Genetic profiles and invasion ability of Listeria monocytogenes isolated from bovine carcasses in Southern Brazil

The goals of this study were to evaluate the persistence and the virulence potential of Listeria monocytogenes isolated from beef carcasses obtained in processing facilities in the Southern region of Rio Grande do Sul, Brazil, based on pulsed field gel electrophoresis (PFGE), invasion ability in human colorectal carcinoma cells (HCT-116), InlA expression by western blot (WB) and identification of mutation points in the inlA . PFGE profiles demonstrated that L. monocytogenes isolates were grouped based on their previously identified lineages and serogroups (lineage I: serogroups IIb, n = 2, and IVb, n = 5; lineage II, serogroup IIc, n = 5), isolates with indistinguishable genetic profiles by this method were obtained from different slaughterhouses and sampling steps, with up to 3-year interval. Seven isolates showed high invasion ability (2.4 to 7.4%, lineage I, n = 6, lineage II, n = 1) in HCT and expressed InlA. Five isolates showed low cell invasion ability (0.6 to 1.4%, lineage I, n = 1, lineage II, n = 4) and did not express InlA, and two of them (lineage II, serogroup IIc) presented mutations in inlA leading to a premature stop codon (PMSC) type 19, at position 326 (GAA → TAA). The results demonstrated that most of L. monocytogenes isolates from Lineage I expressed InlA and were the most invasive in HCT indicating their high virulence potential, while most isolates from Lineage II showed attenuated invasion due to non-expression of InlA and the presence of PMSC type 19 in inlA . The obtained results demonstrated that L. monocytogenes with indistinguishable PFGE profiles can be persisting or being reintroduced in beef processing facilities in the studied region and differences on their virulence potential based on their lineages and serogroups.

Psychrotrophic properties, toxigenic characteristics, and PFGE profiles of Bacillus cereus isolated from different foods and spices

ABSTRACT: Bacillus cereus is an aerobic and facultatively anaerobic, spore-forming bacterium, and it is found naturally in soil and poses a risk factor for the contamination of food and foodstuffs including cereals, vegetables, spices, ready-to-eat (RTE) foods, meats, milk, and dairy products. This study determined the prevalence of B. cereus in raw poultry meat, raw cow’s milk, cheese, spices, and RTE foods in Hatay province. The study also analysed the psychrotrophic properties, toxigenic characteristics, and pulsed-field gel electrophoresis (PFGE) profiles of the isolates. The levels of contamination with B. cereus determined for cheese, raw milk, RTE foods, spices, and raw poultry meat were 16.6%, 34.2%, 42.8%, 49%, and 55.5%, respectively. B. cereus was isolated from 84 (42%) of the 200 samples analysed and the 84 isolates were verified by PCR analysis targeting the haemolysin gene specific for B. cereus. Of the total isolates, 64 (76.1%) were psychrotrophic. The toxin gene profiling of B. cereus isolates was determined by amplifying the four genes nhe, hbl, cytK, and ces. The nhe and cytK genes were most frequently detected in the isolates, while the hbl and ces genes were not found. In addition, a high genetic relationship between the isolates was detected at a 92% similarity level by PFGE analysis. In conclusion, the occurrence of both psychrotrophic and toxigenic B. cereus strains in this study indicated a potential risk for food spoilage and food poisoning.

Phylogenetic Analysis and Genome-Wide Association Study Applied to an Italian Listeria monocytogenes Outbreak

From May 2015 to March 2016, a severe outbreak due to Listeria monocytogenes ST7 strain occurred in Central Italy and caused 24 confirmed clinical cases. The epidemic strain was deeply investigated using whole-genome sequencing (WGS) analysis. In the interested area, the foodborne outbreak investigation identified a meat food-producing plant contaminated by the outbreak strain, carried by pork-ready-to-eat products. In the same region, in March 2018, the epidemic strain reemerged causing one listeriosis case in a 10-month-old child. The aim of this study was to investigate the phylogeny of the epidemic and reemergent strains over time and to compare them with a closer ST7 clone, detected during the outbreak and with different pulsed-field gel electrophoresis (PFGE) profiles, in order to identify genomic features linked to the persistence and the reemergence of the outbreak. An approach combining phylogenetic analysis and genome-wide association study (GWAS) revealed that the epidemic and reemergent clones were genetically closer to the ST7 clone with different PFGE profiles and strictly associated with the pork production chain. The repeated detection of both clones was probably correlated with (i) the presence of truly persistent clones and the repeated introduction of new ones and (ii) the contribution of prophage genes in promoting the persistence of the epidemic clones. Despite that no significant genomic differences were detected between the outbreak and the reemergent strain, the two related clones detected during the outbreak can be differentiated by transcriptional factor and phage genes associated with the phage LP-114.

Phage Therapy Experience at the Eliava Phage Therapy Center: Three Cases of Bacterial Persistence

In this retrospective descriptive study we focus on cases of three patients who underwent phage therapy procedures at Eliava Phage Therapy Center (EPTC) in Tbilisi, Georgia. Patients with chronic infectious diseases related to Pseudomonas aeruginosa (two patients, lower respiratory tract infection (LRTI)) and Klebsiella pneumoniae (one patient, urinary tract infection (UTI)) are among those very few EPTC patients whose pathogens persisted through phage therapy. By looking at bacterial strains and personalized phages used against them we tried to point towards possible adaptation strategies that are employed by these pathogens. Genome restriction-based Pulsed Field Gel Electrophoresis (PFGE) profiling of strains isolated before and after phage therapy hints towards two strategies of adaptation. In one patient case (Pseudomonas aeruginosa related lung infection) bacterial strains before and after phage therapy were indistinguishable according to their PFGE profiles, but differed in their phage susceptibility properties. On the other hand, in two other patient cases (Pseudomonas aeruginosa related LRTI and Klebsiella pneumoniae related UTI) bacterial adaptation strategy seemed to have resulted in diversification of infecting strains of the same species. With this work we want to attract more attention to phage resistance in general as well as to its role in phage therapy.

PulseNet international: the missing link between PFGE and WGS

IntroductionDNA-based surveillance of bacterial diseases has been using pulsed field gel electrophoresis (PFGE) since 1996. Currently, the international surveillance network (PulseNet international) is turning toward whole genome sequencing (WGS). ATCGs of WGS are compared using several sequence alignment methods. While patterns of horizontal lines of PFGE profiles are being compared using relative positioning of bands within a range of tolerance. A recently suggested image analysis algorithm and a deployed database (geltowgs.uofk.edu) collectively invented a promising method for comparing PFGE to in-silico obtained digestion models (DMs) derived from WGS. The database requires a parameter that determines PFGE resolution. Here, the author suggests a new method for calculating this factor. Epidemiological and molecular conclusions returned by the database are evaluated.Methodologytwo PFGE profiles representing XbaI digests of E. coli and Salmonella enterica analyzed by the suggested image analysis algorithm were submitted to the database after calculating resolution of PFGE using Dice percentage of difference between the closest PFGE bands in length. E. coli and Salmonella enterica test subjects were compared to 489 and 401 DMs respectively. The three data sets returned were analyzed.Results and conclusionsaccording to modified PFGE evaluation criteria; a single DM is possibly related to E. coli test subject. It belonged to the same serovar. No epidemiologically related DM was shown for S. enterica test subject. Conclusions mentioned earlier could never be made ignoring co-migration. Standardization of both; suggested image analysis and database algorithms will deepen our understanding of bacterial epidemiology by means of possible qualitative approach built upon identification of fragment sequences and their locations within chromosomes.

Determining the DNA Fingerprinting Profiles of Salmonella spp. Isolated from Raw Poultry Meats and Human Clinical Samples from the Same Geographic Area Using Pulsed Field Gel Electrophoresis.

Foods of animal origin, such as poultry, eggs, and pork, are recognized sources of infection with Salmonella , but determining the proportion of foodborne infections resulting from different food sources has been challenging. In the current study, 141 Salmonella strains isolated from poultry products (n=1322) purchased over a period of one year from retail stores across Seattle, Washington, were subtyped by Pulsed Field Gel Electrophoresis (PFGE) using restriction enzyme Xba I. The objectives of the study were: I) to analyze the longitudinal distribution of Salmonella PFGE profiles throughout the sampling period and their clonality within and between different poultry processing establishments; II) to determine the association between PFGE profiles of Salmonella isolated from locally distributed poultry products to those of clinical isolates submitted to the Washington State Department of Health Laboratories (WA-DOH); and III) to compare the PFGE profiles of Salmonella isolated from the National Antimicrobial Resistance Monitoring System (NARMS) retail meats program. Over the sampling period of one year, multiple indistinguishable PFGE patterns were found across multiple poultry processing establishments. There were 12 out of 30 unique PFGE profiles of Salmonella isolated from locally purchased poultry products that were indistinguishable from PFGE profiles of Salmonella from clinical isolates submitted to the WA-DOH. When the PFGE profiles from the poultry samples were compared to those found in NARMS’ database, there were eight indistinguishable PFGE matches isolated from chicken breasts, ground turkey, and ground beef from multiple states. While this study found some association between PFGE profiles from raw poultry products and those of clinical isolates from the same geographical area, it does not prove that all of those clinical isolates were infections that resulted from the consumption or handling of poultry.

Genetic Diversity and The Presence of Circular Plasmids in Bacillus Cereus Isolates of Clinical and Food Origin

The diversity of 61 Bacillus cereus strains isolated from different clinical specimens, food including raw milk and milk products, and water was evaluated. PFGE analysis could discriminate 61 distinct pulsotypes with similarity levels from 25 to 82%, which were divided into 13 clonal complexes. The similarity between clonal complexes was at least 40%. Clinical strains were divided into 10 clonal complexes, while the strains, isolated from milk, food and water were included in 9, 6 and 6 clonal complexes, respectively. Three clonal complexes were dominated by clinical isolates, while they were absent in two complexes. Bacterial isolates from foods, being a probable source of alimentary toxoinfection, showed low similarity to isolates from stool specimens. The isolates from both sources were classified together in only 4 out of 13 clonal complexes. The large circular and linear plasmids with the sizes between 50 and 200 kb were detected in 24 (39.3%) and 14 (23%) B. cereus strains, respectively. Thirteen (21.3%) strains contained only one plasmid, two plasmids were found in 6 (9.8%) of strains, and three or more plasmids were obtained in 5 (8.2%) of tested strains. The plasmids were confirmed in 30.8% and 40% of isolates from clinical specimens and food and milk samples, respectively. No clear correlation between the PFGE profiles, the source as well as plasmid content among all tested strains was observed.

Resistance Profiling and Molecular Characterization of Extended-Spectrum/Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli Isolated from Healthy Broiler Chickens in South Korea

We aimed to identify and characterize extended-spectrum β-lactamase (ESBL)-and/or plasmid-mediated AmpC β-lactamase (pAmpC)-producing Escherichia coli isolated from healthy broiler chickens slaughtered for human consumption in Korea. A total of 332 E. coli isolates were identified from 339 cloacal swabs in 2019. More than 90% of the isolates were resistant to multiple antimicrobials. ESBL/pAmpC-production was noted in 14% (46/332) of the isolates. Six of the CTX-M-β-lactamase-producing isolates were found to co-harbor at least one plasmid-mediated quinolone resistance gene. We observed the co-existence of blaCMY-2 and mcr-1 genes in the same isolate for the first time in Korea. Phylogenetic analysis demonstrated that the majority of blaCMY-2-carrying isolates belonged to subgroup D. Conjugation confirmed the transferability of blaCTX-M and blaCMY-2 genes, as well as non-β-lactam resistance traits from 60.9% (28/46) of the ESBL/pAmpC-producing isolates to a recipient E. coli J53. The ISECP, IS903, and orf477 elements were detected in the upstream or downstream regions. The blaCTX-M and blaCMY-2 genes mainly belonged to the IncI1, IncHI2, and/or IncFII plasmids. Additionally, the majority of ESBL/pAmpC-producing isolates exhibited heterogeneous PFGE profiles. This study showed that healthy chickens act as reservoirs of ESBL/pAmpC-producing E. coli that can potentially be transmitted to humans.