Of 15 extended-spectrum ␤-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae collected from the First Municipal People's Hospital of Guangzhou, in the southern part of the People's Republic of China, 9 were found to produce CTX-M ESBLs, 3 produced SHV-12, and 3 produced both CTX-M and SHV-12. Eleven isolates produced either TEM-1B or SHV-11, in addition to an ESBL. Nucleotide sequence analysis of the 12 isolates carrying bla CTX-M genes revealed that they harbored three different bla CTX-M genes, bla CTX-M-9 (5 isolates), bla CTX-M-13 (1 isolate), and bla CTX-M-14 (6 isolates). These genes have 98% nucleotide homology with bla Toho-2 . The bla CTX-M genes were carried on plasmids that ranged in size from 35 to 150 kb. Plasmid fingerprints and pulsed-field gel electrophoresis showed the dissemination of the bla CTX-M genes through transfer of different antibiotic resistance plasmids to different bacteria, suggesting that these resistance determinants are highly mobile. Insertion sequence ISEcp1, found on the upstream region of these genes, may be involved in the translocation of the bla CTX-M genes. This is the first report of the occurrence of SHV-12 and CTX-M ESBLs in China. The presence of strains with these ESBLs shows both the evolution of bla CTX-M genes and their dissemination among at least three species of the family Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, isolated within a single hospital. The predominance of CTX-M type enzymes seen in this area of China appears to be similar to that seen in South America but is different from those seen in Europe and North America, suggesting different evolutionary routes and selective pressures. A more comprehensive survey of the ESBL types from China is urgently needed.Until 1994, SHV-2 was the only extended-spectrum ␤-lactamase (ESBL) described in bacteria originating from the People's Republic of China (10). Recently, ESBL-producing bacteria have been reported from China, but molecular characterization of these ESBLs has not yet been undertaken (38,(45)(46)(47). During an antimicrobial resistance-monitoring project in the First Municipal People's Hospital of Guangzhou, which is in the southern part of China, in 1997, ESBL production rates in Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Citrobacter freundii were 18, 19, 16, and 18%, respectively (45). In 1998, the rates of resistance due to the production of ESBLs rose dramatically, to 33% for E. coli, 37% for K. pneumoniae, 18% for E. cloacae, and 25% for C. freundii (46). A limited number of these ESBL-producing isolates have been selected for investigation in the present study.(A part of this report was presented at the 21st International Congress of Chemotherapy, Birmingham, United Kingdom, 4 to 7 July 1999, and the 10th European Congress of Clinical Microbiology and Infectious Diseases, Stockholm, Sweden, 28 to 31 May 2000.) MATERIALS AND METHODS Bacterial strains. Fifteen nonduplicate ESBL-producing isolates of the familyEn...

A technically simple method-the MAST double disc (MDD) test-for the detection of extended-spectrum beta-lactamase (ESBL) production by bacteria is described. A wide range of ESBL, non-ESBL and Class 1 beta-lactamase-producing isolates was examined. The MDD test, which uses discs containing ceftazidime and a complementary disc containing ceftazidime and clavulanate and a second pair containing cefotaxime and cefotaxime and clavulanate was compared with the standard double disc diffusion test and an Etest method. Both the Etest and the MDD correctly identified 93% of ESBL producers. The MDD is an inexpensive alternative to current methods for the detection of ESBL production.

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Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand. These included 43 Enterobacteriaceae and 18 Pseudomonadaceae. The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified. Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1). Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids. In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c. 130 kb. Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains. Among the pseudomonad isolates, 16 Pseudomonas aeruginosa and one Pseudomonas putida had bla(VEB-like) and one P. aeruginosa had bla(SHV-12). Nine of the 16 isolates carrying bla(VEB-like) (56.3%) had identical PFGE patterns, suggesting the dissemination of this gene, also by clonal spread. At least six different bla(VEB-like-)containing integrons were found among the 18 isolates. This is the first report of bacteria producing SHV-12 and SHV-2a in Thailand and the first report of SHV-12 in P. aeruginosa, of VEB-1 in Citrobacter freundii and a VEB-1-like beta-lactamase in P. putida. These findings indicate that ESBL genes in the Far East are part of a gene pool capable of broad horizontal gene transfer, in that these genes can transfer between different families of Gram-negative bacilli.

Carbapenem-resistant Enterobacteriaceae isolates by carbapenemase production are being reported globally with increasing frequency, leading to limited therapeutic options. We therefore developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB) for rapid confirmation of bla , bla, bla , bla and bla groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 bla, 9 bla , 2 bla, 1 bla , 1 bla, 1 bla , 1 bla, 4 bla , 1 bla, 1 bla & bla , 7 bla, 3 bla and 3 bla) and 37 non-carbapenemase-producing Enterobacteriaceae isolates as confirmed by the PCR methods were included. Bacterial DNA was extracted by a simple boiling method. The LAMP-HNB method for each target gene was carried out using a set of six primers under isothermal condition at 65 °C in an ordinary water bath within 60 min and visual measurement of reaction by the change from violet to sky blue. This method had high efficiency (100% sensitivity and specificity) for identifying the bla , bla, bla , bla and bla groups compared with the PCR method. The HNB is easy to prepare, inexpensive and provides reliable results. Therefore, this method could be used as a confirmatory carbapenemase test in routine laboratory or for epidemiological purposes.