Phenotypic and Genotypic Characterization of Enterobacteriaceae with Decreased Susceptibility to Carbapenems: Results from Large Hospital-Based Surveillance Studies in China

To date, the emergence of carbapenem-resistant Enterobacteriaceae has been reported in some countries (7, 9, 16, 19). Carbapenemases and porin loss combined with AmpC enzyme hyperproduction are regarded as the main mechanisms of resistance (7, 9, 12, 19). In China, there have been some reports of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae, Serratia marcescens, and Escherichia coli in the city of Hangzhou (2, 17, 20). However, a nationwide survey has not been performed. In this study, 49 Enterobacteriaceae isolates with decreased susceptibility to carbapenems (MIC of imipenem, meropenem, or ertapenem of ≥2 μg/ml) were collected from 16 teaching hospitals in a nationwide distribution, which included 26 K. pneumoniae, 8 E. coli, 10 Enterobacter cloacae, 2 Enterobacter aerogenes, and 3 Citrobacter freundii isolates. Identification of organisms was confirmed by using the API 20E or Vitek2 Compact system (bioMérieux, France). Susceptibility testing was performed by using the agar dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (3, 4). Breakpoints for tigecycline were as defined by the FDA (susceptible, ≤2 μg/ml; resistant, ≥8 μg/ml). Forty-nine isolates were nonsusceptible to most antibiotics except to tigecycline (to which 45 of 49 isolates were susceptible) and polymyxin B (to which 47 of 49 isolates were susceptible).

Conjugation experiments were carried out in mixed broth cultures, as described previously (2). Plasmid DNAs of all carbapenemase-producing isolates were obtained with a QIAfilter midikit (Qiagen, Hilden, Germany). Resistance genes were successfully transferred from 23 of 49 isolates to the recipient E. coli C600. Among the 16 carbapenemase-producing clinical isolates, carbapenemase genes of 13 isolates were successfully transferred to E. coli C600, except for three IMP-4-producing E. cloacae isolates. The 13 carbapenemase-producing transconjugants showed 8- to 64-fold increases in the MIC of imipenem, 32- to 512-fold increases in the MIC of meropenem, and 256- to 4,096-fold increases in the MIC of ertapenem relative to those of the recipient. Most of the carbapenemase-producing transconjugants harbored a single plasmid, while only one transconjugant (GZ64T) harbored four different plasmids (Table 1).

PCR of β-lactamase genes for the transconjugants and respective donors was carried in a PTC-200 PCR system (Bio-Rad). The primers used in this study were described previously (1, 5, 11, 12, 14, 18, 19). PCR products were purified with a QIAquick PCR purification kit (Qiagen) and were sequenced on an ABI PRISM 3730XL sequencer analyzer. Carbapenemase genes were detected in 16 of 49 clinical isolates, which involved the bla_KPC-2 gene from four K. pneumoniae and two E. coli isolates, the bla_IMP-4 gene from three K. pneumoniae, three E. cloacae, and two C. freundii isolates, and the bla_IMP-8 gene from two K. pneumoniae isolates. Among 49 clinical isolates, 23 carried bla_TEM-1, 21 carried bla_SHV, and 26 carried bla_CTX-M, while bla_CTX-M-14 and bla_CTX-M-3 were the predominant genotypes among CTX-M-producing isolates. Fourteen isolates carried bla_DHA-1, and seven carried bla_CMY-2. Other β-lactamase genes (bla_NMC, bla_SME, bla_IMI, bla_GES, bla_VIM, bla_SPM, bla_SIM, bla_GIM, and bla_OXA) were not detected in any of the 49 isolates.

All 49 isolates and their transconjugants were screened for the qnr (qnrA, qnrB, and qnrS) genes by multiplex PCR (13) and for aac(6′)-Ib-cr by PCR and sequencing (10). Among 49 isolates, 14 carried qnr genes, and qnrS1 (9/14) and qnrB (5/14) were the predominant qnr genotypes. Seventeen of 49 isolates carried an aac(6′)-Ib gene, and 9 of them were determined to be aac(6′)-Ib-cr.

Class 1 integrons were detected in the 49 clinical isolates and corresponding transconjugants by PCR and sequencing (8). Nine different structures of class 1 integrons were found in these isolates (Tables 1 and 2). The most common gene cassettes contained resistance determinants to aminoglycosides (aadA5, aadA2, and aadA1) and trimethoprim (dfrA17 and dfrA12). K. pneumoniae strain GZ64 gave a 2.2-kb PCR amplicon for class 1 integrons that contained bla_IMP-4 and orfII (putative reverse transcriptase gene).

Pulsed-field gel electrophoresis (PFGE) typing was performed as described previously (15), and it showed that K. pneumoniae ZJ70 and ZJ71 (from Hangzhou), E. coli ZJ86 and ZJ87 (from Hangzhou), and C. freundii SZ62 and SZ63 (from Shenzhen) were clonally related.

Outer membrane proteins (OMPs) were isolated by sarcosyl extraction of total membrane preparations as described previously (6). Expression levels of the two corresponding major porins (OmpK35 and OmpK36 for K. pneumoniae and OmpF and OmpC for E. coli, E. cloacae, E. aerogenes, and C. freundii) were investigated. Thirty-three of 49 isolates, including 19 K. pneumoniae and 14 other Enterobacteriaceae, lost or had lower expression of both major porins, while 12 isolates lost or had lower expression of one porin. Expression of both major porin proteins was normal in only four isolates. Isolates with a combination of carbapenemase and porin loss showed relatively high carbapenem MICs (K. pneumoniae strains ZJ70 and ZJ71). Among the 33 non-carbapenemase-producing isolates, 29 showed loss or lower expression of both major porins, and 28 produced extended-spectrum β-lactamases (ESBLs), AmpC, or both types of enzymes simultaneously (Tables 2 and 3). The MIC ranges of imipenem, meropenem, and ertapenem against these 28 isolates were 2 to 32 μg/ml (20 imipenem-resistant isolates with MICs of ≥16 μg/ml), 2 to 16 μg/ml (9 meropenem-resistant isolates with MICs of 16 μg/ml), and 16 to 128 μg/ml (all resistant to ertapenem), which is relatively higher than those of the isolates with single porin loss (Table 3).

In this study, 16 of 49 isolates produced KPC-2 or IMP-4/8 carbapenemases. K. pneumoniae was the most frequently isolated carbapenemase-producing species (9/16 isolates) and produced KPC-2, IMP-4, and IMP-8 carbapenemases. IMP-4 was the most common carbapenemase type in this study (8/16) and was found in K. pneumoniae, E. cloacae, and C. freundii. KPC-2 has emerged in China but was limited to certain areas, such as the city of Hangzhou. Importantly, this study showed that two-thirds of carbapenemase-nonsusceptible isolates (33/49) did not produce carbapenemases, and most of these isolates (28/33) had lost or had reduced expression of both major porin proteins (OmpK35/36 or OmpF/C), usually in combination with ESBL production (23/28; mainly CTX-M-14, SHV-11, and CTX-M-3) or AmpC (17/28; DHA-1 and CMY-2). This indicated that loss or decreased expression of both of the major porins may play an important part in an increased resistance level to carbapenems. AmpC or ESBL production may contribute to the resistance level among these isolates. These data suggest that the high prevalence rates of ESBLs and AmpC among Enterobacteriaceae may predispose these organisms to carbapenem resistance.

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