Clinical specimens and isolation of E.coli isolates
A total of 148 E.coli isolates previously collected and studied for their virulence profile and phylogroups [16] were used for this study. Samples were collected from February 2011 to August 2013 as described before [16]. Briefly, the first group of E.coli isolates (N = 77) were obtained from the blood culture of sepsis patients and the second group (N = 71) were obtained from the faeces of patients who were admitted in ICU for various reasons for example had undergone cardiovascular surgery, cases of road transport accident etc. but were not diagnosed with sepsis.
E.coli Pathotypes
Pathotyping was performed by a multiplex PCR using the primers uidA, pic, bfp, invE, LT, escV, aggR, stx1a, stx2a, st1b, st1a, astA corresponding to the genes defining the appropriate pathotypes as previously reported [19]. EAEC strains harbour astA, aggR, and pic genes and can be confirmed if found positive in a combination of pic and aggR or aggR and astA. Isolates found positive for either LT toxin (LT) or heat-stable toxin (ST) were designated as ETEC, whereas designated as EIEC if found positive for invE, and as EPEC if found positive for escV and bfp. STEC isolates were positive for stx and negative to bfp however in the presence or absence of escV. A primer pair for the detection of the E.coli-specific uidA gene was also included.
HeLa cells culture & adherence assays
HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; Pan-Biotech, Germany) in the presence of 1% antibiotic mixture (penicillin and streptomycin; Life Technologies, USA) in an environment of 5% CO2 at 37 °C. Adherence assay was performed on the monolayer of HeLa cells, which were upto 50% confluent [18]. Briefly, HeLa cells were first washed with phosphate buffered saline (PBS, pH 7.4). After washing, 1.0 ml of fresh medium (DMEM supplemented with 2% FBS) was added to the cell monolayers. The HeLa cells thereafter were inoculated with approximately 108 CFU/mL suspensions of E.coli grown in LB broth (overnight culture), diluted 1:50 and incubated at 37 °C. After 6 h of incubation, the cells were washed twice with PBS and thereafter fixed with methanol (Merck, Germany) for 1 h. The methanol-fixed cells were then stained with May Grünwald-Giemsa stain for 1 h and destained with 70% ethanol. Cells were observed under inverted microscope after fixation at 20X (Nikon, Eclipse TS100).
Analysis of Enterobacterial repeated intergenic consensus (ERIC) sequences
E.coli isolates were fingerprinted using ERIC-PCR. The primers used for the ERIC-PCR reaction were ERIC-F 5’-AAGTAAGTGACTGGGGTGAGCG-3’ and ERIC-R 5’-ATGTAAGCTCCTGGGGATTCAC-3′ [20]. The gel images were captured using a Gel-documentation system. All the bands obtained were normalized using ImageLab software. Depending upon the molecular weight of the reference marker, a weighted matrix was generated. Using the PyELph v1.4 software, each band in each lane was analysed to obtain the band size with reference to the marker bands. All the band sizes so obtained were treated as an input for further analysis. A binary code of 1 or 0 was introduced to each band subjected to the presence and/or absence of the band respectively. On the basis of such generated binary matrix file, a phylogenetic tree and principle component analysis was constructed through NTSYS-pc 2.02 J software. Band intensity is an important characteristic for this analysis. The bands with very low resolutions were ignored by the ImageLab software as background noise after manually checking each band. The band profiles of the DNA fragments obtained after PCR amplification using specific primers for ERIC sequences were determined. The fingerprints obtained consisted of 5 to 15 bands ranging in size from 100 bp to 1 kb.
Antibiotic susceptibility assay
Individual antimicrobial disks of Amikacin 30mcg (AK), Cefepime 30mcg (CPM), Cefoperazone 75mcg (CPZ), Cefoxitin 30mcg (CX), Ceftazidime 30mcg (CAZ), Ciprofloxacin 5mcg (CIP), Gentamicin 10mcg (GEN) (HiMedia, India) were placed on the surface of the agar using sterile forceps. The disks were in complete contact with the agar surface by pressing down with forceps. 10 disks each were placed on a 150-mm plate and each had a gap of more than 24 mm between them. The plates were thereafter inverted and incubated at 37 °C for 18 h. Diameters of the inhibition zones were measured to the nearest millimetre using calibrated scale.
Screening of ESBL producers
An inert flat circular ring having a disk of Aztreonam (30 μg), Cefpodoxime (10 μg), Cefpodoxime/Clavulanic acid (10/5 μg), and Ceftazidime (30 μg) with a 6 mm diameter on its projections was used. According to the CLSI guidelines [21], isolates showing Cefpodoxime(10 μg) < 17 mm, Ceftazidime(30 μg) < 22 mm, Aztreonam (30 μg) < 27 mm, Cefotaxime (30 μg) < 27 mm, Ceftriaxone (30 μg) < 25 mm in the initial screening were considered as potential ESBL-producer. ESBL producer isolates were further screened with another set of discs having Cefpodoxime (10 μg), Cefpodoxime/Clavulanic acid (10/5 μg), Ceftazidime (30 μg), Ceftazidime/Clavulanic acid (30/10 μg), Cefotaxime (30 μg) and Cefotaxime/Clavulanic acid (30/10 μg). An increase of ≥2 mm in zone diameter for antimicrobial agent that were tested alone versus when tested in combination with Clavulanic acid confirmed the isolate as a potent ESBL producer.
Genotypic characterisation of ESBL genes
The presence of ESBL genes was tested by two multiplex PCRs, the first one detects TEM/SHV/ OXA-1 (Temoneira/ Sulfhydryl variable/ Oxacillin hydrolysing capabilities) group and the second one detects CTX-M groups 1, 2 and 9 [22].
Detection of ST131 gene and Fim H30 and H30-Rx sub-clones by PCR
Detection of the ST131-O16 and ST131-O25b clades was carried out by PCR using the primers previously described [23]. ST131 isolates were further characterized by screening them for ST131-associated SNPs in mdh (i.e., C288T and C525T) and gyrB (i.e., C621T, C729T, and T735C) [24]. Further, ST131-associated Fim H30 and H30-Rx subclone were identified by PCR. All ST131 positive isolates were tested for fimH 30 allele (encoding a variant of the type 1 fimbrial adhesin) corresponding with the main FQ resistance–associated subset within ST131, using the allele-specific primers as decribed before [24, 25]. The H30-Rx sub-clone was identified by detection of a specific single-nucleotide polymorphism (SNP) (G723A) within the allantoin-encoding gene, ybbW using the Primers AP63 and AP66 as described before [26].
Statistical analysis
Fishers Exact test was used to compare pathotypes between the blood and fecal E.coli isolates. Z-test was used to compare the prevalence of ESBL and ST131 clone between the blood and fecal E.coli isolates. P value of p < 0.05 was considered significant.