Horizontol dissemination of TEM- and SHV-typr beta-lactamase genes-carrying resistance plasmids amongst clonical isolates of Enterobacteriaceae / Disseminação horizontal de plasmídios de resistência contendo genes de beta-lactamase dos tipos TEM e SHV entre isolados clínicos de Enterobacteriaceae

The extended-spectrum â-lactamase (ESBL)-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS) testing. Twenty ESBL producing strains (15 percent) including Escherichia coli (n = 9), Klebsiella pneumoniae (n = 7), Klebsiella oxytoca (n = 2) and Enterobacter aerogenes (n = 2) were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR) and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L) were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospital

O isolamento de bactérias produtoras de beta-lactamases de espectro expandido (ESBL) está aumentando no mundo todo. Freqüentemente, a expressão de ESBL está associada com resistência a múltiplas drogas e disseminação por plasmídios de resistência. Durante um período de dois meses em 2000, 133 isolados clínicos de cepas de enterobactérias foram obtidos aleatoriamente de pacientes internos e externos de um hospital universitário na Turquia. As cepas produtoras de ESBL foram identificadas pelo teste de sinergia em disco-duplo (DDS). Foram detectadas vinte cepas produtoras de ESBL, entre as quais Escherichia coli (n=9), Klebsiella pneumoniae (n=7), Klebsiella oxytoca (n=2) e Enterobacter aerogenes (n=2), que foram posteriormente analisadas quanto a suas características de transferência de resistência, perfil plasmidial e natureza dos genes de resistência. Os testes de transferência de plasmídios foram realizados empregando técnicas de conjugação em caldo. Os genes TEM e SHV foram analisados pela reação da polimerase em cadeia (PCR) e hibridização com sondas especificas. A detecção de plasmídios epidêmicos foi feita por análise dos plasmídios R com a enzima de restrição EcoRI. Através desta análise, foram obtidos catorze perfis plasmidiais (A, B1, B2, C1 e C2 até L).Observou-se pela PCR que a maioria dos plasmidios carregavam genes derivados de TEM e SHV, confirmados através da detecção dos genes pelos testes de hibridização. As evidencias epidemiológicas indicaram que havia uma aparente transferência horizontal dos plasmídios R conjugativos entre as enterobactérias multiresistentes neste hospital.

Evaluation of product analogues for purification of penicillinase by affinity chromatography.

Among different matrices prepared, ampicilloic acid-polymer matrix offered 86.7% adsorption, 95% elution and 82.4% overall recovery of penicillinase. The structure of both the side chain and penicilloic or cephalosporoic acid moieties contribute to the affinity interactions.

Usage of penicillinase as an enzyme marker in enzyme linked immunosorbent assay for the detection of IgG antibodies to Toxoplasma gondii.

An Enzyme linked immunosorbent assay system has been developed using penicillinase as an enzyme marker for the detection of IgG antibodies to Toxoplasma gondii. This system is simple (end result assessed visually) reliable (the results are matching with other available commercial ELISA based kit), sensitive (detection of antitoxoplasma IgG antibodies to a level of 15 IU/ml) and reproducible (4.3 & 6.7% intra and inter coefficient of variation respectively). Experimental results with human serum samples using our system has shown consistent results and were in total agreement with other commercially available diagnostic kits. It is therefore suggested that ELISA system using penicillinase can be a simple, sensitive and reliable method for the diagnosis of toxoplasmosis in human.

Detection of toxoplasma IgM antibodies by ELISA method: a comparative study of different enzymes as markers.

Detection of toxoplasma IgM antibodies by employing enzyme linked immunosorbent assay (ELISA) technique was developed using different enzymes viz, horseradish peroxidase (HRP) (EC. 1.11.17), urease (EC. 3.5.15) and penicillinase (EC 3.5.2.6) as markers. Of these enzymes, HRP is light sensitive and needs dark chamber, also inactivated by preservative sodium azide. Similarly urease test system is extremely pH sensitive and demands special care during ELISA technique. Whereas penicillinase showed certain distinct advantages viz. stable at room temperature, high specific activity and economical. In the present studies it was observed that the sensitivity of penicillinase is similar to HRP and urease, marker enzymes used in commercially available diagnostic kit. The prominent feature of detection of toxoplasma IgM antibodies involving these three enzymes are: a) Shorter incubation time (About 2.5 hours) b) No false positive reaction. Moreover, these enzyme conjugates were prepared from F (ab')2 fragments of antitoxoplasma rabbit serum to elicit specific interaction with IgM antibodies only, avoiding cross interaction with other non-specific proteins like compliment systems and rheumatoid factor.

Importancia del método iodométrico en medio sólido en la detección de Beta Lactamas en Neisseria gonorrhoeae / Importance of the iodometric solid medium method for beta lactamasa detection in Neisseria gonorrhoeae

Se realizó la determinación de Beta Lactamas (B.L. sobre 137 cepas de Neisseria gonorrhoeae por los métodos de la Cefalosporina Cromogénica (C.C.) (Nitrocefin) y método lodométrico en medio sólido (I.M.S>). Notamos una concidencia del 100% entre ambos métodos tanto en resultados positivos (detección de B.L.) como en resultados negativos (no detección de B.L.) y una mayor rapidez con la técnica de la C.C. (promèdio 5 minutos) en comparación con el I.M.S. (promedio 2 horas). Debido a la difícil provisión en nuestro medio de la C.C. y su elevado valor comercial, la técnica I.M.S. descripta adquiere mayor importancia por ser confiable, sencilla y de bajo costo

Beta-lactamase diversity in Shigella spp

Some properties of the beta-lactamases produced by one strain of Shigella flexneri, one strain of Shigella sonnei, and one strain of Shigella boydii are studied. Susceptibility of these microorganisms to ampicillin and to cephalotin is investigated before and after a curing treatment with acridine oragne. The substrate profiles of these beta-lactamases, as well as their inducibility, their release to the extracellular environment by osmotic shcok,their susceptibility to enzime inhibitors, and their isoelectric points are also investigated. Transference of ampicillin resistance is tried by bacterial conjugation using a recipient strain of Escherichia coli K-12. The extrachromosomal DNA of the strain is also investigated. Through the analysis of the results the classification of these beta of these beta-lactamases is attempted in relation to the main groups of enzymes which are known at the present time. The three strains proved to be resistant to ampicillin and cephalotin but the transference of this resistance by conjugation was positive only for Sh. flexneri. Several bands of extrachromosomal DNA were observed in these microorganisms. It is concluded that, according to the properties of the enzymes, the beta-lactamase produced by Sh. flexneri belong to the TEM group of enzymes (plasmid-coded beta-lactamases). In relation to the beta-lactamase of Sh. sonnei, its general properties agree with those previously described for enzymes of the same bacterial species and allow the classification of this beta-lactamase as a constitutive cephalosporinase. It is also concluded that the bata-lactamase produced by Sh. boydii is plasmic-coded, since a curing effect was obtained with acridine orange and enzyme activity was released by osmotic shock procedure to the extracellualar environment in more than 80%. However, according to other properties (isolectric point and inhibition profile), this enzyme does not seem to belong to any of the know groups of plasmid-coded beta-lactamases...

Neisseria gonorrhoeae and penicillinase producing N. gonorrhoeae among some patients in Jakarta Indonesia.

Neisseria gonorrhoeae was isolated from 14 out of 50 (28%) of one group and 35 out of 60 (58%) of a second group of females in different areas of Jakarta, Indonesia. four (7%) of the patients in the second group were infected with penicillinase producing Neisseria gonorrhoeae (PPNG). This may be the first reported isolation of PPNG in Indonesia.