Abstract
BackgroundTreatment 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 β-lactam stress.MethodsScreening of AmpC β-lactamase phenotypically by modified three dimensional extract method followed by Antimicrobial Susceptibility and MIC determination. Genotyping screening of β-lactamase genes was performed by PCR assay followed by their sequencing. The blaDHA-1 transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of blaDHA gene was performed by transformation and conjugation and plasmid incompatibility typing, DNA fingerprinting by enterobacterial repetitive intergenic consensus sequences PCR.Results16 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 β-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 blaDHA-1 was carried mostly by the FIA and L/M Inc group.ConclusionThis study demonstrates the prevalence of DHA-1 gene in this region and highlights high transcription of DHA-1 when induced with different β-lactam antibiotics. Therefore, cephalosporin treatment must be restricted for the patients infected with pathogen expressing this resistance determinant.
Highlights
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
By performing Polymerase chain reaction (PCR), 16 isolates were detected for DHA genes and showed a sequence identical to that of DHA-1 (Table 1)
It was observed that there was a significant increase in the expression of DHA-1 gene in response to cefotaxime, ceftriaxone, ceftazidime but not as high as those for cefoxitin when compared with the basal level without antibiotic pressure (Fig. 1)
Summary
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. DHA-1, another plasmid-mediated AmpC β-lactamase, belonging to DHA family was found increasingly among Enterobacteriaceae in many parts of the world and was a growing concerned in the medical world as it leads to treatment failure [7] It was first characterized in a Salmonella enteritidis which has the ability to hydrolyze penicillins, cephamycin, including broad spectrum cephalosporin leaving physicians with limited antibiotic choices. It was the first plasmid-encoded β-lactamase found to be inducible and can be expressed in high levels [11, 12]. The regulation of this β-lactamase expression is closely linked to cell wall recycling and involves at least three genes: ampR (codes for a transcriptional regulator of the LysR family), ampG (codes for a transmembrane permease) and ampD (codes for a cytosolic N-acetyl-anhydromuramyl- l-alanine amidase) [13]
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