Genetic characteristics, antimicrobial susceptibility, and virulence genes distribution of Campylobacter isolated from local dual-purpose chickens in central China.

Food-borne antibiotic-resistant Campylobacter poses a serious threat to public health. To understand the prevalence and genetic characteristics of Campylobacter in Chinese local dual-purpose (meat and eggs) chickens, the genomes of 30 Campylobacter isolates, including 13 C. jejuni and 17 C. coli from Jianghan-chickens in central China, were sequenced and tested for antibiotic susceptibility. The results showed that CC-354 and CC-828 were the dominant clonal complexes of C. jejuni and C. coli, respectively, and a phylogenetic analysis showed that three unclassified multilocus sequence types of C. coli were more closely genetically related to C. jejuni than to other C. coli in this study. Of the six antibiotics tested, the highest resistance rates were to ciprofloxacin and tetracycline (100%), followed by lincomycin (63.3%), erythromycin (30.0%), amikacin (26.7%), and cefotaxime (20.0%). The antibiotic resistance rate of C. coli was higher than that of C. jejuni. The GyrA T86I mutation and 15 acquired resistance genes were detected with whole-genome sequencing (WGS). Among those, the GyrA T86I mutation and tet(O) were most prevalent (both 96.7%), followed by the blaOXA-type gene (90.0%), ant(6)-Ia (26.7%), aac(6')-aph(3'') (23.3%), erm(B) (13.3%), and other genes (3.3%). The ciprofloxacin and tetracycline resistance phenotypes correlated strongly with the GyrA T86I mutation and tet(O)/tet(L), respectively, but for other antibiotics, the correlation between genes and resistance phenotypes were weak, indicating that there may be resistance mechanisms other than the resistance genes detected in this study. Virulence gene analysis showed that several genes related to adhesion, colonization, and invasion (including cadF, porA, ciaB, and jlpA) and cytolethal distending toxin (cdtABC) were only present in C. jejuni. Overall, this study extends our knowledge of the epidemiology and antibiotic resistance of Campylobacter in local Chinese dual-purpose chickens.

The Feed Additive Potassium Diformate Prevents Salmonella enterica Serovar Pullorum Infection and Affects Intestinal Flora in Chickens.

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.

Droplet Digital PCR-Based Detection and Quantification of GyrA Thr-86-Ile Mutation Based Fluoroquinolone-Resistant Campylobacter jejuni.

Fluoroquinolone (FQ)-resistant Campylobacter jejuni is a serious problem worldwide that limits effective treatment of infections. The traditional detection method depends on bacterial isolation and MIC testing, or traditional PCR, which is time-consuming and hard to identify the FQ-resistant C. jejuni in a high abundance wild-type background. This study aimed to develop a rapid and accurate ddPCR assay to detect FQ-resistant C. jejuni mutants based on the crucial resistance mutation C257T (Thr-86-Ile) in gyrA. Our ddPCR gyrA assay showed high specificity and accuracy. Sanger sequencing and the qPCR assay could only recognize gyrA mutant sequences when the ratios of wild-type/mutant were 1:1 or 10:1, respectively. Our ddPCR gyrA assay was able to detect gyrA mutant sequences in the mixtures with up to at least 1000:1 wild-type/mutant ratios, which suggested a significant advantage to distinguish the low mutant signal from the wild-type background. We further monitored the occurrence of gyrA mutations under ciprofloxacin pressure using our ddPCR gyrA assay, and clearly showed that the transition of a dominant C. jejuni subpopulation from wild-type to gyrA C257T mutant, resulting in FQ-resistance. We tested 52 samples from live chickens and retail chicken meat and showed that four samples contained wild-type/mutant mixtures comprising 1.7%, 28.6%, 53.3%, and 87.0% gyrA C257T mutants, respectively. These results demonstrated that the ddPCR gyrA assay was a highly sensitive alternative method to distinguish and quantify FQ-resistant C. jejuni infections that could help guide the appropriate use of FQs in clinical practice. IMPORTANCE Campylobacter jejuni is considered to be the leading cause of human bacterial gastroenteritis worldwide, and fluoroquinolones (FQs) are the main choices for the treatment of bacterial gastroenteritis in clinical practice. In theory, antimicrobial susceptibility testing should help us to choose the most appropriate drugs for the treatment. However, to test the susceptibility of C. jejuni to FQs, the standardized method is bacteria isolation and MIC measurement, which will take more than 4 days. In addition, a low abundance of FQ-resistant C. jejuni is also hardly distinguished from a high abundance of wild-type background in the mixed infection. Therefore, the development of rapid and accurate detection technology for FQ-resistant C. jejuni is very important. This study provided a ddPCR gyrA assay, which is a highly sensitive alternative method to distinguish and quantify FQ-resistant C. jejuni infections that may help guide the appropriate use of FQs both in veterinary and human clinical practice.

Prevalence of colistin resistance gene mcr-1 in Escherichia coli isolated from chickens in central China, 2014 to 2019.

OBJECTIVES: This study investigated the prevalence and characteristics of mcr-1-harbouring Escherichia coli isolated from chickens in central China from 2014 to 2019. METHODS: A total of 1132 E. coli isolated from 1647 chicken swabs were analysed for colistin susceptibility by broth microdilution method and prevalence of mcr-1 gene by PCR. The colistin-resistant E. coli isolates were typed by multi-locus sequence typing (MLST) and tested with 12 antimicrobial agents. The transconjugation assay was conducted for the mcr-1-positive isolates using the transconjugant E. coli C600. RESULTS: Of the 1132 E. coli isolated from chickens, 131 isolates (11.6%) exhibited colistin resistance, and 51 isolates (4.5%) were mcr-1 positive. The mcr-1-positive rate was quite low in 2014 (2.3%) and 2015 (1.7%), increased to peak in 2016 (12.6%) and 2017 (11.4%), and then decreased significantly in 2018 (1.7%) and 2019 (0.9%). The 131 colistin resistant isolates were assigned to 66 unique sequence types (STs), 27 of which contained mcr-1-positive isolates. Compared with mcr-1-negative E. coli, mcr-1-positive E. coli showed higher resistance rates to nalidixic acid, ciprofloxacin, ceftriaxone, cefotaxime, and tetracycline. Furthermore, 30 of the 51 mcr-1 positive isolates transduced their mcr-1 gene into E. coli C600, and 13 of the 30 transconjugants carried more than one replicon types. CONCLUSION: The mcr-1 positive rate varied enormously during 2014-2019 in central China. The ban on colistin likely decreased the dissemination of mcr-1 in E. coli isolates from chickens. Multidrug-resistant trait is observed in mcr-1 positive E. coli isolates and can be transferred into other transconjugants.

(p)ppGpp synthetases are required for the pathogenicity of Salmonella Pullorum in chickens.

Salmonella Pullorum is a pathogen specific to birds that can cause Pullorum disease in young chickens and lead to considerable economic losses in the poultry industry. During transmission and infection, S. Pullorum will encounter various environmental stresses and host defenses. The stringent response is an important adaptation response induced by (p)ppGpp, and in Salmonella, (p)ppGpp is synthesized by two (p)ppGpp synthetases, RelA and SpoT. To investigate the role of (p)ppGpp synthetases in the adaptation and pathogenicity of S. Pullorum, a (p)ppGpp synthetases mutant (ΔrelAΔspoT) was constructed, and its physiological phenotypes and pathogenicity, as well as transcription profiling, were compared with the parent strain. The ΔrelAΔspoT mutant showed decreased ability to form biofilms, and reduced resistance to acidic, alkaline, high osmolarity and H2O2 conditions. The internalization of the ΔrelAΔspoT mutant into host cells in vitro and its lethality and colonization abilities within young chickens were also significantly reduced. RNA sequencing showed that the (p)ppGpp synthetases did not only affect the classic stringent response, such as inhibition of DNA replication and protein synthesis, but also controlled the expression of many virulence factors, in particular, the Salmonella pathogenicity island 1 (SPI-1) and SPI-2 type III secretion systems (T3SSs), and adhesion factors. These results suggest that the (p)ppGpp synthetases are required for the pathogenicity of S. Pullorum by affecting its stress response and the expression of the virulence factors.

Characterization of Salmonella spp. isolated from chickens in Central China.

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.

Evaluation of young chickens challenged with aerosolized Salmonella Pullorum.

Salmonella enterica serovar Pullorum (S. Pullorum) is an important pathogen specific to avian species, which poses a serious threat to the poultry industry. The transmission of S. Pullorum occurs both horizontally and vertically but the airborne transmission of S. Pullorum has been neglected historically. In this study, the effects of aerosolized S. Pullorum on young chickens were investigated. The results showed that the colonization and morbidity induced by bioaerosol infection are dose dependent. The bacteria colonized in chicken lung for more than 14 days following the exposure to ≥ 1.25 × 106 CFU/m3 of aerosolized S. Pullorum. Tachypnoea and depression were present in all the chickens between 5 and 7 days after infection, and some died, following the exposure to ≥1.25 × 108 CFU/m3 of aerosolized S. Pullorum. RT-PCR results showed that significant expressions of inflammatory cytokines, including tumour necrosis factor α, interleukin 1ß (IL-1ß), IL-6, and IL-8 were noted in the lung and spleen. Histopathological examination showed lung swelling, with obvious lesions, including inflammatory cell infiltration, tissue injury, and acute haemorrhage. These results suggest that uncontrolled and detrimental inflammation is caused by a high dose of aerosolized S. Pullorum. These results further extend our understanding of the pathogenicity of air-transmitted S. Pullorum on chickens. RESEARCH HIGHLIGHTS Aerosolized S. Pullorum caused tachypnoea, depression, and lung swelling in chickens. The colonization and morbidity caused by aerosolized S. Pullorum are dose dependent. Detrimental inflammation is caused by high doses of aerosolized S. Pullorum in lung.

Point Deletion or Insertion in CmeR-Box, A2075G Substitution in 23S rRNA, and Presence of erm(B) Are Key Factors of Erythromycin Resistance in Campylobacter jejuni and Campylobacter coli Isolated From Central China.

Campylobacter jejuni and Campylobacter coli are major food-borne pathogens that cause bacterial gastroenteritis in humans, and poultry is considered as their most important reservoir. Macrolides, such as erythromycin, are the first-line choice for treatment of campylobacteriosis. In this study, of the 143 Campylobacter isolates recovered from poultry in central China during 2015-2017, 25.2% were erythromycin resistant. A2075G substitution in 23S ribosomal RNA (rRNA) and ribosomal methylase encoded by erm(B) were found in 4.2 and 4.9% isolates, respectively, and correlated with erythromycin resistance. The polymorphisms of CmeR-Box were also analyzed in our isolates. Among them, 9.1% isolates harbored a point deletion or insertion within the CmeR-Box, and we first showed that point deletion or insertion, but not substitution, in CmeR-Box led to high expression of cmeABC, which was significantly associated with erythromycin resistance (p < 0.05). These results suggest that point deletion or insertion in CmeR-Box, A2075G substitution in 23S rRNA, and presence of erm(B) are three main factors to erythromycin resistance in C. jejuni and C. coli.

High concentration of coagulase-negative staphylococci carriage among bioaerosols of henhouses in Central China.

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)-1ß, 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.

Draft Genome Sequences of Multidrug-Resistant Campylobacter jejuni Strains Isolated from Chickens in Central China.

Campylobacter jejuni is a major foodborne pathogen that plays an important role in spreading drug resistance. We report the draft genome sequences of two multidrug-resistant C. jejuni isolates which contained similar mutations in the CmeR box. This will improve the understanding of C. jejuni antimicrobial resistance and genetic characteristics.

Quinolone resistance phenotype and genetic characterization of Salmonella enterica serovar Pullorum isolates in China, during 2011 to 2016.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Genotypic diversity, antimicrobial resistance and biofilm-forming abilities of Campylobacter isolated from chicken in Central China.

BACKGROUND: 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. RESULTS: 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, ß-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). CONCLUSIONS: 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.

Correlation between gyrA and CmeR Box Polymorphism and Fluoroquinolone Resistance in Campylobacter jejuni Isolates in China.

Sequence analysis of 79 ciprofloxacin-resistant Campylobacter jejuni isolates collected in China showed resistance-related sequence variations in gyrA and CmeR-Box. All the isolates contain an identical Thr-86-Ile substitution in GyrA. Several novel CmeR-Box variations, including point substitutions, deletion, and insertion, were identified. The point insertion or deletion led to dramatically reduced binding of CmeR to the cmeABC promoter, which significantly increases the expression of cmeABC and contributes to the high fluoroquinolone resistance.