Genotypic correlation between Salmonella Enteritidis isolates from broiler breeders and hatchery flocks

In this study, Salmonella Enteritidis strains isolated from dust and environmental materials from different flocks located in Turkey’s Western Black Sea region were examined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 59 S. Enteritidis strains isolated from broiler breeder and hatchery flocks, and one S. Enteritidis strain isolated from a stool sample of a farm worker were examined. PFGE analysis revealed two major PFGE groups and nine different macro restriction profiles. It was determined that 85% (51/60) of the strains were close to each other and comprised Group I. All S. Enteritidis strains had the same sequence type (ST): ST11. Isolation of strains with a single genotype suggests that there may be a cross transmission between the flocks.

High prevalence of carbapenem-resistant Klebsiella pneumoniae ST307 recovered from fecal samples in an Italian hospital

Aim: This study reports the characterization of carbapenem-resistant colonizing strains of K. pneumoniae. Methods: 650 stool samples were screened for carbapenem-resistant K. pneumoniae (CR-Kp). All strains were characterized for antibiotic susceptibility, typing features, main carbapenemases and extended-spectrum ß-lactamases. The carbapenemase transferability was assessed by interspecific conjugation. Results: Eighteen CR-Kp were multidrug resistant, five were KPC producing. A predominance of ST307 isolates, constituting the predominant cluster by PFGE analysis, was identified (50% were KPC-2 producers). Conjugation data showed the co-transfer of blaKPC-2, blaTEM-1, blaOXA-1, blaCTX-M-15 in a single large pKPN3-like plasmid. Conclusion: Our data pointed out the diversity of colonizing K. pneumoniae strains compared with clinical ones. The predominance of ST307 strains suggested an increased spreading, even in our area, of this high-risk clone.

Genetic Listeria monocytogenes Types in the Pork Processing Plant Environment: From Occasional Introduction to Plausible Persistence in Harborage Sites

The purpose of this study was to investigate the L. monocytogenes occurrence and genetic diversity in three Belgian pork cutting plants. We specifically aim to identify harborage sites and niche locations where this pathogen might occur. A total of 868 samples were taken from a large diversity of food and non-food contact surfaces after cleaning and disinfection (C&D) and during processing. A total of 13% (110/868) of environmental samples tested positive for L. monocytogenes. When looking in more detail, zone 3 non-food contact surfaces were contaminated more often (26%; 72/278) at typical harborage sites, such as floors, drains, and cleaning materials. Food contact surfaces (zone 1) were less frequently contaminated (6%; 25/436), also after C&D. PFGE analysis exhibited low genetic heterogeneity, revealing 11 assigned clonal complexes (CC), four of which (CC8, CC9, CC31, and CC121) were predominant and widespread. Our data suggest (i) the occasional introduction and repeated contamination and/or (ii) the establishment of some persistent meat-adapted clones in all cutting plants. Further, we highlight the importance of well-designed extensive sampling programs combined with genetic characterization to help these facilities take corrective actions to prevent transfer of this pathogen from the environment to the meat.

mcr-1 Identified in Fecal Escherichia coli and Avian Pathogenic E. coli (APEC) From Brazil

Colisitin-associated resistance in bacteria of food producing animals has gained significant attention with the mcr gene being linked with resistance. Recently, newer variants of mcr have emerged with more than nine variants currently recognized. Reports of mcr associated resistance in Escherichia coli of poultry appear to be relatively limited, but its prevalence requires assessment since poultry is one of the most important and cheapest sources of the world’s protein and the emergence of resistance could limit our ability to treat disease outbreaks. Here, 107 E. coli isolates from production poultry were screened for the presence of mcr 1–9. The isolates were collected between April 2015 and June 2016 from broiler chickens and free-range layer hens in Rio de Janeiro, Brazil. All isolates were recovered from the trachea and cloaca of healthy birds and an additional two isolates were recovered from sick birds diagnosed with colibacillosis. All isolates were screened for the presence of mcr-1 to 9 using PCR and Sanger sequencing for confirmation of positive genes. Additionally, pulse field gel electrophoresis (PFGE) analysis, avian fecal E. coli (APEC) virulence associated gene screening, plasmid replicon typing and antimicrobial resistance phenotype and resistance gene screening, were also carried out to further characterize these isolates. The mcr-1 gene was detected in 62 (57.9%) isolates (61 healthy and 1 APEC) and the mcr-5 gene was detected in 3 (2.8%) isolates; mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, and mcr-9 were not detected in any isolate. In addition, mcr 1 and 5 positive isolates were phenotypically resistant to colistin using the agar dilution assay (> 8ug/ml). PFGE analysis found that most of the isolates screened had unique fingerprints suggesting that the emergence of colistin resistance was not the result of clonal dissemination. Plasmid replicon types IncI2, FIB, and B/O were found in 38, 36, and 34% of the mcr positive isolates and were the most prevalent replicon types detected; tetA and tetB (32 and 26%, respectively) were the most prevalent antimicrobial resistance genes detected and iutA, was the most prevalent APEC virulence associated gene, detected in 50% of the isolates. Approximately 32% of the isolates examined could be classified as APEC-like, based on the presence of 3 or more genes of APEC virulence associated path panel (iroN, ompT, hlyF, iss, iutA). This study has identified a high prevalence of mcr-1 in poultry isolates in Brazil, suggesting that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. Emergence of the mcr gene and associated colisitin resistance in production poultry warrants continued monitoring from the animal health and human health perspective.

Analysis of Yersinia pseudotuberculosis isolates recovered from deceased mammals of a German zoo animal collection

Yersinia (Y.) pseudotuberculosis is an important pathogen for both humans and animals. It can infect livestock as well as pets and wild animals. During the last years, a number of reports describe the isolation of Y. pseudotuberculosis from zoo animals, mainly birds and mammals, for which the infection was mostly lethal. Between 2005 and 2019, there were at least 17 cases of deceased mammals belonging to five different species at the Zoo Wuppertal, Germany, which suffered from a Y. pseudotuberculosis infection. Since only scarce information exists on properties of Y. pseudotuberculosis from zoo animals, we characterized eight isolates, covering all infected species, in detail. All of them are members of biotype 1, but belong to five serotypes, five sequence types (STs) and even seven cgMLST types. Using Pulsed-Field Gel Electrophoresis (PFGE) analysis and whole-genome sequencing (WGS), seven isolates could be discriminated from each other. They differ significantly regarding their virulence genes and mobile genetic elements. While the virulence plasmid pYV existed in all serotypes (five isolates), a complete high pathogenicity island (HPI) was detected only in the serotypes O:1a, O:1b and O:13 (four isolates), but not in O:2a and O:2b. Similarly, the content of other plasmids and prophages varies greatly between the isolates. The data demonstrates that the deceased mammals were infected by seven individual isolates and not by a single type predominating in the zoo animals.

Genetic Diversity of Clostridium perfringens Strains Isolated from Broiler Chickens Revealed by PFGE Analysis in China and Pakistan

Clostridium perfringens (C. perfringens) is widely distributed in broiler chickens causing clinical and subclinical enteritis and is especially known for causing necrotic enteritis (NE). There are numerous reports of NE outbreaks in Pakistan as well as China but there is a lack of information related to PFGE profile from both the countries. To close this gap, we designed this study and obtained samples from broiler chicken farms located in 3 different regions of Pakistan and 4 different regions of China. A total of 79 fecal swabs (Pakistan=29; China=50) were collected and grown on FTA media. Further, isolates were grown on TSE agar and black colonies were selected for DNA extraction. All 79 isolates were tested for toxin profiles by PCR (α-gene; beta-2; netB gene) and PFGE profiling (pulsotypes analysis). Toxinotyping results revealed that all the isolates (n=50) from China were type A (α-toxin positive) while 23 and 6 isolates (n=29) from Pakistan were type A (α-toxin positive) and type G (α-toxin, NetB positive), respectively. Toxinotyping revealed α-toxin is highly prevalent in both the countries while from Pakistani isolates, NetB toxin was also detected. PFGE discriminated 79 isolates into 45 different PFGE patterns (pulsotypes). The analysis further showed different pulsotypes originating from China and Pakistan and isolates were subtyped by SmaI. The results showed high genetic polymorphism in C. perfringens even within the same strain. These preliminary findings of genetic variations will further help to design control strategies

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.

Contamination, persistence and dissemination of Chronobacter during the production of powdered infant formula in China in 2016

A total of 620 samples collected from two factories in China producing powdered infant formula (PIF) between July and November 2016 were analyzed for Cronobacter. Antimicrobial susceptibility, pulsed-field gel electropho-resis (PFGE), and biofilm formation of Cronobacter were carried out. The results showed that 2.26% samples were positive for Cronobacter among the 33 isolates that were identified. All Cronobacter isolates were susceptible to 12 antimicrobial agents tested except one isolate which showed intermediate resistance to Chloramphenicol. PFGE analysis showed that nine clusters comprising the 33 isolates were identified, among which C8, C4, and C5 were the predominant types. All 33 isolates were capable of forming biofilm, and particularly, C. malonaticus isolates showed a good biofilm-forming ability at both 28°C and 37°C. The results illustrated that it is necessary for PIF manufactures to develop control measures for reducing Cronobacter contamination and its associated foodborne illness among infants.

Emergence of blaNDM-5-producing Escherichia coli ST410 in Companion Dogs Treated with Meropenem

Carbapenem-resistant Escherichia coli (CRE) with a multidrug resistant phenotype was isolated from four clinically ill dogs treated with meropenem in different local animal hospitals between 2017 and 2019. IncX3-type plasmids of ca. 46 kb in size carrying blaNDM-5 were present in all CRE strains and their transconjugants. High genetic similarity (>90%) by PFGE analysis was observed among the CRE strains, which were identified as ST410.To the best of our knowledge, blaNDM-5-producing E. coli ST410 clones are emerging sporadically in companion dogs treated with meropenem. The spread of Enterobacteriaceae harboring the NDM-5 gene in companion animals can pose a threat to public health; therefore, extensive monitoring in veterinary hospitals using carbapenem and careful antibiotic use are crucial for managing and monitoring these resistant strains

Detection of the Phenicol–Oxazolidinone Resistance Gene poxtA in Enterococcus faecium and Enterococcus faecalis from Food-Producing Animals during 2008–2018 in Korea

We aimed to investigate the presence of the phenicol–oxazolidinone resistance gene poxtA in linezolid-resistant enterococci from food-producing animals and analyze its molecular characteristics. We collected 3941 Enterococcus faecium and 5088 E. faecalis isolates from all provinces of South Korea from 2008 to 2018. We found linezolid resistance in 0.79% (94/3941) of E. faecium and 1.22% (62/5088) of E. faecalis isolates. Overall, 23.1% (36/156) of the linezolid-resistant isolates had the poxtA gene, including 31 E. faecium and five E. faecalis isolates. The poxtA-positive enterococci were mainly isolated from chicken (86.1%; 26/36). Fifteen poxtA-harboring isolates co-carried another linezolid-resistance gene, optrA. Eight E. faecium isolates had an N130K mutation in the ribosomal protein L4, while no mutations were observed in E. faecalis isolates. The poxtA gene was transferred into 10 enterococci by conjugation. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis indicated that poxtA-carrying isolates were heterogeneous. Three E. faecium isolates belonged to CC17 (ST32, ST121, and ST491). To our knowledge, this is the first report on the poxtA gene in Korea. Prudent use of antimicrobials and active surveillance on antimicrobial resistance are urgently needed to reduce the risk of dissemination of the linezolid-resistant isolates in humans and animals.