Jia Chang Cai

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.

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 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.

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.

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.

Detection of OmpK36 porin loss in Klebsiella spp. by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry

The extensive use of carbapenems has led to the rapid dissemination of carbapenem-resistant Enterobacteriaceae ([9][1]), especially KPC-2-producing Klebsiella pneumoniae ([3][2], [8][3], [12][4], [13][5]). In addition to carbapenemase production, porin loss plays an important role in carbapenem

Serotypes and extended-spectrum β-lactamase types of clinical isolates of Shigella spp. from the Zhejiang province of China

To understand the distribution of the serotypes of Shigella spp. and to investigate the drug-resistant genes of extended-spectrum β-lactamases (ESBLs) in Shigella in Zhejiang province, we have collected clinically isolated Shigella isolates from 2 hospitals in Zhejiang province during August to December of 2006. There are 11 Shigella flexneri and 13 Shigella sonnei isolates, respectively. Among the 11 S. flexneri, 9 are serotype F4 and the remaining 2 are F1a and F2a. Antimicrobial susceptibility tests revealed that 20.8% of the isolates were resistant to cefotaxime and 20.8% of the isolates were intermediate to cefotaxime. Isoelectric focusing demonstrated that the ESBL-positive isolates and their transconjugants produce a single β-lactamase with a pI of 7.9 or 9.0. DNA sequence analysis demonstrated that they harbor either CTX-M-14 or CTX-M-15 gene. Pulsed-field gel electrophoresis analysis showed that 9 F4 S. flexneri isolates belong to the same clone and 13 S. sonnei isolates from different regions in Zhejiang province belong to different subclones of the same clone. In summary, resistance and reduced susceptibility to β-lactam antibiotics were mainly caused by the production of CTX-M-type ESBLs. This is the first report of CTX-M-15-type ESBLs in S. sonnei in China.

Dissemination of the Same cfr-Carrying Plasmid among Methicillin-Resistant Staphylococcus aureus and Coagulase-Negative Staphylococcal Isolates in China

ABSTRACT Six cfr-harboring methicillin-resistant Staphylococcus aureus (MRSA) isolates, which belonged to the same clone of sequence type 5 (ST5)-staphylococcal cassette chromosome mec element II (SCCmec II)-spa t311, were investigated in this study. Complete sequencing of a cfr-carrying plasmid, pLRSA417, revealed an 8,487-bp fragment containing a Tn4001-like transposon, cfr, orf1, and ISEnfa4. This segment, first identified in an animal plasmid, pSS-01, was observed in several plasmids from clinical coagulase-negative staphylococci in China, suggesting that the cfr gene, which might originate from livestock, was located in the same mobile element and disseminated among different clinical staphylococcal species.

Detection of KPC-2 and qnrS1 in clinical isolates of Morganella morganii from China

Seven carbapenem-nonsusceptible Morganella morganii isolates, which have similar antibiotic susceptibility profiles, were isolated over a 5-month period. MICs of imipenem, meropenem, and ertapenem were 8, 1, and 0.25 to 0.5 μg/mL, respectively. Pulsed-field gel electrophoresis indicated that 6 isolates were indistinguishable or closely related. Carbapenem resistance can be transferred from M. morganii to Escherichia coli by conjugation. All M. morganii isolates and E. coli transconjugants produced KPC-2 and carried the qnrS1 gene. Production of KPC-2 mainly contributed to the carbapenem resistance in M. morganii. KPC-2-producing M. morganii clonally spread in a hospital in China.