Of the 230 analyzed samples, 91 (39.5%) produced E. coli isolates and 32 (35.1%) were identified as DEC, based on a combination of culture and PCR. All the culture-positive samples were confirmed as positive by PCR. The female-to-male DEC infection ratio was 1.1:1 (Table 1).
Table 1. Total number, sex, and age characteristics of the patients with E. coli pathotypes
We showed that DEC (n=32, 13.9%) was second only to Campylobacter jejuni (n=45, 19.5%; p = 0.003) as the most abundant agent of diarrhea in pediatric patients, whereas in Shiraz (Iran) and India, DEC was the most common agent found (14-16). Pediatricians should therefore request standard microbiological methods for determination of the pathogens involved and for designing subsequent therapy.
Phenotypic and genotypic investigation: The isolates showed a greater frequency of the EAEC (11/32; 34.3%) and EPEC (9/32; 28.1%) pathotypes than of the other E. coli pathotypes. (Table 1). The PCR results were consistently in accordance with those obtained from culture. Table 5 shows the abundance of DEC and E. coli pathotypes in the present study and the pathotypes reported in previous studies. The differences in the frequencies of DEC and E. coli pathotypes may be related to a variety of factors, including differences in climate and other environmental conditions, the level of economic development, individual hygiene practices, lack of/improper medical health care, poor quality medical care, and contamination of food or of food manufacturing or processing facilities (17).
The patients showed a greater frequency of mucus in the stool (28/32; 87.5%), abdominal pain (24/32; 75%), and vomiting (19/32; 59.3%) than of other clinical symptoms (Table 2). Table 5 shows the numerous clinical symptoms identified in this study and in other published papers. Our analysis showed differences in the clinical symptoms between various bacterial infections, but many overlaps were also evident. Hence, a laboratory-based identification of DEC is still required for etiological verification of this diarrheal disease. Nevertheless, pediatricians should seriously consider EHEC as an etiological agent of diarrhea when blood is found in stool specimens.
Table 2: Frequency of clinical symptoms in pediatric patients with E. coli pathotypes
Phenotypic and genotypic antibiotic resistance determination: The CLSI 2017 guidelines indicated greater antibiotic resistance rates for nalidixic acid (30/32; 93.7%), ampicillin (29/32; 90.6%), and tetracycline (25/32; 78.1%) than for any of the other antibiotics. All DEC isolates were susceptible to gentamicin and imipenem. DEC is a common agent in developing countries and is spread by contaminated water and food; therefore, its frequency and antimicrobial resistance are public health concerns (17). Full descriptions of the antibiotic resistance of DEC and E. coli pathotypes, from this study and others, are provided in Table 5. These results strongly suggest that nalidixic acid, ampicillin, tetracycline, and cotrimoxazole can no longer be empirically prescribed for the treatment of severe diarrhea and dysentery in central Iran. Since 1960, cotrimoxazole has been one of the World Health Organization’s essential medicines, as it is widely available and easily affordable and shows a broad spectrum of activity against an extensive range of infections, including diarrhea.
High levels of MDR (25/32; 78.1%), ESBL (18/32; 56.2%), and AmpC (9/32; 28.1%) gene expression were observed for the DEC isolates (Table 3). The most extensive MDR strains for our collection of DEC showed combined resistance to nalidixic acid, ampicillin, tetracycline, and third-generation cephalosporins. The frequency of ESBL positivity was higher in the EAEC pathotype (81.8%) than in the other ESBL-positive E. coli pathotypes.
Table 3. Phenotypic antibiotic resistance rates in E. coli pathotypes
MDR DEC has quickly spread worldwide and represents a serious menace to the proper management of diarrhea and dysentery in developing countries (19). The frequencies of MDR DEC in the present study and in others are summarized in Table 5. The high rate of MDR-resistant isolates in pediatric patients with diarrhea can lead to more frequent treatment failures. In addition, resistant isolates demand the prescription of broad-spectrum antibiotics for the empiric treatment of infections, adding tremendous costs to treatment of these infections (20).
The isolates showed a higher frequency of the ESBL genes blaTEM (18/18; 100%), blaCTX-M15 (17/18; 94.4%), and AmpC [blaCIT (4/9; 44.4%) and blaDHA (4/9; 44.4%)] than of the other ESBL and AmpC genes (Table 4). Table 5 shows the frequency of ESBL and its encoding genes in DEC and the other E. coli pathotype isolates. The differences in the frequencies of MDR, ESBL, and related genes across various regions may reflect different antibiotic usage patterns and different DEC community compositions (21).
Table 4. Frequency of antibiotic resistance genes among E. coli pathotypes
Among the PMQR determinants, qnrS, and qnrA were positive in 18/30 (60%) and 2/30 (6.6%) in nalidixic acid-resistant DEC strains, respectively and no case of qnrB was found. 20/30 (66.6%) isolates carrying PMQR contain similar mutations in gyrA at amino acid 83 (replacement of serine with leucine) and 18/30 (60%) parC at amino acid 80 (replacement of serine with isoleucine; GenBank accession no. HM068910). Although quinolones/fluoroquinolones are intended to be appropriate drugs against resistant isolates, the enhancement in antimicrobial resistance is a burden in controlling infections caused by DEC (22). In India, qnrS, qnrB, and qnrA were found at 46.6%, 40%, and 0% in the DEC strains, respectively (18). In Tehran (Iran), qnr was found at 72% in the DEC strains (23). These studies indicate that diffusion of qnr resistance genes among these DEC isolates is variable.
Class 1 and 2 integrons were observed in a total of 19/32 (59.3%) and 13/32 (40.6%) DEC strains: were observed. No Class 3 integrons were found (Table 4). Of the three categories of integrons pertinent to antimicrobial resistance, the class I integron is the one most frequently obtained from Gram-negative bacteria (24). The prevalence of integrons in the enterobacteriacae family has been shown to vary and has played a significant role in the spread of drug-resistance (25). In India Int1 had a prevalence of 41.6%, and no cases of Int2 or Int3 were found from DEC isolates (18). The class 1 integron is overcoming in outspreading the MDR genes among these DEC isolates despite the advent of class 2 and 3 integrons. In this study, 16/25 (64%) and 36% of MDR isolates of DEC contained Int1 and Int2 genes, respectively. In Iran (Southwest) MDR in intestinal pathogenic bacteria such as DEC is known to be related with presence (21).
Table 5. Comparison of the frequency, clinical symptoms, and antibiotic resistance of DEC and E. coli pathotypes in this study and in other studies