| Since extended-spectrumβ-lactamases (ESBLs) were initially reported to be in Klebsiella ozaenae in the first half of the 1980s in Germany, ESBLs have been spreading globally. Gram-negative bacteria are the present most prevalent pathogens causing infection, within the last 30 years, selective pressure exerted by the widespread use of the extended-spectrumβ-lactamases antibiotics made the incidence rate of ESBLs-producing increased year by year. The production of extended-spectrumβ-lactamases (ESBLs) is the most common mechanism of resistance to the third generation cephalosporins. ESBL production is the major emerging mechanism of resistance to expanded-spectrum cephalosporins , and also is resistant to other antibiotic. Most ESBLs are derived from TEM-1, 2 or SHV-1 enzymes and are capable of hydrolyzing oximino-cephalosporins, the number of CTX-M group has been increasing recently. Vertical and plasmid-mediated horizontal transfer played an important role in the dissemination of those ESBLs determinant, which can disseminate among the same and different classes of bacterium, leading to large scale break-out and spread. Study on ESBLs of the nosocomial infections have been reported greatly and few studies regarding this topic on diarrhea disease pathogenic bacterium have been reported in China. We investigated the prevalence, resistance of ESBL producers, gene type of ESBLs in diarrhea disease pathogenic bacterium collected in 307th hoipital, Renmin hoipital and community hosipital of Daxin in 2006. The results are presented as follows.1. To investigate the prevalence, resistance of the diarrhea disease pathogenic bacterium, we isolated the bacterium from those collecting diarrhea disease samples, and ESBL-producing isolates were detected by the CLSI Comfirmatory Test, drug resistance was tested by K-B. ESBL-producing strains were 9.09%(37/406), 64 percentage of them were Escherichia coli(24). ESBL-producing strains were highly resistant to mostβ-lactams and non-β-lactams (such as quinolones, aminoglycosides, tetracycline, and chloramphenicol), they were multi-drug-resistance, resistance rates of those ESBL producers to piperacillin, cefotaxime, cefazolin, cefpirome, nalidixic acid were about 90%. Resistance rates to comb- antibiotics (amoxacillin/clavulanic acid, ampicillin/sulbactam) were rising, 51.35% and 70.27% respectively, all were susceptible to meropenem. Most ESBL-producing strains were isolated from the person younger than 7 and elder than 45, who were low immunity, they had no relationgship with the collection place. Those data show that ESBLs is the main drug resistant mechanism in the diarrhea disease pathogenic bacterium following the drug resistance of quinolones.2. Detection and study of extended-spectrumβ-lactamases in the diarrhea disease. To investigate the prevalence gene-subtype prevalence of ESBLs in the diarrhea disease pathogenic bacterium, the partial blagene of ESBL producer was amplified by PCR using universal primers for TEM, SHV, and CTX-M respectively. The molecular typing of ESBL producers isolated from the diarrhea disease samples was determined by PFGE. CTX-M type was the most common extended-spectrumβ-lactamases found and TEM , SHV type the next, CTX-M-15 was the most common subtype, some strains produced more than one type of extended-spectrumβ-lactamases. There were a few ESBL producers produced non-CTX-M, non-TEM, non-SHV group. 7 PFGE patters in 24 ESBL-produing E.coli strains found by PFGE, the most patter had 8 strains; 2 PFGE patters in 5 ESBL-produing Shigella sonnel strains , only one PFGE patter in ESBL-produing Bacillus proteus and Klebsiellae strains. Those results show that ESBLs can exist not only in different species bacterium but also in different gene-types of the same bacteria, it is important to detect ESBL producers for controlling their spread.3. Conjugation experiments and detection of ESBL producers, resistance, plasmids of extended-spectrumβ-lactamases in transconjugants. To investigate the location of genes encoding ESBLs and their spread, and provide material for next study , transconjugants from 37 strains were obtained by conjugation experiments using E.coli.J53FOXRLAC- as the recipient strain; ESBL producers in transconjugants were detected by double disk test(CLSI,2006), drug resistance was tested by K-B, the partial blagene of transconjugant was amplified by PCR using universal primers for TEM, SHV, and CTX-M respectively. The rates of conjugation transfer were 56.76% in 37 ESBL-producers, those transcinjugants were obtained at frequency of 2.5×10-5～5×10-4. ESBLs production of all transconjugants were detected by CLSI Confirmatory Test. The transconjugants were highly resistant to cephalosporins just same as their donor strains, Clavulanic acid could greatly decrease drug resistance to cephalosporins. Compared with those donor strains, the transconjugants were lowly resistant to Aztreonam, Amoxacillin/Clavulanic Acid, Ampicillin/Sulbactam, Ceftazidime, Cefepime. Agarose electrophoresis showed that all these transconjugants acquired a plasmid of more than 21.3 kb from the donor strains. CTX-M type enzyme was still the most common type of extended-spectrumβ-lactamases , TEM type took second and SHV was not detected in transconjugants, some strains did not transfer. The gene type of a few ESBL-producing strains were of non-TEM, non-SHV, non-CTX-M.1. ESBL-producing strains are highly detected in the diarrhea disease pathogenic bacterium, most of them were isolated from the old and the child; they are common in hospital and community and may cause epidemic outbreak. ESBLs are now being the important mechanism of drug resistance in the diarrhea disease pathogenic bacterium.2. ESBL-producing isolates are usually multiply drug resistant strains, which resistant to most commonly usedβ-lactams and non-β-lactams antimicrobial agents.3. Most ESBL-producing isolates produce more than one type ofβ-lactamase; CTX-M(CTX-M-15)β-lactamase is the most common one and CTX-M-3, TEM-1 are commonly found ESBLs.4. Besides vertical spread, the ESBL-producing strains may be spread by horizontal transmission of the ESBL gene-carrying plasmid, ESBLs are most often encoded on large plasmid. Most gene-carrying plasmids bearing resistant genes to other antibiotics at the same time, then become multi-resistant, the resistance gene to non-β-lactams can be cotransferred with the ESBL bearing plasmid.5 ESBLs can exist not only in different species bacterium but also in different gene-types of the same bacteria, it is important to monitor, detect ESBL producers for controlling their spread.
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