A systematic review and meta-analysis on the efficacy of vaccination against colibacillosis in broiler production.

Colibacillosis, a disease caused by Escherichia coli in broiler chickens has serious implications on food safety, security, and economic sustainability. Antibiotics are required for treating the disease, while vaccination and biosecurity are used for its prevention. This systematic review and meta-analysis, conducted under the COST Action CA18217-European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), aimed to assess the efficacy of E. coli vaccination in broiler production and provide evidence-based recommendations. A comprehensive search of bibliographic databases, including, PubMed, CAB Abstracts, Web of Science and Agricola, yielded 2,722 articles. Following a defined protocol, 39 studies were selected for data extraction. Most of the studies were experimental infection trials, with only three field studies identified, underscoring the need for more field-based research. The selected studies reported various types of vaccines, including killed (n = 5), subunit (n = 8), outer membrane vesicles/protein-based (n = 4), live/live-attenuated (n = 16), and CpG oligodeoxynucleotides (ODN) (n = 6) vaccines. The risk of bias assessment revealed that a significant proportion of studies reporting mortality (92.3%) or feed conversion ratio (94.8%) as outcomes, had "unclear" regarding bias. The meta-analysis, focused on live-attenuated and CpG ODN vaccines, demonstrated a significant trend favoring both vaccination types in reducing mortality. However, the review also highlighted the challenges in reproducing colibacillosis in experimental setups, due to considerable variation in challenge models involving different routes of infection, predisposing factors, and challenge doses. This highlights the need for standardizing the challenge model to facilitate comparisons between studies and ensure consistent evaluation of vaccine candidates. While progress has been made in the development of E. coli vaccines for broilers, further research is needed to address concerns such as limited heterologous protection, practicability for application, evaluation of efficacy in field conditions and adoption of novel approaches.

Genomic and Phenotypic Characterization of Mastitis-Causing Staphylococci and Probiotic Lactic Acid Bacteria Isolated from Raw Sheep's Milk.

Dairy products play a crucial role in human nutrition as they provide essential nutrients. However, the presence of diverse microorganisms in these products can pose challenges to food safety and quality. Here, we provide a comprehensive molecular characterization of a diverse collection of lactic acid bacteria (LAB) and staphylococci isolated from raw sheep's milk. Whole-genome sequencing, phenotypic characterization, and bioinformatics were employed to gain insight into the genetic composition and functional attributes of these bacteria. Bioinformatics analysis revealed the presence of various genetic elements. Important toxin-related genes in staphylococci that contribute to their pathogenic potential were identified and confirmed using phenotypic assays, while adherence-related genes, which are essential for attachment to host tissues, surfaces in the dairy environment, and the creation of biofilms, were also present. Interestingly, the Staphylococcus aureus isolates belonged to sequence type 5, which largely consists of methicillin-susceptible isolates that have been involved in severe nosocomial infections. Although genes encoding methicillin resistance were not identified, multiple resistance genes (RGs) conferring resistance to aminoglycosides, macrolides, and fluroquinolones were found. In contrast, LAB had few inherently present RGs and no virulence genes, suggesting their likely safe status as food additives in dairy products. LAB were also richer in bacteriocins and carbohydrate-active enzymes, indicating their potential to suppress pathogens and effectively utilize carbohydrate substrates, respectively. Additionally, mobile genetic elements, present in both LAB and staphylococci, may facilitate the acquisition and dissemination of genetic traits, including RGs, virulence genes, and metabolic factors, with implications for food quality and public health. The molecular and phenotypic characterization presented herein contributes to the effort to mitigate risks and infections (e.g., mastitis) and enhance the safety and quality of milk and products thereof.

The genetic network underlying the evolution of pathogenicity in avian Escherichia coli.

Introduction: Colibacillosis is a worldwide prevalent disease in poultry production linked to Escherichia coli strains that belong to the avian pathogenic E. coli (APEC) pathotype. While many virulence factors have been linked to APEC isolates, no single gene or set of genes has been found to be exclusively associated with the pathotype. Moreover, a comprehensive description of the biological processes linked to APEC pathogenicity is currently lacking. Methods: In this study, we compiled a dataset of 2015 high-quality avian E. coli genomes from pathogenic and commensal isolates, based on publications from 2000 to 2021. We then conducted a genome-wide association study (GWAS) and integrated candidate gene identification with available protein-protein interaction data to decipher the genetic network underlying the biological processes connected to APEC pathogenicity. Results: Our GWAS identified variations in gene content for 13 genes and SNPs in 3 different genes associated with APEC isolates, suggesting both gene-level and SNP-level variations contribute to APEC pathogenicity. Integrating protein-protein interaction data, we found that 15 of these genes clustered in the same genetic network, suggesting the pathogenicity of APEC might be due to the interplay of different regulated pathways. We also found novel candidate genes including an uncharacterized multi-pass membrane protein (yciC) and the outer membrane porin (ompD) as linked to APEC isolates. Discussion: Our findings suggest that convergent pathways related to nutrient uptake from host cells and defense from host immune system play a major role in APEC pathogenicity. In addition, the dataset curated in this study represents a comprehensive historical genomic collection of avian E. coli isolates and constitutes a valuable resource for their comparative genomics investigations.

Comparative Genomic Analysis Reveals the Functional Traits and Safety Status of Lactic Acid Bacteria Retrieved from Artisanal Cheeses and Raw Sheep Milk.

Lactic acid bacteria (LAB) are valuable for the production of fermented dairy products. We investigated the functional traits of LAB isolated from artisanal cheeses and raw sheep milk, assessed their safety status, and explored the genetic processes underlying the fermentation of carbohydrates. Lactiplantibacillus plantarum had the largest and more functional genome compared to all other LAB, while most of its protein-encoding genes had unknown functions. A key finding of our analysis was the overall absence of acquired resistance genes (RGs), virulence genes (VGs), and prophages, denoting that all LAB isolates fulfill safety criteria and can be used as starter or adjunct cultures. In this regard, the identified mobile genetic elements found in LAB, rather than enabling the integration of RGs or VGs, they likely facilitate the uptake of genes involved in beneficial functions and in the adaptation of LAB in dairy matrices. Another important finding of our study was that bacteriocins and CAZymes were abundant in LAB though each species was associated with specific genes, which in turn had different activity spectrums and identified applications. Additionally, all isolates were able to metabolize glucose, lactose, maltose, and sucrose, but Lactiplantibacillus plantarum was strongly associated with the fermentation of rhamnose, mannose, cellobiose, and trehalose whereas Levilactobacillus brevis with the utilization of arabinose and xylose. Altogether these results suggest that to fully exploit the beneficial properties of LAB, a combination of strains as food additives may be necessary. Interestingly, biological processes involved in the metabolism of carbohydrates that are not of direct interest for the dairy industry may yield valuable metabolites or activate pathways associated with beneficial health effects. Our results provide useful information for the development of new probiotic artisanal cheeses and probiotic starter cultures.

Biofilm Formation Ability of ESBL/pAmpC-Producing Escherichia coli Isolated from the Broiler Production Pyramid.

Escherichia coli able to produce extended spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpCs) represents a serious threat to public health, since these genes confer resistance to critically important antimicrobials (i.e., third generation cephalosporins) and can be transferred to non-resistant bacteria via plasmids. E. coli are known to be able to form a biofilm, which represents a favorable environment for the exchange of resistance determinants. Here, we assessed the ability of 102 ESBL/pAmpC-producing E. coli isolated from the broiler production pyramid to form a biofilm and to identify genetic factors involved in biofilm formation. All but one of the ESBL/pAmpC-producing E. coli were able to form a biofilm, and this represents a great concern to public health. E. coli belonging to phylogroups D, E, and F, as well as strains harboring the blaCTX-M-type gene, seem to be associated with an increased biofilm capability (p < 0.05). Furthermore, virulence genes involved in adherence and invasion (i.e., csgBAC, csgDEFG, matABCDEF, and sfaX) seem to enhance biofilm formation in E. coli. Efforts should be made to reduce the presence of ESBL/pAmpC- and biofilm-producing E. coli in the broiler production pyramid and, therefore, the risk of dissemination of resistant bacteria and genes.

Third-generation cephalosporin (3GC) resistance and its association with Extra-intestinal pathogenic Escherichia coli (ExPEC). Focus on broiler carcasses.

The worldwide spread of Extra-intestinal Pathogenic Escherichia coli (ExPEC), together with the antimicrobial resistance linked with extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpCs) are pressing threats for public health. This study aimed to investigate the presence of ExPEC genes in third-generation cephalosporin (3 GC)-resistant E. coli and to study their distribution in broiler carcasses at the slaughterhouse after the chilling process. To this purpose, isolates from a collection of 3 GC-resistant E. coli from carcasses of broilers originating from twelve broiler farms and three production chains were investigated. Several multivariate statistical approaches were adopted to elucidate the relationships among features. Phylogroup F was predominant in all broiler batches and was mainly associated with blaTEM and ESBL genes but less correlated to ExPEC genes. Another remarkable finding was the predominance of ExPEC strains assigned to uncommon phylogroups, such as B2, D, E and Clade I, commonly found into the environment. This study represents a first step for a comprehensive characterization of ExPEC genes harboured by 3 GC-resistant E. coli. These findings may be valuable for the identification of potential risks associated to broiler carcasses as source of uncommon E. coli phylogroups.

Dry Fermented Sausages with Total Replacement of Fat by Extra Virgin Olive Oil Emulsion and Indigenous Lactic Acid Bacteria.

RESEARCH BACKGROUND: Formulations based on vegetable or fish oil and modifications in the production technology of dry fermented sausages have emerged in recent years aiming to achieve the desirable target of reducing the fat content of these meat products. However, previous efforts have confronted many difficulties, such as high mass loss and unacceptable appearance due to intensely wrinkled surfaces and case hardening. The objective of this study is to produce and evaluate dry fermented sausages by utilising a meat protein-olive oil emulsion as fat substitute and indigenous lactic acid bacteria (LAB) with probiotic properties isolated from traditional Greek meat products. EXPERIMENTAL APPROACH: A novel formulation with extra virgin olive oil and turkey protein was developed to totally replace the conventionally added pork fat. Probiotic and safety characteristics of autochthonous LAB isolates from spontaneously fermented sausages were evaluated and three LAB isolates were finally selected as starter cultures. Physicochemical, microbiological and sensory analyses were carried out in all treatments (control, Lactobacillus acidophilus, L. casei, L. sakei and Pediococcus pentosaceus) during fermentation. RESULTS AND CONCLUSIONS: Ready-to-eat sausages were found to be microbiologically stable. The olive oil-based formulation produced in this study generated a mosaic pattern visible in the sliced product simulating the fat in conventional fermented sausages and was regarded as an ideal fat substitute for the production of fermented sausages. An autochthonous isolate of Lactobacillus casei adapted the best to the final products as it was molecularly identified to be present in the highest counts among the LAB isolates used as starter cultures. NOVELTY AND SCIENTIFIC CONTRIBUTION: Α novel and high-quality dry fermented meat product was produced by replacing the added pork fat with a fat substitute based on a meat protein-olive oil emulsion. Autochthonous LAB with in vitro probiotic properties could have a potential use in large-scale novel dry fermented sausage production. Such isolates could be used as starters in an effort to standardise the production process and retain the typical organoleptic and sensory characteristics. Moreover, isolates like L. casei 62 that survived in high counts in the final products can increase the safety of fermented sausages by competing not only with pathogens but also with the indigenous microbiota and could have a potential functional value for the consumer.

Occurrence of Colibacillosis in Broilers and Its Relationship With Avian Pathogenic Escherichia coli (APEC) Population Structure and Molecular Characteristics.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the disease with the highest economic loss for the broiler industry. However, studies focusing on the prevalence and population structure of APEC in the broiler production pyramid are scarce. Here, we used genotyping and serotyping data to elucidate the APEC population structure and its changes in different broiler production stages along with whole-genome sequencing (WGS) in a subset of APEC isolates to determine transmission patterns amongst dominant APEC sequence types (STs) and characterize them in detail. Comparison of genotypes encountered in both APEC and avian fecal E. coli (AFEC) provided further insights. Overall, APEC-related mortality, as the proportion of the total sampled mortality in the broiler production, was high (35%), while phylogroup C and serogroup O78 were predominant amongst APEC isolates. We found a low (34.0%) and high (53.3%) incidence of colibacillosis in chicks and end-cycle broilers, respectively, which may be related to a shift in APEC genotypes, suggesting a trend from commensalism to pathogenicity across different broiler production stages. Despite considerable APEC genotypic diversity, there was substantial genotype overlap (40.9%, overall) over the production stages and convergence of STs to the four clusters. Within these clusters, WGS data provided evidence of clonal transmission events and revealed an enriched virulence and resistance APEC repertoire. More specifically, sequenced APEC were assigned to defined pathotypes based on their virulence gene content while the majority (86%) was genotypically multi-drug resistant. Interestingly, WGS-based phylogeny showed that a subset of APEC, which are cephalosporin-resistant, may originate directly from cephalosporin-resistant AFEC. Finally, exploration of the APEC plasmidome indicated that the small fraction of the APEC virulome carried by IncF plasmids is pivotal for the manifestation of the APEC pathotype; thus, plasmid exchange can promote pathogenicity in strains that are at the edge of the commensal and pathogenic states.

High-resolution characterisation of ESBL/pAmpC-producing Escherichia coli isolated from the broiler production pyramid.

The presence of extended-spectrum ß-lactamase (ESBL) or plasmid-mediated AmpC ß-lactamase (pAmpC)-producing Escherichia coli (ESBL/pAmpC-EC) in livestock is a public health risk given the likelihood of their transmission to humans via the food chain. We conducted whole genome sequencing on 100 ESBL/pAmpC-EC isolated from the broiler production to explore their resistance and virulence gene repertoire, characterise their plasmids and identify transmission events derived from their phylogeny. Sequenced isolates carried resistance genes to four antimicrobial classes in addition to cephalosporins. Virulence gene analysis assigned the majority of ESBL/pAmpC-EC to defined pathotypes. In the complex genetic background of ESBL/pAmpC-EC, clusters of closely related isolates from various production stages were identified and indicated clonal transmission. Phylogenetic comparison with publicly available genomes suggested that previously uncommon ESBL/pAmpC-EC lineages could emerge in poultry, while others might contribute to the maintenance and dissemination of ESBL/pAmpC genes in broilers. The majority of isolates from diverse E. coli lineages shared four dominant plasmids (IncK2, IncI1, IncX3 and IncFIB/FII) with identical ESBL/pAmpC gene insertion sites. These plasmids have been previously reported in diverse hosts, including humans. Our findings underline the importance of specific plasmid groups in the dissemination of cephalosporin resistance genes within the broiler industry and across different reservoirs.

Occurrence and diversity of Campylobacter species in captive chelonians.

The aim of this study was to investigate the occurrence and diversity of Campylobacter species in chelonians. From July 2016 to September 2017, a total of 452 individuals from a large variety of tortoises (n = 366) and turtles/terrapins (n = 86) kept in private collections and breeding centres, wildlife rescue centres, zoos, pet shops, and veterinary clinics from Northern Italy was sampled and subjected to microbiological examination. Campylobacter genus and species confirmation was performed by single and multiplex PCRs. Out of 452 samples, five (1.1%) tested positive: three for C. iguaniorum (two Testudo graeca and one Testudo hermanni), one for C. fetus subsp. testudinum (Stigmochelys pardalis) and one for C. geochelonis (Testudo hermanni). This study suggests that Campylobacter spp. are not common in chelonians, but a variety of species can be detected in these hosts, including those potentially pathogenic for humans. Further studies are needed to understand the epidemiology and the pathogenic potential for both animals and humans of reptile-associated Campylobacter spp.

Impact of selective and non-selective media on prevalence and genetic makeup of ESBL/pAmpC-producing Escherichia coli in the broiler production pyramid.

Presence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase (pAmpC)-producing Escherichia coli (ESBL/pAmpC-EC) in humans and animals is alarming due to the associated risks of antibiotic therapy failure. ESBL/pAmpC-EC transmission between the human and animal compartments remains controversial. Using cefotaxime-supplemented (selective) media, we recently showed high sample prevalence of ESBL/pAmpC-EC in an integrated broiler chain [i.e. Parent Stock (PS), offspring broilers and their carcasses]. Here, we used a different approach. In parallel with the selective isolation, samples were processed on non-selective media. E. coli isolates were tested for ESBL/pAmpC-production and those found positive were genotyped. For carcasses, total E. coli were enumerated. This approach enabled us to estimate prevalence at the isolate level, which mirrors ESBL/pAmpC-EC colonisation levels. We showed that although present in many animals, ESBL/pAmpC-EC were overall subdominant to intestinal E. coli, indicating that high sample prevalence is not associated with high levels of resistance in individual hosts. This is a relevant aspect for risk assessments, especially regarding the immediate exposure of farm personnel. An exception was a particularly dominant B2/blaCMY-2 lineage in the gut of imported PS chicks. This predominance obscured presence of latent genotypes, however bias towards particular ESBL/pAmpC-EC genotypes from the selective method or underestimation by the non-selective approach did not occur. At the slaughterhouse, we showed a link between total E. coli and ESBL/pAmpC-EC on carcasses. Mitigation strategies for reducing consumers' exposure should aim at suppressing ESBL/pAmpC-EC in the broiler gut as well as controlling critical points in the processing line.

Assessing the occurrence and transfer dynamics of ESBL/pAmpC-producing Escherichia coli across the broiler production pyramid.

Extended-spectrum ß-lactamase (ESBL)- and plasmid mediated AmpC-type cephalosporinase (pAmpC)-producing Escherichia coli (ESBL/pAmpC E. coli) in food-producing animals is a major public health concern. This study aimed at quantifying ESBL/pAmpC-E. coli occurrence and transfer in Italy's broiler production pyramid. Three production chains of an integrated broiler company were investigated. Cloacal swabs were taken from parent stock chickens and offspring broiler flocks in four fattening farms per chain. Carcasses from sampled broiler flocks were collected at slaughterhouse. Samples were processed on selective media, and E. coli colonies were screened for ESBL/pAmpC production. ESBL/pAmpC genes and E. coli phylogroups were determined by PCR and sequencing. Average pairwise overlap of ESBL/pAmpC E. coli gene and phylogroup occurrences between subsequent production stages was estimated using the proportional similarity index, modelling uncertainty in a Monte Carlo simulation setting. In total, 820 samples were processed, from which 513 ESBL/pAmpC E. coli isolates were obtained. We found a high prevalence (92.5%, 95%CI 72.1-98.3%) in day-old parent stock chicks, in which blaCMY-2 predominated; prevalence then dropped to 20% (12.9-29.6%) at laying phase. In fattening broilers, prevalence was 69.2% (53.6-81.3%) at the start of production, 54.2% (38.9-68.6%) at slaughter time, and 61.3% (48.1-72.9%) in carcasses. Significantly decreasing and increasing trends for respectively blaCMY-2 and blaCTX-M-1 gene occurrences were found across subsequent production stages. ESBL/pAmpC E. coli genetic background appeared complex and bla-gene/phylogroup associations indicated clonal and horizontal transmission. Modelling revealed that the average transfer of ESBL/pAmpC E. coli genes between subsequent production stages was 47.7% (42.3-53.4%). We concluded that ESBL/pAmpC E. coli in the broiler production pyramid is prevalent, with substantial transfer between subsequent production levels.

A review on the current situation and challenges of colistin resistance in poultry production.

Colistin has been re-assessed as a critically important antimicrobial in humans due to its efficacy against multi-drug resistant Gram-negative bacteria, in particular P. aeruginosa, A. baumannii, and K. pneumoniae. The recent discovery of mobile colistin resistance (mcr) determinants in humans and animals has brought concerns regarding the future of this antimicrobial. In this paper, we aim to highlight the current challenges with colistin resistant bacteria and to summarize reliable global data on colistin resistance in poultry production. In addition, we present and compare data from a screening for colistin resistance carried out on a collection of clinical Escherichia coli isolated from poultry in Italy. In Europe, resistance rates for Salmonella and E. coli are in general low with sporadic incidence of high colistin resistance levels. Absence of resistance or very low rates have been recorded in countries where colistin is either not employed (e.g. Norway) or used in minimal amounts (e.g. Denmark) in food-producing animals. In large poultry meat producing countries, such as China and Brazil, the widespread use of colistin has resulted in the dissemination of resistance determinants in diverse bacterial species. Worryingly, these bacteria are often co-resistant to other critically important antimicrobials, such as extended-spectrum cephalosporins. The data gap for many countries and for zoonotic bacteria, the role of the "phantom resistome" and the circulation of mcr-carriers expressing resistance phenotypes close or below the current ECOFF values, should be considered in future investigations. The importance of poultry as a cheap protein source and the global effort to mitigate colistin resistance and preserve this essential antimicrobial require a thorough re-assessment of colistin use in poultry.

Occurrence and molecular characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses in an equine clinic.

Objectives: To investigate the occurrence and characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses at one equine clinic in the Netherlands. Methods: A total of 91 horses, including residents and patients, were sampled. ESBL/AmpC-producing E. coli were identified by a combination disc diffusion test. Phylogenetic groups and MLST were determined. ESBL/AmpC genes were analysed using PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid MLST. Results: At least one E. coli isolate with a confirmed ESBL/AmpC gene was found in samples from 76 horses (84%). Although phylogenetic group B1 E. coli bla CTX-M-1 predominated, a diverse E. coli population was found, indicating that clonal nosocomial spread was not the only reason for the high occurrence found. MLST analysis revealed the presence of 47 E. coli STs, organized in four clusters of genetically related strains. ST10, ST641, ST1079 and ST1250 were most commonly found. With regard to the genes, bla CTX-M-1 was most prevalent ( n = 91), followed by bla CTX-M-2 ( n = 26). The most frequently found plasmid type was IncHI1, but plasmids belonging to the IncF, IncI1 and IncN groups were also identified. Conclusions: A high occurrence of ESBL-producing E. coli in faecal samples was found among horses in an equine clinic and the variety of STs, ESBL genes and plasmid types suggests nosocomial transmission. ESBL E. coli can cause difficult-to-treat infections in horses and prudent use of antimicrobials is warranted. A further assessment of the risks of transmission to persons in close contact with horses, such as caretakers or veterinarians, is crucial.