Chicken products and chickens with colibacillosis are often reported to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC) causing several diseases in humans. Such pathogens in healthy chickens can also contaminate chicken carcasses at the slaughter and then are transmitted to humans via food supply; however, reports about the ExPEC in healthy chickens are still rare. In this study, we determined the prevalence and characteristics of ExPEC isolates in healthy chickens in China. A total of 926 E. coli isolates from seven layer farms (371 isolates), one white-feather broiler farm (78 isolates) and 17 live poultry markets (477 isolates from yellow-feather broilers) in 10 cities in China, were isolated and analyzed for antibiotic resistance phenotypes and genotypes. The molecular detection of ExPEC among these healthy chicken E. coli isolates was performed by PCRs, and the serogroups and antibiotic resistance characteristics of ExPEC were also analyzed. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were used to analyze the genetic relatedness of these ExPEC isolates. We found that the resistance rate for each of the 15 antimicrobials tested among E. coli from white-feather broilers was significantly higher than that from brown-egg layers and that from yellow-feather broilers in live poultry markets (p < 0.05). A total of 22 of the 926 E. coli isolates (2.4%) from healthy chickens were qualified as ExPEC, and the detection rate (7.7%, 6/78) of ExPEC among white-feather broilers was significantly higher than that (1.6%, 6/371) from brown-egg layers and that (2.1%, 10/477) from yellow-feather broilers (p < 0.05). PFGE and MLST analysis indicated that clonal dissemination of these ExPEC isolates was unlikely. Serogroup O78 was the most predominant type among the six serogroups identified in this study, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant (MDR) and the resistance rates to ampicillin (100%) and sulfamethoxazole-trimethoprim (100%) were the highest, followed by tetracycline (95.5%) and doxycycline (90.9%). bla_CTX-M was found in 15 of the 22 ExPEC isolates including 10 harboring additional fosfomycin resistance gene fosA3. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates in this study. Worryingly, two ExPEC isolates were found to carry both mcr-1 and bla_NDM, compromising both the efficacies of carbapenems and colistin. The presence of ExPEC isolates in healthy chickens, especially those carrying mcr-1 and/or bla_NDM, is alarming and will pose a threat to the health of consumers. To our knowledge, this is the first report of mcr-1-positive ExPEC isolates harboring bla_NDM from healthy chickens.

Riemerella anatipestifer causes septicemia in waterfowl, especially in ducks worldwide, while it is becoming a threat to chicken farming in China. In this study, a total of 27,136 samples from septicemic chickens and dead embryos were collected in 29 provinces from 2021 to 2024 to characterize the transmission dynamics of R. anatipestifer and guide evidence-based control strategies. A total of 752 R. anatipestifer strains were isolated and identified by PCR, showing rapid geographical expansion (from 10 to 19 provinces) and rapidly increasing isolation rates (from 1.03 % to 4.56 %), with peaking in winter/spring. Infection shifted toward younger chickens, infection rates in 3-6-week-old white-feathered broilers increased dramatically from 0.99 % to 13.24 % (mainly leg lesions), while 50-100-day-old layers showed 2.16 % salpingitis. Tissue-specific colonization showed high loads in oviducts (21.68 %) and hocks (10.89 %). Although R. anatipestifer was detected in 20.18 % of early "jelly-like embryos," it was not found in semen/ovarian follicles. Serotyping showed dominance of type 1 (70.82 %) and type 10 (15.86 %), with 2.83 % untypable strains. Alarmingly, R. anatipestifer had a severe resistance to enrofloxacin (91.54 %), polymyxin (88.22 %), and amikacin (86.10 %). As the first large-scale survey of R. anatipestifer in chickens, these findings highlight its cross-species adaptation, vertical transmission risk, and increasing prevalence in young broilers, which contributes to the understanding of the epidemiology of R. anatipestifer in chickens and provides scientific guidance for the treatment and control of the disease.

Introduction: The emergence of multidrug-resistant (MDR) strains of Salmonella, which is a genus of important zoonotic pathogens, has aroused great public health concern worldwide.Methods: In this study, 167 strains of Salmonella were isolated from 947 samples from broiler farms, slaughterhouses, and markets in Shandong Province. Antibiotic sensitivity testing was performed, and 70 strains of Salmonella were screened out by whole-genome sequencing (WGS) to evaluate serotypes, antimicrobial resistance genes (ARGs), the prevalence of class 1 integrons, the plasmid carriage rate, and phylogenetic characteristics and for multilocus sequence typing (MLST).Results: The results showed that the 167 isolates showed the highest resistance to ampicillin (AMP, 87.4%), sulfamethoxazole (SF, 87.4%), compound sulfamethoxazole (SXT, 81.4%), nalidixic acid (NAL, 80.2%), and amoxicillin/clavulanic acid (A/C, 77.8%). All the strains were sensitive to meropenem (MEM), and 91.0% of the isolates were MDR strains. We screened a total of 45 ARGs, with the highest detection rate observed for the tetracycline (TET) resistance gene tet (A) (81.4%). A total of 21 types of plasmid replicons were detected in Salmonella, of which IncX1 was the most common (74.3%), and 62.9% of the isolates carried a class 1 integron. In addition, a total of 11 different serotypes were detected, with S. enteritidis as the predominant serovar., followed by S. infantis and S. Newport. Twelve different sequence types (STs) were detected, among which ST11 was the main type. There was a strong correspondence between serotypes and STs. We also found that S. Indiana and S. Kentucky had extremely high rates of resistance to ciprofloxacin (CIP) and third-generation cephalosporins. System-wide genome analysis showed the occurrence of long-distance transmission across fields.Conclusion: In conclusion, the detection of multidrug resistance and isolates carrying multidrug resistance genes is the main problem, and emergency strategies should be implemented to address this issue.

Riemerella anatipestifer (R. anatipestifer) is a pathogenic bacterium belonging to the genus Riemerella within the family Flavobacteriaceae, which has multidrug resistance (MDR) and can cause high pathogenicity in waterfowl. The aim of this study was to investigate the antimicrobial resistance and genomic characteristics of R. anatipestifer strains isolated from several regions of China from 2023 to 2024. Two strains were selected for challenge tests, and virulence protection tests were conducted on florfenicol-resistant and florfenicol-sensitive strains. A total of 88 strains of R. anatipestifer were collected from the Shandong, Jiangsu, Guangdong, Hebei and Henan regions. The results showed that the 88 strains included serotypes 1, 2, 5, 6, 7 and 10. Serotype 5 was the most prevalent in the Shandong region. All strains were multidrug-resistant, with the hexaresistance accounting for the highest proportion (42.1%). A total of five resistance genes (tet(X), floR, ermF, qnrS, rmtB) and seven virulence genes were found (ompA, camp, AS87_04050, SIP, Fur, TbdR1, luxE). The challenge test showed that the LD₅₀ of RA12 was 2.75 × 10⁷ CFU/mL, and that of RA26 was 2.57 × 10⁷ CFU/mL. Phylogenetic tree analysis revealed that strain RA26 was closely related to strain 20190403E1-1, and strain RA16 was closely related to strain JW1. In addition, serotypes 2 and 7 identified in this study have been undergoing clonal transmission in China. Virulence protection tests indicated that the results of in vitro drug susceptibility tests were consistent with the therapeutic effects after in vivo treatment, and no R. anatipestifer was found in the visceral tissues of surviving ducklings. This study provides a reference for the rational use of antibiotics.

Considering that the strip method is simple and convenient for users, a Europium nanosphere-based time-resolved fluorescent immunochromatographic assay (TRFICA) for the rapid screening of 4,4′-dinitrocarbanilide (DNC) was developed to improve the performance of strip assays. After optimization, TRFICA showed IC₅₀, the limit of detection, and cut-off values of 0.4, 0.07, and 5.0 ng mL⁻¹, respectively. No significant cross-reactivity (CR < 0.1%) with 15 DNC analogs was observed in the developed method. TRFICA was validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 77.3% to 92.7%, with coefficients of variation of <14.9%. Moreover, the time needed for the detection procedure, including the sample pre-treatment, was less than 30 min for TRFICA, which had never been achieved before in other immunoassays. The newly developed strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique for DNC analysis in chicken muscle.