Poultry Isolates Research Articles

Occurrence and antimicrobial resistance of Campylobacter jejuni isolated from poultry and hospitalized diarrheal patients in Pakistan

The leading human enteropathogen Campylobacter jejuni (C. jejuni) is recognized as a major public-health concern, due to emerging multidrug resistant (MDR) strains, and associated post-infectious complications. Therefore, the present study determined the occurrence of C. jejuni in diarrheal cases and poultry, in Khyber Pakhtunkhwa Province, Pakistan, and associated antimicrobial resistance patterns. In total, 840 samples were collected from clinical and different poultry sources between May 2019 to April 2020, to determine the occurrence, seasonal variations, bacterial loads in diverse poultry samples, age-related variations in human infections as well as associated antimicrobial resistance patterns of C. jejuni, highlighting its “One Health significance” at the human-food interface. Clinical samples (n = 240) from five age-groups of hospitalized diarrhoea patients, showed occurrence rate of 39 %, which peaked in the summer and was highest in children under 5. In poultry samples, 57 % (342/600) were positive for C. jejuni, varied from 45 % (109/240) in chicken meat to 65 % (233/360) in farm samples. Except frozen meat, C. jejuni-positive poultry samples showed seasonal contamination levels, with summer having the highest bacterial loads. In chicken meat, 2 % of positive samples (2/109) had bacterial loads <1 logCFU/g, while 20 % (22/109) exceeding 3log CFU/g. Grocery store samples showed lower bacterial contamination. All human and poultry isolates (n = 436) were resistant to nalidixic acid (100 %) and none to amikacin (0 %). Of these, 66 % showed multidrug resistance, with 9 % (38/436) resistant to panel of seven out of eight antibiotics tested. This study highlights the high occurrence of and concerns about MDR C. jejuni in Khyber Pakhtunkhwa, Pakistan, particularly in young children.

Genetic Characterization of Antibiotic-Resistant Staphylococcus spp. and Mammaliicoccus sciuri from Healthy Humans and Poultry in Nigeria.

Staphylococcus spp. poses a significant threat to human and animal health due to their capacity to cause a wide range of infections in both. In this study, resistance genes conferring antibiotic resistance in Staphylococcus spp. and Mammaliicoccus sciuri isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 61 Staphylococcus spp. isolates were obtained from urine, wounds, nasal and chicken fecal samples. Species identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was performed using the Kirby-Bauer method for 16 antibiotics. Whole-genome sequencing was used for characterization of the isolates. The 61 investigated isolates included Staphylococcus aureus, S. arlettae, M. sciuri, S. haemolyticus, and S. epidermidis. A total of 47 isolates (77%) belonged to human samples and 14 (23%) isolates were collected from poultry samples. All were phenotypically resistant to at least three antimicrobial(s). Multiple resistance determinants were detected in the human and poultry isolates analyzed. Phylogenetic analysis revealed close relatedness among the isolates within each species for S. arlettae, M. sciuri, and S. haemolyticus, respectively. This study delivered comprehensive genomic insights into antibiotic-resistant Staphylococcus species and M. sciuri isolates from human and poultry sources in Edo state, Nigeria, from a One Health perspective.

In Vitro Efficacy of Silver Carbene Complexes, SCC1 and SCC22, Against Some Enteric Animal Pathogens

Silver carbene complexes (SCCs), a group of novel silver-based compounds capable of gradually ‎releasing ‎silver ions, have shown significant antimicrobial activity against a wide range of ‎bacterial pathogens mainly ‎isolated from human cases. The antimicrobial activity against ‎animal isolated pathogens has yet been done. ‎The in vitro efficacy of two SCCs with different ‎carrier molecules (SCC1 with a methylated caffeine backbone ‎and SCC22 with a ‎dichloroimidazolium backbone) was investigated against three important animal and ‎human ‎pathogen species. SCC1 and SCC22 exhibited bacteriostatic and bactericidal effects against ‎multidrug ‎resistant Salmonella Typhimurium (poultry isolate), E. coli 843 and E. coli 1568 ‎‎(swine isolates), and the ‎poultry field isolates Salmonella Heidelberg, Salmonella Enteritidis, and ‎Salmonella Montevideo with MICs ‎and MBCs ranged from 16-21 µM (6-8 µg/mL) and 16-32 µM ‎‎(6-12 µg/mL), respectively. Clostridium ‎perfringens type A was sensitive to both SCC1 and ‎SCC22 with the MICs being 11 (4 µg/mL) and 21 µM ‎‎(8 µg/mL), respectively. These values were ‎comparable to the MICs and MBCs for silver acetate. The MBCs ‎against C. ‎perfringens was &gt;85 µM for ‎SCCs and &gt;192 µM for silver acetate (&gt;32 µg/mL for all compounds). Ten hours incubation ‎of C. ‎perfringens with ‎‎40 µg/mL of all three products showed down regulation of virulence genes plc and netB, ‎‎suggesting viable cells and silver can modulate the virulence. Treating the C. ‎perfringens with higher ‎concentration (100 ‎‎µg/mL) of each SCC for 10 hours inhibited more bacteria compared to the ‎untreated bacterial cells, however, no ‎differences in the ultrastructure of lysed bacteria were seen ‎and this concentration might not induce viable ‎but non-culturable (VBNC) state as suggested by ‎transmission electron microscopy findings. SCCs showed a ‎broad antimicrobial activity against ‎all bacterial species tested including multidrug resistant pathogens. Both ‎SCCs demonstrated ‎inhibitory effect against the Gram-positive anaerobic C. perfringens type A ‎which ‎could have a high accumulation capacity for silver ion. These data suggest that SCCs may ‎represent a ‎novel class of broad-spectrum antimicrobial agents, which may be used to reduce the ‎burden of pathogenic ‎bacteria in the gastrointestinal tract of poultry‎‎‎.

Pediatric urinary tract infections caused by poultry-associated Escherichia coli.

Escherichia coli is the leading cause of urinary tract infections (UTIs) in children and adults. The gastrointestinal tract is the primary reservoir of uropathogenic E. coli, which can be acquired from a variety of environmental exposures, including retail meat. In the current study, we used a novel statistical-genomic approach to estimate the proportion of pediatric UTIs caused by foodborne zoonotic E. coli strains. E. coli urine isolates were collected from DC residents aged 2 months to 17 years from the Children's National Medical Center Laboratory, 2013-2014. During the same period, E. coli isolates were collected from retail poultry products purchased from 15 sites throughout DC. A total of 52 urine and 56 poultry isolates underwent whole-genome sequencing, core genome phylogenetic analysis, and host-origin prediction by a Bayesian latent class model that incorporated data on the presence of mobile genetic elements (MGEs) among E. coli isolates from multiple vertebrate hosts. A total of 56 multilocus sequence types were identified among the isolates. Five sequence types-ST10, ST38, ST69, ST117, and ST131-were observed among both urine and poultry isolates. Using the Bayesian latent class model, we estimated that 19% (10/52) of the clinical E. coli isolates in our population were foodborne zoonotic strains. These data suggest that a substantial portion of pediatric UTIs in the Washington DC region may be caused by E. coli strains originating in food animals and likely transmitted via contaminated poultry meat.IMPORTANCEEscherichia coli UTIs are a heavy public health burden and can have long-term negative health consequences for pediatric patients. E. coli has an extremely broad host range, including humans, chickens, turkeys, pigs, and cattle. E. coli derived from food animals is a frequent contaminant of retail meat products, but little is known about the risk these strains pose to pediatric populations. Quantifying the proportion of pediatric UTIs caused by food-animal-derived E. coli, characterizing the highest-risk strains, and identifying their primary reservoir species could inform novel intervention strategies to reduce UTI burden in this vulnerable population. Our results suggest that retail poultry meat may be an important vehicle for pediatric exposure to zoonotic E. coli strains capable of causing UTIs. Vaccinating poultry against the highest-risk strains could potentially reduce poultry colonization, poultry meat contamination, and downstream pediatric infections.

Campylobacter occurrence and antimicrobial resistance profile in under five-year-old diarrheal children, backyard farm animals, and companion pets.

Campylobacteriosis disproportionately affects children under five in low-income countries. However, epidemiological and antimicrobial resistance (AMR) information at the children-animal interface is lacking. We hypothesized that Campylobacter is a major cause of enteritis in children in Ethiopia, and contact with animals is a potential source of transmission. The objective of the study was to determine Campylobacter occurrence and its AMR in children under five with diarrhea, backyard farm animals, and companion pets. Stool from 303 children and feces from 711 animals were sampled. Campylobacter was isolated through membrane filtration on modified charcoal cefoperazone deoxycholate agar plates under microaerobic incubation, and the technique showed to be feasible for use in regions lacking organized laboratories. Typical isolates were characterized with MALDI-TOF MS and multiplex PCR. Of 303 children, 20% (n = 59) were infected, with a higher proportion in the 6 to 11-month age group. Campylobacter occurred in 64% (n = 14) of dogs and 44% (n = 112) of poultry. Campylobacter jejuni was present in both a child and animal species in 15% (n = 23) of 149 households positive for Campylobacter. MICs using the gradient strip diffusion test of 128 isolates displayed resistance rates of 20% to ciprofloxacin and 11% to doxycycline. MICs of ciprofloxacin and doxycycline varied between C. coli and C. jejuni, with higher resistance in C. coli and poultry isolates. Campylobacter infection in children and its prevalent excretion from backyard poultry and dogs is a understudied concern. The co-occurrence of C. jejuni in animals and children suggest household-level transmission As resistance to ciprofloxacin and doxycycline was observed, therapy of severe campylobacteriosis should consider susceptibility testing. Findings from this study can support evidence-based diagnosis, antimicrobial treatment, and further investigations on the spread of AMR mechanisms for informed One Health intervention.

Prevalence and Molecular Detection of Virulence Genes among Multidrug-Resistant Escherichia coli from Human Clinical Samples and Poultry in Duhok City, Iraq

Abstract Background: Extended-spectrum beta-lactamase (ESBL)-producing, carbapenem-resistant Escherichia coli has increased virulence and multidrug resistance (MDR). Objectives: This study was designed to ascertain the frequency of some virulence factor genes, antibiotic susceptibility patterns, ESBLs, and MDR, focused on colistin-resistant E. coli strains of human and animal origin in Duhok city, Iraq. Materials and Methods: Between December 2020 and April 2021, a total of 150 E. coli isolates (110 from human clinical specimens and 40 from poultry cloacal swabs) were included in this study. The isolates underwent screening for antibiotic susceptibility, MDR, ESBL, and molecular detection of four virulence genes (fimA, cnfL, crL, and hlyA) was conducted using the polymerase chain reaction. Results: Urine specimens (77.2%) compared to blood, wound, vaginal swab, sputum, and semen from outpatients (71.8%). All strains from humans and poultry showed high resistance to ampicillin (86%–97%), ceftriaxone (74%–47%), tetracycline (72%–85%), ciprofloxacin (48%–97%), and colistin (17%–12%). The lowest levels of resistance were found for carbapenems (4%–4%), and the MDR for the isolates was 63%–93%. Apart from carbapenems, colistin-resistant isolates, especially those from poultry, exhibited significant resistance to other antibiotics, and 57% of these isolates being ESBL producers. Three virulence genes (fimA, cnfL, and crL) were highly prevalent (92%) in human isolates, with the crL gene being predominant (100%). Among poultry isolates, fimA was more prevalent (94%) while crL was less common (6%). Conclusion: The predominance of isolates of colistin-resistant poultry origin and the virulence of isolates of human E. coli origin indicate that both strains are currently experiencing an increase in antibiotic resistance.

Characterization of the prevalence of Salmonella in different retail chicken supply modes using genome-wide and machine-learning analyses

Salmonella is a foodborne pathogen that causes salmonellosis, of which retail chicken meat is a major source. However, the prevalence of Salmonella in different retail chicken supply modes and the threat posed to consumers remains unclear. The prevalence, serotype distribution, antibiotic resistance, and genomic characteristics of Salmonella in three supply modes of retail chicken (live poultry, frozen, and chilled) were investigated using whole-genome sequencing (WGS) and machine learning (ML). In this study, 480 retail chicken samples from live poultry, frozen, and chilled supply modes in Guangzhou from 2020 to 2021, as well as 253 Salmonella isolates (total isolation rate = 53.1 %), were collected. The prevalence of isolates in the live poultry mode (67.5 %, 81/120) was statistically higher than in the frozen (50.0 %, 120/240) and chilled (43.3 %, 52/120) (P < 0.05) modes. Serotype identification showed significant differences in the serotype distribution of Salmonella in different supply modes. S. Enteritis (46.7 %) and S. Indiana (14.2 %) were predominant in the frozen mode. S. Agona (23.5 %) and S. Saintpaul (13.6 %) were predominant in live poultry, while S. Enteritis (40.4 %) and S. Kentucky (17.3 %) were predominant in chilled mode. Antibiotic testing showed that frozen mode isolates were more resistant; the multidrug-resistant (MDR) rate of isolates in the frozen mode reached 91.8 %, significantly higher than in the chilled (86.5 %) and live (74.1 %) (P < 0.05) modes. WGS was performed on 155 top serotypes (S. Enteritidis, S. Kentucky, S. Indiana, and S. Agona). The antibiotic resistance gene analysis showed that the abundance and carrying rate of antibiotic resistance genes of Salmonella in the frozen mode (54 types, 16.1 %) were significantly higher than in other modes (live poultry: 36 types, 9.4 %, P < 0.05; chilled: 31 types, 11.6 %). The blaNDM-1 and blaNDM-9 genes encoding carbapenem resistance were found in frozen mode isolates on a complex transposon consisting of TnAS3-IS26. Virulence factors and plasmid replicons were abundant in the studied frozen mode isolates. In addition, single nucleotide polymorphism (SNP) phylogenetic tree results showed that in the frozen supply mode, the S. Enteritidis clonal clade continued to contaminate retail chicken meat and was homologous to S. Enteritidis strains found in farm chicken embryos, slaughterhouse chicken carcasses, and patients from hospitals in China (SNP 0 = 10). Notably, the pan-genome-based ML model showed that characteristic genes in frozen and live poultry isolates differed. The narZ gene was a key characteristic gene in frozen isolates, encoding nitrate reductase, relating to anaerobic bacterial growth. The ydgJ gene is a key characteristic gene in the live mode and encodes an oxidoreductase related to oxidative function in bacteria. The high prevalence of live poultry mode Salmonella and the transmission of frozen mode MDR Salmonella in this study pose serious risks to food safety and public health, emphasizing the importance of improving disinfection and cold storage measures to reduce Salmonella contamination and transmission. In conclusion, the continued surveillance of Salmonella across different supply models and the development of an epidemiological surveillance system based on WGS is necessary.

Phenotypic and molecular characterisation of Salmonella spp. isolates in healthy poultry

ABSTRACT 1. Epidemiological surveillance of Salmonella spp. serves as a primary tool for maintaining the health of poultry flocks. Characterising circulating serotypes is crucial for implementing control and prevention measures. This study conducted phenotypic and molecular characterisation of S. enterica Pullorum, S. enterica Heidelberg, and S. enterica Corvalis isolated from broiler chickens during slaughtering. 2. All strains were susceptible to gentamicin, neomycin and norfloxacin. However, resistance rates exceeded 50% for ciprofloxacin and tiamulin, irrespective of the serotype. Approximately 64% of strains were classified as multidrug-resistant, with S. enterica Heidelberg strains exhibiting significantly higher overall resistance. The isolates demonstrated the ability to adhere and produce biofilm at a minimum of three temperatures, with S. enterica Pullorum capable of biofilm production at all temperatures encountered during poultry rearing. 3. Each strain possessed between two and seven different virulence-associated genes. Genetic similarity, as indicated by pulsed field gel electrophoresis, exceeded 90% for all three serotypes and strains were classified in the R5 ribotype by PCR, regardless of serotype. Sequencing revealed high similarity among all strains, with homology ranging from 99.61 to 100% and all were classified to a single cluster. 4. The results suggested a clonal relationship among the strains, indicating the possible circulation of a unique clonal group of S. enterica Pullorum in the southern region of Brazil.

A novel variant in Salmonella genomic island 1 of multidrug-resistant Salmonella enterica serovar Kentucky ST198.

Salmonella enterica serovar Kentucky ST198 is a major health threat due to its resistance to ciprofloxacin and several other drugs, including third-generation cephalosporins. Many drug-resistant genes have been identified in the Salmonella genomic island 1 variant K (SGI1-K). In this study, we investigated the antimicrobial resistance (AMR) profile and genotypic relatedness of two isolates of ciprofloxacin-resistant (CIPR) S. Kentucky ST198 from poultry in Northeastern Thailand. We successfully assembled the complete genomes of both isolates, namely SSSE-01 and SSSE-03, using hybrid de novo assembly of both short- and long-read sequence data. The complete genomes revealed their highly similar genomic structures and a novel variant of SGI1-K underlying multidrug-resistant (MDR) patterns, including the presence of blaTEM-1b, which confers resistance to beta-lactams, including cephalosporins and lnu(F) which confers resistance to lincomycin and other lincosamides. In addition, the chromosomal mutations in the quinolone resistance-determining region (QRDR) were found at positions 83 (Ser83Phe) and 87 (Asp87Asn) of GyrA and at positions 57 (Thr57Ser) and 80 (Ser80Ile) of ParC suggesting high resistance to ciprofloxacin. We also compared SSSE-01 and SSSE-03 with publicly available complete genome data and revealed significant variations in SGI1-K genetic structures and variable relationships to antibiotic resistance. In comparison to the other isolates, SGI1-K of SSSE-01 and SSSE-03 had a relatively large deletion in the backbone, spanning from S011 (traG∆) to S027 (resG), and the inversion of the IS26-S044∆-yidY segment. Their MDR region was characterized by the inversion of a large segment, including the mer operon and the relocation of IntI1 and several resistance genes downstream of the IS26-S044∆-yidY segment. These structural changes were likely mediated by the recombination of IS26. The findings broaden our understanding of the possible evolution pathway of SGI1-K in fostering drug resistance, which may provide opportunities to control these MDR strains.IMPORTANCEThe emergence of ciprofloxacin-resistant (CIPR) Salmonella Kentucky ST198 globally has raised significant concerns. This study focuses on two poultry isolates from Thailand, revealing a distinct Salmonella genomic island 1 variant K (SGI1-K) genetic structure. Remarkably, multiple antibiotic resistance genes (ARGs) were identified within the SGI1-K as well as other locations in the chromosome, but not in plasmids. Comparing the SGI1-K genetic structures among global and even within-country isolates unveiled substantial variations. Intriguingly, certain isolates lacked ARGs within the SGI1-K, while others had ARGs relocated outside. The presence of chromosomal extended-spectrum β-lactamase (ESBL) genes and lincosamide resistance, lnu(F), gene, could potentially inform the choices of the treatment of CIPRS. Kentucky ST198 infections in humans. This study highlights the importance of understanding the diverse genetic structures of SGI1-K and emphasizes the role of animals and humans in the emergence of antimicrobial resistance.

Comparison of IncK-blaCMY-2 Plasmids in Extended-Spectrum Cephalosporin-Resistant Escherichia coli Isolated from Poultry and Humans in Denmark, Finland, and Germany.

Escherichia coli carrying IncK-blaCMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-blaCMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the blaCMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the blaCMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0-60 and 13-90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-blaCMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-blaCMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-blaCMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.

A One Health Perspective on Salmonella enterica Serovar Infantis, an Emerging Human Multidrug-Resistant Pathogen.

Salmonella enterica serovar Infantis presents an ever-increasing threat to public health because of its spread throughout many countries and association with high levels of antimicrobial resistance (AMR). We analyzed whole-genome sequences of 5,284 Salmonella Infantis strains from 74 countries, isolated during 1989-2020 from a wide variety of human, animal, and food sources, to compare genetic phylogeny, AMR determinants, and plasmid presence. The global Salmonella Infantis population structure diverged into 3 clusters: a North American cluster, a European cluster, and a global cluster. The levels of AMR varied by Salmonella Infantis cluster and by isolation source; 73% of poultry isolates were multidrug resistant, compared with 35% of human isolates. This finding correlated with the presence of the pESI megaplasmid; 71% of poultry isolates contained pESI, compared with 32% of human isolates. This study provides key information for public health teams engaged in reducing the spread of this pathogen.

Phylogeographic Dynamics of H9N2 Avian Influenza Viruses in Tunisia

Avian influenza virus subtype H9N2 is endemic in commercial poultry in Tunisia. This subtype affects poultry and wild birds in Tunisia and poses a potential zoonotic risk. Tunisian H9N2 strains carry, in their hemagglutinins, the human-like marker 226 L that is most influential in avian-to-human viral transmission. For a better understanding of how ecological aspects of the H9N2 virus and its circulation in poultry, migratory birds and environment shapes the spread of the dissemination of H9N2 in Tunisia, herein, we investigate the epidemiological, evolutionary and zoonotic potential of seven H9N2 poultry isolates and sequence their whole genome. Phylogeographic and phylodymanic analysis were used to examine viral spread within and among wild birds, poultry and environment at geographical scales. Genetic evolution results showed that the eight gene sequences of Tunisian H9N2 AIV were characterized by molecular markers involved with virulence and mammalian infections.The geographical distribution of avian influenza virus appears as a network interconnecting countries in Europe, Asia, North Africa and West Africa. The spatiotemporal dynamics analysis showed that the H9N2 virus was transmitted from Tunisia to neighboring countries notably Libya and Algeria. Interestingly, this study also revealed, for the first time, that there was a virus transmission between Tunisia and Morocco. Bayesian analysis showed exchanges between H9N2 strains of Tunisia and those of the Middle Eastern countries, analysis of host traits showed that duck, wild birds and environment were ancestry related to chicken. The subtypes phylodynamic showed that PB1 segment was under multiple inter-subtype reassortment events with H10N7, H12N5, H5N2 and H6N1 and that PB2 was also a subject of inter-subtype reassortment with H10N4.

Antibiotics Susceptibility Profile of Non-Typhoidal Salmonella Species Using Selected 3rd Generation Cephalosporins Antibiotics Against Humans and Poultry Faeces in South West, Nigeria

Introduction: Non-typhoidal Salmonella (NTS) isolates are among the most common bacterial pathogens associated with antimicrobial resistance (AMR) where animals are known to be the major reservoir of NTS despite improvements in hygiene and sanitation. Aim: This study was conducted to investigate the prevalence of non-typhoidal salmonella species from humans and poultry faeces in Ido-Ekiti using selected 3rd generation cephalosporin antibiotics. Study Location and Duration: The study was carried out at Federal Teaching Hospital Ido Ekiti for 3months between July 2023 and September 2023. Methodology: A total of 300 faecal samples were collected (100 from humans and 200 from poultry) by random sampling and analyzed by enriching on Selenite F broth and cultured on XLD agar using standard method. Faecal samples were identified using morphology, microscopic and biochemical characteristics. Antibiotic susceptibility test determined using disc diffusion method. Results: The prevalence of NTS in human and poultry sources were 9% and 11.5% respectively. Antibiotic susceptibility test revealed high resistance rates to 3rd generation Cephalosporins antibiotics among the poultry isolates, especially to Cefuroxime, Cefotaxime and ceftriaxone at 86.9%, 65.2% and 82.6% respectively while Ceftazidime at 88.9% only had an high resistance from human samples as compared to the poultry samples. Conclusion: The study confirmed that poultries in Ido Ekiti are likely major sources of resistant NTS, and this poses a potential hazard to the public, especially those living close to the poultry and those rearing them.

Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

BackgroundCampylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide.MethodsWe systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids.ResultsOverall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3’)-IIIa, aph(2’’)-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter.ConclusionsThe results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli in Humans, Food, and Environment in Kathmandu, Nepal: Findings From ESBL E. coli Tricycle Project.

The need to address antimicrobial resistance (AMR) through a One Health (OH) approach is now well recognized. There is, however, limited guidance on how AMR surveillance should be implemented across sectors to generate meaningful AMR and AMU data for decision-making. Using a sympatric approach to cross-sector sample collection, Nepal adopted the WHO extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) Tricycle Project as a step toward OH surveillance for assessing the prevalence of ESBL-producing E. coli across human, veterinary, and environment sectors. This involved a three-stage approach: identification of human hotspots (Stage 1) and sample collection sites for poultry (Stage 2) and wastewater (Stage 3). A total of 53 blood cultures from patients with bloodstream infections (BSIs), 100 stool samples from healthy pregnant women, 220 poultry ceca from slaughterhouses and live markets, and 48 wastewater samples were processed for bacterial culture and analyzed for the presence of ESBL-producing E. coli. The prevalence of ESBL-producing E. coli among isolated E. coli was the highest in wastewater samples (91%) followed by human BSIs (49%), poultry (38.6%), and fecal carriage isolates from healthy pregnant females (15%). A statistically significant association was seen in the prevalence of multidrug resistance among ESBL producers (52%) and nonproducers (26%). ESBL-producing E. coli was detected in all wastewater samples tested except for the upstream river. The findings of the study showed a high prevalence of ESBL-producing E. coli in samples from all three sectors and provided baseline data based upon which strategies for the safe disposal of communal and hospital waste, integrated AMR surveillance, and control strategies could be planned and implemented.

Characterization of Salmonella enterica Biofilms and Antibiofilm Effect of Carvacrol and 2-Aminobenzimidazole.

Biofilm-associated foodborne Salmonella infections in poultry have become increasingly challenging for veterinarians, particularly in developing countries, and warrant thorough investigation. We assessed the biofilm-forming tendency of poultry isolates of Salmonella enterica, namely Salmonella Typhimurium (n = 23), Salmonella Infantis (n = 28), and Salmonella Heidelberg (n = 18), in nutrient-rich Rappaport-Vassiliadis Soya (RVS) peptone broth and nutrient-deficient diluted Tryptone Soya Broth (TSB). Seven of the tested isolates exhibited moderate biofilm formation in diluted TSB, whereas two showed such formation in RVS. In addition, the Congo red agar assay revealed curli and cellulose production in seven isolates. Fourteen specific biofilm-associated genes were analyzed identifying sdiA and seqA to be the most prevalent (100%), and glyA the least prevalent (69.5%). The prevalence of the genes bcsA and csgA was significantly lower in moderate and weak biofilm formers, respectively, as compared with nonbiofilm formers in RVS peptone broth. Furthermore, the compounds carvacrol and 2-aminobenzimidazole (2-ABI) effectively inhibited biofilm formation by Salmonella serovars in RVS peptone and TSB media, respectively. Whereas the antibiofilm activity of 2-ABI against Salmonella has not been reported previously, we determined its most effective concentration at 1.5 mM among tested antibiofilm treatments. These findings indicate that Salmonella strains prevalent in poultry farms have the potential to form biofilms, and the tested compounds should be further explored as supportive or alternative antimicrobials.

Fecal carriage of extended spectrum beta-lactamase and fluoroquinolone resistant gene in non-typhoidal Salmonella enterica isolates from food-producing animals and humans

This study seeks to determine the feacal carriage of extended spectrum beta-lactamase and fluoroquinolone resistant non-typhoidal Salmonella enterica isolates from food-producing animals and humans. A total of three hundred (300) fecal samples were collected using sterile universal containers from food-producing animals namely (Chicken [100], Pig [100] and humans (100) from Onicha Local Government Area of Ebonyi State and analyzed for the presence of non-typhoidal Salmonella enterica using standard microbiological techniques. Phenotypic detection of extended-spectrum beta-lactamase (ESBL) were done by disc diffusion and Double Disk Synergy Test. Molecular characterization for ESBL and fluoroquinolone-resistant genes were done by PCR with specific primers. The result shows that non-typhoidal Salmonella species (NTS) accounted for 25 % and 17 % in poultry and pig fecal sample respectively while 60 % and 40% were phenotypic ESBL producers respectively. When compared statistically there is significant difference among isolates confirmed ESBL-positive (P˂ 0.05). Also, none of the 16 (58 %) NTS isolated from humans harbored ESBL phenotype. PCR analysis with β-lactam specific primer detected the presence of blaOXA 50 % and 50 %, blaSHV 36 %, and 64 %, blaTEM 43 % and 57 %, blaCTX-M 36 % and 64 % in poultry and pig respectively. Fluoroquinolone resistant gene QnrA was present in 0 and 100 % of poultry and pig respectively. QnrB was 40 % and 60 % in poultry and pig isolates respectively. QnrS was present in 64 % isolates of poultry and 13 % isolates in pig. The high prevalence of genes encoding beta-lactamases and fluoroquinolone resistance (TEM, SHV, CTX-M and OXA, (qnrA, qnrB and qnrS) were present more in poultry and pig than in humans and demonstrate a significant public health threat from consumption of food-producing animal harboring such pathogenic resistant genotype if not properly controlled.&#x0D; Keywords: Extended spectrum beta-lactamase, Fluoroquinolone, non-typhoidal Salmonella enterica, feacal carriage

Clonal diversity and zoonotic potential of MDR Escherichia coli isolated from poultry at different age intervals

ABSTRACT 1. A pool of 480 E. coli isolates of poultry (broilers and ducks) representing different time intervals (0, 10, 20 and 30 days) was selected for ribotyping and used to determine polymorphism of 16-23S ribosomal RNA intergenic space. All the isolates were multidrug-resistant (MDR). 2. Out of these, 10 isolates were tested for MultiLocus Sequence Typing (MLST) among which novel allelic combinations and therefore new sequence types were identified in seven isolates. 3. This work showed the changes in E. coli strains structure at farm level and individual bird level in host species raised on organised farms with similar parental lineage and environmental housing. The statistical results showed that the structure of variation is very different by farm, supporting a strong effect of location, which confirms the temporal clustering. 4. There were significant differences between E. coli strains in chickens and ducks, indicating host specificity of the E. coli strains. 5. Some of the pathogenic E. coli strains found using MLST belonged to ST735, ST2796 and a pandemic clone ST752 of ST10 clonal complex. The results strongly suggested the clonal expansion and establishment of specific MDR clones that have zoonotic relevance.

Genetic characterization of Escherichia coli and Klebsiella spp. from humans and poultry in Nigeria.

The emergence of antibiotic resistance in livestock, especially food-producing animals, is of major public health importance as a result of the possibility of these bacteria entering the food chain. In this study, the genetic characteristics of antibiotic-resistant Escherichia coli and Klebsiella spp. isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 45 Klebsiella spp. and 46 E. coli isolates were obtained from urine, clinical wounds, nasal and chicken faecal samples. Isolates were recovered and identified as previously described. Species identification was achieved by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and ribosomal multilocus sequence typing. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer method for 12 antibiotics. A double disc synergy test was used to screen for extended-spectrum beta-lactamse (ESBL) production. Whole genome sequencing was performed for strain characterization of the isolates. Thirteen Klebsiella spp. isolates yielded positive results by the ESBL phenotypic test and harboured ESBL genes. Of the 46 E. coli isolates, 21 human and 13 poultry isolates were resistant to at least one of the tested antibiotics. Four human E. coli isolates harboured ESBL genes and revealed positive results when applying ESBL double disc synergy tests. ESBL genes in the Klebsiella spp. and E. coli isolates include bla CTX-M-15 and bla SHV-28. Whole genome-based core gene multilocus sequence typing of the Klebsiella spp. and E. coli isolates revealed a close relatedness among the isolates. An integrated 'One Health' surveillance system is required to monitor transmission of antimicrobial resistance in Nigeria.

Isolation and Genomic Characterization of a Heat-Labile Enterotoxin 1-Producing Escherichia fergusonii Strain from a Human.

Escherichia fergusonii strains have been isolated from patients with diarrhea, but their virulence determinant has not been well elucidated. Here, we report the first isolation of a heat-labile enterotoxin 1 (LT1)-producing E. fergusonii strain (strain 30038) from a patient in Japan. The complete genome sequence of strain 30038 was determined and subjected to comparative genomics and phylogenetic analyses with 195 publicly available genomes of E. fergusonii. In addition to strain 30038, the elt1 gene was also identified in an E. fergusonii strain that is phylogenetically distinct and which was isolated from poultry in the United Kingdom. Fine genomic comparison revealed that these two strains share comparable elt1-bearing plasmids. However, an intriguing distinction arises in strain 30038, wherein the plasmid has integrated into the chromosome via a recombination process mediated by an insertion sequence. The production of active LT1 toxin by strain 30038 was verified through an in vitro assay using cultured cells. A large plasmid carrying 11 antimicrobial resistance genes was also identified in strain 30038. Our results indicate that extensive surveillance of elt1-positive E. fergusonii strains as diarrheagenic pathogens is needed. IMPORTANCE Escherichia fergusonii, a species closely related to Escherichia coli, is known to cause sporadic conditions in humans, including diarrhea. However, the critical virulence factors in E. fergusonii clinical isolates remain to be identified. This study shows the first isolation of an E. fergusonii strain carrying the elt1 gene, which encodes heat-labile enterotoxin 1, from a patient with diarrhea. Our analysis of public databases also revealed the presence of elt1-positive E. fergusonii strains isolated from poultry in the United Kingdom. Interestingly, while the elt1 gene in the poultry isolate was present on a large plasmid, in the human isolate it was integrated into the chromosome, which may confer stability on the elt1-carrying genetic element. Our findings highlight the need for extensive surveillance of elt1-positive E. fergusonii strains in livestock animals.