Ethics
The ethics of the study was confirmed by the Ethics Committee of Shahid Chamran University of Ahvaz according to Declaration of Helsinki (EE/98.24.3.26336/scu.ac.ir). Before collecting information, participants or parents (for children cases) were asked to read, accept and sign an informed consent form.
Sample collection and identification
A cross-sectional study was performed from October 2018 to December 2019. The sample size was estimated using a single population proportion formula based on the prevalence of 0.15 [1], 95% confidence interval, and margin error of 5%. With considering a 10% non-response rate, the minimum samples size was 225; however, to be conservative we collected 427 samples.
The samples were obtained from hospitals and laboratories in Ahvaz city; Iran. E. coli isolates of patients who had recently consumed antimicrobial drugs were excluded from the study. Briefly, the isolates were cultured onto MacConkey (Biolife Italiana; Italy) and Eosin Methylene Blue (EMB, Merck; Germany), and subsequently were incubated at 37°C for 24 h. Lactose-fermenting colonies on MacConkey, or colonies with metallic sheen on EMB were investigated by conventional biochemical tests including production of lysine decarboxylase, oxidase, Sulfur Indole Motility (SIM), Simmon’s Citrate and Methyl Red / Voges-Proskauer (MR/VP). Finally, purified isolates were analyzed by PCR for the presence of uspA gene, which is the highly specific gene of E. coli. Detection of uspA gene was performed as described previously [18].
Antimicrobial susceptibility pattern
Kirby-Bauer disc diffusion method was performed to evaluate the resistance and susceptibility of isolates to antimicrobial agents recommended by Clinical & Laboratory Standards Institute 2018 (CLSI-2018) [19]. The antimicrobial discs included Nalidixic-acid, Ampicillin, Tetracycline, Streptomycin, Sulfamethoxazole-trimethoprim, Ciprofloxacin, Kanamycin, Gentamycin, Fosfomycin, Imipenem, Meropenem, Cefotaxime, Ceftazidime, Cefazolin, and Nitrofurantoin. The isolates resistance to three or more different antimicrobial families were considered multidrug resistant (MDR).
As meropenem or imipenem resistant UPEC isolates were the focus of present study, the resistant isolates were subjected to further analyses. The minimum inhibitory concentration (MICs) for imipenem or meropenem were determined as recommended by CLSI-2018. MIC breakpoints for both antibiotics were defined ≥4 μg/ml.
Combined-disc (CD) and double disc synergy (DDS) tests
Phenotypic CD and DDS tests were performed to identify MBL-producing isolates. For CD test, two discs of imipenem (10 µg) and imipenem-EDTA (10 µg-1460 µg) were placed on the Muller-Hinton agar (MHA; Biolife Italiana; Italy) inoculated by 0.5-McFarland test isolates. After incubation of the plates at 35 °C for 16-18 h, the inhibition zone around discs were measured. The isolates were considered MBL-producer when the diameter of inhibition zone around the imipenem-EDTA disc increased by ≥ 7 mm compared to imipenem disc alone [20].
For DDS test, two discs of imipenem and EDTA (1460 µg) were placed on MHA plates inoculated with test isolates. The distance between two discs was considered 15 mm. After incubation of the plates at 35 °C for 16-18 h, the zone around discs were measured. Increasing of the inhibition zone or the formation of a phantom zone between the two discs indicated the MBL production [20].
Modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM)
Either mCIM and eCIM tests are extensively used for the epidemiological or infection prevention aims. The mCIM test could identify the bacteria that produce all carbapenemases, while the eCIM test was performed to differentiate MBL-producers from the serine carbapenemases. To perform mCIM, 1 µL loopful of the isolates were emulsified in 2 mL of tryptone soya broth (TSB). Then, one meropenem disc was immersed in the suspension for 4 hours at 37 °C. A MHA plate was inoculated by 0.5-McFarland standard E. coli ATCC25922. Meropenem disc was removed from the suspension and excess liquid was expelled. Meropenem disc was placed on the inoculated plate and incubated at 37 °C for 24 hours. Inhibition zone diameter of 6-15 mm or appearance of pinpoint colonies within a 16-18 mm zone around imipenem disc indicate the presence of carbapenemase [19].
eCIM test was performed when the mCIM test was positive. This test was done as similar to mCIM, except that after adding test isolate to the TSB, 20 µL of 0.5 M EDTA was added; then meropenem disc was immersed. Meropenem discs of eCIM and mCIM tests were placed on one plate and analyzed simultaneously. An increase of ≥5 mm in inhibition zone for eCIM versus mCIM was considered MBL-positive, while no change in zone diameter or an increase of ≤ 4 mm indicated the presence of carbapenemase [19].
Phenotypic differentiation of MLBs and class A KPC carbapenemases
To differentiate MBLs- and class A KPC carbapenemase-producing isolates, phenyl boronic acid (PBA) disc test was applied. The test was performed as a combined-disc of meropenem with and without PBA. Phenyl boronic acid was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/mL. Then meropenem disc was inoculated by 20 µL of PBA solution (400 µg PBA/disc). The test was performed as given for the standard disc diffusion method. An increase of ≥5 mm in inhibition zone around meropenem-PBA disc versus meropenem was considered class A KPC carbapenemase producer [21, 22].
Detection of resistance genes
Imipenem or meropenem-resistant isolates were analyzed for the presence of MBL-genes including blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-2, blaSPM-1, blaNDM, blaSIM, and blaGIM. The other CR genes of blaKPC, blaOXA-23 and blaOXA-48 were also investigated. Genomic DNA of isolates was extracted using the boiling lysis procedure. PCR reactions were performed as previously described [23-30] .
Virulence genotyping and phylogenetic grouping
The isolates were assayed for the presence of 30 virulence traits using five multiplex-PCR panel as previously described [31]. The investigated VGs were including papEF, papA, fimH, papG allele I-III, papG allele I, papG allele II, papG allele III, kspMTIII, gafD, focG, sfa/focDE, nfaE, papC, afa/draBC, sfaS, bmaE, ibeA, traT, cvaC, cdtB, hlyA, cnf1, fyuA, chuA, iutA, K1, K5, kpsMTII, and rfc. Determination of four major E. coli phylogroups was performed based on the triplex PCR as described by Clermont et al [32]. According to the presence of two genes of chuA and yjaA, and TSPE4.C2 DNA fragment, the isolates assigned to one of A, B1, B2, or D phylogenetic groups.
Enterobacterial intergenic repetitive element sequence (ERIC)-PCR
Fingerprinting of imipenem or meropenem resistant isolates was performed using ERIC-PCR based on conditions and primers described previously [33]. To evaluate the relationship between isolates, the presence or absence of bands compared to the standard DNA molecular marker was assessed. The clustering of the isolates was done based on Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis using the SAHN NTSYS program version 2.02e. A Dice similarity index was used for the definition of ERIC clusters.
Plasmid profiling and conjugation
Carbapenem resistant isolates were assayed for plasmid content. Plasmids were extracted using alkaline lysis method [34], then electrophoresed on agarose gel (1%). Estimation of plasmid sizes was acquired using a molecular weight marker, made from a lambda/Hind III digest.
The MBLs- or carbapenemase-producing isolates were conjugated with a lactose-negative enteroinvasive E. coli (EIEC) strain that was susceptible to imipenem and meropenem. The donors and recipient bacteria were cultured in nutrient broth for 16 hours; then mixed in a ratio of 1:10 (donor: recipient) [34]. After incubation for 48 h at 37 °C, the mixtures were inoculated on MCA containing imipenem or meropenem (4 μg/mL). The plates were incubated overnight at 37 °C. The resistant lactose-negative isolates were analyzed for the presence of inv and resistance genes via PCR reaction.