A novel plasmid carrying carbapenem-resistant gene bla KPC-2 in Pseudomonas aeruginosa.

A carbapenem-resistant Pseudomonas aeruginosa strain PA1011 (ST463) was isolated from a patient in a surgical intensive care unit. PCR detection showed that PA1011 carried the bla KPC-2 gene. A plasmid was isolated and sequenced using the Illumina NextSeq 500 and PacBio RSII sequencing platforms. The plasmid was named pPA1011 and carried the carbapenem-resistant gene bla KPC-2. pPA1011 was a 62,793 bp in length with an average G+C content of 58.8%. It was identified as a novel plasmid and encoded a novel genetic environment of bla KPC-2 gene (ΔIS6-Tn3-ISKpn8-bla KPC-2-ISKpn6-IS26).

Dynamic Colonization of Klebsiella pneumoniae Isolates in Gastrointestinal Tract of Intensive Care Patients.

Gastrointestinal carriage is regarded as a major reservoir of K. pneumoniae infections, especially in intensive care patients. A total of 101 (95.3%) KPC-producing carbapenem-resistant K. pneumoniae (CRKP) isolates were identified among 106 CRKP isolates collected from stool samples of inpatients performing active rectal screening for carbapenem-resistant Enterobacteriaceae during hospitalization in the ICUs of a tertiary hospital between 2016 and 2017. Among them, six KPC-producing CRKP isolates from three patients (two isolates for each patient) were identified with distinct antibacterial susceptibility. Our findings showed that: (1) bla KPC-2 gene is predominant in CRKP strains isolated from the intensive care patients and can be incorporated into various plasmids that are transmissible among multiple bacterial hosts in the human gastrointestinal tract; (2) the human gastrointestinal tract has a capacity to dynamically colonize multiple clones of CRKP strains with varied plasmids, diverse antimicrobial resistance genes and virulence genes. K. pneumoniae colonization is an important step in progression to extraintestinal infection, which provides the rationale for establishing intervention measures to prevent subsequent infection. Thus, close surveillance on CRKP colonization, together with effective infection prevention and control measures, should be put into practice.

Colistin resistance gene mcr-1 in gut flora of children.

The aim of this study was to determine the prevalence and transmission mechanism(s) of mcr-1 in the gut flora of children. Faecal samples (n = 173) were obtained from non-diarrhoea patients at the Children's Hospital of Zhejiang University (Hangzhou, China). PCR-based analysis indicated that 17 isolates from 9.8% of the samples were positive for mcr-1, comprising 16 Escherichia coli and 1 Citrobacter freundii. Nine mcr-1-bearing isolates co-expressed extended-spectrum ß-lactamase (ESBL) genes, but plasmid-mediated quinolone resistance (PMQR) genes were not detected. Transconjugation followed by Southern hybridisation analysis revealed that 14 of the E. coli isolates were able to transfer their colistin-resistant phenotype to E. coli EC600. All 14 of these E. coli strains contained a major mcr-1-containing conjugative plasmid with a size of ca. 33 kb or 55 kb. All but two of the E. coli isolates presented distinct pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing (MLST) analysis revealed 11 sequence types (STs) among the E. coli 16 isolates, with ST117 being the most common. The finding of a high prevalence of mcr-1 in the intestinal flora of children, with the majority of mcr-1-positive isolates being E. coli, highlights the need for more rational use of polymyxins to prevent polymyxin resistance from becoming disseminated among different microbial pathogens. Given the high detection rate of mcr-1 in children, we recommend that polymyxin is no longer used as a last-resort antimicrobial agent and that alternative strategies are developed to treat infections caused by such pathogens.

Occurrence of Plasmid- and Chromosome-Carried mcr-1 in Waterborne Enterobacteriaceae in China.

The aim of this study was to investigate the prevalence of the polymyxin resistance gene mcr-1 in Enterobacteriaceae from environmental water sources in Hangzhou, China. Colistin-resistant bacteria were isolated from environmental water samples using an enrichment broth culture method, were screened for mcr-1, and then were analyzed for the location and transferability of mcr-1 Isolates positive for mcr-1 were further examined to determine their susceptibility profiles and were screened for the presence of additional resistance genes. Twenty-three mcr-1-positive isolates (16 Escherichia coli, two Citrobacter freundii, two Klebsiella oxytoca, two Citrobacter braakii, and one Enterobacter cloacae) were isolated from 7/9 sampling locations; of those, eight mcr-1-positive isolates also contained ß-lactamase-resistance genes, eight contained qnrS, and 10 contained oqx No mcr-2-positive isolates were identified. The majority of isolates demonstrated a low to moderate level of colistin resistance. Transconjugation was successfully conducted from 14 of the 23 mcr-1-positive isolates, and mcr-1 was identified on plasmids ranging from 60 to 220 kb in these isolates. Conjugation and hybridization experiments revealed that mcr-1 was chromosome-borne in only three isolates. Pulsed-field gel electrophoresis showed that the majority of E. coli isolates belonged to different clonal lineages. Multilocus sequence typing analysis revealed that sequence type 10 (ST10) was the most prevalent, followed by ST181 and ST206. This study demonstrates the utility of enrichment broth culture for identifying environmental mcr-1-positive isolates. Furthermore, it highlights the importance of responsible agriculture and clinical use of polymyxins to prevent further widespread dissemination of polymyxin-resistant pathogens.

Comparative genetic characterization of Enteroaggregative Escherichia coli strains recovered from clinical and non-clinical settings.

The origin of pathogenic Enteroaggregative Escherichia coli (EAEC), a major causative agent of childhood diarrhea worldwide, remains ill-defined. The objective of this study was to determine the relative prevalence of EAEC in clinical and non-clinical sources and compare their genetic characteristics in order to identify strains that rarely and commonly cause human diarrhea. The virulence gene astA was commonly detectable in both clinical and non-clinical EAEC, while clinical isolates, but not the non-clinical strains, were consistently found to harbor other virulence factors such as aap (32%), aatA (18%) and aggR (11%). MLST analysis revealed the extremely high diversity of EAEC ST types, which can be grouped into three categories including: (i) non-clinical EAEC that rarely cause human infections; (ii) virulent strains recoverable in diarrhea patients that are also commonly found in the non-clinical sources; (iii) organisms causing human infections but rarely recoverable in the non-clinical setting. In addition, the high resistance in these EAEC isolates in particular resistance to fluoroquinolones and cephalosporins raised a huge concern for clinical EAEC infection control. The data from this study suggests that EAEC strains were diversely distributed in non-clinical and clinical setting and some of the clinical isolates may originate from the non-clinical setting.

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

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 bla(KPC-2) gene. These findings indicate that K1 hvKP is simultaneously hypervirulent, multidrug resistant, and transmissible.

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.

Substitutions of Ser83Leu in GyrA and Ser80Leu in ParC Associated with Quinolone Resistance in Acinetobacter pittii.

To investigate the prevalence and the mechanism of quinolone-resistant Acinetobacter pittii, 634 Acinetobacter calcoaceticus-Acinetobacter baumannii complex isolates were collected throughout Zhejiang Province. Identification of isolates was conducted by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS), blaOXA-51-like gene, and partial RNA polymerase ß-subunit (rpoB) amplification. Twenty-seven isolates of A. pittii were identified. Among the 634 isolates, A. baumannii, A. pittii, Acinetobacter nosocomialis, and A. calcoaceticus counted for 87.22%, 4.26%, 8.20%, and 0.32%, respectively. Antimicrobial susceptibility of nalidixic acid, ofloxacin, enoxacin, ciprofloxacin, lomefloxacin, levofloxacin, sparfloxacin, moxifloxacin, and gatifloxacin for 27 A. pittii were determined by the agar dilution method. Detection of quinolone-resistant determining regions of gyrA, gyrB, parC, and parE was performed for the A. pittii isolates. In addition, plasmid-mediated quinolone resistance (PMQR) determinants (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, qepA, oqxA, and oqxB) were investigated. All the 27 isolates demonstrated a higher minimum inhibitory concentration (MIC) to old quinolones than the new fluoroquinolones. No mutation in gyrA, gyrB, parC, or parE was detected in 20 ciprofloxacin-susceptible isolates. Seven ciprofloxacin-resistant A. pittii were identified with a Ser83Leu mutation in GyrA. Among them, six isolates with simultaneous Ser83Leu amino acid substitution in GyrA and Ser80Leu in ParC displayed higher MIC values against ciprofloxacin. Additionally, three were identified with a Met370Ile substitution in ParE, and two were detected with a Tyr317His mutation in ParE, which were reported for the first time. No PMQR determinants were identified in the 27 A. pittii isolates. In conclusion, mutations in chromosome play a major role in quinolone resistance in A. pittii, while resistance mechanisms mediated by plasmid have not been found. Ser83Leu substitution in GyrA and Ser80Leu substitution in ParC are associated with quinolone resistance in A. pittii. Whether Met370Ile and Tyr317His substitutions in ParE play a minor role requires further investigation.

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.

Detection of the Smqnr quinolone protection gene and its prevalence in clinical isolates of Stenotrophomonas maltophilia in China.

The aim of this study was to detect novel variants of the Stenotrophomonas maltophilia Smqnr gene family and analyse the prevalence of Smqnr genes in clinical isolates of S. maltophilia in China. In total, 442 clinical isolates of S. maltophilia were collected from nine hospitals in four provinces in China. Antimicrobial susceptibility testing against six commonly used antibiotics was performed on these isolates. The sequences of the Smqnr genes amplified by PCR were aligned with those of known Smqnr genes in GenBank and an Smqnr database. The resistance rate against co-trimoxazole was highest at 48.6 %, followed by resistance rates against ceftazidime, chloramphenicol, ticarcillin/clavulanate and tigecycline at 28.7, 21.3, 19.0 and 16.1 %, respectively. The highest susceptibility was shown to levofloxacin, with a resistance rate of just 6.1 %. Smqnr genes were detected in 114 isolates, and comprised 11 previously identified genes and 20 new variants, bringing the total number of known Smqnr genes to 47. The 20 novel Smqnr genes were designated Smqnr28-47 and the encoded proteins showed only 1-12 amino acid differences among each other. The most common Smqnr genes in China were Smqnr8 and its variant Smqnr35 with prevalences of 17.5 % (20/114) and 13.2 % (15/114), respectively. Both the known and the novel Smqnr genes were discovered in both quinolone non-sensitive and sensitive isolates with similar frequency, suggesting that the Smqnr gene makes little contribution to quinolone resistance in this organism.

A ten years (2000-2009) surveillance of resistant Enterobacteriaceae in Zhejiang Province, China.

OBJECTIVE: In Zhejiang Province, there are several highly developed cities near the coast and several relatively under-developed mountain areas. Analysis of the composition of bacteria isolated from patients as well as their antibiotic resistance profile from various areas of this province, and tracing of such data year-by-year, will help to delineate the bacterial resistance profile of these areas and to understand how the stage of socio-economical development impacts on the composition of clinical micro-flora and their resistance profile. METHODS: In order to investigate variation in resistance rates and isolation rates of Enterobacteriaceae, from 2000 to 2009 in Zhejiang Province, China, Enterobacteriaceae isolated from 15 hospitals located in different regions of the province were surveyed. RESULTS: The total numbers of the Enterobacteriaceae isolated increased more than 20-fold from 2000 to 2009. Among the Enterobacteriaceae, Escherichia coli and Klebsiella pneumoniae were the dominant isolates. The percentage of E. coli and K. pneumoniae that produced detectable extended-spectrum ß-lactamases (ESBLs) increased from 2000 to 2007, and then declined slightly in 2008 and 2009. The percentages of K. pneumoniae and E. coli that were resistant to ceftazidime increased sharply from 2000 to 2009. There were remarkable increases in the carbapenem resistant rates during the decade, but they were much higher for the isolates from the developed cities than from the rural areas. In 2002, carbapenem-resistant E. coli was first found in Hangzhou, one of the highly developed cities in Zhejiang Province. By 2009, carbapenem-resistant bacteria were found for all species of Enterobacteriaceae surveyed in almost all areas of the province, although they were more frequently identified in developed areas than in rural areas. CONCLUSION: Much restrictive actions have to be taken in terms of rational use of antibiotics and nosocomial control to prevent the further spread of the drug-resistant pathogens.

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.