Characterization of a Novel Plasmid Type and Various Genetic Contexts of bla OXA-58 in Acinetobacter spp. from Multiple Cities in China

Background/Objective

Several studies have described the epidemiological distribution of bla _OXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of bla _OXA-58-carrying plasmids and the genetic context surrounding bla _OXA-58 in Acinetobacter spp. in China.

Methodology/Principal Findings

Twelve non-duplicated bla _OXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of bla _OXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of bla _OXA-58-harboring plasmids. The genetic structure surrounding bla _OXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype), three Acinetobacter nosocomialis isolates (belong to two pulsotypes) and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types). A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both bla _OXA-58 and bla _OXA-23. bla _OXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in bla _OXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of bla _OXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15)-ΔISAba3-like element-bla _OXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element-bla _OXA-58 were carbapenem susceptible.

Conclusion

The study revealed the unique features of bla _OXA-58-carrying plasmids in Acinetobacter spp. in China, which were different from that of Acinetobacter spp. found in European countries. The diversity of the genetic contexts of bla _OXA-58 contributed to various antibiotics resistance profiles.     

Inverse PCR for subtyping of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> carrying IS<Emphasis Type="Italic">Aba</Emphasis>1

Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla_OXA-51-like, bla_OXA-23-like, and bla_ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes — bla_ampC, bla_OXA-66-like, and csuD — were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.     

Emergence of OXA-48-Producing Klebsiella pneumoniae in Taiwan

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four bla _OXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured bla _CTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the bla _OXA-48 gene in all four isolates was identical to pKP_OXA-48N1, a bla _OXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify bla _OXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of bla _OXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern.     

Epidemiological Characteristics of bla NDM-1 in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii Complex in China from 2011 to 2012

Objectives

The study aimed to investigate the prevalence and epidemiological characteristics of bla _NDM-1 (encoding New Delhi metallo-β-lactamase 1) in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ABC) in China from July 2011 to June 2012.

Methods

PCR was used to screen for the presence of bla _NDM-1 in all organisms studied. For bla _NDM-1-positive strains, 16S rRNA analysis and Analytical Profile Index (API) strips were used to identify the bacterial genus and species. The ABCs were reconfirmed by PCR detection of bla _OXA-51-like. Antibiotic susceptibilities of the bacteria were assessed by determining minimum inhibitory concentration (MIC) of them using two-fold agar dilution test, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot hybridization were conducted to ascertain the gene location of bla _NDM-1. Conjugation experiments were conducted to determine the transmission of bla _NDM-1-positive strains.

Results

Among 2,170 Enterobacteriaceae and 600 ABCs, seven Enterobacteriaceae strains and two A. calcoaceticus isolates from five different cities carried the bla _NDM-1 gene. The seven Enterobacteriaceae strains comprised four Klebsiella pneumoniae, one Enterobacter cloacae, one Enterobacter aerogen and one Citrobacter freundii. All seven were non-susceptible to imipenem, meropenem or ertapenem. Two A. calcoaceticus species were resistant to imipenem and meropenem. Three K. pneumoniae showed the same PFGE profiles. The bla _NDM-1 genes of eight strains were localized on plasmids, while one was chromosomal.

Conclusions

Compared with previous reports, the numbers and species containing the bla _NDM-1 in Enterobacteriaceae have significantly increased in China. Most of them are able to disseminate the gene, which is cause for concern. Consecutive surveillance should be implemented and should also focus on the dissemination of bla _NDM-1 among gram-negative clinical isolates.     

Within-Host and Population Transmission of bla OXA-48 in K. pneumoniae and E. coli

During a large hospital outbreak of OXA-48 producing bacteria, most K. pneumoniae _OXA-48 isolates were phenotypically resistant to meropenem or imipenem, whereas most E. coli _OXA-48 isolates were phenotypically susceptible to these antibiotics. In the absence of molecular gene-detection E. coli _OXA-48 could remain undetected, facilitating cross-transmission and horizontal gene transfer of bla _OXA-48. Based on 868 longitudinal molecular microbiological screening results from patients carrying K. pneumoniae _OXA-48 (n = 24), E. coli _OXA-48 (n = 17), or both (n = 40) and mathematical modelling we determined mean durations of colonisation (278 and 225 days for K. pneumoniae _OXA-48 and E. coli _OXA-48, respectively), and horizontal gene transfer rates (0.0091/day from K. pneumoniae to E. coli and 0.0015/day vice versa). Based on these findings the maximum effect of horizontal gene transfer of bla _OXA-48 originating from E. coli _OXA-48 on the basic reproduction number (R ₀) is 1.9%, and it is, therefore, unlikely that phenotypically susceptible E. coli _OXA-48 will contribute significantly to the spread of bla _OXA-48.     

First detection of OXA-24 carbapenemase-producing Acinetobacter baumannii isolates in Bulgaria

This report describes the first identification of OXA-24 carbapenemase-producing Acinetobacter baumannii isolates from Bulgaria. According to national surveillance data A. baumannii along with Pseudomonas aeruginosa are the most troublesome microorganisms in hospital environment with high rates of acquired carbapenem resistance. In the present study real-time multiplex PCR was performed to identify the most common carbapenemase genes in 15 non-duplicate carbapenem-resistant A. baumannii isolates collected in 2012. The results showed lack of KPC, GES, VIM, IMP-type enzymes. Four A. baumannii isolates tested positive by PCR for the acquired OXA-24 together with the intrinsic OXA-51 carbapenemase. OXA-24 and OXA-23 were determined as co-existent in one isolate. Two isolates were identified with OXA-23 in addition to the OXA-51 carbapenemase.     

Carbapenem-Resistant Acinetobacter baumannii from Serbia: Revision of CarO Classification

Carbapenem-resistant A. baumannii present a significant therapeutic challenge for the treatment of nosocomial infections in many European countries. Although it is known that the gradient of A. baumannii prevalence increases from northern to southern Europe, this study provides the first data from Serbia. Twenty-eight carbapenem-resistant A. baumannii clinical isolates were collected at a Serbian pediatric hospital during a 2-year period. The majority of isolates (67.68%) belonged to the sequence type Group 1, European clonal complex II. All isolates harbored intrinsic OXA-51 and AmpC cephalosporinase. OXA-23 was detected in 16 isolates (57.14%), OXA-24 in 23 isolates (82.14%) and OXA-58 in 11 isolates (39.29%). Six of the isolates (21.43%) harbored all of the analyzed oxacillinases, except OXA-143 and OXA-235 that were not detected in this study. Production of oxacillinases was detected in different pulsotypes indicating the presence of horizontal gene transfer. NDM-1, VIM and IMP were not detected in analyzed clinical A. baumannii isolates. ISAba1 insertion sequence was present upstream of OXA-51 in one isolate, upstream of AmpC in 13 isolates and upstream of OXA-23 in 10 isolates. In silico analysis of carO sequences from analyzed A. baumannii isolates revealed the existence of two out of six highly polymorphic CarO variants. The phylogenetic analysis of CarO protein among Acinetobacter species revised the previous classification CarO variants into three groups based on strong bootstraps scores in the tree analysis. Group I comprises four variants (I-IV) while Groups II and III contain only one variant each. One half of the Serbian clinical isolates belong to Group I variant I, while the other half belongs to Group I variant III.     

Distribution of Allelic Variants of the Chromosomal Gene bla OXA-114-like in Achromobacter xylosoxidans Clinical Isolates

Achromobacter xylosoxidans is increasingly being documented in cystic fibrosis patients. The bla _OXA-114 gene has been recognized as a naturally occurring chromosomal gene, exhibiting different allelic variants. In the population under study, the bla _OXA-114-like gene was found in 19/19 non-epidemiological-related clinical isolates of A. xylosoxidans with ten different alleles including 1 novel OXA-114 variant.     

Epidemic Plasmid Carrying bla CTX-M-15 in Klebsiella penumoniae in China

Objective

To investigate the local epidemiology of Klebsiella penumoniae carrying bla _CTX-M-15 in southern China and to characterize the genetic environment of bla _CTX-M-15.

Methods

PCR and DNA sequencing were used to detect and characterize the genetic contexts of bla _CTX-M-15. The clonal relatedness of isolates carrying bla _CTX-M-15 was determined by pulse-field gel electrophoresis. Conjugative plasmids carrying bla _CTX-M-15 were obtained by mating and were further subject to restriction analysis and replicon typing.

Results

A total of 47CTX-M-15 ESBL-producing isolates of K. pneumoniae were collected from nine hospitals in China from October 2007 to October 2008. Isolates were clustered into various clonal groups. The local spread of bla _CTX-M-15 was mainly mediated by one major conjugative plasmid as determined by S1-PFGE and restriction analysis. A 90-kb plasmid belonging to incompatible group FII was the major carrier of bla _CTX-M-15 in K. pneumoniae. Except bla _TEM-1, the resistance genes such as bla _SHV, bla _DHA-1, bla _OXA-1, qnrB, qnrS, aac(3)-II, and aac(6′)-Ib were not found in the plasmid. In the comparing of conjugative gene sequence, it is 100% identical with the plasmid pKF3–94, which was found in K. pneumonia from Zhejiang province of china previously.

Conclusions

bla _CTX-M-15 was prevalent in K. pneumonia of southern China. The dissemination of bla _CTX-M-15 appeared to be due to the horizontal transfer of a 90-kb epidemic plasmid.     

Antimicrobial Combinations against Pan-Resistant Acinetobacter baumannii Isolates with Different Resistance Mechanisms

The study investigated the effect of antibiotic combinations against 20 clinical isolates of A. baumannii (seven colistin-resistant and 13 colistin-susceptible) with different resistance mechanisms. Clinical data, treatment, and patient mortality were evaluated. The following methods were used: MIC, PCRs, and outer membrane protein (OMP) analysis. Synergy was investigated using the checkerboard and time-kill methods. Clonality was evaluated by PFGE. Based on clonality, the whole genome sequence of six A. baumannii isolates was analyzed. All isolates were resistant to meropenem, rifampicin, and fosfomycin. OXA-23 and OXA-143 were the most frequent carbapenemases found. Four isolates showed loss of a 43kDa OMP_. The colistin-susceptible isolates belonged to different clones and showed the highest synergistic effect with fosfomycin-amikacin. Among colistin-resistant isolates, the highest synergistic effect was observed with the combinations of colistin-rifampicin followed by colistin-vancomycin. All colistin-resistant isolates harbored bla_OXA-23-like and belonged to CC113. Clinical and demographic data were available for 18 of 20 patients. Fourteen received treatment and eight patients died during treatment. The most frequent site of infection was the blood in 13 of 14 patients. Seven patients received vancomycin plus an active drug against A. baumannii; however, mortality did not differ in this group. The synergistic effect was similar for colistin-susceptible isolates of distinct clonal origin presenting with the same resistance mechanism. Overall mortality and death during treatment was high, and despite the high synergism in vitro with vancomycin, death did not differ comparing the use or not of vancomycin plus an active drug against A. baumannii.     

Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France

Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene bla _OXA-23 was the most frequently detected (82%), followed by bla _OXA-24 (11%) and bla _OXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones.     

Prevalence of Plasmid-Mediated Quinolone Resistance and Aminoglycoside Resistance Determinants among Carbapeneme Non-Susceptible Enterobacter cloacae

Background

Simultaneous resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS) isolates will inevitably create problems. The present study was performed to characterize the prevalence of the plasmid-mediated quinolone resistance determinants (QRDs) and aminoglycoside resistance determinants (ARDs) among the CNS Enterobacter cloacae (E. cloacae) isolates in a Chinese teaching hospital, and to acquire their molecular epidemiological characteristics.

Methods

The β-lactamases genes (including class A carbapenemase genes bla_KPC and bla_SME, metallo-β-lactamase genes (MBLs) bla_IMP, bla_VIM and bla_NDM, and extended spectrum β-lactamases (ESBLs),bla_CTX-M, bla_TEM and bla_SHV), QRDs (including qnrA, qnrB, qnrS and aac(6′)-Ib-cr) and ARDs (including aac(6′)-Ib, armA and rmtB) of these 35 isolates were determined by PCR and sequenced bidirectionally. The clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE).

Results

Of the 35 isolates, 9 (25.7%) harbored a carbapenemase gene; 23 (65.7%) carried ESBLs; 24 (68.6%) were QRD positive; and 27 (77.1%) were ARD positive. Among the 5 bla_IMP-8 positive strains, 4 (80%) contained both ESBL and QRD genes, and all the 5 (100%) harbored ARD genes. Of the 23 ESBLs positive isolates, 6 (26.1%) were carbapenemase positive, 14 (60.9%) were QRD positive, and 18 (78.3%) were ARD positive. PFGE revealed genetic diversity among the 35 isolates, indicating that the high prevalence of CNS E. cloacae isolates was not caused by clonal dissemination.

Conclusion

QRD and ARD genes were highly prevalent among the CNS E. cloacae isolates. Multiple resistant genes were co-expressed in the same isolates. The CNS E. cloacae isolate co-expressing bla_NDM-1, bla_IMP-26, qnrA1 and qnrS1 was first reported.     

Epidemiology and resistance features of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> isolates from the ward environment and patients in the burn ICU of a Chinese hospital

Acinetobacter baumannii is an important opportunistic pathogen that causes severe nosocomial infections, especially in intensive care units (ICUs). Over the past decades, an everincreasing number of hospital outbreaks caused by A. baumannii have been reported worldwide. However, little attention has been directed toward the relationship between A. baumannii isolates from the ward environment and patients in the burn ICU. In this study, 88 A. baumannii isolates (26 from the ward environment and 62 from patients) were collected from the burn ICU of the Southwest Hospital in Chongqing, China, from July through December 2013. Antimicrobial susceptibility testing results showed that drug resistance was more severe in isolates from patients than from the ward environment, with all of the patient isolates being fully resistant to 10 out of 19 antimicrobials tested. Isolations from both the ward environment and patients possessed the β-lactamase genes bla_OXA-51, bla_OXA-23, bla_AmpC, bla_VIM, and bla_PER. Using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), these isolates could be clustered into 4 major PFGE types and 4 main sequence types (ST368, ST369, ST195, and ST191) among which, ST368 was the dominant genotype. Epidemiologic and molecular typing data also revealed that a small-scale outbreak of A. baumannii infection was underway in the burn ICU of our hospital during the sampling period. These results suggest that dissemination of β-lactamase genes in the burn ICU might be closely associated with the high-level resistance of A. baumannii, and the ICU environment places these patients at a high risk for nosocomial infection. Cross-contamination should be an important concern in clinical activities to reduce hospitalacquired infections caused by A. baumannii.     

Co-Carriage of bla KPC-2 and bla NDM-1 in Clinical Isolates of Pseudomonas aeruginosa Associated with Hospital Infections from India

Global spread of KPC poses to be a serious threat complicating treatment options in hospital settings. The present study investigates the genetic environment of bla _KPC-2 among clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital of India. The study isolates were collected from different wards and clinics of Silchar Medical College and Hospital, India, from 2012–2013. The presence of bla _KPC was confirmed by genotypic characterization followed by sequencing. Cloning of the bla _KPC-2 gene was performed and the genetic environment of this gene was characterized as well. Transferability of the resistance gene was determined by transformation assay and Southern hybridization. Additionally, restriction mapping was also carried out. Two isolates of P. aeruginosa were found to harbor bla _KPC-2, were resistant towards aminoglycosides, quinolone and β-lactam-β-lactamase inhibitor combination. In both the isolates, the resistance determinant was associated with class 1 integron and horizontally transferable. Both the isolates were co-harboring bla _NDM-1. The first detection of this integron mediated bla _KPC-2 coexisting with bla _NDM-1 in P. aeruginosa from India is worrisome, and further investigation is required to track the gene cassette mediated bla _KPC-2 in terms of infection control and to prevent the spread of this gene in hospitals as well as in the community.     

Characterization of an IncFII plasmid encoding NDM-1 from Escherichia coli ST131

Background

The current spread of the gene encoding the metallo-ß-lactamase NDM-1 in Enterobacteriaceae is linked to a variety of surrounding genetic structures and plasmid scaffolds.

Methodology

The whole sequence of plasmid pGUE-NDM carrying the bla _NDM-1 gene was determined by high-density pyrosequencing and a genomic comparative analysis with other bla _NDM-1-negative IncFII was performed.

Principal Findings

Plasmid pGUE-NDM replicating in Escherichia coli confers resistance to many antibiotic molecules including β-lactams, aminoglycosides, trimethoprim, and sulfonamides. It is 87,022 bp in-size and carries the two β-lactamase genes bla _NDM-1 and bla _OXA-1, together with three aminoglycoside resistance genes aacA4, aadA2, and aacC2. Comparative analysis of the multidrug resistance locus contained a module encompassing the bla _NDM-1 gene that is actually conserved among different structures identified in other enterobacterial isolates. This module was constituted by the bla _NDM-1 gene, a fragment of insertion sequence ISAba125 and a bleomycin resistance encoding gene.

Significance

This is the first characterized bla _NDM-1-carrying IncFII-type plasmid. Such association between the bla _NDM-1 gene and an IncFII-type plasmid backbone is extremely worrisome considering that this plasmid type is known to spread efficiently, as examplified with the worldwide dissemination of bla _CTX-M-15-borne IncFII plasmids.     

Clonal Dissemination of KPC-2, VIM-1, OXA-48-Producing Klebsiella pneumoniae ST147 in Katowice,Poland

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important bacterium of nosocomial infections. In this study, CRKP strains, which were mainly isolated from fecal samples of 14 patients in three wards of the hospital in the Silesia Voivodship, rapidly increased from February to August 2018. Therefore, we conducted microbiological and molecular studies of the CRKP isolates analyzed. Colonized patients had critical underlying diseases and comorbidities; one developed bloodstream infection, and five died (33.3%). Antibiotic susceptibilities were determined by the E-test method. A disc synergy test confirmed carbapenemase production. CTX-Mplex PCR evaluated the presence of resistance genes bla_CTX-M-type, bla_CTX-M-1, bla_CTX-M-9, and the genes bla_SHV, bla_TEM, bla_KPC-2, bla_NDM-1, bla_OXA-48, bla_IMP, and bla_VIM-1 was detected with the PCR method. Clonality was evaluated by Multi Locus Sequence Typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Six (40%) strains were of XDR (Extensively Drug-Resistant) phenotype, and nine (60%) of the isolates exhibited MDR (Multidrug-Resistant) phenotype. The range of carbapenem minimal inhibitory concentrations (MICs, μg/mL) was as follows doripenem (16 to >32), ertapenem (> 32), imipenem (4 to > 32), and meropenem (> 32). PCR and sequencing confirmed the bla_CTX-M-15, bla_KPC-2, bla_OXA-48, and bla_VIM-1 genes in all strains. The isolates formed one large PFGE cluster (clone A). MLST assigned them to the emerging high-risk clone of ST147 (CC147) pandemic lineage harboring the bla_OXA-48 gene. This study showed that the K. pneumoniae isolates detected in the multi-profile medical centre in Katowice represented a single strain of the microorganism spreading in the hospital environment.     

National Surveillance Study on Carbapenem Non-Susceptible Klebsiella pneumoniae in Taiwan: The Emergence and Rapid Dissemination of KPC-2 Carbapenemase

Objectives

The global spread and increasing incidence of carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) has made its treatment difficult, increasing the mortality. To establish nationwide data on CnSKP spread and carbapenem-resistance mechanisms, we conducted a national surveillance study in Taiwanese hospitals.

Methods

We collected 100 and 247 CnSKP isolates in 2010 and 2012, respectively. The tests performed included antibiotic susceptibility tests; detection of carbapenemase, extended-spectrum β-lactamases (ESBL), and AmpC β-lactamases genes; outer membrane porin profiles; and genetic relationship with pulsed-field gel electrophoresis and multilocus sequence type.

Results

The resistance rate of CnSKP isolates to cefazolin, cefotaxime, cefoxitin, ceftazidime, and ciprofloxacin was over 90%. Susceptibility rate to tigecycline and colistin in 2010 was 91.0% and 83.0%, respectively; in 2012, it was 91.9% and 87.9%, respectively. In 2010, carbapenemase genes were detected in only 6.0% of isolates (4 bla _IMP-8 and 2 bla _VIM-1). In 2012, carbapenemase genes were detected in 22.3% of isolates (41 bla _KPC-2, 7 bla _VIM-1, 6 bla _IMP-8, and 1 bla _NDM-1). More than 95% of isolates exhibited either OmpK35 or OmpK36 porin loss or both. Impermeability due to porin mutation coupled with AmpC β-lactamases or ESBLs were major carbapenem-resistance mechanisms. Among 41 KPC-2-producing K. pneumoniae isolates, all were ST11 with 1 major pulsotype.

Conclusions

In 2010 and 2012, the major mechanisms of CnSKP in Taiwan were the concomitance of AmpC with OmpK35/36 loss. KPC-2-KP dissemination with the same ST11 were observed in 2012. The emergence and rapid spread of KPC-2-KP is becoming an endemic problem in Taiwan. The identification of NDM-1 K. pneumoniae case is alarming.     

Virulence of Klebsiella pneumoniae Isolates Harboring bla KPC-2 Carbapenemase Gene in a Caenorhabditis elegans Model

Klebsiella pneumoniae carbapenemase (KPC) is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i) five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii) seven transformants obtained after electroporation of either these natural KPC plasmids or a recombinant plasmid harboring only the bla _KPC-2 gene into reference strains K. pneumoniae ATCC10031/{"type":"entrez-protein","attrs":{"text":"CIP53153","term_id":"878514309","term_text":"CIP53153"}}CIP53153; and (iii) five clinical strains cured of plasmids. The virulence of K. pneumoniae isolates was evaluated in the Caenorhabditis elegans model. The clinical KPC producers and transformants were significantly less virulent (LT50: 5.5 days) than K. pneumoniae reference strain (LT50: 4.3 days) (p<0.01). However, the worldwide spread KPC-2 positive K. pneumoniae ST258 strains and reference strains containing plasmids extracted from K. pneumoniae ST258 strains had a higher virulence than KPC-2 strains belonging to other ST types (LT50: 5 days vs. 6 days, p<0.01). The increased virulence observed in cured strains confirmed this trend. The bla _KPC-2 gene itself was not associated to increased virulence.