Production of a new tetravalent vaccine targeting fimbriae and enterotoxin of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an important type of pathogenic bacteria that causes diarrhea in pigs. The objective of this study was to prepare a novel tetravalent vaccine to effectively prevent piglet diarrhea caused by E. coli. In order to realize the production of K88ac-K99-ST1-LTB tetravalent inactivated vaccine, the biological characteristics, stability, preservation conditions, and safety of the recombinant strain BL21(DE3) (pXKKSL4) were studied, and the vaccine efficacy and minimum immune dose were measured. The results indicated that the biological characteristics, target protein expression, and immunogenicity of the 1st to 10th generations of the strain were stable. Therefore, the basic seed generation was preliminarily set as the 1st to 10th generations. The results of the efficacy tests showed that the immune protection rate could reach 90% with 1 minimum lethal dose (MLD) virulent strain attack in mice. The immunogenicity was stable, and the minimum immune dose was 0.1 mL per mouse. Our research showed that the genetically engineered vaccine developed in this way could prevent piglet diarrhea caused by enterotoxigenic E. coli through adhesin and enterotoxin. In order to realize industrial production of the vaccine as soon as possible, we conducted immunological tests and production process research on the constructed K88ac-K99-ST1-LTB tetravalent inactivated vaccine. The results of this study provide scientific experimental data for the commercial production of vaccines and lay a solid foundation for their industrial production.

Escherichia coli entérotoxinogènes (ETEC) est un type important de bactéries pathogènes qui cause de la diarrhée chez les porcs. L'objectif de l'étude était de préparer un nouveau vaccin tétravalent pour prévenir efficacement la diarrhée causée par E. coli chez les porcelets. Afin de réaliser la production du vaccin tétravalent inactivé K88ac-K99-ST1-LTB, les caractéristiques biologiques, la stabilité, les conditions de conservation, et la sécurité de la souche recombinante (BL21(DE3)(pXKKSL4) ont été étudiées et l'efficacité du vaccin et la dose immunitaire minimum ont été mesurées. Les résultats indiquent que les caractéristiques biologiques, l'expression des protéines cibles, et l'immunogénicité de la 1ère à la 10e génération de la souche étaient stables. Ainsi, la génération germinale de base a été établie de manière préliminaire comme étant de la 1ère à la 10e générations. Les résultats des tests d'efficacité ont démontré que le taux de protection immunitaire pouvait atteindre 90 % avec une attaque au moyen de 1 dose léthale minimale (MLD) d'une souche virulente chez les souris. L'immunogénicité était stable et la dose immunitaire minimum était de 0,1 mL par souris. Nos travaux ont démontré que le vaccin génétiquement élaboré développé de cette façon pourrait prévenir la diarrhée chez les porcelets causée par des E. coli entérotoxigénique via les adhésines et les entérotoxines. Afin d'atteindre la production industrielle de ce vaccin aussitôt que possible, nous avons mené des tests immunologiques et de la recherche sur le processus de production du vaccin tétravalent inactivé K88ac-K99-ST1-LTB. Les résultats de la présente étude fournissent des données scientifiques expérimentales pour la production commerciale de vaccins et jettent une base solide pour leur production industrielle.(Traduit par Docteur Serge Messier).

Characterization of diarrheagenic Escherichia coli from different cattle production systems in Brazil.

Diarrheagenic E. coli (DEC) can cause severe diarrhea and is a public health concern worldwide. Cattle are an important reservoir for this group of pathogens, and once introduced into the abattoir environment, these microorganisms can contaminate consumer products. This study aimed to characterize the distribution of DEC [Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)] from extensive and intensive cattle production systems in Brazil. Samples (n = 919) were collected from animal feces (n = 200), carcasses (n = 600), meat cuts (n = 90), employee feces (n = 9), and slaughterhouse water (n = 20). Virulence genes were detected by PCR in 10% of animal samples (94/919), with STEC (n = 81) as the higher prevalence, followed by EIEC (n = 8), and lastly EPEC (n = 5). Animals raised in an extensive system had a higher prevalence of STEC (average 48%, sd = 2.04) when compared to animals raised in an intensive system (23%, sd = 1.95) (Chi-square test, P < 0.001). From these animals, most STEC isolates only harbored stx2 (58%), and 7% were STEC LEE-positive isolates that were further identified as O157:H7. This study provides further evidence that cattle are potential sources of DEC, especially STEC, and that potentially pathogenic E. coli isolates are widely distributed in feces and carcasses during the slaughter process.

Phenotypic and genotypic characterization of extended spectrum beta-lactamase producing E. coli harboring carbapenem and colistin-resistant genes from poultry farms in Egypt.

Background: eEscherichia coli (E. coli) bacteria that produce extended spectrum beta-lactamase (ESBL) is associated with a high prevalence of human illnesses worldwide. The emergence of resistance to carbapenem and colistin compounds poses further challenges to the treatment options for these illnesses. This study aimed to evaluate the phenotypic and genotypic pattern of resistance to carbapenem and colistin in ESBL-producing E. coli. Escherichia coli isolates collected from the respiratory tract of chickens in El-Sharkia government, Egypt. Methods: A total of 250 lung samples were collected from 50 poultry farms. These samples were then subjected to isolation, identification, and serotyping of E. coli. The presence of antimicrobial resistance was identified by disc diffusion testing. The occurrence of ESBL phenotypes was also assessed using the double disc synergy method. PCR/sequencing techniques were employed to examine the presence of ESBL (ß-lactamase (bla)-TEM, blaSHV, and blaCTX-M), colistin (mcr-1), and carbapenem (blaNDM, blaVIM, and blaKPC) resistance genes. Results: The findings revealed that 140 out of 250 (56%) were identified as E. coli. All E. coli isolates had a high level of multi-antimicrobial resistance (MAR) with an index value greater than 0.2, and 65.7% of them were confirmed to produce ESBL. Out of the 92 ESBL phenotypes, 55 (59.7%), 32 (34.7%), 18 (19.6%), and 37 (40.2%) isolates harbor b laTEM-3, b laSHV-4, b laCTX-M-1, a nd blaCTX-M-14 genes, respectively. The blaNDM-1 gene was identified in all 40 phenotypes that exhibited resistance to carbapenem, accounting for 28.5% of all strains of E. coli and 43.4% of ESBL isolates. The VIM and KPC genes were not detected in any of the samples. Furthermore, there was a significant prevalence of the mobilized colistin resistance (mcr)-1 gene, with 64 (69.5%) of the ESBL isolates exhibiting this gene. Conclusion: The prevalence of ESBL-producing E. coli, particularly those resistant to carbapenem and colistin, poses a significant public health risk in society.

Antibiotic resistance profiling and phylotyping of human-diarrheagenic Escherichia coli pathotypes detected from diarrheic and non-diarrheic calves in Iran.

BACKGROUND: Escherichia coli (E. coli) serves as a common indicator of gut microbiota and is utilized for monitoring antimicrobial resistance determinants in food-producing animals. This study aimed to investigate antimicrobial resistance patterns in virulence gene-positive E. coli isolates obtained from 340 healthy and diarrheic calves. METHODS AND RESULTS: A total of 340 fecal swab samples were obtained from diarrheic (n = 170) and healthy (n = 170) calves for 12 months from different farms in Kerman, Iran. The samples were phenotypically analyzed to detect E. coli isolates and antibiotic resistance. Also, antimicrobial resistance genes, diarrheagenic E. coli pathotypes, and phylogenetic background were screened by PCR. Fifteen percent (51/340) of E. coli isolates were positive for at least one of the examined virulence genes (VGs); the prevalence of VGs in E. coli isolates from healthy calves (36/170; 21.17%) was higher than that in diarrheic cases (15/170; 8.82%). Out of the 51 VG-positive isolates, six pathotypes including Shiga toxin-producing E. coli (STEC; 27.45%), enterotoxigenic E. coli (ETEC; 23.52%), enterohemorrhagic E. coli (EHEC; 19.6%), necrotoxigenic E. coli (NTEC; 19.6%), enteropathogenic E. coli (EPEC; 15.68%), enteroinvasive E. coli (EIEC; 1.96%) and three hybrid pathotypes including ETEC/STEC, ETEC/EHEC, and STEC/EIEC were detected among the strains. Antimicrobial resistance (AR) was observed in 98.03% of the VG-positive isolates, which was the same for both healthy and diarrheic calves. The maximum prevalence rate of AR was found against trimethoprim/sulfamethoxazole (49.01%; 3/51), while the minimum prevalence rate was against gentamycin (5.88%; 25/51). Among the VG-positives, phylotype A was found to be the most prevalent followed by B1 and D phylotypes. CONCLUSIONS: The prevalence of VG-positive E. coli isolates was higher in healthy calves compared to diarrheic cases. AR was widespread among VG-positive isolates. These findings suggest that calves may serve as potential reservoirs of antimicrobial-resistant hybrid pathotypes of E. coli.

Resistant Escherichia coli isolated from wild mammals from two rescue and rehabilitation centers in Costa Rica: characterization and public health relevance.

This study aimed to characterize the antimicrobial resistance (AMR) and virulence profiles of 67 Escherichia coli isolates obtained from faecal samples of 77 wild mammals from 19 different species, admitted in two rescue and rehabilitation centers in Costa Rica. It was possible to classify 48% (n = 32) of the isolates as multidrug-resistant, and while the highest resistance levels were found towards commonly prescribed antimicrobials, resistance to fluoroquinolones and third generation cephalosporins were also observed. Isolates obtained from samples of rehabilitated animals or animals treated with antibiotics were found to have significantly higher AMR levels, with the former also having a significant association with a multidrug-resistance profile. Additionally, the isolates displayed the capacity to produce α-haemolysins (n = 64, 96%), biofilms (n = 51, 76%) and protease (n = 21, 31%). Our results showed that AMR might be a widespread phenomenon within Costa Rican wildlife and that both free-ranging and rehabilitated wild mammals are potential carriers of bacteria with important resistance and virulence profiles. These results highlight the need to study potential sources of resistance determinants to wildlife, and to determine if wild animals can disseminate resistant bacteria in the environment, potentially posing a significant threat to public health and hindering the implementation of a "One Health" approach.

Comparison of Three Preharvest Sampling Strategies to Monitor Pathogens in Cattle Lairage Areas.

The objective of this study was to compare preharvest monitoring strategies by evaluating three different sampling methods in the lairage area to determine pathogen recovery for each sampling method and incoming pathogen prevalence from the cattle to inform in-plant decision making. Samples were gathered over a 5-month period, from February to June 2022, at a harvesting and processing facility located in Eastern Nebraska. Sampling methods included (i) fecal pats, (ii) boot swabs, and (iii) MicroTally swab. A total of 329 samples were collected over the study period (fecal pats: n = 105, boot swabs: n = 104, and MicroTally swabs: n = 120). Specific media combinations, an incubation temperature of 42°C, and incubation timepoints (18-24 h) were utilized for each matrix and the prevalence of Salmonella, Escherichia coli O157:H7, and six non-O157 Shiga-toxin producing E. coli (STEC) was evaluated using the BAX system Real-Time PCR assay. Overall, results from the study concluded that boot swabs were an effective sampling method for pathogen detection in the cattle lairage area. Boot swabs (97.1%) were statistically more likely to detect for Salmonella (p < 0.05) when compared to fecal pats (67.6%) and MicroTally swab (77.5%) methods. For E. coli O157:H7 and STEC - O26, O121, O45, and O103 prevalence, boot swabs were significantly better at detecting for these pathogens (p < 0.05) than MicroTally swabs (OR = 3.16 - 11.95) and a comparable sampling method to fecal pats (OR = 0.93 - 2.01, p > 0.05). Lastly, all three sampling methods detected a very low prevalence for E. coli O111 and O145; therefore, no further analysis was conducted. The boot swab sampling method was strongly favored because they require little training to implement, are inexpensive, and they do not require much sampling labor; therefore, would be a simple and effective sampling method to implement within the industry to evaluate pathogen prevalence preharvest.

Molecular detection of extended-spectrum ß-lactamase-producing Escherichia coli from bat caves on Lombok Island.

Background: The discovery of antibiotic-resistant Enterobacteriaceae bacteria in wild animals is an indication of their potential for wildlife as a reservoir. Bats are natural reservoir hosts and a source of infection for several microorganisms and have the potential to become vectors for the spread of zoonotic diseases. Aim: A study was conducted based on these characteristics to identify and detect the blaTEM gene in Eschericia coli isolated from bat excrements in Tanjung Ringgit Cave, East Lombok. Methods: Bat fecal samples were firstly inoculated onto eosin methylene blue agar media. Recovered bacterial isolates were further characterized using standard microbiological techniques. Antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method. blaTEM gene detection was carried out using polymerase chain reaction (PCR). Results: Out of the 150 bat fecal samples obtained from Tanjung Ringgit cave, Lombok Island, Indonesia, 56 (37%) were positive for E. coli. Eight (8) out of the 56 E. coli isolates that underwent antimicrobial susceptibility testing using the disc diffusion method were confirmed to be multidrug-resistant as they exhibited resistance to at least three different classes of antibiotics. Out of the eight (8) multidrug resistance E. coli isolates recovered from fecal samples of bats, 2 (two) harbored the blaTEM gene. Conclusion: The discovery of the blaTEM gene in bat fecal samples indicates the potential for wild animals, especially bats, to spread ESBL resistance genes to the environment and to humans.

Antimicrobial resistance, ß-lactamase genotypes, and plasmid replicon types of Shiga toxin-producing Escherichia coli isolated from different animal hosts.

AIMS: The primary objective of this study was to analyze antimicrobial resistance (AMR), with a particular focus on ß-lactamase genotypes and plasmid replicon types of Shiga toxin-producing Escherichia coli (STEC) strains originating from various animal hosts. METHODS AND RESULTS: A total of 84 STEC strains were isolated from cattle (n = 32), sheep/goats (n = 26), pigeons (n = 20), and wild animals (n = 6) between 2010 and 2018 in various regions of Iran. The Kirby-Bauer susceptibility test and multiple polymerase chain reaction (PCR) panels were employed to elucidate the correlation between AMR and plasmid replicon types in STEC isolates. The predominant replicon types were IncFIC and IncFIB in cattle (46.8%), IncFIC in sheep/goats (46.1%), IncA/C in pigeons (90%), and IncP in wild animals (50%). STEC of serogroups O113, O26, and O111 harbored the IncFIB (100%), IncI1 (80%), and IncFIC + IncA/C (100%) plasmids, respectively. A remarkable AMR association was found between ciprofloxacin (100%), neomycin (68.7%), and tetracycline (61.7%) resistance with IncFIC; amoxicillin + clavulanic acid (88.8%) and tetracycline (61.7%) with IncA/C; ciprofloxacin (100%) with IncFIB; fosfomycin (85.7%) and sulfamethoxazole + trimethoprim (80%) with IncI1. IncI1 appeared in 83.3%, 50%, and 100% of the isolates harboring blaCTX-M, blaTEM, and blaOXA ß-lactamase genes, respectively. CONCLUSIONS: The emergence of O26/IncI1/blaCTX-M STEC in cattle farms poses a potential risk to public health.

Antimicrobial resistance and public and animal health risks associated with pathogenic Escherichia coli isolated from calves.

We aimed to determine the antimicrobial susceptibility profile of pathogenic Escherichia coli strains isolated from fecal samples of calves and buffalo calves (2008-2013), in Minas Gerais, Brazil, as well as the frequency of O157 gene and strains carrying extended-spectrum beta-lactamases (ESBL) and mobile colistin resistance (mcr) genes. E. coli strains (n=518) were tested for susceptibility against ten antimicrobials. Tetracycline was the antimicrobial with the highest resistance rate (382/518), followed by ampicillin (321/518), sulfamethoxazole/trimethoprim (312/518), chloramphenicol (192/518), gentamicin (126/518), ciprofloxacin (148/518), cefazolin (89/518), colistin (54/518) and cefoxitin (34/518). Multidrug resistance (MDR) was observed in 381/518 isolates. No strain harbored mcr or O157 genes, whereas 19/99 were ESBL positive. The most prevalent pathotype and phylogroup were STEC and B1, respectively. Age, EHEC pathotype and resistance to aminoglycoside and cephem were significantly associated with MDR in the multivariate model. Overall, E. coli strains showed high rates of resistance to penicillin, tetracyclines and folate inhibitors, in addition to an alarming rate of MDR and ESBL-producing strains.

Unexpected role of pig nostrils in the clonal and plasmidic dissemination of extended-spectrum beta-lactamase-producing Escherichia coli at farm level.

The presence of methicillin-resistant or -susceptible S. aureus in pig nostrils has been known for a long time, but the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli has hardly been investigated. Here, we collected 25 E. coli recovered from nasal samples of 40 pigs/10 farmers of four farms. Nine ESBL-producing isolates belonging to ST48, ST117, ST847, ST5440, ST14914 and ST10 were retrieved from seven pigs. All blaESBL genes (blaCTX-M-32,blaCTX-M-14,blaCTX-M-1,blaCTX-M-65, and blaSHV-12) were horizontally transferable by conjugation through plasmids belonging to IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1) types. IncI1-plasmids displayed different genetic environments: i) IS26-blaSHV-12-deoR-IS26, ii) wbuC-blaCTX-M-32-ISKpn26 (IS5), and iii) IS930-blaCTX-M-14-IS26. The IncHI2-plasmid contained the genetic environment IS903-blaCTX-M-65-fipA with multiple resistance genes associated either to: a) Tn21-like transposon harbouring genes conferring aminoglycosides/beta-lactams/chloramphenicol/macrolides resistance located on two atypical class 1 integrons with an embedded ΔTn5393; or b) Tn1721-derived transposon displaying an atypical class 1 integron harbouring aadA2-arr3-cmlA5-blaOXA-10-aadA24-dfrA14, preceding the genetic platform IS26-blaTEM-95-tet(A)-lysR-floR-virD2-ISVsa3-IS3075-IS26-qnrS1, as well as the tellurite resistance module. Other plasmids harbouring clinically relevant genes were detected, such as a ColE-type plasmid carrying the mcr-4.5 gene. Chromosomally encoded genes (fosA7) or integrons (intI1-dfrA1-aadA1-qacE-sul1/intI1-IS15-dfrA1-aadA2) were also identified. Finally, an IncY plasmid harbouring a class 2 integron (intI2-dfrA1-sat2-aadA1-qacL-IS406-sul3) was detected but not associated with a blaESBL gene. Our results evidence that pig nostrils might favour the spread of ESBL-E. coli and mcr-mediated colistin-resistance. Therefore, enhanced monitoring should be considered, especially in a sector where close contact between animals in intensive farming increases the risk of spreading antimicrobial resistance.

The spontaneously produced lysogenic prophage phi456 promotes bacterial resistance to adverse environments and enhances the colonization ability of avian pathogenic Escherichia coli strain DE456.

In the last decade, prophages that possess the ability of lysogenic transformation have become increasingly significant. Their transfer and subsequent activity in the host have a significant impact on the evolution of bacteria. Here, we investigate the role of prophage phi456 with high spontaneous induction in the bacterial genome of Avian pathogenic Escherichia coli (APEC) DE456. The phage particles, phi456, that were released from DE456 were isolated, purified, and sequenced. Additionally, phage particles were no longer observed either during normal growth or induced by nalidixic acid in DE456Δphi456. This indicated that the released phage particles from DE456 were only phi456. We demonstrated that phi456 contributed to biofilm formation through spontaneous induction of the accompanying increase in the eDNA content. The survival ability of DE456Δphi456 was decreased in avian macrophage HD11 under oxidative stress and acidic conditions. This is likely due to a decrease in the transcription levels of three crucial genes-rpoS, katE, and oxyR-which are needed to help the bacteria adapt to and survive in adverse environments. It has been observed through animal experiments that the presence of phi456 in the DE456 genome enhances colonization ability in vivo. Additionally, the number of type I fimbriae in DE456Δphi456 was observed to be reduced under transmission electron microscopy when compared to the wild-type strain. The qRT-PCR results indicated that the expression levels of the subunit of I fimbriae (fimA) and its apical adhesin (fimH) were significantly lower in DE456Δphi456. Therefore, it can be concluded that phi456 plays a crucial role in helping bacterial hosts survive in unfavorable conditions and enhancing the colonization ability in DE456.

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.

Antibacterial activity of the antimicrobial peptide PMAP-36 in combination with tetracycline against porcine extraintestinal pathogenic Escherichia coli in vitro and in vivo.

The increase in the emergence of antimicrobial resistance has led to great challenges in controlling porcine extraintestinal pathogenic Escherichia coli (ExPEC) infections. Combinations of antimicrobial peptides (AMPs) and antibiotics can synergistically improve antimicrobial efficacy and reduce bacterial resistance. In this study, we investigated the antibacterial activity of porcine myeloid antimicrobial peptide 36 (PMAP-36) in combination with tetracycline against porcine ExPEC PCN033 both in vitro and in vivo. The minimum bactericidal concentrations (MBCs) of AMPs (PMAP-36 and PR-39) against the ExPEC strains PCN033 and RS218 were 10 µM and 5 µM, respectively. Results of the checkerboard assay and the time-kill assay showed that PMAP-36 and antibiotics (tetracycline and gentamicin) had synergistic bactericidal effects against PCN033. PMAP-36 and tetracycline in combination led to PCN033 cell wall shrinkage, as was shown by scanning electron microscopy. Furthermore, PMAP-36 delayed the emergence of PCN033 resistance to tetracycline by inhibiting the expression of the tetracycline resistance gene tetB. In a mouse model of systemic infection of PCN033, treatment with PMAP-36 combined with tetracycline significantly increased the survival rate, reduced the bacterial load and dampened the inflammatory response in mice. In addition, detection of immune cells in the peritoneal lavage fluid using flow cytometry revealed that the combination of PMAP-36 and tetracycline promoted the migration of monocytes/macrophages to the infection site. Our results suggest that AMPs in combination with antibiotics may provide more therapeutic options against multidrug-resistant porcine ExPEC.

Genetic insights of antibiotic resistance, pathogenicity (virulence) and phylogenetic relationship of Escherichia coli strains isolated from livestock, poultry and their handlers - a one health snapshot.

BACKGROUND: Pathogenic and non-pathogenic strains of Escherichia coli harbouring antibiotic resistance genes (ARGs) from any source (clinical samples, animal settings, or environment) might be transmitted and contribute to the spread and increase of antibiotic resistance in the biosphere. The goal of this study was to investigate the genome to decipher the repertoire of ARGs, virulence genes carried by E. coli strains isolated from livestock, poultry, and their handlers (humans), and then unveil the genetic relatedness between the strains. METHODS: Whole genome sequencing was done to investigate the genetic makeup of E. coli isolates (n = 20) [swine (n = 2), cattle (n = 2), sheep (n = 4), poultry (n = 7), and animal handlers (n = 5)] from southern India. The detection of resistome, virulome, biofilm forming genes, mobile genetic elements (MGE), followed by multilocus sequence typing (MLST) and phylogenetic analyses, were performed. RESULTS: E. coli strains were found to be multi drug resistant, with a resistome encompassing > 20 ARGs, the virulome-17-22 genes, and > 20 key biofilm genes. MGE analysis showed four E. coli isolates (host: poultry, swine and cattle) harbouring composite transposons with ARGs/virulence genes (blaTEM, dfr, qnr/nleB, tir, eae,and esp) with the potential for horizontal transfer. MLST analyses revealed the presence of ST937 and ST3107 in both livestock/poultry and their handlers. Phylogenomic analyses with global E. coli isolates (human/livestock/poultry hosts) showed close relatedness with strains originating from different parts of the world (the United States, China, etc.). CONCLUSION: The current study emphasizes the circulation of strains of pathogenic sequence types of clinical importance, carrying a diverse repertoire of genes associated with antibiotic resistance, biofilm formation and virulence properties in animal settings, necessitating immediate mitigation measures to reduce the risk of spread across the biosphere.

Bacillus halotolerans SW207 alleviates enterotoxigenic Escherichia coli-induced inflammatory responses in weaned piglets by modulating the intestinal epithelial barrier, the TLR4/MyD88/NF-κB pathway, and intestinal microbiota.

Enterotoxigenic Escherichia coli (ETEC) is one of the major pathogens contributing to piglet diarrhea, with significant implications for both piglet health and the economic aspects of the livestock industry. SW207 is an isolate of Bacillus halotolerans isolated from the cold- and disease-resistant Leixiang pigs in Northeastern China. We have discovered that SW207 can survive in the pig's gastrointestinal fluid and under conditions of high bile salt concentration, displaying potent antagonistic activity against ETEC. In this study, we established a weaned piglet diarrhea model infected with ETEC to investigate the role of SW207 in preventing diarrhea and improving intestinal health. Results indicate that SW207 upregulates the expression of tight junction proteins, including claudin-1, occludin, and zonula occludens-1, at both the transcriptional and translational levels. Furthermore, SW207 reduces serum endotoxin, D-lactic acid, and various oxidative stress markers while enhancing piglet mechanical barrier function. In terms of immune barrier, SW207 suppressed the activation of the TLR4/MyD88/NF-κB pathway, reducing the expression of various inflammatory factors and upregulating the expression of small intestine mucosal sIgA. Concerning the biological barrier, SW207 significantly reduces the content of E. coli in the intestines and promotes the abundance of beneficial bacteria, thereby mitigating the microbiota imbalance caused by ETEC. In summary, SW207 has the potential to prevent weaned piglet diarrhea caused by ETEC, alleviate intestinal inflammation and epithelial damage, and facilitate potential beneficial changes in the intestinal microbiota. This contributes to elucidating the potential mechanisms of host-microbe interactions in preventing pathogen infections.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC) has consistently been one of the significant pathogens causing mortality in weaned piglets in pig farming. The industry has traditionally relied on antibiotic administration to control ETEC-induced diarrhea. However, the overuse of antibiotics has led to the emergence of drug-resistant zoonotic bacterial pathogens, posing a threat to public health. Therefore, there is an urgent need to identify alternatives to control pathogens and reduce antibiotic usage. In this study, we assessed the protective effect of a novel probiotic in a weaned piglet model infected with ETEC and analyzed its mechanisms both in vivo and in vitro. The study results provide theoretical support and reference for implementing interventions in the gut microbiota to alleviate early weaned piglet diarrhea and improve intestinal health.

Prevalence and molecular characterization of multi-resistant Escherichia coli isolates from clinical bovine mastitis in China.

Different antibiotics are used to treat mastitis in dairy cows that is caused by Escherichia coli (E. coli). Antimicrobial resistance in food-producing animals in China has been monitored since 2000. Surveillance data have shown that the prevalence of multiresistant E. coli in animals has increased significantly. This study aimed to investigate the occurrence and molecular characteristics of resistance determinants in E. coli strains (n = 105) obtained from lactating cows with clinical bovine mastitis (CBM) in China. A total of 220 cows with clinical mastitis, which has swollen mammary udder with reduced and red or gangrenous milk, were selected from 5000 cows. The results showed 94.3% of the isolates were recognized as multidrug resistant. The isolates (30.5%) were positive for the class I integrase gene along with seven gene cassettes that were accountable for resistance to trimethoprim resistance (dfrA17, dfr2d and dfrA1), aminoglycosides resistance (aadA1 and aadA5) and chloramphenicol resistance (catB3 and catB2), respectively. The blaTEM gene was present in all the isolates, and these carried the blaCTX gene. A double mutation in gyrA (i.e., Ser83Leu and Asp87Asn) was observed in all fluoroquinolone-resistant isolates. In total, nine fluoroquinolone-resistant E. coli isolates were identified with five different types of mutations in parC. In four (44.4%) isolates, Ser458Ala was present in parE, and in all nine (9/9) fluoroquinolone-resistant isolates, Pro385Ala was present in gyrB. Meanwhile, fluoroquinolone was observed as highly resistant, especially in isolates with gyrA and parC mutations. In summary, the findings of this research recognize the fluoroquinolone resistance mechanism and disclose integron prevalence and ESBLs in E. coli isolates from lactating cattle with CBM.

Prevalence and characteristics of ESBL-producing Escherichia coli in clinically healthy pigs: implications for antibiotic resistance spread in livestock.

AIM: This study aimed to compare and characterize the resistance profile and the presence of extended-spectrum beta-lactamase (ESBL) related genes in Escherichia coli isolated from healthy finishing pigs fed with or without antibiotics in their diets. METHODS AND RESULTS: A total of 27 ceftiofur-resistant E. coli isolates were obtained from 96 healthy pigs. The antibiotic resistance profile was tested, and all 27 isolates were classified as multidrug-resistant (MDR). A high proportion of isolates were resistant to cephalosporins, ampicillin, ciprofloxacin, and tetracyclines. The ESBL production was observed in 85% of isolates by double-disc synergy test. The MDR-E. coli isolates harbored ESBL genes, such as blaTEM, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-8,25. In addition, other antibiotics resistance genes (ARGs) were also detected, such as sul2, ant(3″)-I, tetA, and mcr-1. The mobilization of the blaCTX-M gene was confirmed for nine E. coli isolates by conjugation assays. The presence of blaCTX-M on mobile genetic elements in these isolates was demonstrated by Southern blot hybridization, and the resistance to cephalosporins was confirmed in the transconjugants. Our results indicate the prevalence of CTX-M-producing E. coli strains harboring mobile genetic elements in the normal microbiota of healthy pigs. CONCLUSIONS: These findings highlight the significance of ESBL genes as a global health concern in livestock and the potential spread of antimicrobial resistance to other members of the gastrointestinal tract microbiota.

Whole-genome sequencing-based antimicrobial resistance and shedding dynamics of Escherichia coli isolated from calves before and after antimicrobial group treatments.

The fattening of calves is often associated with high antimicrobial use and the selection of antimicrobial resistance (AMR). The objective of this observational longitudinal study was to describe the AMR and strain dynamics, using whole-genome sequencing (WGS), of fecal Escherichia coli in a cohort of 22 calves. All calves received antimicrobial group treatments on Day (D) 1 (oxytetracycline, intramuscularly) and on D4 through D12 (doxycycline, in-feed). Additionally, eight calves received individual parenteral treatments between D7 and D59, including florfenicol, amoxicillin, marbofloxacin, and gamithromycin. Rectal swabs were collected from all calves on D1 (prior to treatment), D2, D9, and D82. The swabs were spread onto Enterobacterales-selective agar, and three E. coli colonies per plate were subjected to WGS. Out of 264 isolates across all calves and sampling times, 80 unique strains were identified, a majority of which harbored genes conferring resistance to tetracyclines, streptomycin, and sulfonamides. The diversity of strains decreased during the in-feed antimicrobial group treatment of the calves. On D82, 90% of isolates were strains that were not isolated at previous sampling times, and the median number per strain of AMR determinants to tetracyclines, florfenicol, ß-lactams, quinolones, or macrolides decreased compared to D9. Additionally, clonal dissemination of some strains represented the main transmission route of AMR determinants. In this study, WGS revealed important variations in strain diversity and genotypic AMR of fecal E. coli over time in calves subjected to group antimicrobial treatments. IMPORTANCE: The continued emergence and spread of antimicrobial resistance (AMR) determinants are serious global concerns. The dynamics of AMR spread and persistence in bacterial and animal host populations are complex and not solely driven by antimicrobial selection pressure. In calf fattening, both antimicrobial use and carriage prevalence of antimicrobial-resistant bacteria are generally recognized as high. This study provides new insights into the short-term, within-farm dynamics and transmission of AMR determinants in Escherichia coli from the dominant fecal flora of calves subjected to antimicrobial group treatments during the rearing period. The diversity of E. coli strains decreased over time, although, in contrast to previous observations in extended-spectrum ß-lactamase-producing Enterobacterales, the predominance of a few clones was not observed. The spread of AMR determinants occurred through the dissemination of clonal strains among calves. The median number per strain of AMR determinants conferring resistance to selected antimicrobials decreased toward the end of the rearing period.

Comparison of the safety and efficacy of the wild-type and lpxL/lpxM mutant inactivated vaccine against the avian pathogenic Escherichia coli O1, O2, and O78 challenge.

Avian pathogenic Escherichia coli (APEC) is primarily responsible for causing septicemia, pneumonitis, peritonitis, swollen head syndrome, and salpingitis in poultry, leading to significant losses in the poultry sector, particularly within the broiler industry. The removal of the lpxL and lpxM genes led to an eightfold decrease in the endotoxin levels of wild APEC strains. In this study, mutant strains of lpxL/lpxM and their O1, O2, and O78 wild-type strains were developed for an inactivated vaccine (referred to as the mutant vaccine and the wild-type vaccine, respectively), and the safety and effectiveness of these two prototype vaccines were assessed in white Leghorn chickens. Findings indicated that chickens immunized with the mutant vaccine showed a return of appetite sooner post-immunization and experienced earlier disappearance of nodules at the injection site compared to those immunized with the wild-type vaccine. Pathological examinations revealed that lesions were still present in the liver, lung, and injection site in chickens vaccinated with the wild-type vaccine 14 days post-vaccination (dpv), whereas no lesions were found in chickens vaccinated with the mutant vaccine at 14 dpv. There were no significant differences in antibody levels on the challenge day or in mortality or lesion scores between challenged birds immunized with either the mutant vaccine or the wild-type vaccine at the same dose. In this study, the safety of a single dose or overdose of the mutant vaccine and its efficacy at one dose were evaluated in broilers, and the results showed that the mutant vaccine had no adverse effects on or protected vaccinated broilers from challenge with the APEC O1, O2, or O78 strains. These results demonstrated that the mutant polyvalent inactivated vaccine is a competitive candidate against APEC O1, O2, and O78 infection compared to the wild-type vaccine.