Identification and Distribution of the Clinical Isolates of Imipenem-resistant Pseudomonas aeruginosa Carrying Metallo-β-lactamase and/or Class 1 Integron Genes

To investigate the distribution of the genes of two major metallo-β-1actamases （MBL;i.e.,IMP and VIM） and class 1 integrons （intI） in the clinical imipenem-resistant Pseudomonas aeruginosa, a total of 65 isolates, from a university hospital in Sichuan between December 2004 and April 2005 were screened for MBL genes by PCR using primers specific for blaIMP-1, blaVIM and blaVIM-2 genes. The MBL-positive isolates were further assessed for class 1 integrons by PCRusing specific primers. The nucleotide sequences of several PCR products were also determined. The results revealed that the blaVIM gene was found in 81.5% （53/65） of all isolates, blaVIM-2 gene was found in only 1 isolate and the intl gene was observed in 45.3% （24/53） of blaVIM-positive isolates. One isolate carried simultaneously both blaIMP-1 and intl genes, and to the best of our knowledge this is the first report of such isolate in southwest China. These observations highlight that the genes for VIM β-1actamase and class 1 integrons were predominantly present among the imipenem-resistant P. aeruginosa tested, confirming the current widespread threat of imipenem-resistant, integron-borne P.aeruginosa.

Occurrence of Extended-Spectrum β-1actamase Producing Enterobacteriaceae （ESBLPE） among Primary School Pupil in Obafemi-Owode, Nigeria

Occurrence of extended-β-1actamase producing enterobacteriaceae （ESBLPE）, which has reduced the antibacterial efficacy and potency of many 3rd generation cephalosporins, was investigated among the primary school pupils. 88 primary school pupils in Obafemi-Owode Local Government, Southwestern Nigeria, including 49 males （55.7%） and 39 females （44,3%） （mean age 12 ± 3） were screened for ESBLPE isolates with exclusion criterion of antimicrobial use in the preceding 2 weeks either as therapy for gastro-intestinal complication or prophylaxis. ESBLPE detected include 4.5% of Eschericia coli, 2.3% of Enterobacter cloaca, 0% Proteus mirabilis, 2.3% Pseudomonas aeruginosa, 1.1% Staphylococcus aureus and 4.5% of Klebsiella oxytoca. 10 （76.9%） of ESBLPE isolates were resistant to disc of cefuroxime （30 μg）, 8 （61.5%） susceptible to amoxicillin/clavulanic （20/10 lag） and low susceptibility of 7 （53.8%） was recorded for ceftazidime （30 lag）. 0% susceptibility was recorded for the ESBLPE isolates to cefuroxime MIC 〉 8 gg/mL and ampicillin MIC 〉 8 lag/mL while E, coli and E. cloca each show 50.0% and P. aeruginosa and K. oxytoca show 100.0% and 75.0% susceptibility to augmentin （MIC 〈 8）. This study has shown a 14.7% proportion of the pupil to harbour ESBLPE from enteric source with increased resistant to most new generation cefuroximes. Therefore, transfer of virulent and antibiotic resistant ESBLPE could be aided by sharing feeding materials while fecal-oral route of transmission cannot be ruled out as hygiene level is very low thereby increasing emergence of virulent resistant enteric strains leading to treatment failure.

Total Alkaloids from Sophora Alopecuroides L.Increase Susceptibility of Extended-Spectrum β-Lactamases Producing Escherichia coli Isolates to Cefotaxime and Ceftazidime

Objective： To evaluate the antimicrobial activity of total alkaloids extracted from Sophorea alopecuroides L. （TASA） against clinical isolated extended-spectrum beta-lactamases （ESBLs） producing Escherichia coil （E.. coil） strains. Methods： The antibacterial activity of TASA either itself or in combination with cefotaxime （CTX） or ceftazidime （CAZ） was investigated by using the microbroth dilution method and phenotypic confirmatory disk diffusion test against three clinical isolated ESBLs-producing E. coil strains; the interactions of TASA and C＇I＇X or CAZ were ascertained by evaluating the fractional inhibitory concentration index （FICI）. Results： The antibacterial activity of either TASA itself or in combination with C＇IX or CAZ was found. The minimum inhibitory concentration （MICs） of TASA against the ESBLs producing isolates was 12.5 mg/mL. In the combinations with a sub-inhibitory concentration of TASA, a synergistic effect on CTX and CAZ against the ESBLs producing isolates was observed. Similarly, the isolates exposed to lower dose of TASA yielded an increased susceptibility to CTX and CAZ by 8-16 folds determined by microdilution assay. Moreover, enzymatic detection of ESBLs demonstrated that TASA induced reversal resistance to CTX and CAZ partially by a mechanism of inhibition of ESBLs activity in these isolates. Additionally, in the tested isolates following the exposure of TASA, molecular analysis verified the SHV-type beta-lactamase encoding ESBL gene in these isolates, and no mutation was introduced into the ESBL gene. Conclusions： These results suggest that TASA could be used as a source of natural compound with pharmacological activity of reversal resistance to antimicrobial agent. These findings also indicated that the application of the TASA in combination with antibiotics might prove useful in the control and treatment of infectious diseases caused by the ESBLs producing enterobacteriaceae.

Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples

We developed a colorimetric assay to quantify clavulanic acid （CA） in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-1actamase-catalyzed reaction, in which the yellow substrate nitrocefin （λmax=390 nm） is converted to a red product （λmax=486 nm）. Since CA can irreversibly inhibit β-1actamase activity, the level of CA in a sample can be measured as a function of the A390]A486 ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L -1 and 50 μg L to 10 mg L-1, respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.