Campylobacter species are the major food-borne pathogens which could cause bacterial gastroenteritis in humans. Contaminated chicken products have been recognized as the primary vehicles of Campylobacter transmission to human beings. In this study, the prevalence of Campylobacter in retail chicken meat in Central China was investigated, and the isolates were further characterized using molecular approaches and tested for antibiotic resistance. A total of 302 chicken samples purchased from April 2014 to April 2015 were tested. The level of Campylobacter contamination was enumerated by most probable number-PCR (MPN-PCR). The Campylobacter positive rate was 17.2% (52/302), with bacterial count varying from 3.6 to 360 MPN/g in positive samples. A total of 52 Campylobacter strains, including 40 Campylobacter jejuni and 12 Campylobacter coli, were isolated from the positive samples. To examine the genetic diversity of the isolates, multilocus sequence typing (MLST) technology was applied, which identified 23 sequence types (STs) belonging to seven clonal complexes (CCs) and unassigned. Among them, the dominant CCs of C. jejuni included CC-353 and CC-464, and the dominant CCs of C. coli were CC-828 and CC-1150. Antibiotic resistance analysis showed that all of the isolates were resistant to norfloxacin and ciprofloxacin. 23 virulence-associated genes were tested in the isolates, which showed that the number of virulence-associated genes detected in the C. jejuni isolates ranged from 16 to 21, while in most of the C. coli isolates ranged from 12 to 16. Virulence-associated genes, flaA, flgB, flgE2, fliM, fliY and cadF were detected in all isolates. VirB11, however, was not detected in any of the isolates. To the best of our knowledge, this is the first report on the contamination level and molecular biological features of Campylobacter strains in retail chicken meat in Central China, which showed high genetic diversity and remarkable antibiotic resistance. This study provided scientific data for the risk assessment and evaluation of Campylobacter contamination in retail chicken products.

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.

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.

The emergence and dissemination of Escherichia coli (E. coli) strains that produce extended-spectrum beta-lactamases (ESBLs) represents a major public health threat. The present study was designed to evaluate the prevalence and characteristics of ESBL-producing Escherichia coli isolates from chickens in central China during 2016–2019. A total of 407 E. coli strains isolated from 581 chicken swabs were identified conventionally and analyzed for various cephalosporin susceptibility by disk-diffusion assay. ESBL-producing strains were screened using the double=disk synergy test and ESBL-encoding genes were carried out by PCR/sequencing. A total of 402 E. coli isolates exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, especially cefazolin (60.69%), cefuroxime (54.05%), cefepime (35.14%), ceftriaxone (54.30%), and aztreonam (40.29%). Piperacillin/tazobactam (1.72%) was the most effective drug against the strains, but the resistance rates increased each year. Among the isolates, 262 were identified as ESBL producers and the isolation rates for the ESBL producers increased from 63.37% to 67.35% over the four years. CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) and SHV (3.05%). The most common ESBL genotype combination was bla_TEM + bla_CTX-M (74.46%), in which the frequency of carriers increased steadily, followed by bla_CTX-M + bla_SHV (3.05%). In addition, the most predominant specific CTX-M subtypes were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). Moreover, a novel multiplex qPCR assay was developed to detect bla_CTX-M, bla_TEM, and bla_SHV, with limits of detection of 2.06 × 10¹ copies/μL, 1.10 × 10¹ copies/μL, and 1.86 × 10¹ copies/μL, respectively, and no cross-reactivity with other ESBL genes and avian pathogens. The assays exhibited 100% sensitivity and specificities of 85%, 100%, and 100% for bla_CTX-M, bla_TEM, and bla_SHV, respectively. In conclusion, our findings indicated that ESBL-producing E.coli strains isolated from chickens in central China were highly resistant to cephalosporins and frequently harbored diversity in ESBL-encoding genes. These isolates can pose a significant public health risk. The novel multiplex qPCR method developed in this study may be a useful tool for molecular epidemiology and surveillance studies of ESBL genes.

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.

Avian leukosis (AL), caused by avian leukosis virus (ALV), is a contagious tumor disease that results in significant economic losses for the poultry industry. Currently, ALV-A, B, J, and K subgroups are the most common in commercial poultry and cause possible coinfections. Therefore, close monitoring is necessary to avoid greater economic losses. In this study, a novel multiplex quantitative polymerase chain reaction (qPCR) assay was developed to detect ALV-A, ALV-B, ALV-J, and ALV-K with limits of detection of 40, 11, 13.7, and 96 copies/µL, respectively, and no cross-reactivity with other ALV subtypes and avian pathogens. We detected 852 cell cultures inoculated with clinical samples using this method, showing good consistency with conventional PCR and ELISA. The most prevalent ALV strain in Hubei Province, China, was still ALV-J (11.74%). Although single infections with ALV-A, ALV-B, and ALV-K were not found, coinfections with different subgroup strains were identified: 0.7% for ALV-A/J, 0.35% for ALV-B/J, 0.25% for ALV-J/K, and 0.12% for ALV-A/B/K and ALV-A/B/J. Therefore, our novel multiplex qPCR may be a useful tool for molecular epidemiology, clinical detection of ALV, and ALV eradication programs.