Complete nucleotide sequences of two KPC-2-encoding plasmids from the same Citrobacter freundii isolate

Complete nucleotide sequences of two KPC-2-encoding plasmids from the same Citrobacter freundii... Sir, Large amounts of antibiotics are released from humans and animals into aquatic environments and lead to an increased abundance of environmental MDR bacteria, which pose a potential threat to public health.1 It is worrisome that the entry of carbapenemase-producing Enterobacteriaceae (CPE) into the environment is increasingly reported;2–4 these carbapenem-resistant bacteria pose a severe health threat as few therapeutic options are available for such pathogens.5 Although culture-independent approaches are capable of revealing the vast genetic diversity of the environmental resistome, there are few data regarding deeper characterization of mechanisms of environmental CPE isolates. Here, we describe the complete sequences of two blaKPC-2-containing plasmids present in the same Citrobacter freundii isolated from river sediment. C. freundii 18-1 (C18-1) was isolated from Shifeng River in Zhejiang Province, China (H. Xu and B. Zheng, unpublished data). C18-1 was non-susceptible to carbapenems and the presence of the blaKPC-2 gene was confirmed by PCR and sequencing. Subsequently, Southern blot hybridization with a blaKPC-2-specific probe showed two distinguishable DNA bands of ∼40 and ∼140 kb, respectively. The genomic DNA of C18-1 was extracted using a Gentra Puregene Yeast/Bact. Kit (Qiagen, Hilden, Germany) and sequenced using Pacbio RS II (Pacific Biosciences, Menlo Park, CA, USA) and the Illumina HiSeq 2500-PE150 platform (Illumina, San Diego, CA, USA). The reads were assembled using SMRT 2.3.0. Bioinformatics analysis was conducted as described previously.6 WGS confirmed the presence of two blaKPC-2-bearing plasmids, which is in line with the result of Southern hybridization. The ∼144 kb IncF plasmid pBKPC18-1 possesses an average GC content of 54.3%, with 166 predicted ORFs (Figure S1, available as Supplementary data at JAC Online). The overall structure of pBKPC18-1 is most similar to pKPC2_ECIY2403 present in Enterobacter cloacae (KY399973) and pK516_KPC from Klebsiella michiganensis (Figure S1). Of note, plasmid pK516_KPC (CP022349) was carried by a clinical K. michiganensis isolate, which was also isolated from Zhejiang Province (B. Zheng and Y. Xiao, unpublished data). Repeated conjugation experiments with pBKPC18-1 were unsuccessful, although pBKPC18-1 harboured 16 tra genes, 4 trb genes and 15 other putative transfer-associated genes. Interestingly, this phenomenon was also observed in pK516_KPC. It may be explained by the fact that the traABC operon is far away from the primary transcriptional region, impairing synthesis of primary RNA transcript.7 In addition, the distinction between pBKPC18-1 and pK516_KPC is the fragment (nt 12262–32995) covering two integrases, three hypothetical proteins and several multidrug transporters, with IS5 inserted between acrR and acrA (Figure 1a and Figure S1). The sequence of this segment shows high similarity to p12969-DIM (KU130294) and RIVM-EMC2982 (CP016955), with 53% and 58% query coverage, respectively (Figure 1a). A plausible hypothesis regarding the formation of this region is that the persistent exposure to antibiotics in aquatic environments may provoke SOS activation in response to DNA damage.8 The integrase expression and rearrangements may contribute to the acquisition of antibiotic resistance genes from other plasmids, subsequently forming a fusion structure, given that umuC and umuD are located upstream of this region (Figure 1a).8 Figure 1 View largeDownload slide Linear plasmid characterization of two KPC-2-bearing plasmids from C18-1 with closely related plasmids. (a) Comparative analysis of genetic structures of pBKPC18-1 (CP022275), p12969-DIM (KU130294) and RIVM-EMC2982 (CP016955). (b) Major structural features of plasmid pCKPC18-1 compared with KPC-2-positive plasmids pKP048 (FJ628167), p1 (CP006657), pKPC-ECN49 (KP726894) and pHS062105-3 (KF623109). Grey shading indicates shared regions with a high degree of nucleotide identity (70%–100%). Arrows indicate predicted ORFs and are coloured according to their putative functions. Blue arrows indicate replication-associated genes. Yellow arrows indicate genes involved in conjugal transfer. Orange arrows indicate genes associated with plasmid stability. Antimicrobial resistance genes and mobile element genes are indicated by red and green arrows, respectively. Grey arrows indicate genes encoding hypothetical proteins as well as proteins with unknown function. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1 View largeDownload slide Linear plasmid characterization of two KPC-2-bearing plasmids from C18-1 with closely related plasmids. (a) Comparative analysis of genetic structures of pBKPC18-1 (CP022275), p12969-DIM (KU130294) and RIVM-EMC2982 (CP016955). (b) Major structural features of plasmid pCKPC18-1 compared with KPC-2-positive plasmids pKP048 (FJ628167), p1 (CP006657), pKPC-ECN49 (KP726894) and pHS062105-3 (KF623109). Grey shading indicates shared regions with a high degree of nucleotide identity (70%–100%). Arrows indicate predicted ORFs and are coloured according to their putative functions. Blue arrows indicate replication-associated genes. Yellow arrows indicate genes involved in conjugal transfer. Orange arrows indicate genes associated with plasmid stability. Antimicrobial resistance genes and mobile element genes are indicated by red and green arrows, respectively. Grey arrows indicate genes encoding hypothetical proteins as well as proteins with unknown function. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. The second blaKPC-2-bearing plasmid, pCKPC18-1, is 47164 bp in length, harbouring a total of 55 predicted ORFs with an average GC content of 50.2%. It could not be assigned to any known incompatibility group. BLASTn comparisons revealed that pCKPC18-1 shares 99% nucleotide identity with pKPC-ECN49 (KP726894) from E. cloacae with 92% query coverage and 99% nucleotide identity with pHS062105-3 (KF623109) from K. pneumoniae with 88% query coverage, which were both isolated from China (Figure 1b). The nearly identical backbone of such non-typeable plasmids among different species in different regions implies that such plasmids may be important vehicles for the dissemination of blaKPC-2 in China. Moreover, the transfer region of pCKPC18-1 also shares high identity with IncN plasmid p1 (CP006657), except for the insertion of a Tn3 transposon element on the right flank of blaKPC-2 in pCKPC18-1 (Figure 1b). p1 is carried by a K. pneumoniae strain, which was also recovered from Zhejiang Province, China. The genetic context of blaKPC-2 on pCKPC18-1 is Tn3-tnpA, Tn3-tnpR, ISKpn27, blaKPC-2, ΔISKpn6, korC, klcA, repB and Tn1722, which is almost identical to pKP048 isolated from K. pneumoniae in Zhejiang Province (Figure 1b).9 Notably, this structure has also been widely detected in other Enterobacteriaceae in China, implying the role of horizontal transfer of Tn1722 transposons in different species.10 In contrast, blaKPC-2 was located on a Tn3 transposon element in pBKPC18-1 with the linear structure Tn3-ISKpn27-blaKPC-2-IS5-korC-klcA (Figure S1), suggesting that the dissemination of blaKPC-2 is mediated by diverse mechanisms in this isolate. In summary, to the best of our knowledge, we report the first case of C. freundii harbouring two blaKPC-2-encoding plasmids. Furthermore, the fact that we identified two blaKPC-2 genetic contexts in the same environmental isolate underscores the high promiscuity of the mobile element harbouring blaKPC-2. However, in both plasmids, the Tn3 transposon might have contributed to the acquisition of the blaKPC-2 gene. These findings highlight the difficulty of controlling CPE dissemination and the significance of multisectoral surveillance for CPE in China. Accession numbers The complete sequences of C18-1, pBKPC18-1, pCKPC18-1 and other plasmids have been deposited in GenBank under the accession numbers CP022273–CP022277. Funding This study was partially funded by grants from: the National Key R&D Program of China (No. 2016YFD0501105); the National Natural Science Foundation of China (Nos. 81361138021, 81711530049 and 81301461); the Zhejiang Provincial Key Research and Development Program (No. 2015C03032); and the Zhejiang Provincial Natural Science Foundation of China (No. LY17H190003). Transparency declarations None to declare. Supplementary data Figure S1 is available as Supplementary data at JAC Online. References 1 Montezzi LF, Campana EH, Correa LL et al.   Occurrence of carbapenemase-producing bacteria in coastal recreational waters. Int J Antimicrob Agents  2015; 45: 174– 7. Google Scholar CrossRef Search ADS PubMed  2 Oliveira S, Moura RA, Silva KC et al.   Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11 (ST437 and ST340) in urban rivers. J Antimicrob Chemother  2014; 69: 849– 52. Google Scholar CrossRef Search ADS PubMed  3 Yang F, Huang L, Li L et al.   Discharge of KPC-2 genes from the WWTPs contributed to their enriched abundance in the receiving river. Sci Total Environ  2017; 581–582: 136– 43. Google Scholar CrossRef Search ADS PubMed  4 Ahammad ZS, Sreekrishnan TR, Hands CL et al.   Increased waterborne blaNDM-1 resistance gene abundances associated with seasonal human pilgrimages to the Upper Ganges River. Environ Sci Technol  2014; 48: 3014– 20. Google Scholar CrossRef Search ADS PubMed  5 Giacobbe DR, Del Bono V, Trecarichi EM et al.   Risk factors for bloodstream infections due to colistin-resistant KPC-producing Klebsiella pneumoniae: results from a multicenter case-control-control study. Clin Microbiol Infect  2015; 21: 1106.e1– 8. Google Scholar CrossRef Search ADS   6 Wang J, Yuan M, Chen H et al.   First report of Klebsiella oxytoca strain simultaneously producing NDM-1, IMP-4 and KPC-2 carbapenemases. Antimicrob Agents Chemother  2017; 61: e00877-17. Google Scholar CrossRef Search ADS PubMed  7 Feng J, Qiu Y, Yin Z et al.   Coexistence of a novel KPC-2-encoding MDR plasmid and an NDM-1-encoding pNDM-HN380-like plasmid in a clinical isolate of Citrobacter freundii. J Antimicrob Chemother  2015; 70: 2987– 91. Google Scholar CrossRef Search ADS PubMed  8 Hocquet D, Llanes C, Thouverez M et al.   Evidence for induction of integron-based antibiotic resistance by the SOS response in a clinical setting. PLoS Pathog  2012; 8: e1002778. Google Scholar CrossRef Search ADS PubMed  9 Shen P, Wei Z, Jiang Y et al.   Novel genetic environment of the carbapenem-hydrolyzing β-lactamase KPC-2 among Enterobacteriaceae in China. Antimicrob Agents Chemother  2009; 53: 4333– 8. Google Scholar CrossRef Search ADS PubMed  10 Wang L, Fang H, Feng J et al.   Complete sequences of KPC-2-encoding plasmid p628-KPC and CTX-M-55-encoding p628-CTXM coexisted in Klebsiella pneumoniae. Front Microbiol  2015; 6: 838. Google Scholar PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Complete nucleotide sequences of two KPC-2-encoding plasmids from the same Citrobacter freundii isolate

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Oxford University Press
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© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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0305-7453
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10.1093/jac/dkx381
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Abstract

Sir, Large amounts of antibiotics are released from humans and animals into aquatic environments and lead to an increased abundance of environmental MDR bacteria, which pose a potential threat to public health.1 It is worrisome that the entry of carbapenemase-producing Enterobacteriaceae (CPE) into the environment is increasingly reported;2–4 these carbapenem-resistant bacteria pose a severe health threat as few therapeutic options are available for such pathogens.5 Although culture-independent approaches are capable of revealing the vast genetic diversity of the environmental resistome, there are few data regarding deeper characterization of mechanisms of environmental CPE isolates. Here, we describe the complete sequences of two blaKPC-2-containing plasmids present in the same Citrobacter freundii isolated from river sediment. C. freundii 18-1 (C18-1) was isolated from Shifeng River in Zhejiang Province, China (H. Xu and B. Zheng, unpublished data). C18-1 was non-susceptible to carbapenems and the presence of the blaKPC-2 gene was confirmed by PCR and sequencing. Subsequently, Southern blot hybridization with a blaKPC-2-specific probe showed two distinguishable DNA bands of ∼40 and ∼140 kb, respectively. The genomic DNA of C18-1 was extracted using a Gentra Puregene Yeast/Bact. Kit (Qiagen, Hilden, Germany) and sequenced using Pacbio RS II (Pacific Biosciences, Menlo Park, CA, USA) and the Illumina HiSeq 2500-PE150 platform (Illumina, San Diego, CA, USA). The reads were assembled using SMRT 2.3.0. Bioinformatics analysis was conducted as described previously.6 WGS confirmed the presence of two blaKPC-2-bearing plasmids, which is in line with the result of Southern hybridization. The ∼144 kb IncF plasmid pBKPC18-1 possesses an average GC content of 54.3%, with 166 predicted ORFs (Figure S1, available as Supplementary data at JAC Online). The overall structure of pBKPC18-1 is most similar to pKPC2_ECIY2403 present in Enterobacter cloacae (KY399973) and pK516_KPC from Klebsiella michiganensis (Figure S1). Of note, plasmid pK516_KPC (CP022349) was carried by a clinical K. michiganensis isolate, which was also isolated from Zhejiang Province (B. Zheng and Y. Xiao, unpublished data). Repeated conjugation experiments with pBKPC18-1 were unsuccessful, although pBKPC18-1 harboured 16 tra genes, 4 trb genes and 15 other putative transfer-associated genes. Interestingly, this phenomenon was also observed in pK516_KPC. It may be explained by the fact that the traABC operon is far away from the primary transcriptional region, impairing synthesis of primary RNA transcript.7 In addition, the distinction between pBKPC18-1 and pK516_KPC is the fragment (nt 12262–32995) covering two integrases, three hypothetical proteins and several multidrug transporters, with IS5 inserted between acrR and acrA (Figure 1a and Figure S1). The sequence of this segment shows high similarity to p12969-DIM (KU130294) and RIVM-EMC2982 (CP016955), with 53% and 58% query coverage, respectively (Figure 1a). A plausible hypothesis regarding the formation of this region is that the persistent exposure to antibiotics in aquatic environments may provoke SOS activation in response to DNA damage.8 The integrase expression and rearrangements may contribute to the acquisition of antibiotic resistance genes from other plasmids, subsequently forming a fusion structure, given that umuC and umuD are located upstream of this region (Figure 1a).8 Figure 1 View largeDownload slide Linear plasmid characterization of two KPC-2-bearing plasmids from C18-1 with closely related plasmids. (a) Comparative analysis of genetic structures of pBKPC18-1 (CP022275), p12969-DIM (KU130294) and RIVM-EMC2982 (CP016955). (b) Major structural features of plasmid pCKPC18-1 compared with KPC-2-positive plasmids pKP048 (FJ628167), p1 (CP006657), pKPC-ECN49 (KP726894) and pHS062105-3 (KF623109). Grey shading indicates shared regions with a high degree of nucleotide identity (70%–100%). Arrows indicate predicted ORFs and are coloured according to their putative functions. Blue arrows indicate replication-associated genes. Yellow arrows indicate genes involved in conjugal transfer. Orange arrows indicate genes associated with plasmid stability. Antimicrobial resistance genes and mobile element genes are indicated by red and green arrows, respectively. Grey arrows indicate genes encoding hypothetical proteins as well as proteins with unknown function. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1 View largeDownload slide Linear plasmid characterization of two KPC-2-bearing plasmids from C18-1 with closely related plasmids. (a) Comparative analysis of genetic structures of pBKPC18-1 (CP022275), p12969-DIM (KU130294) and RIVM-EMC2982 (CP016955). (b) Major structural features of plasmid pCKPC18-1 compared with KPC-2-positive plasmids pKP048 (FJ628167), p1 (CP006657), pKPC-ECN49 (KP726894) and pHS062105-3 (KF623109). Grey shading indicates shared regions with a high degree of nucleotide identity (70%–100%). Arrows indicate predicted ORFs and are coloured according to their putative functions. Blue arrows indicate replication-associated genes. Yellow arrows indicate genes involved in conjugal transfer. Orange arrows indicate genes associated with plasmid stability. Antimicrobial resistance genes and mobile element genes are indicated by red and green arrows, respectively. Grey arrows indicate genes encoding hypothetical proteins as well as proteins with unknown function. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. The second blaKPC-2-bearing plasmid, pCKPC18-1, is 47164 bp in length, harbouring a total of 55 predicted ORFs with an average GC content of 50.2%. It could not be assigned to any known incompatibility group. BLASTn comparisons revealed that pCKPC18-1 shares 99% nucleotide identity with pKPC-ECN49 (KP726894) from E. cloacae with 92% query coverage and 99% nucleotide identity with pHS062105-3 (KF623109) from K. pneumoniae with 88% query coverage, which were both isolated from China (Figure 1b). The nearly identical backbone of such non-typeable plasmids among different species in different regions implies that such plasmids may be important vehicles for the dissemination of blaKPC-2 in China. Moreover, the transfer region of pCKPC18-1 also shares high identity with IncN plasmid p1 (CP006657), except for the insertion of a Tn3 transposon element on the right flank of blaKPC-2 in pCKPC18-1 (Figure 1b). p1 is carried by a K. pneumoniae strain, which was also recovered from Zhejiang Province, China. The genetic context of blaKPC-2 on pCKPC18-1 is Tn3-tnpA, Tn3-tnpR, ISKpn27, blaKPC-2, ΔISKpn6, korC, klcA, repB and Tn1722, which is almost identical to pKP048 isolated from K. pneumoniae in Zhejiang Province (Figure 1b).9 Notably, this structure has also been widely detected in other Enterobacteriaceae in China, implying the role of horizontal transfer of Tn1722 transposons in different species.10 In contrast, blaKPC-2 was located on a Tn3 transposon element in pBKPC18-1 with the linear structure Tn3-ISKpn27-blaKPC-2-IS5-korC-klcA (Figure S1), suggesting that the dissemination of blaKPC-2 is mediated by diverse mechanisms in this isolate. In summary, to the best of our knowledge, we report the first case of C. freundii harbouring two blaKPC-2-encoding plasmids. Furthermore, the fact that we identified two blaKPC-2 genetic contexts in the same environmental isolate underscores the high promiscuity of the mobile element harbouring blaKPC-2. However, in both plasmids, the Tn3 transposon might have contributed to the acquisition of the blaKPC-2 gene. These findings highlight the difficulty of controlling CPE dissemination and the significance of multisectoral surveillance for CPE in China. Accession numbers The complete sequences of C18-1, pBKPC18-1, pCKPC18-1 and other plasmids have been deposited in GenBank under the accession numbers CP022273–CP022277. Funding This study was partially funded by grants from: the National Key R&D Program of China (No. 2016YFD0501105); the National Natural Science Foundation of China (Nos. 81361138021, 81711530049 and 81301461); the Zhejiang Provincial Key Research and Development Program (No. 2015C03032); and the Zhejiang Provincial Natural Science Foundation of China (No. LY17H190003). Transparency declarations None to declare. Supplementary data Figure S1 is available as Supplementary data at JAC Online. References 1 Montezzi LF, Campana EH, Correa LL et al.   Occurrence of carbapenemase-producing bacteria in coastal recreational waters. Int J Antimicrob Agents  2015; 45: 174– 7. Google Scholar CrossRef Search ADS PubMed  2 Oliveira S, Moura RA, Silva KC et al.   Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11 (ST437 and ST340) in urban rivers. J Antimicrob Chemother  2014; 69: 849– 52. Google Scholar CrossRef Search ADS PubMed  3 Yang F, Huang L, Li L et al.   Discharge of KPC-2 genes from the WWTPs contributed to their enriched abundance in the receiving river. Sci Total Environ  2017; 581–582: 136– 43. Google Scholar CrossRef Search ADS PubMed  4 Ahammad ZS, Sreekrishnan TR, Hands CL et al.   Increased waterborne blaNDM-1 resistance gene abundances associated with seasonal human pilgrimages to the Upper Ganges River. Environ Sci Technol  2014; 48: 3014– 20. Google Scholar CrossRef Search ADS PubMed  5 Giacobbe DR, Del Bono V, Trecarichi EM et al.   Risk factors for bloodstream infections due to colistin-resistant KPC-producing Klebsiella pneumoniae: results from a multicenter case-control-control study. Clin Microbiol Infect  2015; 21: 1106.e1– 8. Google Scholar CrossRef Search ADS   6 Wang J, Yuan M, Chen H et al.   First report of Klebsiella oxytoca strain simultaneously producing NDM-1, IMP-4 and KPC-2 carbapenemases. Antimicrob Agents Chemother  2017; 61: e00877-17. Google Scholar CrossRef Search ADS PubMed  7 Feng J, Qiu Y, Yin Z et al.   Coexistence of a novel KPC-2-encoding MDR plasmid and an NDM-1-encoding pNDM-HN380-like plasmid in a clinical isolate of Citrobacter freundii. J Antimicrob Chemother  2015; 70: 2987– 91. Google Scholar CrossRef Search ADS PubMed  8 Hocquet D, Llanes C, Thouverez M et al.   Evidence for induction of integron-based antibiotic resistance by the SOS response in a clinical setting. PLoS Pathog  2012; 8: e1002778. Google Scholar CrossRef Search ADS PubMed  9 Shen P, Wei Z, Jiang Y et al.   Novel genetic environment of the carbapenem-hydrolyzing β-lactamase KPC-2 among Enterobacteriaceae in China. Antimicrob Agents Chemother  2009; 53: 4333– 8. Google Scholar CrossRef Search ADS PubMed  10 Wang L, Fang H, Feng J et al.   Complete sequences of KPC-2-encoding plasmid p628-KPC and CTX-M-55-encoding p628-CTXM coexisted in Klebsiella pneumoniae. Front Microbiol  2015; 6: 838. Google Scholar PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Feb 1, 2018

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