A Systematic Approach to Identify and Characterize the Effectiveness and Safety of Novel Probiotic Strains to Control Foodborne Pathogens

A total of 44 lactic acid bacteria (LAB) strains originally isolated from cattle feces and different food sources were screened for their potential probiotic features. The antimicrobial activity of all isolates was tested by well-diffusion assay and competitive exclusion on broth against Salmonella Montevideo, Escherichia coli O157:H7 and Listeria monocytogenes strain N1-002. Thirty-eight LAB strains showed antagonistic effect against at least one of the pathogens tested in this study. Improved inhibitory effect was observed against L. monocytogenes with zones of inhibition up to 24 mm when LAB overnight cultures were used, and up to 21 mm when cell-free filtrates were used. For E. coli O157:H7 and Salmonella maximum inhibitions of 12 and 11.5 mm were observed, respectively. On broth, 43 strains reduced L. monocytogenes up to 9.06 log10 CFU/ml, 41 reduced E. coli O157:H7 up to 0.84 log10 CFU/ml, and 32 reduced Salmonella up to 0.94 log10 CFU/ml 24 h after co-inoculation. Twenty-eight LAB isolates that exhibited the highest inhibitory effect among pathogens were further analyzed to determine their antimicrobial resistance profile, adhesion potential, and cytotoxicity to Caco-2 cells. All LAB strains tested were susceptible to ampicillin, linezolid, and penicillin. Twenty-six were able to adhere to Caco-2 cells, five were classified as highly adhesive with > 40 bacterial cells/Caco-2 cells. Low cytotoxicity percentages were observed for the candidate LAB strains with values ranging from -5 to 8%. Genotypic identification by whole genome sequencing confirmed all as members of the LAB group; Enterococcus was the genus most frequently isolated with 21 isolates, followed by Pediococcus with 4, and Lactobacillus with 3. In this study, a systematic approach was used for the improved identification of novel LAB strains able to exert antagonistic effect against important foodborne pathogens. Our findings suggest that the selected panel of LAB probiotic strains can be used as biocontrol cultures to inhibit and/or reduce the growth of L. monocytogenes, Salmonella, and E. coli O157:H7 in different matrices, and environments

Outbreaks of virulent diarrheagenic Escherichia coli - are we in control?

Shiga toxin-producing Escherichia coli (STEC) are the most virulent diarrheagenic E. coli known to date. They can be spread with alarming ease via food as exemplified by a large sprout-borne outbreak of STEC O104:H4 in 2011 that was centered in northern Germany and affected several countries. Effective control of such outbreaks is an important public health task and necessitates early outbreak detection, fast identification of the outbreak vehicle and immediate removal of the suspected food from the market, flanked by consumer advice and measures to prevent secondary spread

Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR®Green Real-Time PCR Methods and High Resolution Melting Analysis

Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the “top-five” serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform

Mainstreams of Horizontal Gene Exchange in Enterobacteria: Consideration of the Outbreak of Enterohemorrhagic E. coli O104:H4 in Germany in 2011

Escherichia coli O104:H4 caused a severe outbreak in Europe in 2011. The strain TY-2482 sequenced from this outbreak allowed the discovery of its closest relatives but failed to resolve ways in which it originated and evolved. On account of the previous statement, may we expect similar upcoming outbreaks to occur recurrently or spontaneously in the future? The inability to answer these questions shows limitations of the current comparative and evolutionary genomics methods.status: publishe

Low density macro-array targeting non-LEE effectors (nle genes) and main virulence factors of Shiga toxin-encoding Escherichia coli (STEC) as a new approach for the molecular risk assessment of STEC isolates

Rapid and specific detection of Shiga toxin-producing Escherichia coli (STEC) strains with high virulence for human has become a priority for public health authorities. This study reports on the development of a low density macro-array for simultaneously testing the genes stx1, stx2, eae, ehxA, and six different nle genes issued from genomic islands OI#122 (ent, nleB, nleE) and OI#71 (nleF, nleH1-2, nleA). Various strains of E. coli isolated from the environment, food, animals and healthy children have been compared with clinical isolates of various seropathotypes. The eae gene was detected in all enteropathogenic E. coli (EPEC) strains as well as in enterohaemorrhagic E. coli (EHEC) strains, excepted in EHEC O91:H21 and EHEC O113:H21. The gene ehxA was more prevalent (90%) in EHEC than in STEC (42.66%) strains in which it was unequally distributed. The nle genes were detected only in some EPEC and EHEC strains but with various distributions, showing that nle are either strain and/or serotype specific, probably reflecting adaptation of the strains to different host or environmental niche. One characteristic nle gene distribution in EHEC O157:[H7], O111:[H8], O26:[H11], O103:H25, O118:[H16], O121:[H19], O112:H19, O5:HNM, O55:H7, O123:H11, O172:H25 and O165:H25 was [ent/espL2, nleB, nleE, nleF, nleH1-2, nleA]. A second nle pattern [ent/espL2, nleB, nleE, nleH1-2] was characteristic of EHEC O103:H2, O145:[H28], O45:H2 and O15:H2. The presence of eae, ent/espL2, nleB, nleE, and nleH1-2 genes is a clear signature of STEC strains with high virulence for humans

Two Draft Genome Sequences of a New Serovar of \u3ci\u3eSalmonella enterica\u3c/i\u3e, Serovar Lubbock

Salmonella enterica is principally a foodborne pathogen that shows considerable serovar diversity. In this report, we present two draft genome sequences of Salmonella enterica subsp. enterica serovar Lubbock, a novel serovar

Isolation and phenotypic characterization of antibiotic resistant E.coli and Salmonella from food animals v1

This protocol was designed to help researchers with limited laboratory resources to generate valuable information on the status of antibiotic resistance among indicator E.coli and pathogenic Salmonella from food producing animals. The protocol uses less technology intensive methods such the disk diffusion and the combination disk test to estimate isolate- and sample-level prevalence of E.coli and Salmonella resistance to most critical important antibiotics. In this protocol, agar-based media without antibiotic-supplements are used to isolate E. coli and Salmonella from fecal samples. Besides, agar-based media supplemented with antibiotics are used to screen for E. coli and Salmonella resistant to third-generation cephalosporins and to screen for E. coli and Salmonella with low susceptibility to quinolones. Furthermore, all bacterial isolates are tested for their susceptibility to a panel of twelve antibiotic disks using the disk diffusion. Finally, the combination disk test is used to test for phenotypic production of extended spectrum beta-lactamases (ESBLs) or AmpC among all bacterial isolates resistant to third-generation cephalosporins using the a second panel of 12 antibiotic disks. </p

Isolation and phenotypic characterization of antibiotic resistant E.coli and Salmonella from food animal feces v1

This protocol was designed to help researchers with limited laboratory resources to generate valuable information on the status of antibiotic resistance among indicator E.coli and pathogenic Salmonella from food producing animals. The protocol uses less technology intensive methods such the disk diffusion and the combination disk test to estimate isolate- and sample-level prevalence of E.coli and Salmonella resistance to most critical important antibiotics. In this protocol, agar-based media without antibiotic-supplements are used to isolate E. coli and Salmonella from fecal samples. Besides, agar-based media supplemented with antibiotics are used to screen for E. coli and Salmonella resistant to third-generation cephalosporins and to screen for E. coli and Salmonella with low susceptibility to quinolones. Furthermore, all bacterial isolates are tested for their susceptibility to a panel of twelve antibiotic disks using the disk diffusion. Finally, the combination disk test is used to test for phenotypic production of extended spectrum beta-lactamases (ESBLs) or AmpC among all bacterial isolates resistant to third-generation cephalosporins using a second panel of 12 antibiotic disks. </p