The hidden threat: Comprehensive assessment of antibiotic and disinfectant resistance in commercial pig slaughterhouses.

The presence of antibiotic resistance genes (ARGs), disinfectant resistance genes (DRGs), and pathogens in animal food processing environments (FAPE) poses a significant risk to human health. However, knowledge of the contamination and risk profiles of a typical commercial pig slaughterhouse with periodic disinfectant applications is limited. By creating the overall metagenomics-based behavior and risk profiles of ARGs, DRGs, and microbiomes in a nine-section pig slaughterhouse, an important FAPE in China. A total of 454 ARGs and 84 DRGs were detected in the slaughterhouse with resistance genes for aminoglycosides and quaternary ammonium compounds, respectively. The entire slaughtering chain is a hotspot for pathogens, including 83 human pathogenic bacteria (HPB), with 47 core HPB. In addition, 68 high-risk ARGs were significantly correlated with 55 HPB, 30 of which were recognized as potential bacteria co-resistant to antibiotics and disinfectants, confirm a three-fold risk of ARGs, DRGs, and pathogens prevailing throughout the chain. Pre-slaughter pig house (PSPH) was the major risk source for ARGs, DRGs, and HPB. Moreover, 75 Escherichia coli and 47 Proteus mirabilis isolates showed sensitivity to potassium monopersulfate and sodium hypochlorite, suggesting that slaughterhouses should use such related disinfectants. By using whole genome multi-locus sequence typing and single nucleotide polymorphism analyses, genetically closely related bacteria were identified across distinct slaughter sections, suggesting bacterial transmission across the slaughter chain. Overall, this study underscores the critical role of the PSPH section as a major source of HPB, ARGs, and DRGs contamination in commercial pig slaughterhouses. Moreover, it highlights the importance of addressing clonal transmission and cross-contamination of antibiotic- and disinfectant-resistant bacteria within and between slaughter sections. These issues are primarily attributed to the microbial load carried by animals before slaughter, carcass handling, and content exposure during visceral treatment. Our findings provide valuable insights for One Health-oriented slaughterhouse management practices.

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection.

Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 °C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5'-/6-FAM/CCCCCCCC/BHQ1/-3') presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection.

siRNA targeting Atp5a1 gene encoding ATPase α, the ligand of Peg fimbriae, reduced Salmonella Enteritidis adhesion.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a zoonotic pathogen that can infect both humans and animals. Among the 13 types of fimbrial operons in S. Enteritidis, the highly conserved Peg fimbriae play a crucial role in the adhesion and invasion of S. Enteritidis into host cells but are not well studied. In this study, we identified the ATP synthase subunit alpha (ATPase α) as a ligand of Peg fimbriae using ligand blotting and mass spectrometry techniques. We confirmed the in vitro binding of ATPase α to the purified adhesion protein (PegD). Furthermore, we used siRNA to suppress the expression of ATPase α gene Atp5a1 in Leghorn male hepatoma (LMH) cells, which resulted in a significant reduction in the adhesion rate of S. Enteritidis to the cells (P < 0.05). The findings in this study provide insight into the mechanism of S. Enteritidis infection through Peg fimbriae and highlight the importance of ATPase α in the adhesion process.RESEARCH HIGHLIGHTS Ligand blotting was performed to screen the ligand of S. Enteritidis Peg fimbriae.Binding assay confirmed that ATPase α is the ligand of the Peg fimbriae.siRNA targeting ATPase α gene (Atp5a1) significantly reduced S. Enteritidis adhesion.

Machine learning combined with MALDI-TOF MS has the potential ability to identify serotypes of the avian pathogen Riemerella anatipestifer.

AIM: Combining MALDI-TOF MS and machine learning to establish a new rapid method to identify two important serotypes of Rimerella anatipestifer. METHODS AND RESULTS: MALDI-TOF MS was performed on 115 R. anatipestifer strains (serotype 1, serotype 2, and other serotypes) to explore its ability to identify serotypes of R. anatipestifer. Raw spectral data were generated in diagnostic mode; these data were preprocessed, clustered, and analysed using principal component analysis. The results indicated that MALDI-TOF MS completely differentiated serotype 1 from serotype 2 of R. anatipestifer; the potential serotype-associated m/z loci are listed. Furthermore, Random Forest and Support Vector Machine were used for modelling to identify the two important serotypes, and the results of cross-validation indicated that they had ∼80% confidence to make the right classification. CONCLUSION: We proved that MALDI-TOF MS can differentiate serotype 1 from serotype 2 of R. anatipestifer. Additionally, the identification models established in this study have high confidence to screen out these two important serotypes from other serotypes.

Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of goose astrovirus genotypes 1 and 2.

Goose astrovirus (GAstV) is a novel pathogen that was discovered in 2018. It has two genotypes, GAstV-1 and GAstV-2, and both can cause visceral gout of goslings and result in significant economic losses. The present work aimed to develop a duplex TaqMan real-time quantitative reverse transcription PCR (RT-qPCR) assay to distinguish the two genotypes. MegAlign software was used to design two pairs of primers and a pair of matched probes based on the open reading frame 2 (ORF2) sequence with the greatest difference between GAstV-1 and GAstV-2, and primer and probe concentrations and annealing temperatures were optimised. Fluorescence signals were obtained for GAstV-1 and GAstV-2 in the FAM and VIC channels, respectively, but no fluorescent signal was observed for other pathogens. The detection limit for GAstV-1 and GAstV-2 was 33.3 and 33.7 DNA copies/µL, respectively. Intra- and inter-assay variability tests revealed excellent reproducibility. Furthermore, the assay detected GAstV-1 and GAstV-2 in allantoic fluids (100% positive) spiked with viruses, and 70 clinical gout gosling samples were examined, of which 11.4% were positive for GAstV-1, 74.3% were positive for GAstV-2%, and 5.7% were positive for mixed infection. In summary, the developed duplex RT-qPCR assay has high specificity, sensitivity, and reproducibility, and can be used in the clinic for detection of GAstV-1 and GAstV-2.

O145 may be emerging as a predominant serogroup of Avian pathogenic Escherichia coli (APEC) in China.

Among the numerous serotypes of Avian pathogenic Escherichia coli (APEC), O1, O2 and O78 have long been considered the predominant serogroups. O145, a pivotal serogroup in non-O157 Shiga toxin-producing Escherichia coli, has never been considered an important serogroup among APEC. The prevalence of APEC O145 was determined from the results of molecular serogrouping based on 42 sequenced isolates from Jiangsu and Guangxi Provinces in China. After realizing the potential importance of O145, 224 APEC isolates isolated from Jiangsu, Guangxi, Anhui, Shandong, Henan, Yunnan and Fujian provinces were screened using PCR amplification. The results showed that the proportion of O145 detected was 37.9 % (85/224), which was higher than those of the three traditional APEC serogroups. The virulence evaluation experiment showed that this serogroup may have stronger pathogenicity. Here, we report for the first time that O145 may be emerging as a predominant serogroup of APEC in China. The possible reasons for its prevalence and oversight were analyzed through genomic analysis. Furthermore, pangenome analysis with STEC O145 was performed to assess the potential threat to humans. The discovery of the ubiquity of O145 may not be coincidental, which may also account for the failure of vaccines that target the three major serogroups. Therefore, this newly predominant serogroup should be paid more attention and the focus should not be limited to the so-called three major APEC serogroups.

Combining pangenome analysis to identify potential cross-protective antigens against avian pathogenic Escherichia coli.

RESEARCH HIGHLIGHTSPan-RV analysis was used for the first time in the discovery of APEC-protective proteins.A total of 53 potential protective proteins were screened out.Four proteins were verified as potential vaccine candidates using western blotting.

Characterization and genome analysis of a broad lytic spectrum bacteriophage P479 against multidrug-resistant Escherichia coli.

The increase of multi-drug resistant and multi-serotypes of pathogenic Escherichia coli has brought more severe challenge to control infection. Nowadays, bacteriophage is a promising tool to treat colibacillosis as an alternative of antibiotics. A coliphage P479, isolated from sewage of poultry farm, could lyse multiple serotypes, including not only O1, O2, O8, O9, O21, O78, O83, O145 of Avian pathogenic E. coli, but O157:H7 of Enterohaemorrhagic E. coli and O18:K1:H7 Neonatal meningitis E. coli. Additionally, P479 could also lyse multi-drug resistant E. coli. These indicated that P479 had good lytic ability. One-step growth curve revealed that the latent time period of P479 was 10 min and the burst size was about 318 PFU/cell. Stability tests demonstrated that P479 had good stability under various temperature (4 to 50 °C) and pH (3 to11) conditions. P479 contained of a linear, double-stranded DNA molecule of 172,033 bp with 40.3% GC content. P479 contained 296 putative coding sequences (CDSs) and two tRNA genes. Based on genomic comparison, P479 was classified as a member of genus Gaprivervirus, subfamily Tevenvirinae, family Myoviridae, order Caudovirales. No known virulent or lysogenic genes were detected in the genome of P479, manifesting P479 was safe to adhibit. Antibacterial activity in vitro manifested that P479 has varying degrees bacteriostatic activity against different bacteria. According to the above properties, P479 has the potential to be applied in phage therapy in the future.

[A simulation model for predicting the dry matter allocation in cut lily plants under effects of substrate water potential].

Dry matter allocation and translocation is the base of the formation of appearance quality of ornamental plants, and strongly affected by water supply. Taking cut lily cultivar 'Sorbonne' as test material, a culture experiment of different planting dates and water supply levels was conducted in a multi-span greenhouse in Nanjing from March 2009 to January 2010 to quantitatively analyze the seasonal changes of the dry matter allocation and translocation in 'Sorbonne' plants and the effects of substrate water potential on the dry matter allocation indices for different organs (flower, stem, leaf, bulb, and root), aimed to define the critical substrate water potential for the normal growth of the cultivar, and establish a simulation model for predicting the dry matter allocation in cut lily plants under effects of substrate water potential. The model established in this study gave a good prediction on the dry mass of plant organs, with the coefficient of determination and the relative root mean square error between the simulated and measured values of the cultivar' s flower dry mass, stem dry mass, leaf dry mass, bulb dry mass, and root dry mass being 0.96 and 19.2%, 0.95 and 12.4%, 0.86 and 19.4%, 0.95 and 12.2%, and 0.85 and 31.7%, respectively. The critical water potential for the water management of cut lily could be -15 kPa.