Characterisation of plasmid-mediated rmtB-1 in Enterobacteriaceae clinical isolates from São Paulo, Brazil

OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.

Prevalence of Plasmid-Mediated ampC Genes in Clinical Isolates of Enterobacteriaceae from Cairo, Egypt.

Aims: To determine the prevalence of acquired pAmpCs in clinically important and relevant enterobacterial species and to characterize the molecular types of pAmpC present in our geographic area. Methodology: Sixty Enterobacterial clinical isolates resistant to third generation cephalosporins and to cephamycins were included in the study. Samples were collected for a period of 6 months between July 2008 and December 2008 from Theodor Bilharz Research Institute (TBRI), Egypt. Bacterial species were identified using API E20. AmpC genes clusters: (bla ACC, bla EBC, bla FOX, bla CMY, bla MOX, and bla DHA) were tested by PCR and DNA sequencing. Clonal relatedness of AmpC-producing Klebsiellae isolates was determined by Pulsed Field Gel Electrophoresis (PFGE). Results: AmpC genes were detected in 28.3% (17/60) of the study population including E. coli, Klebsiella and Proteus mirabilis (P mirabilis). CMY-2 enzyme was found disseminating in all 6 AmpC-positive Escherichia coli (E. coli) and in 6/10 of Klebsiellae species. Only one Klebsiella pneumonia (K. pneumonia) isolate harbored CMY-4 while DHA-1 was detected in 3 Klebsiellae and in one P. mirabilis isolate. PFGE patterns showed no clonal relatedness among the 6 CMY-2-positive Klebsiella isolates. Conclusion: Plasmid-mediated AmpC enzymes are important mechanisms of resistance to ß- lactam drugs. CMY-2 and DHA-1 are the most common gene clusters of pAmpC in our region. AmpC-type resistance in our hospital setting is not due to the dissemination of clonal strains but due to the spread of resistant genes. This is the first report from Egypt identifying DHA-1 and CMY-4 in enterobacterial isolates.

Production and partial purification of protease by selected bacterial strains using raw milk as substrate

Aims: The present study was investigated to optimize and partially purify the proteases produced by the food borne bacterial strains. Methodology and Results: Four bacterial strains such as Bacillus cereus, Proteus vulgaris, P. mirabilis and Enterobacter aerogenes were isolated from food wastes. These strains were individually inoculated in to the formulated culture media supplied with three different concentrations (1:1 to 1:3) of raw milk as major substrate. Among the concentrations, 1:2 ratio of substrate supplied medium showed maximum (0.133 to 8.000 IU/mL) protease production by all the tested organisms. After optimization, the organisms were tested for protease production at various pH (3 to 9), and temperature (30 to 80 °C). The result showed that all the organisms were capable of producing maximum protease at pH 6 (8.533 to 10.133 IU/mL) and at 50 °C (8.666 to 10.666 IU/mL). The crude enzymes produced by the tested organisms were individually purified by two different methods viz sodium alginate and ammonium sulphate-butanol methods. The purity of the protease determined in these two methods was ranged between 3.24 to 5.44 I and 3.13 to 5.55 IU/mL respectively. The partially purified enzymes were further analysed through SDS-PAGE; accordingly the molecular weight of protein produced by the test organisms was determined in between 49.44 and 50.98 kDa. Conclusion, significance and impact of study: Among the tested strains P. vulgaris was identified as the major protease producer in optimized culture condition of 50o C and pH6. The molecular mass of the partially purified protease of P. vulgaris was 50.32 KDa. Further research on optimization of other fermentation parameters using statistical tools with P. vulgaris is needed to scale up the process.