Antimicrobial susceptibility and integrons detection among extended-spectrum β-lactamase producing Enterobacteriaceae isolates in patients with urinary tract infection

Subjects

This cross-sectional study involved 400 patients (inpatients and outpatients) with the signs and symptoms of UTI who were admitted to Shar Teaching Hospital, Sulaimani Teaching hospital, and Anwar Shexa Medical City Hospital Sulaimaniyah, Iraq, from September 2021 to January 2022.

Inclusion criteria

Patients with confirmed UTI, regardless of gender, ethnicity, and nationality.

Exclusion criteria

Pediatric age group (less than 7 years old) and pregnant ladies.

Questionnaire

A well-designed, self-prepared questionnaire was used to collect the patient’s sociodemographic data such as age, gender, catheterization condition, hospitalization type, UTI recurrence and type, with their medical history and comorbidities.

Bacterial isolation

The early morning midstream urine specimens were requested to be inoculated into different culture media and then incubated for 24–48 h at 37 °C in aerobic situations (Karah et al., 2020). VITEK 2 compact (BioMerieux, Marcy-l′Étoile, France) was used to identify isolated Enterobacteriaceae that produces a pink color colony with green metallic sheen property on Eosin Methylene Blue (EMB) agar according to the manufacturer’s recommendation. The bacterial stock was prepared in an Eppendorf tube and stored in a deep freezer at −40 °C for further analysis (Sprouffske, Aguilar-Rodríguez & Wagner, 2016).

Antibiotic susceptibility test (AST)

Using commonly available antibiotic discs, all the bacterial isolates were tested using the Kirby-Bauer disc diffusion technique according to what was fixed by Clinical, & Institute, Laboratory Standards (2020). The commonly used discs were amoxiclav (AMC) (20 μg amoxicillin [AMOX] + 10 μg clavulanic acid [CLA]), ceftazidime (CAZ; 30 μg), ceftriaxone (CRO, 30 μg), cefotaxime (CTX; 30 μg), cefixime (CFM; 5.0 μg), cefepime (CPM; 30 μg), ciprofloxacin (CIP; 5.0 μg), trimethoprim-sulfamethoxazole (SXT; 5.0 μg), nalidixic acid (NA; 10 μg), nitrofurantoin (NFN; 10 μg), gentamicin (GEN; 10 μg), imipenem (IPM; 10 μg), and meropenem (MEM; 10 μg).

ESBL screen test

A double-disk synergy test (DDST) was used to screen ESBL with the positive keyhole phenomenon (Kaur et al., 2013) and confirmed by two combined discs (CD) test methods such as cefotaxime-clavulanic acid disc and cefepime-clavulanic acid disc (Anoar, Ali & Omer, 2014; Teklu, 2019) and using AST GN74 cards (BioMerieux, Marcy-l′Étoile, France).

Klebsiella pneumoniae (ATCC 700603) was utilized as a positive control for ESBL, while E. coli (ATCC 25922) was used as a negative control.

Molecular detection of integron classes

Genomic DNA was extracted using the boiling method (colony PCR). Briefly, 2–3 pure isolated colonies from an overnight cultured bacterial growth in blood agar were taken and suspended in 200 μL of sterile distilled water, boiled at 95 °C for 10 min in the water bath. Then, the suspensions were centrifuged at 13,000 rpm for 10 min to remove cellular debris. The supernatant was used as the template for amplification and stored in an Eppendorf tube at −40 °C until use (Lan et al., 2019).

All isolates were screened for classes I, II, and III integrons using conventional polymerase chain reaction (PCR) according to specific conditions and primers applied previously (Kargar et al., 2014). Briefly, the PCR reaction was done based on the manufacturer’s instructions using the Add Star Taq master mix PCR kit (Add Bio, Gyeongbuk, Republic of Korea), which contains 20 mM Tris-HCL (pH 8.8), 100 mM KCL, 0.2% Triton X100, 4.0 MgCl₂, a protein stabilizer, loading dye and 0.5 mM each of dATP, dCTP, dGTP, and dTTP. Then, PCR reaction was accomplished to 20 µL by DEPC-H₂O (3.0 µL), 1.0 µL of 10 pmol forward/reverse primers, and 5.0 µL of a DNA sample. Next, the thermocycler (ESCO Thermocycler; ESCO, Singapore) was set for denaturation, annealing, and a final extension phase. Table 1 illustrates the PCR condition of primers and their sequences.

The amplicons were analyzed by electrophoresis on 1.0% w/v agarose gel in TBE buffer for 60 min and a voltage of 90. Then, the results were assessed under UV light on the UV transilluminator.

Sequencing of PCR products

The purified PCR products of four samples (two for integron class I and two for integron class II) were sequenced by the Macrogen Genome Center (Seoul, Republic of Korea). Then, the sequences were analyzed using Chromas Technelysium with online BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/).

Ethical approval

All measures accomplished in this study are based on the ethical standards of the National Research Committee and the Helsinki declaration, 1964 and its later amendments or comparable ethical standards. Simultaneously, written informed consent was obtained from the patients to publish their data. Therefore, the Scientific and Ethical Committee of the Sulaimani Directorate of Health and the Ethics Committee of the College of Medicine, University of Sulaimani, Iraq, approved the research protocol (No. 170-CoM-UoS on September 14, 2021).

Statistical analysis

Statistical Package performed data analysis for Social Science (SPSS Inc., Chicago, IL, USA, version 26). The Chi-square test was used for the association between variables. Descriptive statistics were presented as mean ± standard deviation (SD) and frequency/percentages for categorical variables. The significance level was defined at p ≤ 0.05, while highly significant was set at p ≤ 0.001.

Sociodemographic characteristics of the patients

The participant’s ages in this study ranged from 7–84 years, with a mean of 45.5 years. The primary age groups that attended hospitals with UTI symptoms were elderly patients (>60 years), and females were more affected (69%) than males. Among the studied patients, 110 were hospitalized, and 17% were catheterized for different conditions. On the other hand, most patients had UTI for the first time (69.5%), mainly due to cystitis (56.3%). In comparison, the most prevalent comorbidities among patients were diabetes (35%), and 42% were without medical history. Consequently, to examine if the positive culture rate was associated with any patient socio-demographics, catheterization status, hospitalization condition, UTI status/type, and medical history of the patients were investigated, and a highly significant (p < 0.001) correlation was found between the variables and the rate of positive cultured samples (Table 2).

Patient’s urine culture results

Among 400 urine samples, 171 (42.75%) were positive for urine culture, including Enterobacteriaceae (120 samples), followed by Gram-positive bacteria (no. = 16) and non-fermenter Gram-positive bacteria (no. = 16), then Pseudomonas species (no. = 15), fungi (no. = 11) with Acinetobacter species (no. = 9). Among Enterobacteriaceae, 86 (71.7%) isolates were E. coli, 32 (26.7%) were Klebsiella pneumoniae, and 2 (1.7%) were Proteus mirabilis (Fig. 1).

Bacterial AST outcomes

The most resistant antibiotics in this study for all isolated Enterobacteriaceae species (E. coli, Klebsiella pneumoniae, and Proteus mirabilis) were 3^rd generation cephalosporins (CTR, CAZ, CTX), followed by NA (78.35%), SXT (70%), then CIP (55.8%), and CPM (46.7%). At the same time, the most sensitive antibiotic for all three isolates was IPM (85.8%), followed by MEM (67.5%) and NFN (64.2%) (Table 3).

Bacterial isolates ESBL production

All isolated Enterobacteriaceae bacteria were produced ESBL (p > 0.05) using a double disc synergy test (DDST). The zone of inhibition towards the amoxicillin-clavulanate (AMC) disc is shown in Fig. 2.

Then, two phenotypic methods were used to confirm ESBL production. CD1 recorded 56.6% against all species, and the value decreased to 53.3% by automated method (VITEK System). E. coli (71.7%) was the highest ESBL producer, followed by Klebsiella pneumoniae (26.7%), while Proteus mirabilis (1.7%) was the minor producer (Table 4). Regarding the ESBL screen through the antibiotic’s profile, 74.2% of isolated Gram-negative bacteria were resistant to the 3^rd generation of cephalosporins (CTR, CAZ, CTX). Out of these isolates, different ranges were found using DDST. Additionally, CD2 was used to confirm fourth-generation cephalosporins (cefepime) resistance among isolated bacteria, which was 15.8% (p = 0.108) (Table 5).

The prevalence rate of integrons classes among isolated Enterobacteriaceae

All classes of integrons (I, II, III) were screened among isolated Enterobacteriaceae by PCR, and clear, joyous bands for classes I and II were observed (Fig. 3). The prevalence rate of class I was 54.2%, while for class II, integrons were 15.8%, and no isolate was positive for class III integrons (Table 6). Class I integron was among the commonest ESBL producers (66.2%) and non-ESBL producers (38.5%) of Enterobacteriaceae (p = 0.023).

Sequence analysis of integron classes

When sequence data from two samples of class I integron products were aligned using the Chromas Technelysium program, the amplified PCR of class I integron product belongs to one of several resistance genes integrated into E. coli and Klebsiella pneumoniae. When the forward primer data sequence was uploaded into the BLAST query, the same result of integron class I was obtained. In addition, class II products were aligned using Chromas Technelysium programs, and class II was accepted, Both sequence data were accepted by NCBI provided by GenBank nucleotide sequence accession numbers (ON745429, ON745430, ON745431, and ON745432).