Gong-Xiang Chen

Emergence of Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli Isolates Possessing the Plasmid-Mediated Carbapenem-Hydrolyzing β-Lactamase KPC-2 in Intensive Care Units of a Chinese Hospital

ABSTRACT Twenty-one Serratia marcescens, ten Klebsiella pneumoniae, and one Escherichia coli isolate with carbapenem resistance or reduced carbapenem susceptibility were recovered from intensive care units (ICUs) in our hospital. Enterobacterial repetitive intergenic consensus-PCR and pulsed-field gel electrophoresis demonstrated that all the S. marcescens isolates belonged to a clonal strain and the 10 K. pneumoniae isolates were indistinguishable or closely related to each other. The MICs of imipenem, meropenem, and ertapenem for all isolates were 2 to 8 μg/ml, except for K. pneumoniae K10 (MICs of 128, 256, and >256 μg/ml). Isoelectric focusing, PCRs, and DNA sequencing indicated that all S. marcescens isolates produced KPC-2 and a β-lactamase with a pI of 6.5. All K. pneumoniae isolates produced TEM-1, KPC-2, CTX-M-14, and a β-lactamase with a pI of 7.3. The E. coli E1 isolate produced KPC-2, CTX-M-15, and a β-lactamase with a pI of 7.3. Conjugation studies with E. coli (EC600) resulted in the transfer of reduced carbapenem susceptibility compared to that of the original isolates, and only the blaKPC-2 gene was detected in E. coli transconjugants. Plasmid restriction analysis showed identical restriction patterns among all E. coli transconjugants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ompK35/36 gene sequence analysis of outer membrane proteins revealed that K. pneumoniae K10 failed to express OmpK36, because of insertional inactivation by an insertion sequence ISEcp1. All these results indicate that KPC-2-producing S. marcescens, K. pneumoniae, and E. coli isolates emerged in ICUs in our hospital. KPC-2 combined with porin deficiency results in high-level carbapenem resistance in K. pneumoniae. The same blaKPC-2-encoding plasmid was spread among the three different genera.

Molecular Typing of CTX-M-Producing Escherichia coli Isolates from Environmental Water, Swine Feces, Specimens from Healthy Humans, and Human Patients

ABSTRACT CTX-M-producing Escherichia coli is the predominant type of extended-spectrum β-lactamase (ESBL)-producing E. coli worldwide. In this study, molecular typing was conducted for 139 CTX-M-producing E. coli isolates, phenotypically positive for ESBLs, isolated from environmental water, swine, healthy humans, and hospitalized patients in Hangzhou, China. The antibiotic resistance profiles of the isolates for the cephalosporins and fluoroquinolones were determined. The isolates showed 100% resistance to cefotaxime and ceftriaxone while maintaining relatively high susceptibility to cefoxitin, cefepime, and ceftazidime. A total of 61.9% (86/139) of the isolates, regardless of origin, showed high resistance to fluoroquinolones. PCRs and DNA sequencing indicated that bla CTX-M-14 was the most prevalent CTX-M-9 group gene and that bla CTX-M-15 and bla CTX-M-55 were the dominant CTX-M-1 group genes. Isolates from all sources with CTX-M types belonging to the CTX-M-1 or CTX-M-9 group were most frequently associated with epidemics. Molecular homology analysis of the isolates, conducted by phylogenetic grouping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), demonstrated that the dominant clones belonged to B2-ST131, D-ST648, D-ST38, or A-CC10. These four sequence types (STs) were discovered in E. coli isolates both from humans and from environmental water, suggesting frequent and continuous intercompartment transmission between humans and the aquatic environment. Seven novel sequence types were identified in the current study. In conclusion, this study is the first to report the molecular homology analysis of CTX-M-producing E. coli isolates collected from water, swine, and healthy and hospitalized humans, suggesting that pathogens in the environment might originate both from humans and from animals.

High-level carbapenem resistance in a Citrobacter freundii clinical isolate is due to a combination of KPC-2 production and decreased porin expression

An imipenem-resistant isolate of Citrobacter freundii ZJ163 (MIC 256 microg ml(-1)) isolated from a Chinese hospital was investigated. The C. freundii ZJ163 isolate exhibited high-level resistance to carbapenems, penicillins, cephalosporins, cefoxitin, aztreonam, quinolones and aminoglycosides. Isoelectric focusing (IEF) demonstrated three beta-lactamases with pIs of 5.4 (TEM-1), 6.7 (KPC-2) and 7.9 (CTX-M-14). Two different transconjugants (types A and B) were obtained by conjugation studies. The type A transconjugant exhibited reduced susceptibility or resistance to penicillins, cephalosporins and aztreonam, but was susceptible to carbapenems, quinolones and aminoglycosides. The antimicrobial susceptibility patterns of the type B transconjugant were similar to that of type A, except for its significantly reduced carbapenem susceptibility (imipenem MIC 2 microg ml(-1)). IEF, specific PCRs and DNA sequence analysis indicated that the type A transconjugant produced CTX-M-14 beta-lactamase with a pI of 7.9, that the type B transconjugant produced KPC-2 beta-lactamase with a pI of 6.7 and that the beta-lactamase with a pI of 5.4 was TEM-1. PCR analysis and sequencing confirmed the presence of the ampC gene in the chromosomal DNA from C. freundii ZJ163, although no activity of AmpC beta-lactamase was detected by IEF. Urea/SDS-PAGE analysis of outer-membrane proteins revealed that the levels of the 41 and 38 kDa porins were decreased in C. freundii ZJ163. It was concluded that production of KPC-2 combined with decreased expression of porins contributes to high-level resistance to carbapenems in C. freundii ZJ163.

Reduced susceptibility to carbapenems in Klebsiella pneumoniae clinical isolates associated with plasmid-mediated β-lactamase production and OmpK36 porin deficiency

Two carbapenem-non-susceptible Klebsiella pneumoniae isolates, Z2554 and Z2110, were collected from a hospital in China and analysed by PFGE. K. pneumoniae Z2554 and Z2110 were genetically unrelated and showed resistance to ertapenem, and reduced susceptibility to imipenem and meropenem. Analysis of their beta-lactamases indicated that K. pneumoniae Z2554 produced TEM-1 and CTX-M-14 beta-lactamases, whilst Z2110 produced a plasmid-mediated AmpC beta-lactamase, DHA-1, in addition to TEM-1 and CTX-M-14. SDS-PAGE analysis of the outer-membrane proteins (OMPs) revealed that both isolates lacked an OMP of approximately 39 kDa (OmpK36), whilst Z2110 had an additional protein with an approximate molecular mass of 26 kDa. Analysis of the OMP-encoding genes demonstrated that the ompK35 sequence of K. pneumoniae Z2554 and Z2110 contained a number of silent mutations. In ompK36, several insertions and deletions of short DNA fragments (1-6 bp) were detected in both isolates. The N-terminal sequence of the approximately 26 kDa protein band identified in Z2110 had no similarity to the sequence of OmpK36. Instead, it shared high similarity with hypothetical protein KPN_03267 originating from K. pneumoniae subsp. pneumoniae MGH 78578. It was concluded that beta-lactamase production combined with OmpK36 deficiency results in ertapenem resistance, and reduced imipenem and meropenem susceptibility, in K. pneumoniae Z2554 and Z2110. OmpK36 may play an important role in the resistance or reduced susceptibility to carbapenems in K. pneumoniae producing AmpC, extended-spectrum beta-lactamase or broad-spectrum beta-lactamase.

Emergence of Carbapenem-Resistant Serotype K1 Hypervirulent Klebsiella pneumoniae Strains in China

ABSTRACT We report the emergence of five carbapenem-resistant K1 hypervirulent Klebsiella pneumoniae (hvKP) strains which caused fatal infections in hospital patients in Zhejiang Province, China, upon entry through surgical wounds. Genotyping results revealed the existence of three genetically related strains which exhibited a new sequence type, ST1797, and revealed that all strains harbored the magA and wcaG virulence genes and a plasmid-borne blaKPC-2 gene. These findings indicate that K1 hvKP is simultaneously hypervirulent, multidrug resistant, and transmissible.

Emergence of Escherichia coli Sequence Type 131 Isolates Producing KPC-2 Carbapenemase in China

ABSTRACT Twenty-two KPC-2-producing Escherichia coli isolates were obtained from three hospitals in Hangzhou, China, from 2007 to 2011. One isolate, with OmpC porin deficiency, exhibited high-level carbapenem resistance. Pulsed-field gel electrophoresis showed that few isolates were indistinguishable or closely related. Multilocus sequence typing indicated that sequence type 131 (ST131) was the predominant type (9 isolates, 40.9%), followed by ST648 (5 isolates), ST405 (2 isolates), ST38 (2 isolates), and 4 single STs, ST69, ST2003, ST2179, and ST744. Phylogenetic analysis indicated that 9 group B2 isolates belonged to ST131, and 5 of 11 group D isolates belonged to ST648. Only one group B1 isolate and one group A isolate were identified. A representative plasmid (pE1) was partially sequenced, and a 7,788-bp DNA fragment encoding Tn3 transposase, Tn3 resolvase, ISKpn8 transposase, KPC-2, and ISKpn6-like transposase was obtained. The blaKPC-2-surrounding sequence was amplified by a series of primers. The PCR results showed that 13 isolates were consistent with the genetic environment in pE1. It is the first report of rapid emergence of KPC-2-producing E. coli ST131 in China. The blaKPC-2 gene of most isolates was located on a similar genetic structure.

Emergence of Proteus mirabilis Harboring blaKPC-2 and qnrD in a Chinese Hospital

ABSTRACT Nineteen carbapenem-nonsusceptible Proteus mirabilis isolates were recovered from intensive care units in the Second Affiliated Hospital of Zhejiang University during a 3-month period. The isolates showed a high level of resistance against ciprofloxacin, in addition to their resistance against the carbapenems. Pulsed-field gel electrophoresis (PFGE) analysis showed that these isolates belonged to three clonal strains. PCRs and DNA sequence analysis of the carbapenemase and other β-lactamase genes indicated that all the isolates harbored the blaKPC-2 gene. Twelve of 19 isolates harbored the plasmid-mediated quinolone resistance (PMQR) genes, both the qnrD and aac(6′)-Ib-cr genes. Eight representative isolates with high levels of quinolone resistance carried the similar mutation profiles of S83I in gyrA, E466D in gyrB, and S80I in parC. Reduced carbapenem susceptibility was transferred to Escherichia coli (EC600) in a conjugation experiment, while the quinolone resistance was not. DNA hybridization showed that qnrD was located on a plasmid of approximately 4.5 kb. In summary, large clonally related isolates of KPC-2-producing P. mirabilis emerged in a Chinese hospital, and qnrD was detected in KPC-producing P. mirabilis for the first time.

Outbreak of Klebsiella pneumoniae carbapenemase 2-producing K. pneumoniae with high qnr prevalence in a Chinese hospital

Forty carbapenem-resistant Klebsiella pneumoniae isolates were recovered from 28 patients from various sites in an intensive care unit in Zhejiang Provincial Peoples Hospital, China, over a 6 month period. PFGE analysis indicated that the 40 strains were all closely related. The MICs of carbapenems varied from 16 to >256 µg ml⁻¹. Conjugation studies with Escherichia coli resulted in the transfer of reduced carbapenem susceptibility from the original isolates. All K. pneumoniae and E. coli transconjugants produced K. pneumoniae carbapenemase 2 (KPC-2), and most of them produced TEM, SHV and CTX-M. Additionally, 27 isolates and 27 E. coli transconjugants carried the qnr gene (25 were qnrB2 and 2 were qnrS1). K. pneumoniae harboured several plasmids, and bla(KPC-2) was located on a 55 kb plasmid. SDS-PAGE and ompK35/36 gene sequence analysis of OMPs suggested that porins in K. pneumoniae are expressed normally. The MICs of the carbapenems did not change in the presence of CCCP. Thus, production of KPC-2 appears to play an important role in resistance to carbapenems, although other mechanisms may be involved. The bla(KPC-2) gene is associated with several antibiotic-resistance genes, such as bla(TEM), bla(SHV), bla(CTX-M) and qnr.

Increased prevalence of carbapenem resistant Enterobacteriaceae in hospital setting due to cross-species transmission of the blaNDM-1 element and clonal spread of progenitor resistant strains

This study investigated the transmission characteristics of carbapenem-resistant Enterobacteriaceae (CRE) strains collected from a hospital setting in China, in which consistent emergence of CRE strains were observable during the period of May 2013 to February 2014. Among the 45 CRE isolates tested, 21 (47%) strains were found to harbor the blaNDM-1 element, and the rest of 24 CRE strains were all positive for blaKPC-2. The 21 blaNDM-1—borne strains were found to comprise multiple Enterobacteriaceae species including nine Enterobacter cloacae, three Escherichia coli, three Citrobacter freundii, two Klebsiella pneumoniae, two Klebsiella oxytoca, and two Morganella morganii strains, indicating that cross-species transmission of blaNDM-1 is a common event. Genetic analyses by PFGE and MLST showed that, with the exception of E. coli and E. cloacae, strains belonging to the same species were often genetically unrelated. In addition to blaNDM-1, several CRE strains were also found to harbor the blaKPC-2, blaVIM-1, and blaIMP-4 elements. Conjugations experiments confirmed that the majority of carbapenem resistance determinants were transferable. Taken together, our findings suggest that transmission of mobile resistance elements among members of Enterobacteriaceae and clonal spread of CRE strains may contribute synergistically to a rapid increase in the population of CRE in clinical settings, prompting a need to implement more rigorous infection control measures to arrest such vicious transmission cycle in CRE-prevalent areas.

Linezolid-resistant clinical isolates of meticillin- resistant coagulase-negative staphylococci and Enterococcus faecium from China

Seventeen meticillin-resistant coagulase-negative staphylococci (MRCoNS), including ten Staphylococcus capitis, four Staphylococcus cohnii, two Staphylococcus haemolyticus and one Staphylococcus sciuri, and an Enterococcus faecium isolate with various levels of linezolid resistance were isolated from intensive care units in a Chinese hospital. PFGE indicated that the four S. cohnii isolates belonged to a clonal strain, and that nine of the S. capitis isolates were indistinguishable (clone A1) and the other one was closely related (clone A2). A G2576T mutation was identified in domain V of the 23S rRNA gene in the E. faecium isolate. Besides the G2576T mutation, a novel C2104T mutation was detected in the nine clone A1 S. capitis isolates. The cfr gene was detected in all the staphylococci except an S. sciuri isolate, whose 23S rRNA gene contained the G2576T mutation. There was a clonal dissemination of linezolid-resistant MRCoNS in intensive care units of our hospital, and this is the first report, to our knowledge, of linezolid-resistant staphylococci and enterococci in China.