Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP).

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.

Laboratory studies on host-parasite relationship of Bithynia snails and the liver fluke, Opisthorchis viverrini.

The infection rate of Bithynia snails to Opisthorchis viverrini eggs was studied in relation to exposure intensity, age and species of host. It was found that 50 miracidial eggs per snail yielded the highest percentage of living surviving positive snails. Bithynia funiculata and Bithynia siamensis siamensis were highly susceptible to O. viverrini, about four to seven times higher than Bithynia siamensis goniomphalos. Young snails, 1-3 months old, appeared more susceptible than old snails.

Detection of shared antigens of human liver flukes Opisthorchis viverrini and its snail host, Bithynia spp.

The relationship between Opisthorchis viverrini and its snail intermediate host including Bithynia funiculata, B. siamensis siamensis and B. siamensis goniomphalos was carried out on the correlation of shared antigens and infection rates. B. funiculata and B. s. siamensis were equally susceptible to O. viverrini with relatively high infection rates of 72.2% and 69.9% respectively whereas B. s. goniomphalos gave lowest percentage of infection of only 9.6%. By immunoelectrophoresis, crude extract of O. viverrini adult worm produced the same number of two common precipitin bands of four different electrophoretic mobilities against anti-B. funiculata and anti-B. s. siamensis sera and one precipitin band in the antigen well region against anti-B. s. goniomphalos serum. The three snail antigens produced the same number of two common precipitin bands against anti-O. viverrini serum.

SHV-12, SHV-5, SHV-2a and VEB-1 extended-spectrum beta-lactamases in Gram-negative bacteria isolated in a university hospital in Thailand.

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.

Detection of extended-spectrum beta-lactamases in members of the family enterobacteriaceae: comparison of the MAST DD test, the double disc and the Etest ESBL.

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.

Discrimination of SHV beta-lactamase genes by restriction site insertion-PCR.

Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3' end to modulate a restriction site. We have developed this technique to identify described mutations of the bla(SHV) genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV beta-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly-->Ala) (BsrI), and 240 (NruI) of bla(SHV) genes. All amplimers of the bla(SHV) genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of bla(SHV-6), bla(SHV-8), and 12 novel bla(SHV) variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV beta-lactamases and may be extended to the characterisation of other resistance determinants.