Rapid detection of porins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of Escherichia coli and eight strains of Klebsiella pneumoniae were conducted for both sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the correspondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000, 37,000, and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC, and OmpF with molecular weight of approximately ca. 38, 40, and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000 and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference.

Dissemination of the same cfr-carrying plasmid among methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococcal isolates in China.

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.

Emergence of KPC-2-producing Pseudomonas aeruginosa sequence type 463 isolates in Hangzhou, China.

Thirty-nine Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa isolates, all exhibiting high-level resistance to carbapenems and other ß-lactam antibiotics, were isolated in Hangzhou, China. Molecular epidemiology analysis indicated the presence of two dominant clones, namely, clones A and B, both of which belong to sequence type 463 (ST463). A genetic environment analysis demonstrated that both clones harbor an ISKpn8 transposase, bla(KPC-2), and an ISKpn6-like transposase. These findings depict the features of clonal expansion and transmission of KPC-2-producing P. aeruginosa strains in Hangzhou, China.

Emergence of Escherichia coli sequence type 131 isolates producing KPC-2 carbapenemase in China.

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.

Molecular typing of CTX-M-producing escherichia coli isolates from environmental water, swine feces, specimens from healthy humans, and human patients.

CTX-M-producing Escherichia coli is the predominant type of extended-spectrum ß-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 blaCTX-M-14 was the most prevalent CTX-M-9 group gene and that blaCTX-M-15 and blaCTX-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.

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.

High prevalence of qnr and aac(6')-Ib-cr genes in both water-borne environmental bacteria and clinical isolates of Citrobacter freundii in China.

We investigated the prevalence of qnr and aac(6')-Ib-cr genes in water-borne environmental bacteria and in clinical isolates of Enterobacteriaceae, as well as the subtypes of qnr. Environmental bacteria were isolated from surface water samples obtained from 10 different locations in Hangzhou City, and clinical isolates of Citrobacter freundii were isolated from several hospitals in four cities in China. qnrA, qnrB, qnrS, and aac(6')-Ib-cr genes were screened using PCR, and the genotypes were analyzed by DNA sequencing. Ten of the 78 Gram-negative bacilli isolated from water samples were C. freundii and 80% of these isolates carried the qnrB gene. qnrS1 and aac(6')-Ib-cr genes were detected in two Escherichia coli isolates and qnrS2 was detected in one species, Aeromonas punctata. The qnr and aac(6')-Ib-cr genes were present in 75 (72.8%) and 12 (11.6%) of 103 clinical isolates of C. freundii, respectively. Of the clinical C. freundii isolates with the qnr gene, 65 isolates (63.1%) carried qnrB, but only three (2.9%) and one (1.0%) carried qnrA1 and qnrS2, respectively, while five isolates carried both qnrA1 and qnrB, and one isolate carried both qnrS1 and qnrB. The qnrB9 gene was the dominant qnrB subtype, followed by qnrB8 and qnrB6. Southern hybridization studies indicated that the qnr genes are located on different plasmids. Plasmids isolated from both environmental and clinical C. freundii isolates appeared to be homogenous.

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.

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

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 ß-lactamase genes indicated that all the isolates harbored the bla(KPC-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.

Genotypic characterization and in vitro activities of tigecycline and polymyxin B for members of the Enterobacteriaceae with decreased susceptibility to carbapenems.

Carbapenem resistance in members of the Enterobacteriaceae is increasing. To evaluate the effects of tigecycline and polymyxin B against carbapenem-non-susceptible pathogens, 89 representative clinical carbapenem-non-susceptible Enterobacteriaceae isolates were recovered from seven hospitals from four cities in China during 2006-2009: 30 Serratia marcescens, 35 Klebsiella pneumoniae, seven Enterobacter cloacae, six Enterobacter aerogenes, five Escherichia coli, four Citrobacter freundii and two Klebsiella oxytoca isolates. Twenty-eight S. marcescens isolates were indistinguishable. The 35 K. pneumoniae isolates belonged to 12 clonal strains. Among the 89 Enterobacteriaceae isolates, 82 produced KPC-2, seven produced IMP (three produced KPC-2 simultaneously), three did not produce any carbapenemases and nine were deficient in porins. Polymyxin B was much more active than tigecycline against carbapenem-non-susceptible Enterobacteriaceae. The MIC(50) and MIC(90) of imipenem, meropenem, ertapenem, polymyxin B and tigecycline were 8 and 32 µg ml(-1), 8 and 32 µg ml(-1), 16 and 128 µg ml(-1), 0.5 and 16 µg ml(-1), and 4 and 16 µg ml(-1), respectively. Rates of susceptibility to imipenem, meropenem, ertapenem and polymyxin B were 30.0%, 27.5%, 2.5% and 89.2% by CLSI criteria. The rate of susceptibility to tigecycline was 40% and 17.5% by Food and Drug Administration (MIC ≤2 µg ml(-1)) and European Committee on Antimicrobial Susceptibility Testing (MIC ≤1 µg ml(-1)) criteria, respectively. KPC-2- or IMP-producing E. coli transconjugants exhibited reduced susceptibility to carbapenems but were susceptible to polymyxin B and tigecycline with an MIC range of 0.5-2 µg ml(-1), 0.25-2 µg ml(-1), 0.5-4 µg ml(-1), 0.5 µg ml(-1) and 0.5-1 µg ml(-1). In conclusion, carbapenem resistance in Enterobacteriaceae is mainly due to production of KPC-2, and polymyxin B is active for the carbapenem-resistant Enterobacteriaceae.

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 People's 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.

Serotypes and extended-spectrum ß-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 ß-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 ß-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 ß-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.

Combination of IMP-4 metallo-beta-lactamase production and porin deficiency causes carbapenem resistance in a Klebsiella oxytoca clinical isolate.

This study shows for the first time the mechanism of carbapenem resistance of a Klebsiella oxytoca clinical isolate ZC101 recovered from a Zhejiang University Hospital in Hangzhou, China. MIC values of imipenem, meropenem, and ertapenem for K. oxytoca ZC101 were 16, 16, and 128 microg/mL, respectively. Conjugation experiments demonstrated the transferability of a resistance determinant from K. oxytoca ZC101 to Escherichia coli EC600. Results from isoelectric focusing, polymerase chain reactions, and DNA sequencing confirmed that K. oxytoca ZC101 produced IMP-4 metallo-beta-lactamase (MBL) and CTX-M-14 extended-spectrum beta-lactamase, whereas E. coli transconjugant only produced the IMP-4. Amplification of integron revealed that bla(IMP-4) gene is located within a class I integron that was carried in a plasmid approximately 55 kb in size. Sodium dodecyl sulfate polyacrylamide gel electrophoresis profiling of outer membrane proteins of K. oxytoca ZC101 indicated lack of expression of the OmpK36 porin. DNA sequence analysis of ompK36 gene of K. oxytoca ZC101 showed the gene was disrupted by an insertion sequence IS5. In all, the results show that plasmid-mediated IMP-4 MBL production combined with the loss of OmpK36 porin caused the resistance in K. oxytoca ZC101 to carbapenems.

Reduced susceptibility to carbapenems in Klebsiella pneumoniae clinical isolates associated with plasmid-mediated beta-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.

Detection of plasmid-mediated IMP-1 metallo-beta-lactamase and quinolone resistance determinants in an ertapenem-resistant Enterobacter cloacae isolate.

OBJECTIVE: To investigate the mechanism of carbapenem resistance and the occurrence of plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in a clinical isolate of Enterobacter cloacae. METHODS: An ertapenem-resistant E. cloacae ZY106, which was isolated from liquor puris of a female gastric cancer patient in a Chinese hospital, was investigated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments, isoelectric focusing, polymerase chain reaction (PCR), and DNA sequence analyses of plasmid-mediated carbapenemases and quinolone resistance determinants were preformed to confirm the genotype. Outer membrane proteins (OMPs) were examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Urea-SDS-PAGE). RESULTS: Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem for ZY106 were 2, 4, and 16 microg/ml, respectively. Conjugation studies with Escherichia coli resulted in the transfer of significantly reduced carbapenem susceptibility. ZY106 produced IMP-1 metallo-beta-lactamase and CTX-M-3 extended-spectrum beta-lactamase, and E. coli transconjugant produced IMP-1. Plasmid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrS1-encoding-plasmid into E. coli by conjugation resulted in intermediate resistance to ciprofloxacin in E. coli transconjugant. Urea-SDS-PAGE analysis of OMPs showed that ZY106 lacked an OMP of approximately 38 kDa. CONCLUSION: It is the first IMP-1-producing Enterobacteriaceae in China and the first report of a clinical isolate that harbors both blaIMP and qnrS genes as well. The blaIMP-1, blaCTX-M-3, and qnrS1 are encoded at three different plasmids. IMP-1 combined with the loss of an OMP possibly resulted in ertapenem resistance and reduced imipenem and meropenem susceptibility in E. cloacae.

[Detection of plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in a strain of carbapenem-resistant Enterobacter cloacae].

OBJECTIVE: To investigate the mechanism of carbapenem resistance in Enterobacter cloacae. METHODS: A carbapenem-resistant strain of E. cloacae (strain ZY1465) was isolated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments were carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkaline lysis technique and were digested by various endonucleases; The crude beta-lactamase extracts of E. cloacae and E. coli transconjugant were subjected to analytical isoelectric focusing (IEF). Specific PCR amplification and DNA sequence analysis were preformed to confirm the beta-lactamase type. Outer membrane proteins (OMPs) were isolated and examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: The E. cloacae isolate showed resistance against carbapenems. The MICs of imipenem and meropenem were both 32 microg/ml. The isolate was also resistant strongly against penicillins, cephalosporins, cefoxitin, aztreonam, quinolones, and aminoglycosides. Conjugation studies with E. coli resulted in the transfer of reduced carbapenem susceptibility from E. cloacae isolate. Plasmid restriction analysis showed identical restriction profiles between the transconjugants of E. cloacae ZY1465 and Serratia marcescens ZN008. Isoelectric focusing demonstrated six beta-lactamases, with the isoelectric points (pIs) of 5.4, 6.7, 7.3, 7.8, 7.9, and 8.6, in E. cloacae ZY1465, and only one beta-lactamase with the pI of 6.7 in transconjugant. Specific PCR amplification and DNA sequence analysis confirmed that E. cloacae ZY1465 harbored TEM-1, KPC-2, DHA-1, CTX-M-14, CTX-M-3 and chromosomal AmpC (not detected in IEF) genes. Urea-SDS-PAGE analysis of OMPs showed that E. cloacae ZY1465 lacked an OMP of approximately 38 000 Da which was present in E. cloacae ATCC13047. CONCLUSION: It is the first detection of plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in a clinical isolate of E. cloacae from China. Production of multiple beta-lactamases, especially KPC-2 and loss of an OMP in E. cloacae ZY1465 result in resistance to carbapenems.

Emergence of Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli Isolates possessing the plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in intensive care units of a Chinese hospital.

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 microg/ml, except for K. pneumoniae K10 (MICs of 128, 256, and >256 microg/ml). Isoelectric focusing, PCRs, and DNA sequencing indicated that all S. marcescens isolates produced KPC-2 and a beta-lactamase with a pI of 6.5. All K. pneumoniae isolates produced TEM-1, KPC-2, CTX-M-14, and a beta-lactamase with a pI of 7.3. The E. coli E1 isolate produced KPC-2, CTX-M-15, and a beta-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 bla(KPC-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 bla(KPC-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.