Occurrence of blaDHA-1mediated 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 β-lactam stress.


METHODS
Screening 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-1transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of blaDHAgene 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 × 102fold), ceftriaxone at 2 µg/ml (2.63 × 103fold), ceftazidime at 8 µg/ml (7.06 × 103fold) and cefoxitin at 4 µg/ml (3.60 × 104fold) 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-1was carried mostly by the FIA and L/M Inc group.


CONCLUSIONS
This 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.
Department of Microbiology, Assam University, Silchar, 788011, India.
Birson
B
Ingti
Department of Microbiology, Assam University, Silchar, 788011, India.
Deepjyoti
D
Paul
Department of Microbiology, Assam University, Silchar, 788011, India.
Anand Prakash
AP
Maurya
Department of Statistics, Dibrugarh University, Dibrugarh, India.
Debajyoti
D
Bora
Department of Microbiology, Silchar Medical College and Hospital, Silchar, 788014, India.
Debadatta Dhar
DD
Chanda
Department of Microbiology, Silchar Medical College and Hospital, Silchar, 788014, India.
Atanu
A
Chakravarty
Department of Microbiology, Assam University, Silchar, 788011, India. ab0404@gmail.com.
Amitabha
A
Bhattacharjee
D000328:Adult; D000369:Aged, 80 and over; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018550:Cephalosporin Resistance; D002510:Cephalosporinase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002511:Cephalosporins / Q000494:pharmacology*; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D004927:Escherichia coli Infections / Q000453:epidemiology / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D022761:Gene Transfer, Horizontal; D060005:Genotyping Techniques; D006801:Humans; D007194:India / Q000453:epidemiology; D008297:Male; D008826:Microbial Sensitivity Tests; D015995:Prevalence; D060888:Real-Time Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D029968:Escherichia coli Proteins; D002510:Cephalosporinase; C508952:DHA-1 cephalosporinase, E coli