Characteristics of bacterial pathogens associated with acute diarrhea in children under 5 years of age: a hospital-based cross-sectional study

Acute diarrhea was defined as three or more liquid, loose, mucus, or bloody stools within 24 h, lasting no longer than 14 days. Persistent diarrhea was defined as diarrhea that lasted for more than 14 days at presentation [14, 15]. Patients with persistent diarrhea were excluded from this study. Fever was defined as a temperature of ≥37.5 °C. If the parents or legal guardians accepted participation in the study, children under 5 years of age with acute diarrhea who were outpatients at our hospital were selected. Demographic information for each patient, including age, sex, address and clinical symptoms, was collected.

From May 2014 to Aug 2015, samples were collected consecutively from patients whose parents or legal guardians agreed to take part in the study. All of the parents or guardians of the patients signed the informed consent. A stool sample was collected, prior to treatment with any prescribed antibiotics. A sterilized cotton swab was dipped in the mucus, purulent or bloody part of the stool sample, then immediately placed in Cary-Blair Medium (Oxoid, United Kingdom). The samples were sent to the laboratory for immediate testing. In the laboratory, samples were cultured in different media to detect nine bacteria, these included Salmonella spp., Shigella spp., Vibrio cholerae, diarrheagenic Escherichia coli (DEC), Aeromonas spp., Plesiomonas spp., Vibrio parahaemolyticus, Campylobacter spp. and Yersinia enterocolitica. Xylose lysine deoxycholate medium (XLD), MacConkey agar (Mac) and thiosulfate–citrate–bile salts–sucrose (TCBS) agar were used to isolate the pathogens conventionally (with the exception of Campylobacter). To further characterize Salmonella spp., sulfa enrichment broth (SBG) (Qingdao HopeBio-Technology, China) was used to enhance growth of the bacterium. Cefoperazone deoxycholate agar (CCDA) and Skirrow’s medium were used to culture Campylobacter spp. in a microaerophilic environment at 42 °C. Phosphate buffered saline (PBS) was used for the incubation of Yersinia enterocolitica to increase the bacterium concentration over 10 days at 4 °C.

Strains that could not be easily identified were further investigated by manual biochemical reaction methods and/or instruments (VITEK-2 COMPACT, Biomerieux, France). Suspected Campylobacter colonies were subjected to PCR analysis to confirm the identification, using a method previously reported by Denis et al [16, 17].

Serotype identification was carried out by slide agglutination tests for Salmonella spp., Shigella spp. and Vibrio cholerae (using Salmonella, Shigella spp. and V. cholerae O1, 0139 antiserum Diagnostic Antisera Kit, from Lanzhou Institute of Biological Products Co., Ltd. Lanzhou, China). Five distinct classes of diarrheagenic Escherichia coli (DEC) are recognized as being associated with diarrheal disease: enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC). PCR was used to distinguish these E. coli pathotypes by amplification of the following gene targets: typical EPEC (eae and bfp), atypical EPEC (eae or bfp), STEC (eae and stx1 and/or stx2), ETEC (elt and/or estIa or estIb), EIEC (invE and ipaH) and EAEC (aggR and/or pic or astA) [18, 19].

To test antibiotic resistance in Campylobacter spp., the broth microdilution method was used with 5 % sheep blood. For all other pathogens, antimicrobial susceptibilities were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) Guidelines, 2015 [20]. All isolates of Salmonella spp. were tested for their minimum inhibitory concentrations (MICs) of ampicillin, ampicillin-sulbactam, ceftriaxone, cefotaxime, nalidixic acid, ciprofloxacin, levofloxacin, co-trimoxazole, azithromycin, chloramphenicol and tetracycline (Oxoid); DEC were tested for ampicillin, ampicillin-sulbactam, cefotaxime, ciprofloxacin, levofloxacin, chloramphenicol, tetracycline, cefazolin, cefuroxime, imipenem, amikacin and gentamicin (Oxoid); Campylobacter spp. were tested for ciprofloxacin, azithromycin, tetracycline, erythromycin and doxycycline (Oxoid); and Aeromonas spp. were tested for cefotaxime, ciprofloxacin, levofloxacin, co-trimoxazole, chloramphenicol, tetracycline, cefazolin, cefuroxime, imipenem, amikacin and gentamicin (Oxoid). ATCC 25922, 35218, 700603 and 27853 were used as quality control strains. Antibiotic susceptibility was interpreted according to CLSI guidelines, 2015 [20].

Those strains showing significantly decreased susceptibility to ceftriaxone and cefotaxime (MIC ≥32 mg/Ml) were further studied by PCR amplification and sequencing of the extended-spectrum β-lactamase genes (ESBLs, including blaSHV, blaTEM and blaCTX-M). Those isolates showing high resistance to quinolones (MIC ≥32 mg/mL to ciprofloxacin or levofloxacin) were further tested for amino acid changes in the plasmid-mediated quinolone resistance genes gyrA and gyrB by PCR and sequencing, according to the methods reported by Yenkao et al [21]. The DNA sequences were compared with sequences in the GenBank database (http://​www.​ncbi.​nlm.​nih.​gov/​genbank/​) and the β-lactamase classification system (http://​www.​lahey.​org/​studies/​) to confirm the subtypes of the β-lactamase genes.

The chi-squared (x²) test was used to determine the statistical significance of the data by the software PASW Statistics 18.0 (IBM Corporation, New York). A P-value of <0.05 was considered statistically significant.

During the data collection period (2014.5.1–2015.8.31), a total of 508 children aged 0–59 months who visited our hospital as outpatients were recruited to this study. Of the children, 295 (58.1 %) were male and 213 (41.9 %) were female. All of the parents or legal guardians of the patients agreed to participate in the study. A fecal specimen was collected from each patient. Blood and mucus were uncommon in the feces (1.0 % and 3.7 %, respectively). Of the 508 patients, 64.76 % (329/508) were infants (0–11 months), 22.24 % (113/508) were toddlers (12–23 months), and the other 12.99 % (66/508) were children (24–59 months). Fever and vomiting were the main symptoms accompanying acute diarrhea. Up to 57.9 % (294/508) of patients stayed in hospital for 1–3 days. There was a significant difference in the duration of diarrhea across the three age groups in the all studied pathogens (chi-squared test, p < 0.05), whereas no significant difference was observed in the duration of diarrhea and symptoms across the three age groups with Salmonella infection (chi-squared test, p > 0.05).

In total, 102 strains of bacterial pathogens were isolated from 508 patients. Co-infection of bacteria was not detected in any of the cases. Salmonella spp. accounted for 42.2 % of the detected bacterial pathogens, followed by DEC 23.5 %, Campylobacter spp. 14.7 %, Aeromonas spp. 9.8 % and Shigella spp. 5.9 %. No Vibrio cholerae and Plesiomonas spp. were isolated from the 508 samples (Fig. 2). All of the Campylobacter spp. was confirmed to be C. jejuni by PCR. The 43 strains of Salmonella spp. detected were distributed among 10 serotypes, with S. Typhimurium (O4Hi) being the predominant serotype identified (18/43, 41.9 %). Among 24 strains of DEC, 11 strains of EPEC, 10 strains of STEC and three strains of EAEC were confirmed by PCR typing. Of the six strains of Shigella spp., four were S. flexneri and two were S. sonnei as determined by serum agglutination assays (Table 1).

Of the 43 strains of Salmonella spp. detected, more than 60 % were resistant to ampicillin and tetracycline. The resistance rates to co-trimoxazole and azithromycin were >40 % and to chloramphenicol was >30 %, for all other antibiotics tested the rates were <30 %. For DEC, the highest resistance rate of >60 % was detected for ampicillin and tetracycline, followed by 50 % for cefotaxime, then >30 % for ampicillin/sulbactam, ciprofloxacin, chloramphenicol, cefuroxime and gentamycin, and <30 % for levofloxacin, amikacin and imipenem. Among the 15 strains of C. jejuni detected, nine strains showed resistance to ciprofloxacin and one strain was resistant to azithromycin; the strains were sensitive to all other antibiotics tested. Among the 10 strains of Aeromonas spp., five strains were resistant to cefazolin, four to tetracycline, and three to cefotaxime (Fig. 3). Drug sensitivity tests were not performed for Shigella spp., Vibrio parahaemolyticus and Yersinia enterocolitica because the number of strains was too small (<10) to provide interpretable results.

Three isolates of Salmonella spp. were found to be highly resistant to ciprofloxacin or levofloxacin (MIC ≥32 μg/mL), however, only one isolate possessed the gyrA gene. Twelve isolates of Salmonella spp. were found to be highly resistant to cefotaxime or ceftriaxone (MIC ≥32 μg/mL), three of which possessed the TEM-1 and/or CTX-M-14 genes (Table 2).