Background: Salmonella is an important zoonotic pathogen, and chickens are one of its main hosts. Every year, Salmonella infections pose a serious threat to the poultry industry in developing countries, especially China. In this study, a total of 84 Salmonella isolates recovered from sick and healthy-looking chickens in central China were characterized by serotyping, MLST-based strain typing, presence of potential virulence factors, and antimicrobial resistance profiles. Result: Data showed that the main serotypes of Salmonella isolates in central China were Salmonella enterica serovar Gallinarum biovar Pullorum, Salmonella enterica serovar Gallinarum biovar Gallinarum, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium, and among them, S. Pullorum was the dominant type in both sick and healthy-looking chickens, accounting for 43.9 and 46.5%, respectively, while S. Enteritidis was only found in healthy-looking chickens. All isolates exhibited higher resistance rates to ampicillin (97.6%), tetracycline (58.3%) and colistin (51.2%), and among these isolates, 49.5% were resistant to more than three drugs in different combinations. S. Enteritidis was the most severe multidrug-resistant serotype, which showed higher resistance rates to colistin, meropenem and ciprofloxacin. Multilocus sequence typing (MLST) revealed that S. Gallinarum and S. Enteritidis isolates were clustered in clade 1, which belonged to two and one STs, respectively. All S. Typhimurium isolates were clustered in clade 3, and belonged to three STs. However, S. Pullorum were distributed in three clades, which belonged to 7 STs. Twenty-seven virulence-associated genes were detected, and expected cdtB, which was absent in all the isolates, the other 26 genes were conserved in the closely related Salmonella serogroup D (S. Enteritidis, S. Pullorum, and S. Gallinarum). Conclusion: Salmonella serogroup D was the major subgroup, and S. Pullorum was the most common type in sick and healthy-looking chickens in central China. Drug resistance assays showed serious multiple antimicrobial resistances, and S. Enteritidis was the most severe drug-resistant serotype. MLST showed that there was correlation between serotypes and genotypes in most Salmonella isolates, except S. Pullorum, which showed complicated genetic diversity firstly. These results provide important epidemiological information for us to control Salmonella in chickens.

Pullorum disease, caused by Salmonella enterica serovar Pullorum (S. Pullorum), is one of the most important bacterial infections in the poultry industry in developing countries, including China. To examine the prevalence and characteristics of S. Pullorum, the Multilocus Sequence Typing (MLST) genotypes, fluoroquinolones resistance, and biofilm-forming abilities of S. Pullorum isolates were investigated, collected from 2011 to 2016 in China. Thirty S. Pullorum isolates collected from 2011 to 2016 were analyzed. Quinolones susceptibility testing showed that 90% of the isolates were resistant to the first generation of quinolines nalidixic acid, but the resistance rates to different fluoroquinolones agents were lower than 13.3%; for some there was even no resistance. Multilocus sequence typing (MLST) showed that ST-92 was the dominating genotype, accounting for 90.0% of all S. pullorum strains. The remaining three isolates were of the new reported sequence type ST-2151. Interestingly, the Asp87Gly substitution in quinolone resistance-determining regions (QRDR) of GyrA was only observed in the three strains of ST-2151, suggesting a potential correlation between Asp87Gly substitution and sequence type (p < 0.05). However, Asp87Gly substitution could not confer the resistant to ofloxacin and ciprofloxacin of these isolates. The plasmid-mediated quinolone resistance (PMQR) gene was not found in any of the tested isolates. Furthermore, an assay measuring biofilm-forming abilities showed that 46.7% of the isolates were non-biofilm producers, while 53.3% could form very weak biofilms, which might explain the relatively lower resistance to fluoroquinolones. We reported a high resistance rate to the first generation of quinolines nalidixic acid and relatively low resistance rates to fluoroquinolones in S. Pullorum isolates. In addition, weak biofilm-forming abilities were found, which might be an important reason of the low fluoroquinolones resistance rates of S. Pullorum isolates. ST-92 was the dominating genotype demonstrated by MLST, and the new sequence type ST-2151 showed a potential correlation with Asp87Gly substitution in QRDR of GyrA. We believe the characterization of these S. Pullorum isolates will be helpful to develop prevention and control strategies.

Campylobacter is considered to be the leading cause of human bacterial gastroenteritis, of which poultry is the main reservoir. Campylobacter contaminated chicken products are a major cause of human Campylobacter infection. In this study, the prevalence of Campylobacter in chicken in central China was investigated, and the genotypic diversity, antimicrobial resistance and biofilm of these isolates were characterized. A total of 206 Campylobacter isolates, including 166 C. jejuni and 40 C. coli, were isolated from chicken farms and live poultry markets in central China. Multilocus sequence typing and phylogenetic analysis showed that the Campylobacter isolates had diverse genetic backgrounds, which covered most of the dominant clone complexes (CCs) reported throughout China. The most prevalent CCs were CC-464, CC-1150, CC-353, and CC-828. All the isolates showed resistance to norfloxacin, ciprofloxacin and Cefazolin, and a prevalent resistance to fluoroquinolones, β-lactams and tetracyclines was also observed. Among all the isolates, 133 strains showed the ability to form biofilm, thereinto, the isolates in two genetic branches, mainly including CC-21, CC-48, CC-677 and CC-45, showed a significantly lower ability to form biofilm than other genetic branches (p < 0.05). However, in general, the ability to form biofilm varied among different genetic branches, suggesting a complex genetic background to biofilm formation, but not only the genetic lineages. Compared with the strains unable to form biofilm, biofilm-producing strains possessed a significantly higher resistance to ampicillin, neomycin, sulfamethoxazole, amikacin, clindamycin and erythromycin (p < 0.05). To the best of our knowledge, this is the first report on the relationship of the genotypic diversity, antimicrobial resistance and biofilm-forming abilities of Campylobacter isolated from chicken in Central China, which showed the potential importance of biofilm in antimicrobial resistance. This study will help us better understand the epidemiology and antimicrobial resistance of Campylobacter.

Extensive studies have shown that potassium diformate (KDF), an antibiotic substitute used as a feed additive, improves animal growth performance, although there is less direct evidence of its preventive effect on bacterial infections and its influence on the intestinal flora of animals. In this study, the inhibition effect of KDF on Salmonella enterica serovar Pullorum, an important enteric pathogen causing pullorum disease, was investigated in vitro and on a chicken infection model. The effect of KDF on the diversities and structures of chicken duodenal and cecum flora were also investigated using 16S rRNA gene sequencing. The results showed that addition of 0.5% KDF in feed or 0.1% KDF in drinking water significantly reduced the bacterial loads and the degree of pathological changes in the cecum, improved digestion and reduced the pH of the gastrointestinal tract of chickens infected with S. pullorum. KDF also significantly modified the diversity and abundance of intestinal microflorae in chickens. In particular, it promoted the colonization of several probiotics, such as Bacteroides, Blautia, Ruminococcus_torques_group and Faecalibacteriumm, which are involved in maintenance of the intestinal barrier, modulation of inflammation, energy supply for intestinal cells and pathogen resistance. These results enrich the theoretical basis for the clinical application of KDF in chickens.

Background: Avian colibacillosis is an infectious bacterial disease caused by avian pathogenic Escherichia coli (APEC). APEC causes a wide variety of intestinal and extraintestinal infections, including InPEC and ExPEC, which result in enormous losses in the poultry industry. In this study, we investigated the prevalence of InPEC and ExPEC in Central China, and the isolates were characterized using molecular approaches and tested for virulence factors and antibiotic resistance. Results: A total of 200 chicken-derived E. coli isolates were collected for study from 2019 and 2020. The prevalence of B2 and D phylogenic groups in the 200 chicken-derived E. coli was verified by triplex PCR, which accounted for 50.53% (48/95) and 9.52% (10/105) in ExPEC and InPEC, respectively. Additionally, multilocus sequence typing method was used to examine the genetic diversity of these E. coli isolates, which showed that the dominant STs of ExPEC included ST117 (n = 10, 20.83%), ST297 (n = 5, 10.42%), ST93 (n = 4, 8.33%), ST1426 (n = 4, 8.33%) and ST10 (n = 3, 6.25%), while the dominant ST of InPEC was ST117 (n = 2, 20%). Furthermore, antimicrobial susceptibility tests of 16 antibiotics for those strains were conducted. The result showed that more than 60% of the ExPEC and InPEC were resistant to streptomycin and nalidixic acid. Among these streptomycin resistant isolates (n = 49), 99.76% harbored aminoglycoside resistance gene strA, and 63.27% harbored strB. Among these nalidixic acid resistant isolates (n = 38), 94.74% harbored a S83L mutation in gyrA, and 44.74% harbored a D87N mutation in gyrA. Moreover, the prevalence of multidrug-resistant (MDR) in the isolates of ExPEC and InPEC was 31.25% (15/48) and 20% (2/10), respectively. Alarmingly, 8.33% (4/48) of the ExPEC and 20% (2/10) of the InPEC were extensively drug-resistant (XDR). Finally, the presence of 13 virulence-associated genes was checked in these isolates, which over 95% of the ExPEC and InPEC strains harbored irp2, feoB, fimH, ompT, ompA. 10.42% of the ExPEC and 10% of the InPEC were positive for kpsM. Only ExPEC isolates carried ibeA gene, and the rate was 4.17%. All tested strains were negative to LT and cnf genes. The carrying rate of iss and iutA were significantly different between the InPEC and ExPEC isolates (P < 0.01). Conclusions: To the best of our knowledge, this is the first report on the highly pathogenic groups of InPEC and ExPEC in Central China. We find that 50.53% (48/95) of the ExPEC belong to the D/B2 phylogenic group. The emergence of XDR and MDR strains and potential virulence genes may indicate the complicated treatment of the infections caused by APEC. This study will improve our understanding of the prevalence and pathogenicity of APEC.

Background: Coagulase-negative staphylococci (CoNS) are a group of opportunistic pathogens, which are widely spread in the environment. Animal breeding is an important source of pathogen spreading. However, the concentration and characteristics of CoNS in the bioaerosols of henhouses are unclear. Results: In this study, we showed that CoNS were significantly increased in bioaerosols of henhouses during the first 60 days, and reached 2.0 × 10⁶ CFU/m³, which account for 75.4% of total bacteria. One hundred and two CoNS isolates from bioaerosols and nasal swabs of farmers were further identified, covering seven species. Among these, 41.2% isolates were Staphylococcus sciuri, which was the predominant species, followed by S. equorum, S. saprophyticus, S. haemolyticus, S. xylosus, S. arlettae and S. gallinarum. There were high rates of resistance to oxacillin in CoNS (49.0%), which were defined as Methicillin-Resistant CoNS (MRCoNS), and 36.3% isolates contained resistance gene mecA. Bioaerosol infection models showed that, chickens exposed to aerosolized S. sciuri had significant induction of inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8 and IL-10 at 5 days post-infection (dpi) in lungs and at 7 dpi in spleens. Conclusions: We reported a high concentration of CoNS in henhouses, and S. sciuri was the preponderant CoNS species. Antibiotic resistance analysis and bioaerosols infection of CoNS further highlighted its hazards on resistance and immunological challenge. These results suggested that, CoNS in bioaerosols could be one serious factor in the henhouses for not only poultry industry but also public health.