AcrAB-TolC efflux pump system plays a role in carbapenem non-susceptibility in Escherichia coli.

BACKGROUND: Efflux pump mediated antibiotic resistance is an unnoticed and undetected mechanism in clinical microbiology laboratory. RND efflux systems are known for aminoglycoside and tetracycline resistance whereas their role in carbapenem non-susceptibility is not established. The study was undertaken to investigate the role of efflux pump in providing resistance against carbapenems and their response against concentration gradient carbapenem stress on the transcriptional level of the AcrAB gene in the clinical isolates of Escherichia coli from a tertiary referral hospital of Northeast India. RESULTS: Out of 298 non-susceptible Escherichia coli isolates 98 isolates were found to have efflux pump mediated carbapenem non-susceptibility. Among them thirty-five were non carbapenemase producers and their expressional levels were verified using qRT-PCR under concentration gradient carbapenem stress. In this study, a strong correlation between ertapenem resistance and AcrA overexpression was observed which has not been reported previously. Further, it was observed that imipenem stress increased AcrB expression in Escherichia coli which holds the novelty of this study. Additionally, the transcription of AcrR was insistently increased which is much higher than the transcriptional level of AcrA under concentration gradient carbapenem stress condition. CONCLUSION: The study established that AcrAB pump is a relevant antibiotic resistance determinant in bacterial pathogen, has an important role in developing resistance against carbapenem group of antibiotics.

Occurrence of bla DHA-1 mediated cephalosporin resistance in Escherichia coli and their transcriptional response against cephalosporin stress: a report from India.

BACKGROUND: Treatment alternatives for DHA-1 harboring strains are challenging as it confers resistance to broad spectrum cephalosporins and may further limit treatment option when expressed at higher levels. Therefore, this study was designed to know the prevalence of DHA genes and analyse the transcription level of DHA-1 against different ß-lactam stress. METHODS: Screening of AmpC ß-lactamase phenotypically by modified three dimensional extract method followed by Antimicrobial Susceptibility and MIC determination. Genotyping screening of ß-lactamase genes was performed by PCR assay followed by their sequencing. The bla DHA-1 transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of bla DHA gene was performed by transformation and conjugation and plasmid incompatibility typing, DNA fingerprinting by enterobacterial repetitive intergenic consensus sequences PCR. RESULTS: 16 DHA-1 genes were screened positive from 176 Escherichia coli isolates and primer extension analysis showed a significant increase in DHA-1 mRNA transcription in response to cefotaxime at 8 µg/ml (6.99 × 102 fold), ceftriaxone at 2 µg/ml (2.63 × 103 fold), ceftazidime at 8 µg/ml (7.06 × 103 fold) and cefoxitin at 4 µg/ml (3.60 × 104 fold) when compared with untreated strain. These transcription data were found significant when analyzed statistically using one way ANOVA. Four different ESBL genes were detected in 10 isolates which include CTX-M (n = 6), SHV (n = 4), TEM (n = 3) and OXA-10 (n = 1), whereas, carbapenemase gene (NDM) was detected only in one isolate. Other plasmid mediated AmpC ß-lactamases CIT (n = 9), EBC (n = 2) were detected in nine isolates. All DHA-1 genes detected were encoded in plasmid and incompatibility typing from the transformants indicated that the plasmid encoding bla DHA-1 was carried mostly by the FIA and L/M Inc group. CONCLUSION: This study demonstrates the prevalence of DHA-1 gene in this region and highlights high transcription of DHA-1 when induced with different ß-lactam antibiotics. Therefore, cephalosporin treatment must be restricted for the patients infected with pathogen expressing this resistance determinant.

Distribution of carbapenem resistant Acinetobacter baumannii with blaADC-30 and induction of ADC-30 in response to beta-lactam antibiotics.

A wide range of intrinsic Acinetobacter-derived cephalosporinases (ADC) along with other carbapenemases has now been detected in Acinetobacter baumannii leaving clinicians with few treatment options. The present study reports the spread of ADC-30 co-producing KPC-2 along with other ß-lactamases among carbapenem resistant A. baumannii strains obtained from ICU patients in two Indian hospitals. Primer extension analysis revealed higher transcript level of the ADC gene when induced with cefoxitin at 8 µg/ml (170 fold), ceftriaxone at 8 µg/ml (136 fold), ceftazidime at 4 µg/ml (65 fold), cefepime at 8 µg/ml (77 fold) and aztreonam at 8 µg/ml (21 fold) when compared with the basal level without antibiotic pressure. Slight increase in expression of blaADC-30 when induced with imipenem and meropenem at 0.25 µg/ml (3 and 6 fold) was observed and may help in conferring resistance to carbapenem. MLST analysis revealed the circulation of A. baumannii sequence types ST188, ST386, ST583 and ST390 in these hospitals.

Transcriptional Response of Multiple ESBL Genes Within Escherichia coli Under Oxyimino-Cephalosporin Stress.

The expression of extended-spectrum beta-lactamases directly interferes with the treatment options in a clinical setting. It is not clearly defined why bacteria acquire multiple beta-lactamases and how they are being expressed in antibiotic stress. With this key question, the study was designed to understand the transcriptional response in Escherichia coli harboring multiple blaESBLs against different oxyimino-cephalosporin stress. A total of 169 consecutive, nonduplicate oxyimino-cephalosporin-resistant isolates of E. coli were screened and were ESBL positive. Among them six isolates were found to harbor multiple beta-lactamase genes and we, as per our objective, selected them for this study. Molecular characterization was done by multiplex polymerase chain reaction (PCR) assay. Minimum inhibitory concentration, transcriptional expression, transferability, and plasmid incompatibility typing of multiple blaESBLs were carried out. Plasmid stability and antibiotic susceptibility of donor and transconjugants were performed. A total of six isolates were found to be harboring multiple ESBL genes and MIC above the breakpoint level against all the tested antibiotics. Quantitative real-time PCR showed that in basal level without antibiotic stress, SHV-148 expressed more, but with ceftriaxone stressed, expression of CTX-M-15 and SHV-148 was high. In case of PER-1, expression was high with ceftazidime stress. blaESBLs were horizontally transferable and originated through multiple inc types. Plasmids were stable till 115 serial passages. Pulsed-field gel electrophoresis results showed that multiple ESBL genes were spread through six pulsotypes. Our study concludes that acquisition of multiple ESBL genes in E. coli was a specific adaptation for survival against multiple oxyimino-cephalosporin stress in this clinical setting.