected to distinct selective pressures, which could have resulted in different OMP modifications. In this study, a low frequency of isolates carrying blaBKC-1 was observed and was mainly attributed to the persistence of a K. pneumoniae clone carrying the same plasmid, p60136. In addition, blaBKC-1 was harbored by a small unstable plasmid that could be easily lost in the absence of selective pressure. More studies with
Antimicrobial Agents and Chemotherapy
aac.asm.org
5045
Martins et al.
greater numbers of isolates from all Brazilian states are necessary to assess the real prevalence of isolates carrying blaBKC-1. It is important to note that such new studies must search for blaBKC-1 using molecular techniques, since phenotypic tests may not accurately detect BKC-1-producing isolates (8). In conclusion, prudent use of antimicrobials and strict adherence to infection control measures seem to be the only effective tools for preventing the acquisition and spread of new carbapenemase-encoding genes. ACKNOWLEDGMENTS We are grateful to Ana Carolina Ramos, Ana Paula Streling, Lorena Cristina Fehlberg, and Rodrigo Cayô for their excellent technical contributions to this study. We are grateful to the National Council for Science and Technological Development for providing a grant to A.C.G. (process number 305535/ 2014-5). A.C.G. recently received research funding and/or consultation fees from AstraZeneca, MSD, and Novartis. The other authors have no conflicts to declare.
REFERENCES 1. Pitout JDD, Nordmann P, Poirel L. 2015. Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrob Agents Chemother 59:5873–5884. http://dx.doi.org/10 .1128/AAC.01019-15.
5046
aac.asm.org
2. Nicoletti AG, Marcondes MFM, Martins WMBS, Almeida LGP, Nicolás MF, Vasconcelos ATR, Oliveira V, Gales AC. 2015. Characterization of BKC-1 class A carbapenemase from Klebsiella pneumoniae clinical isolates in Brazil. Antimicrob Agents Chemother 59:5159 –5164. http://dx.doi.org /10.1128/AAC.00158-15. 3. Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. 2005. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 43:4178 – 4182. http://dx.doi.org/10.1128/JCM.43.8.4178-4182 .2005. 4. Clinical and Laboratory Standards Institute. 2015. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard— 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA. 5. Clinical and Laboratory Standards Institute. 2015. Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI document M100-S25. Clinical and Laboratory Standards Institute, Wayne, PA. 6. Poirel L, Jayol A, Bontron S, Villegas MV, Ozdamar M, Türkoglu S, Nordmann P. 2015. The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae. J Antimicrob Chemother 70:75– 80. http: //dx.doi.org/10.1093/jac/dku323. 7. Kieser T. 1984. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid 12:19 –36. http://dx.doi.org/10 .1016/0147-619X(84)90063-5. 8. Martins WMBS, Cordeiro-Moura JR, Ramos AC, Fehlberg LC, Nicoletti AG, Gales AC. 2016. Comparison of phenotypic tests for detecting BKC1-producing Enterobacteriaceae isolates. Diagn Microbiol Infect Dis 84: 246 –248. http://dx.doi.org/10.1016/j.diagmicrobio.2015.11.021.
Antimicrobial Agents and Chemotherapy
August 2016 Volume 60 Number 8
Frequency of BKC-1-Producing Klebsiella Species Isolates Willames M. B. S. Martins,a Adriana G. Nicoletti,a Silvia R. Santos,b Jorge L. M. Sampaio,b,c Ana C. Galesa Laboratório Alerta, Division of Infectious Diseases, Department of Internal Medicine, Escola Paulista de Medicina, Federal University of São Paulo, São Paulo, Brazila; School of Pharmacy, University of São Paulo, São Paulo, Brazilb; Fleury Diagnostic Medicine, Microbiology Section, São Paulo, Brazilc
BKC-1 is a new class A serine carbapenemase that was recently identified in Klebsiella pneumoniae clinical isolates. The principal objective of this study was to evaluate the frequency of blaBKC-1 by testing a collection of Klebsiella isolates. Only 2 of 635 Klebsiella isolates (0.3%) carried blaBKC-1. The two BKC-1-producing isolates belonged to clonal complex 442 and possessed identical pulsed-field gel electrophoresis patterns. The blaBKC-1 gene was inserted into a 10-kb plasmid that was identical to the previously reported plasmid, p60136. The BKC-producing K. pneumoniae isolates presented also possessed other mechanisms for beta-lactam resistance, such as genes encoding extended-spectrum beta-lactamases and mutations in the genes ompK35 and ompK36, encoding the major porins.
I
n the past decade, carbapenemase-encoding genes such as blaKPC-like, blaOXA-48, and blaNDM-like have emerged as the main mechanisms of carbapenem resistance in Enterobacteriaceae. These carbapenem resistance genes are frequently harbored on mobile genetic elements that are highly transmissible, contributing to the increasing frequency of carbapenem-resistant Enterobacteriaceae (1). BKC-1 (Brazilian Klebsiella carbapenemase) is a class A serine carbapenemase that was recently identified in Klebsiella pneumoniae isolates from Brazil that belonged to sequence type 1781 (ST1781) (clonal complex 442 [CC442]) (2). The carbapenemase was encoded by blaBKC-1, which was located on a small nonconjugative plasmid (2). Here we investigated the frequency of blaBKC-1 among Brazilian clinical isolates of Klebsiella spp., and we performed full characterization of BKC-1-producing isolates. A total of 635 Klebsiella strains collected from previous surveillance studies were randomly selected for this study, in order to represent distinct Brazilian geographic regions. Since BKC-1 is a weak carbapenemase and other mechanisms could be associated with carbapenemase production, we selected Klebsiella strains independent of the resistance phenotype. The Klebsiella strains were isolated from different clinical specimens obtained between 2008 and 2014 (Fig. 1A). Among the 635 Klebsiella strains, 523 (82.3%) and 22 (3.4%) were previously identified as K. pneumoniae and Klebsiella oxytoca, respectively. The search for resistance genes was carried out by PCR using specific primers (2), followed by DNA sequencing (ABI 3500 Genetic Analyzer; Applied Biosystems, Foster City, CA). Only 2 BKC-1-producing K. pneumoniae isolates (0.3%), i.e., Kpn252 and KpnJ, were detected. These strains were isolated from blood and rectal swab cultures from two patients admitted to different hospitals located in the same region (the city of São Paulo and the surrounding area) in 2010 and 2014 (2 and 6 years, respectively, after the first isolation of BKC-1 [2]). The clonal relationships among the BKC-1-producing isolates were determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) (3). PFGE was performed using SpeI as the restriction enzyme. Fingerprint patterns were analyzed using BioNumerics software (Applied Maths, Austin, TX), and a dendrogram was generated by unweighted pair group method with arithmetic mean (UPGMA) cluster analyses, using a 1.5% Dice coefficient and 1% tolerance. For comparison purposes, the first reported BKC-producing strains, i.e., KP60134,
5044
aac.asm.org
KP60135, and KP6013, were included in the PFGE experiment (2). The Kpn252 and KpnJ strains showed more than 80% similarity to the KP60134, KP60135, and KP60136 strains and were grouped in the same cluster (Fig. 1B). Despite the clonal relationship, Kpn252 and KpnJ belonged to distinct STs, namely, ST1781 and ST442, respectively. ST1781 is a single-locus variant of ST442, and both are grouped in CC442. To date, all isolates identified as producers of BKC-1 have belonged to CC442. Antimicrobial susceptibility testing was performed by the broth microdilution method, according to CLSI guidelines (4, 5). The Kpn252 and KpnJ strains showed resistance to all beta-lactams and fluoroquinolones. Kpn252 showed susceptibility to tigecycline (MIC, 0.25 g/ml) and polymyxin B (MIC, 0.5 g/ml), while KpnJ was susceptible only to gentamicin (MIC, 0.25 g/ml) (Table 1). KpnJ was resistant to polymyxin B (MIC, 16 g/ml), and molecular investigation revealed disruption of mgrB by IS903. To date, inactivation of mgrB has been the main mechanism of polymyxin resistance observed in K. pneumoniae isolates (6). The genes blaBKC-1, blaSHV-110-like, and aph3A-VI were detected in both strains; in addition, qnrS1 and blaCTX-M-2 were present only in Kpn252 (Table 1). Plasmid extraction of K. pneumoniae isolates was performed using the method described by Kieser (7). The Kpn252 and KpnJ strains showed different plasmid profiles. However, Southern blot analysis revealed that the blaBKC-1 gene was located on the same 10-kb plasmid in both isolates. To determine whether this plasmid was identical to p60136, the first plasmid identified as carrying blaBKC-1, we performed whole-plasmid sequencing of the plasmid recovered from KpnJ. Initially, we performed transformation by electroporation into Escherichia coli Top10. Transformant cells
Received 29 February 2016 Returned for modification 16 March 2016 Accepted 18 May 2016 Accepted manuscript posted online 23 May 2016 Citation Martins WMBS, Nicoletti AG, Santos SR, Sampaio JLM, Gales AC. 2016. Frequency of BKC-1-producing Klebsiella species isolates. Antimicrob Agents Chemother 60:5044 –5046. doi:10.1128/AAC.00470-16. Address correspondence to Willames M. B. S. Martins, [email protected]. W.M.B.S.M. and A.G.N. contributed equally to this work. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Antimicrobial Agents and Chemotherapy
August 2016 Volume 60 Number 8
blaBKC-1 in Klebsiella Isolates
FIG 1 (A) Map of Brazil, showing the geographic distribution of Klebsiella isolates that were investigated for the blaBKC-1 gene. PA, Pará; MA, Maranhão; CE, Ceará; RN, Rio Grande do Norte; BA, Bahia; MG, Minas Gerais; GO, Goiás; SP, São Paulo; RJ, Rio de Janeiro; PR, Paraná; SC, Santa Catarina; RS, Rio Grande do Sul. (B) Dendrogram and computer-generated image of PFGE results, analyzed by BioNumerics, for the BKC-1-producing K. pneumoniae isolates.
were selected on LB agar plates supplemented with 0.5 g/ml imipenem. The sequencing of the plasmid obtained from the transformant strain was carried out using an Ion Torrent PGM system (Thermo Fisher Scientific, Waltham, MA). Newbler v. 2.8 software (Roche Diagnostics GmbH, Penzberg, Germany) was employed for plasmid sequence assembly. The whole-plasmid se-
TABLE 1 Antimicrobial susceptibility profiles of BKC-1-producing K. pneumoniae clinical isolates MIC (g/ml) (susceptibility category)a Antimicrobial
Kpn252
KpnJ
Gentamicin Amikacin Ertapenem Imipenem Meropenem Aztreonam Piperacillin-tazobactam Ceftazidime Ceftriaxone Levofloxacin Ciprofloxacin Tigecycline Polymyxin B
256 (R) 256 (R) 32 (R) 32 (R) 8 (R) ⬎128 (R) ⬎128 (R) ⬎128 (R) ⬎64 (R) 64 (R) ⬎8 (R) 0.25 (S) 0.5 (S)
0.25 (S) 256 (R) 64 (R) 32 (R) 32 (R) ⬎128 (R) ⬎128 (R) 128 (R) ⬎64 (R) 256 (R) ⬎8 (R) 2 (R) 16 (R)
a The resistance genes in Kpn252 were blaCTX-M-2, blaSHV-110-like, blaBKC-1, aph3A-VI, and qnrS1, and the resistance genes in KpnJ were blaSHV-110-like, blaBKC-1, and aph3AVI. S, sensitive; R, resistant.
August 2016 Volume 60 Number 8
quence of the 10-kb KpnJ plasmid was identical to that of the blaBKC-1-carrying plasmid p60136 (GenBank accession number KP689347.1). During the experiments, it was observed that blaBKC-1 was easily lost in the absence of selective pressure. To evaluate the stability of p60136 harbored by the Kpn252 isolate, we performed serial passages of the Kpn252 isolate on Mueller-Hinton agar plates without the addition of antibiotic. PCR was used to search for blaBKC-1 in K. pneumoniae colonies obtained from daily cultures (approximately 50 colonies per day). We verified that, in the absence of selective pressure, the plasmid harboring the blaBKC-1 gene, p60136, was unstable. A total of 20%, 32%, and 34% of colonies grown on the plates lost blaBKC-1 after 3, 4, and 5 passages, respectively. SDS-PAGE of outer membrane proteins (OMPs) and DNA sequencing of the genes coding for porins (ompK35 and ompk36) were performed. By SDS-PAGE, both isolates showed only one of the main OMPs. DNA sequencing revealed mutations in the ompK36 gene of Kpn252 and nucleotide insertion in the ompK35 gene of KpnJ. These data might suggest that, although genetically related, the isolates were probably subjected to distinct selective pressures, which could have resulted in different OMP modifications. In this study, a low frequency of isolates carrying blaBKC-1 was observed and was mainly attributed to the persistence of a K. pneumoniae clone carrying the same plasmid, p60136. In addition, blaBKC-1 was harbored by a small unstable plasmid that could be easily lost in the absence of selective pressure. More studies with
Antimicrobial Agents and Chemotherapy
aac.asm.org
5045
Martins et al.
greater numbers of isolates from all Brazilian states are necessary to assess the real prevalence of isolates carrying blaBKC-1. It is important to note that such new studies must search for blaBKC-1 using molecular techniques, since phenotypic tests may not accurately detect BKC-1-producing isolates (8). In conclusion, prudent use of antimicrobials and strict adherence to infection control measures seem to be the only effective tools for preventing the acquisition and spread of new carbapenemase-encoding genes. ACKNOWLEDGMENTS We are grateful to Ana Carolina Ramos, Ana Paula Streling, Lorena Cristina Fehlberg, and Rodrigo Cayô for their excellent technical contributions to this study. We are grateful to the National Council for Science and Technological Development for providing a grant to A.C.G. (process number 305535/ 2014-5). A.C.G. recently received research funding and/or consultation fees from AstraZeneca, MSD, and Novartis. The other authors have no conflicts to declare.
REFERENCES 1. Pitout JDD, Nordmann P, Poirel L. 2015. Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrob Agents Chemother 59:5873–5884. http://dx.doi.org/10 .1128/AAC.01019-15.
5046
aac.asm.org
2. Nicoletti AG, Marcondes MFM, Martins WMBS, Almeida LGP, Nicolás MF, Vasconcelos ATR, Oliveira V, Gales AC. 2015. Characterization of BKC-1 class A carbapenemase from Klebsiella pneumoniae clinical isolates in Brazil. Antimicrob Agents Chemother 59:5159 –5164. http://dx.doi.org /10.1128/AAC.00158-15. 3. Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. 2005. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 43:4178 – 4182. http://dx.doi.org/10.1128/JCM.43.8.4178-4182 .2005. 4. Clinical and Laboratory Standards Institute. 2015. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard— 10th ed. CLSI document M07-A10. Clinical and Laboratory Standards Institute, Wayne, PA. 5. Clinical and Laboratory Standards Institute. 2015. Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI document M100-S25. Clinical and Laboratory Standards Institute, Wayne, PA. 6. Poirel L, Jayol A, Bontron S, Villegas MV, Ozdamar M, Türkoglu S, Nordmann P. 2015. The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae. J Antimicrob Chemother 70:75– 80. http: //dx.doi.org/10.1093/jac/dku323. 7. Kieser T. 1984. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid 12:19 –36. http://dx.doi.org/10 .1016/0147-619X(84)90063-5. 8. Martins WMBS, Cordeiro-Moura JR, Ramos AC, Fehlberg LC, Nicoletti AG, Gales AC. 2016. Comparison of phenotypic tests for detecting BKC1-producing Enterobacteriaceae isolates. Diagn Microbiol Infect Dis 84: 246 –248. http://dx.doi.org/10.1016/j.diagmicrobio.2015.11.021.
Antimicrobial Agents and Chemotherapy
August 2016 Volume 60 Number 8
