Molecular typing methodologies for microbial source tracking and epidemiological investigations of Gram-negative bacterial foodborne pathogens ☆

Context. — Shigella dysenteriae type 2 is rare in the United States, and outbreaks associated with this pathogen are uncommon. Objective. —To determine the magnitude and source of an outbreak of S dysenteriae type 2. Design. —Retrospective cohort. Setting. —Laboratory of a large medical center. Patients. —Case patients were identified as laboratory workers who had diarrhea on or after October 28 and a positive stool culture or temperature greater than 37.8°C. Laboratory workers with diarrhea only were probable case patients. Main Outcome Measures. —We interviewed laboratory staff and performed identification, serotyping, and pulsed-field gel electrophoresis on isolates from case patients, implicated food, and laboratory stock culture. Results. —From October 29 through November 1, a total of 12 (27%) of 45 laboratory staff developed severe, acute diarrheal illness; 8 had S dysenteriae isolated from stool and 4 were hospitalized. All case patients reported having eaten muffins or doughnuts placed in the staff break room on October 29. Pulsed-field gel electrophoresis showed stool isolates from 9 case patients were indistinguishable from S dysenteriae type 2 recovered from an uneaten muffin and from the laboratory's stock strain, a portion of which was missing. Conclusions. —The source of the outbreak was most likely the laboratory's stock culture, which was used to contaminate the pastries. Results of this investigation underscore the need for adequate precautions to prevent inadvertent or intentional contamination from highly pathogenic laboratory specimens.

https://jamanetwork.com/journals/jama/articlepdf/417894/jama_278_5_034.pdf

An Outbreak of Shigella dysenteriae Type 2 Among Laboratory Workers Due to Intentional Food Contamination

Eighty-six strains of Shigella spp. were isolated during the dry season from stool samples of children under 5 years of age in Ifakara, Tanzania. The epidemiological relationship as well as the antimicrobial susceptibility and mechanisms of resistance to ampicillin, chloramphenicol, and co-trimoxazole were investigated. Four different epidemiological tools, pulsed-field gel electrophoresis (PFGE), repetitive extragenic palindromic (REP)-PCR, plasmid analysis, and antibiogram, were compared for typing Shigella strains. Seventy-eight (90%) strains were Shigella flexneri and were distributed into four groups, by either PFGE or REP-PCR, with 51, 17, 7, and 3 strains. The four strains of Shigella dysenteriae belonged to the same group, and the four strains of Shigella sonnei were distributed in two groups with three and one strain each. Plasmid analysis showed a high level of heterogeneity among strains belonging to the same PFGE group, while the antibiogram was less discriminative. REP-PCR provided an alternative, rapid, powerful genotyping method for Shigella spp. Overall, antimicrobial susceptibility testing showed a high level of resistance to ampicillin (81.8%), chloramphenicol (72.7%), tetracycline (96.9%), and co-trimoxazole (87.9%). Ampicillin resistance was related to an integron-borne OXA-1-type β-lactamase in 85.1% of the cases and to a TEM-1-type β-lactamase in the remaining 14.8%. Resistance to co-trimoxazole was due to the presence of a dhfr Ia gene in all groups except one of S. flexneri, where a dhfr VII gene was found within an integron. Chloramphenicol resistance was associated in every case with positive chloramphenicol acetyltransferase activity. All strains were susceptible to nalidixic acid, ciprofloxacin, ceftazidime, cefotaxime, and cefoxitin. Therefore, these antimicrobial agents may be good alternatives for the treatment of diarrhea caused by Shigella in Tanzania.

Typing and Characterization of Mechanisms of Resistance of Shigella spp. Isolated from Feces of Children under 5 Years of Age from Ifakara, Tanzania

Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate Enterococcus faecalis isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in Streptococcus pneumoniae, was applied to 41 isolates of E. faecalis collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied to 18 selected isolates. Of the 41 isolates examined, 7 were β-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen.

Repetitive Sequence-Based PCR versus Pulsed-Field Gel Electrophoresis for Typing of Enterococcus faecalis at the Subspecies Level

To characterize Shigella clinical strains, we studied 82 Shigella strains recovered from 719 stool samples of patients with bloody diarrhea in Shiraz, Iran, over the period from April to October 2003. Serological assay classified the Shigella isolates as follows: 61 (74.39%) Shigella sonnei isolates, 16 (19.51%) Shigella flexneri isolates, 3 (3.65%) Shigella boydii isolates, and 2 (2.43%) Shigella dysenteriae isolates. In an antibiogram test, all Shigella strains were susceptible to ceftazidime, ciprofloxacin, and ceftriaxone. They showed high degrees of sensitivity to nalidixic acid, gentamicin, cephalothin, and amikacin. Approximately 90.24% of the Shigella isolates were resistant to co-trimoxazole. The plasmid profile patterns of all strains were determined by a modified alkaline lysis method. The average number of plasmid bands for each strain was 9.5. By plasmid profile analysis we identified 56 genotypes among all isolates and 42, 14, 3, and 2 genotypes among the S. sonnei, S. flexneri, S. boydii, and S. dysenteriae strains, respectively. PCR assays showed that all isolates were positive for two virulence genes, ipaBCD and ipaH. In conclusion, these data mandate local monitoring of drug resistance and its consideration in the empirical therapy of Shigella infections. These results also demonstrate that plasmid profile analysis is more reliable than antibiotic susceptibility pattern analysis for the identification of Shigella epidemic strains isolated in Iran.

Characterization of Shigella Strains in Iran by Plasmid Profile Analysis and PCR Amplification of ipa Genes

Shigella sonnei is a major cause of diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared different molecular typing methods, i.e., plasmid profile analysis, restriction endonuclease analysis of plasmids, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis (PFGE), and enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR) for typing 20 clinical isolates of S. sonnei collected from six incidents of infection. PFGE and ERIC-PCR fingerprintings had the highest discriminatory power for discrimination of epidemiologically related isolates from epidemiologically unrelated strains of S. sonnei, and both gave seven distinct strain types among these isolates and the type strain of the species. Plasmid study and ribotyping produced only six and typing techniques demonstrated two distinct patterns, respectively, among these strains. All of these molecular an identical fingerprint for eight temporally related sporadic isolates. It is possible that these temporally related isolates belonged to a single bacterial clone and circulated obscurely through the community. Our results indicate that the ERIC-PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis of plasmids, and ribotyping.

Y H Lin

Papers

Analysis of clonal relationships among isolates of Shigella sonnei by different molecular typing methods.