Poultry Isolates Research Articles

Biofilm Formation and Antimicrobial Susceptibility of E. coli Associated With Colibacillosis Outbreaks in Broiler Chickens From Saskatchewan.

The global poultry industry has grown to the extent that the number of chickens now well exceeds the number of humans on Earth. Escherichia coli infections in poultry cause significant morbidity and economic losses for producers each year. We obtained 94 E. coli isolates from 12 colibacillosis outbreaks on Saskatchewan farms and screened them for antimicrobial resistance and biofilm formation. Fifty-six isolates were from broilers with confirmed colibacillosis, and 38 isolates were from healthy broilers in the same flocks (cecal E. coli). Resistance to penicillins, tetracyclines, and aminoglycosides was common in isolates from all 12 outbreaks, while cephalosporin resistance varied by outbreak. Most E. coli were able to form biofilms in at least one of three growth media (1/2 TSB, M63, and BHI broth). There was an overall trend that disease-causing E. coli had more antibiotic resistance and were more likely to form biofilms in nutrient-rich media (BHI) as compared to cecal strains. However, on an individual strain basis, there was no correlation between antimicrobial resistance and biofilm formation. The 21 strongest biofilm forming strains consisted of both disease-causing and cecal isolates that were either drug resistant or susceptible. Draft whole genome sequencing indicated that many known antimicrobial resistance genes were present on plasmids, with disease-causing E. coli having more plasmids on average than their cecal counterparts. We tested four common disinfectants for their ability to kill 12 of the best biofilm forming strains. All disinfectants killed single cells effectively, but biofilm cells were more resistant, although the difference was less pronounced for the disinfectants that have multiple modes of action. Our results indicate that there is significant diversity and complexity in E. coli poultry isolates, with different lifestyle pressures affecting disease-causing and cecal isolates.

Antimicrobial resistance and genetic diversity of Staphylococcus aureus collected from livestock, poultry and humans

Staphylococcus aureus is one of the most prominent nosocomial, community and farm acquired bacterial infections among animals and human populations. The main purpose of our study was to identify and characterize antimicrobial resistance (AMR) among Staphylococcus aureus isolated from livestock, poultry and humans and to further identify the associated genes. Staphylococcus aureus isolates from human, bovine, swine and poultry were collected from different laboratories across the United States collected between 2003 and 2016. Antimicrobial susceptibility testing for 13 antimicrobials was performed and conventional PCR was used to detect the presence of the nuc gene, mec gene, and to detect int1 gene. Associations between the presence of mec and intl and specific AMR profiles were determined. Antimicrobial resistance was detected in all four host categories, with the highest overall rates found in swine, 100% resistant to tetracycline, 88% to penicillin and 64% clindamycin. The next highest was found among humans with 81.6% of isolates resistant to penicillin followed by 44% to clindamycin and 43% to erythromycin. Among beef cattle isolates, 63.2% were resistant to penicillin, 15.8% resistant to clindamycin and 15.8% to erythromycin. No isolates from any of the hosts were resistant to linezolid. Among poultry isolates, the highest AMR was found to clindamycin, followed by erythromycin and penicillin. Among dairy cattle, highest resistance was found to penicillin, followed by chloramphenicol and gentamicin. Dairy cattle were the only host category with isolates that are resistant to trimethoprim-sulfamethoxazole. Of the 220 isolates detected by latex agglutination, 217 were confirmed to be S. aureus via PCR of the nuc gene, 21.4% were positive for the mecA gene. Swine had the highest prevalence of the mecA gene, followed by humans, poultry and beef cattle. This study has demonstrated a high occurrence of penicillin resistance among all S. aureus isolates. There were differences observed between host species with tetracycline resistance being the highest among swine isolates and clindamycin being highest in poultry isolates. No detection of oxacillin resistance was found in isolates from dairy cattle but was found in isolates from all of the other host species, 94% of which contained the mecA gene.

ESBL Genes, blaTEM, blaOXA, and blaSHV in Poultry Gut Bacteria: An Endemic Public Health Burden in Bangladesh

Background: In Bangladesh, the poultry industry contributes a significant role in the food sector. A vast amount of antibiotic is used as prophylaxis and growth promotion factors in farms. These unconcerned uses of antibiotics ultimately generate resistant bacteria affecting substantial adverse consequences on human health. Extended-spectrum b-lactamase (ESBL) genes are responsible for inactivation of antibiotics containing b-lactam ring, namely, penicillin, cephalosporins, monobactams, and carbapenems. Objectives: This study was designed to analyse the distribution of three ESBL genes and associated antimicrobial susceptibility profile of poultry gut bacteria. Methods: This study was designed to analsze the distribution of three ESBL genes and associated antimicrobial susceptibility profile of poultry gut bacteria. Poultry feces were collected and cultured on cysteine lactose electrolyte deficient (CLED) agar and Salmonella-Shigella (SS) agar to differentiate various isolates based on colony characteristics. Identification of the isolates was made by convention biochemical tests, analytical profile index (API-20E), and 16S rRNA sequence analysis. Antibiotic susceptibility test was done by disc diffusion method using 17 antibiotics from seven groups. Subsequently, polymerase chain reaction (PCR) was employed with a specific primer to identify respective ESBL genes (blaTEM, blaSHV, blaOXA). All data were analysed by SPSS. Results: A total of 113 isolates were identified from 85 poultry feces tested. Most of the bacteria belonged to Enterobacteriaceae family, notably Proteus spp., E. coli, Klebsiella spp., Salmonella spp., and Enterobacter spp. Different bacteria were detected, namely, Kurthia populi, Cronobactersp, and Eikenella corrodens. Most of the poultry isolates were resistant against more than one group of antibiotics. ESBL gene, blaTEM gene was identified most frequently (53.9%), followed by blaOXA (52.2%), and blaSHV (23%). Higher phenotypic resistance was observed in isolates carrying ESBL genes. Conclusion: This study revealed a very high frequency of three ESBL genes with their phenotypic resistance- capacities in Bangladeshi poultry gut microbiota. Excess uses of antibiotics in local poultry farms may result in the emergence of antibiotic resistance that is imposing public health threatening in Bangladesh. Bangladesh Med Res Counc Bull 2021; 47(2): 165-174

Occurrence, antimicrobial resistance and virulence properties of thermophilic Campylobacter coli originating from two different poultry settings

Thermophilic Campylobacter's (Campylobacter coli and Campylobacter jejuni) are usually zoonotic and spread by raw or undercooked meat especially poultry meat, unpasteurized milk, direct animal contact, and contaminated drinking water. In this study, 385 samples from commercial and backyard poultry settings were screened for the presence of Campylobacter spp. which revealed an isolation rate of 3.9% with higher isolation from the chicken caecum (15/113, 13.27%). On antimicrobial susceptibility testing, the highest resistance was observed for tetracycline (11/13, 84.61%) followed by macrolides (8/13, 61.53%), β-lactams (7/13, 53.84%), fluoroquinolones (5/13, 38.45%) and sulphonamide (4/13, 30.76%). Isolates from backyard poultry showed resistance to tetracycline (100%), β-lactam (66.66%), fluoroquinolone (25%), and macrolide (33.33%). Isolates from commercial poultry showed resistance to tetracycline (80%), macrolides (70%), β-lactams (51.66%), fluoroquinolones (42.5%) and sulphonamides (40%). Among the isolates, 92.30% were multi-drug resistant (MDR). Overall, tet(O) gene was observed in 6/11(54.55%) tetracycline resistant isolates. Notably, tet O gene was found in 6/8(75%) isolates from commercial poultry farms and it was absent in backyard poultry isolates. Similarly 23SrRNA and blaOXA-61 genes were detected in 4/8(50%) and 2/8(25%) macrolide and ampicillin resistant isolates, respectively. Among virulence genes, cdtC gene was noticed in all the Campylobacter isolates (100%), cadF in 93.33%, iamA in 80%, flaA in 73.33%, cdtA in 13.33% and, cdtB in 6.66% isolates while dnaJ, ciaB and pldA genes were not detected in any of the isolate. The Campylobacter isolates possessing antimicrobial resistance and virulence genes pose a serious public health threat through food chain.

Clonal relationship between multidrug-resistant Escherichia coli ST69 from poultry and humans in Lusaka, Zambia.

Multidrug-resistant (MDR) Escherichia coli in food animals such as chickens is an emerging public health concern in Zambia. Additionally, the country's high demand for poultry products necessitates further investigation into the link between poultry and human MDR E. coli. Twenty cefotaxime-resistant E. coli isolates collected from poultry in Lusaka, Zambia, were screened for multidrug resistance and sequenced on MiSeq and MinION platforms. Genomes were assembled de novo and compared with 36 previously reported cefotaxime-resistant E. coli isolates from inpatients at the University Teaching Hospital, Lusaka. All (20/20, 100%) poultry isolates exhibited resistance to ampicillin, chloramphenicol and doxycycline. Phylogenetic analysis and hierarchical clustering showed a high degree of genetic relatedness between E. coli O17:H18-ST69 from poultry and humans. The E. coli O17:H18-ST69 clone accounted for 4/20 (20%) poultry- and 9/36 (25%) human-associated isolates that shared two plasmids harboring 14 antimicrobial resistance (AMR) genes. However, comparison analysis showed that the isolates also had other AMR plasmids distinct for each niche. Our results suggested clonal transmission of MDR E. coli between poultry and humans, with the potential acquisition of niche-specific AMR plasmids. Thus, the control of MDR E. coli requires a One Health approach involving both human and animal health sectors.

Molecular detection and differentiation of Mycoplasma gallisepticum field isolates of poultry in Andhra Pradesh

Molecular detection and differentiation of Mycoplasma gallisepticum field isolates of poultry in Andhra Pradesh

Identification of Novel Phage Resistance Mechanisms in Campylobacter jejuni by Comparative Genomics.

Phages infecting Campylobacter jejuni are considered a promising intervention strategy at broiler farms, yet phage sensitivity of naturally occurring poultry isolates is not well studied. Here, we investigated phage sensitivity and identified resistance mechanisms of C. jejuni strains originating from Danish broilers belonging to the most prevalent MLST (ST) types. Determining plaque formation of 51 phages belonging to Fletchervirus or Firehammervirus showed that 21 out of 31 C. jejuni strains were susceptible to at least one phage. While C. jejuni ST-21 strains encoded the common phase variable O-methyl phosphoramidate (MeOPN) receptor of the Fletchervirus and were only infected by these phages, ST-45 strains did not encode this receptor and were exclusively infected by Firehammervirus phages. To identify internal phage resistance mechanism in ST-21 strains, we performed comparative genomics of two strains, CAMSA2002 sensitive to almost all Fletchervirus phages and CAMSA2038, resistant to all 51 phages. The strains encoded diverse clustered regularly interspaced short palindromic repeats (CRISPR) spacers but none matched the tested phages. Sequence divergence was also observed in a predicted SspE homolog and putative restriction modification systems including a methyl-specific McrBC endonuclease. Furthermore, when mcrB was deleted, CAMSA2038 became sensitive to 17 out of 43 phages, three being Firehammervirus phages that otherwise did not infect any ST-21 strains. Yet, 16 phages demonstrated significantly lower efficiencies of plating on the mcrB mutant suggesting additional resistance mechanism still restricting phage propagation in CAMSA2038. Thus, our work demonstrates that C. jejuni isolates originating from broilers may have acquired several resistance mechanisms to successfully prevent phage infection in their natural habitat.

1296. Genomic Analysis of Human-Associated Outbreak Caused by Salmonella enterica serovar Infantis in Cali, Colombia

Abstract Background Salmonella enterica serovar Infantis is emerging as a leading cause of foodborne infections. The Colombian National Health Institute reported 263 Infantis among all received Salmonella(n=12,966) from 1997 to 2018 being the 5th most frequent serovar in human clinical samples during this period. In this study, Infantis outbreak-associated isolates were sequenced and collectively analysed with global strains from different sources to compare their genomic content and phylogeny Figure 1. Genomic Comparisons Methods Between September 15-20 of 2019, 160 patients with gastroenteric symptoms resulting from the ingestion of a common food, arrived at Fundación Valle del Lilí. Salmonella spp were cultured from stool specimens of patients and 15 isolates were sent to the Genomic Unit of Agrosavia for whole-genome sequencing. Genomic DNA was sequenced using an Illumina®MiSeqX10 platform. Genome assembly was performed with standardized bioinformatics pipelines and in-silico serotyping with SISTR. Genomic comparisons included newly-sequenced isolates of Infantis(n=6) from a Colombian poultry farms as well as datasets from Patric(n=441) and EnteroBase(n=54). Results The 15 outbreak isolates were identified by in silico serotyping as S. Infantis, these isolates showed phenotypic sensitivity to all tested antibiotics except by tetracycline. Antimicrobial resistance plasmids IncFIBKpn3, IncX4 and the host-environment persistence plasmid pSL483 were only detected in 2 outbreak-related and 1 in poultry isolates. Out of a total of 511 high-quality sequences, ST32 was the most prevalent and were phylogenetically grouped into a single non-host specific clade. Outbreak-isolates were included in a monophyletic group with some genomes from the US and Chile, suggesting that the parent strain could have originated in those countries. Conclusion The magnitude of the outbreak(288 informed-cases) evidenced a high-virulent potential of outbreak-isolates. Routine sequencing of S.enterica and availability of genomes in Colombia can improve outbreak detection and resolution in the future. The presence of plasmids, although with low frequency, suggests a risk of the appearance of resistant clones. Disclosures All Authors: No reported disclosures

Comparative occurrence of ESBL/AmpC beta-lactamase-producing Escherichia coli and Salmonella in contract farm and backyard broilers.

The present study was conducted to detect the occurrence of beta-lactamase and biofilm-producing Escherchia coli and Salmonella in apparently healthy broiler birds reared in household and contract poultry farms. In total, 150 cloacal swabs were collected from apparently healthy broiler birds of various age groups reared in backyard (n = 100) and contract farms (n = 50) in West Bengal (India). The isolation rate of ESBL producers was significantly more (P < 0·05) reared in contract poultry farms than those reared in backyard. Majority of the E.coli isolates possessed blaCTX-M followed by blaSHV and blaTEM . Majority of the Salmonella strains possessed blaTEM followed by blaCTX-M , and no blaSHV was detected. The selected sequences of the PCR products were found cognate with blaCTX-M-1 , blaCTX-M-2 , blaCTX-M-9 , blaCTX-M-14 , blaCTX-M-15 , blaSHV-12 and blaTEM-52 . The study detected 46·8% of E.coli and 42·5% of Salmonella strains as biofilm producers. Beta-lactamase-producing Enterobacteriaceae strains showed resistance against tetracycline, chloramphenicol, doxycycline, co-trimoxazole and ampicillin and sensitivity to imipenem-EDTA, colistin and gentamicin. The study revealed the partial clonal relationship of ESBL sequences possessed by the poultry isolates of the present study and local clinical isolates available in the database. The study made consumer awareness about careful handling of live birds or poultry meat to avoid the zoonotic transmission of antimicrobial-resistant Enterobacteriaceae.

Potential sources and characteristic occurrence of mobile colistin resistance (mcr) gene-harbouring bacteria recovered from the poultry sector: a literature synthesis specific to high-income countries.

Understanding the sources, prevalence, phenotypic and genotypic characteristics of mcr gene-harbouring bacteria (MGHB) in the poultry sector is crucial to supplement existing information. Through this, the plasmid-mediated colistin resistance (PMCR) could be tackled to improve food safety and reduce public health risks. Therefore, we conducted a literature synthesis of potential sources and characteristic occurrence of MGHB recovered from the poultry sector specific to the high-income countries (HICs). Colistin (COL) is a last-resort antibiotic used for treating deadly infections. For more than 60 years, COL has been used in the poultry sector globally, including the HICs. The emergence and rapid spread of mobile COL resistance (mcr) genes threaten the clinical use of COL. Currently, ten mcr genes (mcr-1 to mcr-10) have been described. By horizontal and vertical transfer, the mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, and mcr-9 genes have disseminated in the poultry sector in HICs, thus posing a grave danger to animal and human health, as harboured by Escherichia coli, Klebsiella pneumoniae, Salmonella species, and Aeromonas isolates. Conjugative and non-conjugative plasmids are the major backbones for mcr in poultry isolates from HICs. The mcr-1, mcr-3 and mcr-9 have been integrated into the chromosome, making them persist among the clones. Transposons, insertion sequences (IS), especially ISApl1 located downstream and upstream of mcr, and integrons also drive the COL resistance in isolates recovered from the poultry sector in HICs. Genes coding multi-and extensive-drug resistance and virulence factors are often co-carried with mcr on chromosome and plasmids in poultry isolates. Transmission of mcr to/among poultry strains in HICs is clonally unrestricted. Additionally, the contact with poultry birds, manure, meat/egg, farmer’s wears/farm equipment, consumption of contaminated poultry meat/egg and associated products, and trade of poultry-related products continue to serve as transmission routes of MGHB in HICs. Indeed, the policymakers, especially those involved in antimicrobial resistance and agricultural and poultry sector stakeholders-clinical microbiologists, farmers, veterinarians, occupational health clinicians and related specialists, consumers, and the general public will find this current literature synthesis very useful.

Occurrence of Escherichia coli producing extended spectrum β-lactamases in food-producing animals.

Multidrug resistance due to the production of extended-spectrum beta-lactamases (ESBLs) is a major problem in human as well as in veterinary medicine. These strains appear in animal and human microbiomes and can be the source of infection both in animal and in human healthcare, in accordance with the One Health theorem. In this study we examined the prevalence of ESBL-producing bacteria in food-producing animals. We collected 100 porcine and 114 poultry samples to examine the prevalence of ESBL producers. Isolates were identified using the MALDI-TOF system and their antibiotic susceptibility was tested using the disk diffusion method. ESBL gene families and phylogroups were detected by polymerase chain reactions. The prevalence of ESBL producers was relatively high in both sample groups: 72 (72.0%) porcine and 39 (34.2%) poultry isolates were ESBL producers. Escherichia coli isolates were chosen for further investigations. The most common ESBL gene was CTX-M-1 (79.3%). Most of the isolates belong to the commensal E. coli phylogroups. The porcine isolates could be divided into three phylogroups, while the distribution of the poultry isolates was more varied. In summary, ESBL-producing bacteria are prevalent in the faecal samples of the examined food-producing animals, with a dominance of the CTX-M-1 group enzymes and commensal E. coli phylogroups.

Functional annotation unravels probiotic properties of a poultry isolate, Bacillus velezensis CGS1.1

Probiotics are an eco-friendly and prospective substitute for antibiotic growth promoters. The present investigation aimed to dissect the probiotic attributes of a poultry isolate CGS1.1 by integrating genomics and phenotypic traits. The strain exhibited bile salt hydrolase activity and ∼73% survival in simulated gastrointestinal juices. It displayed notable aggregation, cell surface hydrophobicity, biofilm formation, and antimicrobial activity against gram-positive and gram-negative bacterial pathogens. Interestingly, the strain CGS1.1 completely inhibited the growth of E. coli and S. enterica after 24 h in liquid co-culture assay. The strain CGS1.1 effectively hydrolyzes starch, cellulose, pectin, tributyrin, casein, and phytate. The in silico genome analysis revealed its identity as Bacillus velezensis. Genome annotation revealed the presence of genes encoding the aforementioned probiotic properties. Furthermore, CGS1.1 was found to be non-hemolytic and sensitive to major classes of antibiotics. No potent virulent factor or toxin encoding genes could be predicted in CGS1.1 genome signifying its safety. The results demonstrate the potential of CGS1.1 as a probiotic to promote chicken growth and control pathogens.

A Pigeon-Derived Sub-Genotype XXI.1.2 Newcastle Disease Virus from Bangladesh Induces High Mortality in Chickens.

Newcastle disease virus (NDV) is a significant pathogen of poultry; however, variants also affect other species, including pigeons. While NDV is endemic in Bangladesh, and poultry isolates have been recently characterized, information about viruses infecting pigeons is limited. Worldwide, pigeon-derived isolates are commonly of low to moderate virulence for chickens. Here, we studied a pigeon-derived NDV isolated in Bangladesh in 2010. To molecularly characterize the isolate, we sequenced its complete fusion gene and performed a comprehensive phylogenetic analysis. We further studied the biological properties of the virus by estimating mean death time (MDT) and by experimentally infecting 5-week-old naïve Sonali chickens. The studied virus clustered in sub-genotype XXI.1.2 with NDV from pigeons from Pakistan isolated during 2014–2018. Deduced amino acid sequence analysis showed a polybasic fusion protein cleavage site motif, typical for virulent NDV. The performed in vivo pathogenicity testing showed a MDT of 40.8 h, and along with previously established intracerebral pathogenicity index of 1.51, these indicated a velogenic pathotype for chickens, which is not typical for pigeon-derived viruses. The experimental infection of chickens resulted in marked neurological signs and high mortality starting at 7 days post infection (dpi). Mild congestion in the thymus and necrosis in the spleen were observed at an advanced stage of infection. Microscopically, lymphoid depletion in the thymus, spleen, and bursa of Fabricius were found at 5 dpi, which progressed to severe in the following days. Mild to moderate proliferation of glial cells was noticed in the brain starting at 2 dpi, which gradually progressed with time, leading to focal nodular aggregation. This study reports the velogenic nature for domestic chickens of a pigeon-derived NDV isolate of sub-genotype XXI.1.2. Our findings show that not all pigeon-derived viruses are of low virulence for chickens and highlight the importance of biologically evaluating the pathogenicity of NDV isolated from pigeons.

High-Resolution Comparative Genomics of Salmonella Kentucky Aids Source Tracing and Detection of ST198 and ST152 Lineage-Specific Mutations

Non-typhoidal Salmonella (NTS) is a major cause of foodborne illness globally. Salmonella Kentucky is a polyphyletic NTS serovar comprised of two predominant multilocus sequence types (STs): ST152 and ST198. Epidemiological studies have revealed that ST152 is most prevalent in US poultry whereas ST198 is more prevalent in international poultry. Interestingly, ST152 is sporadically associated with human illness, whereas ST198 is more commonly associated with human disease. The goal of this study was to develop a better understanding of the epidemiology of ST198 and ST152 in WA State. We compared the antimicrobial resistance phenotypes and genetic relationship, using pulsed-field gel electrophoresis, of 26 clinical strains of S. Kentucky isolated in Washington State between 2004 and 2014, and 140 poultry-associated strains of S. Kentucky mostly recovered from the northwestern USA between 2004 and 2014. We also sequenced whole genomes of representative human clinical and poultry isolates from the northwestern USA. Genome sequences of these isolates were compared with a global database of S. Kentucky genomes representing 400 ST198 and 50 ST152 strains. The results of the phenotypic, genotypic, and case report data on food consumption and travel show that human infections caused by fluoroquinolone-resistant (FluR) S. Kentucky ST198 in WA State originated from outside of North America. In contrast, fluoroquinolone-susceptible (FluS) S. Kentucky ST198 and S. Kentucky ST152 infection have a likely domestic origin, with domestic cattle and poultry being the potential sources. We also identified lineage-specific non-synonymous single nucleotide polymorphisms (SNPs) that distinguish ST198 and ST152. These SNPs may provide good targets for further investigations on lineage-specific traits such as variation in virulence, metabolic adaptation to different environments, and potential for the development of intervention strategies to improve the safety of food.

Detection of Quorum Sensing N-Acyl-Homoserine Lactone Molecules Produced by Different Resistant Klebsiella pneumoniae Isolates Recovered from Poultry and Different Environmental Niches.

This study aimed to detect and identify the N-acyl-homoserine lactones molecules (AHLs) produced by different resistant Klebsiella pneumoniae isolates recovered from poultry and environmental samples using a modified validated high-performance liquid chromatography method. A total of 56 K. pneumoniae isolates were recovered, investigated for their antibiotic susceptibility, and screened for AHLs production using the Agrobacterium tumefaciens NTL4 biosensor system and a validated high-performance liquid chromatography method. The results revealed the detection of different short- and long-chain AHLs molecules among 39 K. pneumoniae isolates recovered from poultry and environmental samples. All environmental isolates produced nine peaks with retention times for C4-HSL, C6-HSL, C12-HSL, C8-HSL, C14-HSL, C8-oxo-HSL, C10-HSL, C6-oxo-HSL, and C7-HSL. The most quantifiable AHL signal molecules in poultry isolates were C4-HSL, C6-HSL, and C12-HSL. No statistical correlation between the AHL-producing ability of K. pneumoniae isolates and antibiotic resistance was reported. To the best of our knowledge, this study provides the first detailed report on the detection and identification of AHLs in K. pneumoniae isolates recovered from poultry and environmental samples. Furthermore, it provides a new insight available tool other than LC-MS/MS for detection and identification of AHL molecules.

Salmonella Newport outbreak in Brazilian parrots: confiscated birds from the illegal pet trade as possible zoonotic sources.

Salmonella has long been linked to zoonotic risks, including exotic pets. Parrots are popular pets, and we here describe a salmonellosis outbreak involving Blue-fronted Amazon parrots (Amazona aestiva) confiscated from the illegal pet trade in Brazil. High mortality was observed during the rehabilitation for which the causative agent was identified by cultures, and VITEK®2 GN identification card as Salmonella enterica. Genome sequencing of two isolates revealed serovar Newport ST45. The isolates were resistant to aminoglycosides and fluoroquinolones, and genomic analyses detected characteristic Salmonella Pathogenicity Islands (SPIs) and virulence factors. A phylogenetic analysis with other 275 S. Newport ST45 from different international sources showed clustering with poultry and vegetables isolates and closely related clades of intermingled animal, human, food/environmental isolates from different countries (Tables S1 and S2). The virulent profiles and phylogenetic connection to multiple sources bring the attention to the non-host specificity of these strains highlighting the zoonotic potential in the illegal wildlife trade for companion animals.

Epitope Peptide-Based Predication and Other Functional Regions of Antigenic F and HN Proteins of Waterfowl and Poultry Avian Avulavirus Serotype-1 Isolates From Uganda.

Uganda is a Newcastle disease (ND) endemic country where the disease is controlled by vaccination using live LaSota (genotype II) and I2 (genotype I) vaccine strains. Resurgent outbreak episodes call for an urgent need to understand the antigenic diversity of circulating wild Avian Avulavirus serotype-1 (AAvV-1) strains. High mutation rates and the continuous emergence of genetic and antigenic variants that evade immunity make non-segmented RNA viruses difficult to control. Antigenic and functional analysis of the key viral surface proteins is a crucial step in understanding the antigen diversity between vaccine lineages and the endemic wild ND viruses in Uganda and designing ND peptide vaccines. In this study, we used computational analysis, phylogenetic characterization, and structural modeling to detect evolutionary forces affecting the predicted immune-dominant fusion (F) and hemagglutinin-neuraminidase (HN) proteins of AAvV-1 isolates from waterfowl and poultry in Uganda compared with that in LaSota vaccine strain. Our findings indicate that mutational amino acid variations at the F protein in LaSota strain, 25 poultry wild-type and 30 waterfowl wild-type isolates were distributed at regions including the functional domains of B-cell epitopes or N-glycosylation sites, cleavage site, fusion site that account for strain variations. Similarly, conserved regions of HN protein in 25 Ugandan domestic fowl isolates and the representative vaccine strain varied at the flanking regions and potential linear B-cell epitope. The fusion sites, signal peptides, cleavage sites, transmembrane domains, potential B-cell epitopes, and other specific regions of the two protein types in vaccine and wild viruses varied considerably at structure by effective online epitope prediction programs. Cleavage site of the waterfowl isolates had a typical avirulent motif of 111GGRQGR'L117 with the exception of one isolate which showed a virulent motif of 111GGRQKR'F117. All the poultry isolates showed the 111GRRQKR'F117 motif corresponding to virulent strains. Amino acid sequence variations in both HN and F proteins of AAvV-1 isolates from poultry, waterfowl, and vaccine strain were distributed over the length of the proteins with no detectable pattern, but using the experimentally derived 3D structure data revealed key-mapped mutations on the surfaces of the predicted conformational epitopes encompassing the experimental major neutralizing epitopes. The phylogenic tree constructed using the full F gene and partial F gene sequences of the isolates from poultry and waterfowl respectively, showed that Ugandan ND aquatic bird and poultry isolates share some functional amino acids in F sequences yet do remain unique at structure and the B-cell epitopes. Recombination analyses showed that the C-terminus and the rest of the F gene in poultry isolates originated from prevalent velogenic strains. Altogether, these could provide rationale for antigenic diversity in wild ND isolates of Uganda compared with the current ND vaccine strains.

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.

First Outbreak of an H5N8 Highly Pathogenic Avian Influenza Virus on a Chicken Farm in Japan in 2020.

On 5 November 2020, a confirmed outbreak due to an H5N8 highly pathogenic avian influenza virus (HPAIV) occurred at an egg-hen farm in Kagawa prefecture (western Japan). This virus, A/chicken/Kagawa/11C/2020 (Kagawa11C2020), was the first HPAI poultry isolate in Japan in 2020 and had multiple basic amino acids—a motif conferring high pathogenicity to chickens—at the hemagglutinin cleavage site. Mortality of chickens was 100% through intravenous inoculation tests performed according to World Organization for Animal Health criteria. Phylogenetic analysis showed that the hemagglutinin of Kagawa11C2020 belongs to clade 2.3.4.4B of the H5 Goose/Guangdong lineage and clusters with H5N8 HPAIVs isolated from wild bird feces collected in Hokkaido (Japan) and Korea in October 2020. These H5N8 HPAIVs are closely related to H5N8 HPAIVs isolated in European countries during the winter of 2019–2020. Intranasal inoculation of chickens with 106 fifty-percent egg infectious doses of Kagawa11C2020 revealed that the 50% chicken lethal dose was 104.63 and the mean time to death was 134.4 h. All infected chickens demonstrated viral shedding beginning on 2 dpi—before clinical signs were observed. These results suggest that affected chickens could transmit Kagawa11C2020 to surrounding chickens in the absence of clinical signs for several days before they died.

Human Colonization with Extended-Spectrum Beta-Lactamase-Producing E. coli in Relation to Animal and Environmental Exposures in Bangladesh: An Observational One Health Study.

Background:Human exposure to intensively farmed livestock is a potential risk for transmission of antibiotic-resistant bacteria (ARB) but few studies have assessed the relative role of animal vs. environmental sources of ARB in low-resource community settings.Objectives:We conducted an observational study to compare ARB colonization and antibiotic-resistant gene prevalence and abundance in humans with high or low exposure to poultry in rural households, commercial poultry farms, and urban markets in Bangladesh.Methods:Extended-spectrum (ESBL)-producing and carbapenem-resistant E. coli were quantified in feces from adults with high or low poultry exposure (, respectively), poultry (), drinking water (), and wastewater () from 40 rural households, 40 poultry farms, and 40 urban markets.Results:ESBL-producing E. coli (ESBL-EC) prevalence was 67.5% (95% CI: 61.0, 74.0) in samples from adults, 68.0% (95% CI: 61.5, 74.5) in samples from poultry, and 92.5% (95% CI: 87.7, 97.3) in wastewater samples. Carbapenem-resistant E. coli prevalence was high in market wastewaters [30% (95% CI: 15.0, 45.0)] but low in humans (1%) and poultry (1%). Human, poultry, and wastewater isolates shared common resistance genes: , qnr, and . Human colonization was not significantly associated with exposure to poultry or setting (rural, farm, or market). Ninety-five percent of commercial poultry farms routinely administered antibiotics. Susceptibility tests were significantly different in household vs. farm and market poultry isolates for four of seven antibiotic classes. In human isolates, there were no differences except aminoglycoside resistance (16.4% high vs. 4.4% low exposure, ). Urban market wastewaters and poultry samples had significantly higher concentrations of ESBL-EC () and () compared with samples from farms and rural households.Discussion:ESBL-EC colonization was high in humans but not significantly associated with exposure to poultry. Bidirectional transmission of antibiotic resistance is likely between humans, poultry, and the environment in these community settings, underlining the importance of One Health mitigation strategies. https://doi.org/10.1289/EHP7670