BackgroundEmergence of atypical enteropathogenic Escherichia coli (EPEC) and hybrid E. coli (harboring genes of more than one DEC pathotypes) strains have complicated the issue of growing antibiotic resistance in diarrhoeagenic Escherichia coli (DEC). This ongoing evolution occurs in nature predominantly via horizontal gene transfers involving the mobile genetic elements like integrons notably class 1 integron. This study was undertaken to determine the virulence pattern and antibiotic resistance among the circulating DEC strains in a tertiary care center in south of India.MethodsDiarrhoeal stool specimens were obtained from 120 children (< 5 years) and 100 adults (> 18 years), subjected to culture and isolation of diarrhoeal pathogens. Conventional PCR was performed to detect 10 virulence and 27 antimicrobial resistance (AMR) genes among the E. coli isolated.ResultsDEC infection was observed in 45 (37.5%) children and 18 (18%) adults, among which [18 (40%), 10 (10%)] atypical EPEC was most commonly detected followed by [6 (13.3%), 4 (4%)] ETEC, [5 (11.1%) 2 (2%)] EAEC, [(3 (6.6%), 0 (0%)] EIEC, [3 (6.6%), 0 (0%] typical EPEC, and [4 (8.8%), 1 (1%)] STEC, and no NTEC and CDEC was detected. DEC co-infection in 3 (6.6%) children, and 1(1%) adult and sole hybrid DEC infection in 3 (6.6%) children was detected. The distribution of sulphonamide resistance genes (sulI, sulII, and sulIII were 83.3 and 21%, 60.41 and 42.1%, and 12.5 and 26.3%, respectively) and class 1 integron (int1) genes (41.6 and 26.31%) was higher in DEC strains isolated from children and adults, respectively. Other AMR genes detected were qnrS, qnrB, aac(6’)Ib-cr, dhfr1, aadB, aac(3)-IV, tetA, tetB, tetD, catI, blaCTX, blaSHV, and blaTEM. None harbored qnrA, qnrC, qepA, tetE, tetC, tetY, ermA, mcr1, int2, and int3 genes.ConclusionsAtypical EPEC was a primary etiological agent of diarrhea in children and adults among the DEC pathotypes. Detection of high numbers of AMR genes and class 1 integron genes indicate the importance of mobile genetic elements in spreading of multidrug resistance genes among these strains.Electronic supplementary materialThe online version of this article (10.1186/s41043-018-0147-z) contains supplementary material, which is available to authorized users.
Ciprofloxacin resistant Shigella sonnei across the globe have been increasing alarmingly. In order to understand the emergence of S.sonnei with respect to ciprofloxacin resistance in our patient population, the following study was carried out. Of the 184 Shigella sp. Isolated from 2012 to 2015, 34 S.sonnei which were confirmed by standard methods and subjected to antimicrobial susceptibility testing were selected. The minimum inhibitory concentrations (MICs) of 16/34 quinolone resistant isolates tested ranged from 4micrograms/ml to 16micrograms/ml for ciprofloxacin, from 16 micrograms/ml to 64 micrograms/ml for ofloxacin and from 16micrograms/ml to 64micrograms/ml for levofloxacin. Sequence determination of the quinolone resistance determining regions of gyrA, gyrB, parC, and parE genes showed mutations in GyrA at Gln69/Trp, Phe71/Ser, Ser72/Pro, Met75/Leu, Ser90/Cys, Met94/Leu, His106/Pro, Asn161/His, Thr163/Ala and in ParC at Ala64/Asp. Among the plasmid-mediated quinolone resistance (PMQRs) targets investigated,qnrB was the most (93.7%) prevalent followed by qnrC (18.7%). None hadqnrA, qnrS and qepA. Two (0.1%) of the isolates harboured theaac(6’)-lb gene. Drug accumulation assay detected the presence of efflux pump activity in 9/15 (60%) among ciprofloxacin resistant isolates. All isolates harboured the ipaH gene followed by ial (17.6%), sen (11.7%), set1A&set1B (5.8%) genes. None had stx1 element. PCR for Enterobacterial repetitive intergenic consensus (ERIC) sequences resulted in 4 unique clusters, of which Type III was the most (44%) dominant but there was no correlation between the ERIC types and the antibiotic resistance pattern or the virulence profile. A documented increase in S.sonnei harbouring the qnrgenes and some unusual genes like set1Aand indicate an ongoing process of horizontal gene transfer. The accumulation of novel mutations in GyrA and ParC in the presence of efflux pump and PMQR genes contributed to the raised MIC to quinolones. These findings are crucial in our understanding of quinolone resistance in these isolates.
Background Rising rates of resistance to antimicrobial drugs among Enterobacteriaceae limit the choice of therapeutic agents to treat urinary tract infections. In this context we assessed the in-vitro effect of fosfomycin against extended-spectrum beta-lactamases, AmpC beta-lactamases and carbapenemase-producing strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter spp, and P seudomonas aeruginosa isolated from the patients with urinary tract infection (UTI) and also studied the effect of fosfomycin on their biofilm formation. Materials and methods A total of 326 multidrug-resistant (MDR) isolates comprising of Escherichia coli, Klebsiella pneumoniae, Enterobacter spp, and P seudomonas aeruginosa from the urine samples of the patients with a diagnosis of UTI were included in the study. MIC 50 and MIC 90 were detected by agar dilution method and the capacity to form biofilm in the presence of fosfomycin by these MDR isolates was assessed by the tissue culture plate method. Results The MIC50 for meropenem (0.5 µgm/mL) and nitrofurantoin (32 µgm/mL) was within the susceptible range only for E. coli . Fosfomycin was the only antibiotic that inhibited 100% E.coli , 70% Klebsiella spp, and 50% Pseudomonas spp and 40% Enterobacter spp which included the extended-spectrum beta-lactamases producers. It showed a similar effect on carbapenemase producers and AmpC producers. Fosfomycin disrupted biofilm in 67% (n=141) E.coli , 74% (n=50) Klebsiella spp, 88% (n=27) Pseudomonas spp and 36% (n=23) Enterobacter spp at 24 hrs of incubation with a concentration of 2 fold dilution lower than that of the MIC. Conclusion Fosfomycin showed a good inhibitory effect on the biofilms produced by the MDR organisms studied here.
During the COVID-19 pandemic, several laboratories used different RNA extraction methods based on the resources available. Hence this study was done to compare the Ct values in qRT-PCR, time taken (sample processing-loading to PCR), manpower requirement, and cost of consumables between manual and automated methods. Materials and methodsA cross-sectional study was done on 120 nasopharyngeal/oropharyngeal swabs received in VRDL for RT-PCR testing. Based on the results of automated RNA extraction (Genetix, HT 96 Purifier) and RT-PCR (Trivitron PCR Kit) detecting E gene (screening) and ORF gene (confirmatory), the division into Group-I (Ct 15-22), Group-II (Ct 23-29), Group-III (Ct 30-36) and Group-IV (Ct >36) was done. Manual RNA extraction was done using magnetic beads (Lab system, Trivitron). Statistical analysisData were analyzed by SPSS 19.0 version software. Ct values obtained in the two methods were compared by paired t-test, GroupWise. Z test was used to compare the other parameters. ResultsThe difference in Ct values for target genes was statistically significant (p<0.05) in Group-I to III; however, no variation in result interpretation. The difference in time, manpower, and cost were statistically significant (p<0.05). The manual method required twice more manpower; 40 minutes more time & automated method cost 3.5 times more for consumables. ConclusionThe study showed that RNA yield was better with automated extraction in comparison to manual extraction. The samples extracted by the automated method detected the virus at a lower Ct range by PCR than the manual method. Automated method processed samples in less time and with less manpower. Considering the cost factor, manual extraction can be preferred in resource-limited settings as there was no difference in the results of the test. The manual method requires more hands-on time with potential chances of crosscontamination and technical errors.
Background: There is limited data on the resistance mechanisms of Diarrhoeagenic Escherichia coli (DEC) from our centre which prompted us to perform this study to determine the antimicrobial resistance pattern.Methods & Materials: DNA extracted from confirmed DEC strains (48) isolated between November 2014 to March 2015 from 120 diarrhoeal stool specimens collected from children less than 5 years from were cultured were subjected to PCR for the detection of antimicrobial resistance genes (gyrA, gyrB, parC, parE, qnrA, qnrB, qnrS, qnrC, qepA, aac(6 )-Ib, sulI, sulII, sulIII, dhfrI, aac(3)-IV, aadB, tetA, tetY, tetD, tetE, tetC, tetB, catI, blaCTX, blaTEM blaSHV, int1, int3, int2 and erm).Results: All (100%) the strains harboured the quinolone resistance gene gyrB, while, gyrA and parE were present in 93.7% each, parC in 85.4%, aac(6 )-Ib and qnrS in 41.6% each, qnrB in 34.7%, qnrC in 31.2%, qepA in 18.7% and qnrA in 12.5%. Genes which are responsible for resistance to aminoglycoside aac(3)-IV (14.5%), aadB (6.2%), sulfonamides (sulI) (77%), (sulII) (31.2%), (sulIII) (6.2%), trimethoprim (dhfrI) (50%) and tetracycline (tetA) (56.25%), (tetY) (20.8%), (tetD) (18.7%), (tetE) (18.7%), (tetC) (16.6%),(tetB) (8.3%), chloramphenicol (catI) (39.58%) and  lactam (blaCTX) (64.5%), (blaTEM) (20.8%) and (blaSHV) (8.3%) were also widely distributed. Integrons were noted in 70.8% (int1), 33.3% (int3) and 31.2% (int2), while none harboured the macrolide resistance gene erm. Conclusion:A diverse pattern was observed with regards to antimicrobial resistance genes in DEC. This study also illustrates the importance of integrons in the epidemiology of antibiotic resistance in DEC strains in our setting.
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