Characterization of Antibiotic Resistance and Virulence Genes in Shigella Species Isolated from Patient Samples from Southern Palestine

Abstract

Shigella is a Gram-negative rod-shaped bacteria from the Enterobacteriaceae family, classified into four species; S. dysenteriae, S. flexneri, S. sonnei, and S. boydii. Shigella species cause shigellosis, which is transmitted through fecal-oral route causing destruction of colonic epithelium resulting in bloody mucous diarrhea. Infected individuals usually recover without medical intervention. However, severe Shigella infections require antibiotic treatment to reduce the severity and duration of illness and to reduce the spread of the disease. For children, ceftriaxone is recommended as first line parenteral therapy, and azithromycin is recommended as oral treatment. While in adults, flouroquinolones are the first choice, in addition to azithromycin and trimethoprim-sulfamethoxazole. In this study, 502 isolates of Shigella species collected from patients seen at Caritas Baby Hospital (CBH) between 2004-2014 were included. The Clinical and Laboratory Standard Institute (CLSI) guidelines were used to determine the antibiograms of the isolates. The presence of macrolide resistance genes {erm(A), erm(B), erm(C), ere(A), ere(B), mph(A), mph(B), mph(D), mef(A), msr(A)}, and ESBL producing genes were determined in the resistant isolates by Polymerase Chain Reaction (PCR). Additionally, the six virulence genes (ipaH, ial, sen, set1A, set1B, stx) were screened in all isolates by PCR. Moreover, subtyping of the bacterial isolates using pulsed-field gel electrophoresis (PFGE) was performed according to the Centers for Disease Control and Prevention (CDC) standard procedure for 15 isolates of S. flexneri which were resistant to azithromycin to investigate their genetic diversity. Of the 502 isolates, 40.4% were resistant to ampicillin, 92.2% resistant to trimethoprim-sulfamethoxazole, 78.3% resistant to tetracycline, 3.2% resistant for cephalosporins, 14.7% resistant to naldixic acid, however, 100% were sensitive to ciprofloxacin. For azithromycin, 62 (12.4%) of the isolates showed reduced susceptibility (Breakpoint≤16 mm). Screening for macrolide resistance genes in these isolates showed that only isolates with breakpoint ≤12 mm (49 isolates) were positive for the mph(A) gene, and negative for the other genes. Ten isolates showed reduced susceptibility to cefotaxime, thus indicating the isolates could be ESBL producers. All of them were positive for the blaTEM gene; 5 positive for the blaCTX gene, 6 were positive for the blaCTX-M2-5 and all were negative for all other ESBL genes evaluated. Virulence genes results showed the presence of invasion plasmid antigen H (ipaH) in 479 (95.8 %) isolates, the invasion-associated locus (ial) in 374 isolates (75.4%), set1B which is responsible for enterotoxins in 39 isolates (7.8%), and exotoxin Shiga toxin (Stx) in 8 (1.6%) of the samples. While the other enterotoxin genes (set1A and sen) were not found in any of the isolates. The high Shigella species resistant patterns to oral antibiotics and the emergence of ESBL producing Shigella species, mandates the Palestinian Ministry of Health to control the misuse of antibiotics. In addition, the results of this study can be used to better interpret azithromycin disk diffusion results as no clear guidelines by the CLSI are currently available.