Yersinia enterocolitica

For evaluation of assay performance, genomic materials or reference strains were obtained from American Tissue and Culture Collection (ATCC, Manassas, VA) or BEI resources for adenovirus 1, 5, 40 and 41, human cytomegalovirus, enterovirus 71, Epstein-Barr virus, Aeromonas hydrophila, Bacteroides fragilis, Campylobacter coli, Campylobacter upsalensis, Campylobacter hyointestinalis, Campylobacter jejuni, Helicobacter pylori, Listeria monocytogenes, Mycobacterium tuberculosis, Plesiomonas shigelloides, Salmonella enterica, Vibrio parahaemolyticus, Yersinia enterocolitica, Blastocystis hominis, Cryptosporidium hominis, Cryptosporidium meleagridis, Schistosoma mansoni. Cryptosporidium parvum and Encephalitozoon intestinalis were purchased from Waterborne Inc. (New Orleans, LA). PCR amplicons were generated from the relevant positive clinical samples for Ancyclostoma duodenale, Necator americanus, Strongyloides stercoralis, Cyclospora cayetanensis, Cystoisospora belli, and Enterocytozoon bieneusi. For comparison between stool and swab (FLOQSwabs; Copan Italia, Brescia, Italy), 129 consecutive swab samples were collected from children under five admitted for acute diarrhea in Haydom Lutheran Hospital, Tanzania. A matched stool sample from the same patient was obtained as soon as feasible within the same day. Raw stool samples were transported with a cold chain to the lab within 6 hours and stored at -80°C until testing. Swabs were stored at room temperature until testing. For comparison between different extraction methods and validation of the newly developed qPCR assays on clinical samples, we chose 246 archived stool samples collected in Tanzania, Bangladesh, Nepal, Pakistan, and India through the MAL-ED project (the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development [6 (link)]) in order to obtain specimens positive for 30 diverse enteropathogens. All sites including Haydom Global Health Institute, Tanzania, Aga Khan University, Pakistan, Armed Forces Research Institute of Medical Sciences, Thailand, International Centre for Diarrhoeal Disease Research, Bangladesh, Christian Medical College, India, received ethical approval from their respective governmental, local institutional, and collaborating institutional ethics review boards. Written informed consent was obtained from the parent or guardian of every child.

Trained field enumerators completed consent procedures and surveys in the participant’s preferred language (Portuguese or Changana) and collected biological sampless from enrolled children (Appendix 1- Consent procedures, survey administration, and sample collection and analysis). At baseline we aimed to visit intervention compounds 2 weeks prior to the opening of the new latrines. We scheduled follow-up visits to be 12 months (±2 weeks) and 24 months (±2 weeks) from the date compound members began using their new latrines, with visits to control compounds made concurrently (±2 weeks).
We collected stool samples independently of reported symptomology. If we were unable to collect a stool sample after multiple attempts, a registered nurse collected a rectal swab after obtaining written consent for the procedure from a parent or guardian. Stool samples were kept cold and delivered to the Laboratory of Molecular Parasitology at the Instituto Nacional de Saúde (INS) within 6 hr of collection for analysis and storage at −80°C.
Samples were shipped frozen with temperatures monitors to the Georgia Institute of Technology (Atlanta, USA) where we used the xTAG GPP (Luminex Corp, Austin, USA), a qualitative multiplex molecular assay, to detect 15 enteric pathogens in stool samples: Campylobacter jejuni/coli/lari; Clostridium difficile, toxin A/B; enterotoxigenic Escherichia coli (ETEC) LT/ST; Shiga-like toxin producing E. coli (STEC) stx1/stx2; E. coli O157; Salmonella; Shigella boydii/sonnei/flexneri/dysenteriae; Vibrio cholerae; Yersinia enterocolitica; Giardia lamblia; Cryptosporidium parvum/hominis; Entamoeba histolytica; adenovirus 40/41; norovirus GI/GII; and rotavirus. The GPP has been rigorously tested and extensively used for stool-based enteric pathogen detection (Chisenga et al., 2018 (link); Claas, 2013 (link); Deng et al., 2015 (link); Duong et al., 2016 (link); Huang et al., 2016 (link); Kellner et al., 2019 (link); Khare et al., 2014 (link); Navidad et al., 2013 (link); Patel et al., 2014 (link)). We analyzed samples according to manufacturer instructions with the addition of elution steps for the pretreatment of rectal swabs and diaper material saturated with liquid stool (Appendix 1- Consent procedures, survey administration, and specimen collection and analysis). Technicians at INS assessed stool samples for the presence of soil-transmitted helminths (STH) using the single-slide Kato-Katz microscope method (Vestergaard Frandsen, Lausanne, Switzerland).
Representatives of the National Deworming Campaign (NDC) at the Mozambican Ministério da Saúde (MISAU) offered single-dose albendazole (400 mg, 200 mg for children aged 6–12 months) to all eligible members of intervention and control compounds following sample collection activities of each phase. Eligibility was defined by the NDC and included compound members older than 6 months who were not pregnant.

Western blotting was performed, as described previously (Pan et al., 2016 (link)). Horseradish peroxidase (HRP)-conjugated 6×His-tag antibody (Abmart, Shanghai, China) (1:3,000) was used to detect 6×His-tag-fused proteins. Monoclonal Yersinia enterocolitica O:9 antibody (Fitzgerald, Acton, MA) (1:400) and Brucella antibody (1:400) to detect glycoproteins. The antibody against Brucella was produced by immunizing rabbits with whole Brucella suis S2 and blocking with E. coli W3110 cell lysates. HRP-conjugated anti-rabbit IgG (TransGen Biotech, Beijing, China) (1:15,000) was used as a secondary antibody.

The commercially available BioFire^® Film Array^® GI Panel system was used per the manufacturer’s instructions (BioFire Diagnostics, Salt Lake City, UT, USA) to analyse all stool specimens. Briefly: only one specimen at a time was processed within a closed pouch where 200 μL of the stool specimen is added together with the supplied hydration solution. The system performs a nucleic acid extraction, multiplex PCR and melting analysis in 60 min and provides a printout of the results. The test simultaneously detects and identifies nucleic acids from the following 22 diarrhoea pathogens:

Bacteria: Campylobacter [C. jejuni; C. coli; C. upsaliansis], Clostridium difficile [toxin A/B], Plesiomonas shigelloides, Salmonella, V. cholerae; Vibrio spp [V. cholerae; V. parahaemolyticus; V. vulnificus], Yersinia enterocolitica, Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC [lt/st]), Shiga-like-toxin-producing E. coli [STEC stx1/stx2], E. coli O157, Shigella-EIEC.

Viruses: Adenovirus F40/41, Sapovirus [I; II; IV; V], Astrovirus [HAstV 1-8], Norovirus [GI; GII], Rotavirus A.

Parasites: Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia [G. intestinalis; G. duodenalis].

S. marcescens CSM-RMT-1 (non-pigmented) (GenBank accession No. OQ254763) and S. marcescens CSM-RMT-II-1 (pigmented) (GenBank accession No. OQ254768) strains were derived from a rugged and not easily accessible surface of a diary-producing plant [24 (link)]. Pure cultures of the potential inhibitory strains were maintained on tryptone soya agar (TSA; Biokar, France) slants at 4 °C and in tryptone soya broth (TSB; Biokar, France) supplemented with 20% glycerol at −80 °C, respectively.
Inhibitory effect of the two S. marcescens strains were screened against four foodborne pathogenic bacteria: Listeria monocytogenes CCM 4699 (Czech Collection of Microorganisms, Brno, Czech Republic), Salmonella enterica subsp. enterica ser. Hartford NCAIM B.01310 (National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary), Yersinia enterocolitica HNCMB 98002 (Hungarian National Collection of Medical Bacteria, Budapest, Hungary) and Escherichia coli NCAIM B.01909 (National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary). Each of them was cultured on TSA at 37 °C for 24 h, except Y. enterocolitica, which was incubated at 25 °C.
In the co-culturing study the growth of a food-derived Salmonella enterica subsp. enterica strain was tested. This bacterium was previously isolated from egg and identified by Sanger sequencing of the 16S rRNA encoding gene and MALDI-TOF MS analyses (data are not shown). The GenBank accession number of the strain is OQ254770. The pathogen was cultured on TSA at 37 °C for 24 h, while its maintenance was performed on TSA slants at 4 °C and in TSB supplemented with 20% glycerol at −80 °C.