Inappropriate use of antibiotic enhances antibiotic resistance dissemination in ESBL-EC: Role of ydcz in outer membrane vesicles biogenesis and protein transport.

Extended-spectrumß-lactam producing Escherichia coli (ESBL-EC) readily colonizes live poultry and serves as a major source of contamination in retail chicken meat, posing significant threats to public health. This study aims to investigate the impact of inappropriate antibiotic use on the dissemination and exacerbation of antibiotic resistance in ESBL-EC and explore the underlying molecular mechanisms. Through experimental analysis, we propose a hypothesis that inappropriate antibiotic use may exacerbate resistance by affecting vesicle formation and protein secretion. Experimental results demonstrate that under the influence of amoxicillin, the concentration of proteins secreted in outer membrane vehicles (OMVs) by ESBL-EC significantly increases, along with a significant upregulation in the expression of the CTX-M-55-type Extended-spectrum beta-lactamase (CTX-M-55). Proteomic analysis and differential gene knockout experiments identified the key protein YdcZ, associated with OMVs formation and protein transportation in ESBL-EC under amoxicillin treatment. Further investigations reveal direct interactions between YdcZ and other proteins (YdiH and BssR). Upon ydcz gene knockout, a significant decrease in protein concentration within OMVs is observed, accompanied by a noticeable reduction in protection against sensitive bacteria. These findings suggest a critical role of YdcZ in regulating the process of protein transportation to OMVs in ESBL-EC under the influence of amoxicillin. In summary, our research uncovers the significant role of inappropriate antibiotic use in promoting the secretion of OMVs by ESBL-EC, aiding the survival of antibiotic-sensitive bacteria in the vicinity of infection sites. These findings provide new insights into the mechanisms underlying antibiotic-induced bacterial resistance dissemination and offer novel avenues for exploring prevention and control strategies against bacterial resistance propagation.

ESBL-Escherichia coli extracellular vesicles mediate bacterial resistance to ß-lactam and mediate horizontal transfer of blaCTX-M-55.

OBJECTIVES: Extracellular vesicles (EVs) have become the focus of research as an emerging method of horizontal gene transfer. In recent years, studies on the association between EVs and the spread of bacterial resistance have emerged, but there is a lack of research on the role of EVs secreted by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in the spread of ß-lactam resistance. Therefore, the aim of this study was to investigate the role of EVs in the transmission of ß-lactam resistance. METHODS: In this study, the role of EVs in the transmission of ß-lactam resistance in E. coli was evaluated by the EVs-mediated bacterial resistance to ß-lactam antibiotics test and the EVs-mediated blaCTX-M-55 transfer experiments using EVs secreted by ESBL-E. coli. RESULTS: The results showed that ESBL-EVs were protective against ß-lactam antibiotic-susceptible bacteria, and this protective effect was dependent on the integrity of the EVs and showed dose- and time-dependent effects. At the same time, ESBL-EVs can also mediate the horizontal transmission of blaCTX-M-55, and EVs-mediated gene transfer is selective, preferring to transfer in more closely related species. CONCLUSIONS: In this study, we demonstrated the important role of EVs in the transmission of ß-lactam resistance in chicken ESBL-E. coli, and evaluated the risk of EVs-mediated horizontal gene transfer, which provided a theoretical basis for elucidating the mechanism of EVs-mediated resistance transmission.

A Longitudinal Study on Campylobacter in Conventionally Reared Commercial Broiler Flocks in the United States: Prevalence and Genetic Diversity.

Poultry meat contaminated with Campylobacter, a major bacterial cause of foodborne gastroenteritis worldwide, is considered the primary source of human campylobacteriosis. Thus, reduction or elimination of Campylobacter in poultry production will have a significant impact on food safety and public health. Despite the significant progress made over the last decades, many puzzles remain about the epidemiology of Campylobacter on poultry farms, hampering the development of an effective control strategy. This longitudinal study was conducted to determine the prevalence and genetic diversity of Campylobacter in a U.S. commercial broiler production farm system. Cecal contents (15 samples/flock) and boot swabs (3 samples/flock) were collected from approximately 6-wk-old birds from 406 conventional broiler flocks reared in 53 houses on 15 farms (located within a relatively close geographic proximity and managed by the same poultry integrator) for up to eight consecutive production cycles and cultured for Campylobacter. Pulsed-field gel electrophoresis was used to investigate the genetic diversity of the Campylobacter jejuni isolates recovered from the cecal contents. The prevalence of Campylobacter at the farm, house, and flock levels were found to be 93% (14/15), 79% (42/53), and 47% (192/406), respectively. Campylobacter prevalence varied remarkably among different farms and flocks, with some farms or houses testing consistently negative while others being positive all the time over the entire study period. Campylobacter isolation rate changed significantly by sample type (higher by cecal contents vs. boot swabs) and season/production cycle (higher in spring vs. other seasons). The majority (88%; 2364/2675) of the isolates were identified as C. jejuni, and almost all the rest (11%; 303/2675) were Campylobacter coli. Genotyping showed limited diversity within a flock and suggested persistence of some C. jejuni clones over multiple production cycles on the same farm. In conclusion, this study indicated that although Campylobacter prevalence was overall high, there were marked differences in the prevalence among the broiler flocks or farms tested. Future studies aimed at identification of potential risk factors associated with differential Campylobacter status are warranted in order to develop effective on-farm interventions.

Estudio longitudinal sobre Campylobacter en parvadas comerciales de pollo de engorde criados convencionalmente en los Estados Unidos: prevalencia y diversidad genética. Los productos cárnicos de origen avícola contaminado con Campylobacter, que es una importante causa bacteriana de gastroenteritis transmitida por alimentos en todo el mundo, se consideran la principal fuente de campilobacteriosis humana. Por lo tanto, la reducción o eliminación de Campylobacter en la producción avícola tendrá un impacto significativo en la seguridad alimentaria y en la salud pública. A pesar de los importantes avances realizados en las últimas décadas, persisten muchos enigmas sobre la epidemiología de Campylobacter en las granjas avícolas, lo que obstaculiza el desarrollo de una estrategia de control eficaz. Este estudio longitudinal se realizó para determinar la prevalencia y la diversidad genética de Campylobacter en un sistema de granja de producción comercial de pollos de engorde en los Estados Unidos. Se recogieron contenidos cecales (15 muestras/parvada) y cubre botas de arrastre (tres muestras/parvada) de aves de aproximadamente seis semanas de edad de 406 parvadas de pollos de engorde convencionales criadas en 53 casetas de 15 granjas (ubicadas dentro de una proximidad geográfica relativamente cercana y manejadas por el mismo integrador avícola) durante ocho ciclos de producción consecutivos y con cultivo para Campylobacter. Se utilizó electroforesis en gel de campo con pulsasiones para investigar la diversidad genética de los aislados de Campylobacter jejuni recuperados del contenido cecal. Se encontró que la prevalencia de Campylobacter a nivel de granja, caseta y parvada era del 93% (14/15), 79% (42/53) y 47% (192/406), respectivamente. La prevalencia de Campylobacter varió notablemente entre diferentes granjas y rebaños, y algunas granjas o casetas dieron resultados consistentemente negativos mientras que otras dieron positivo todo el tiempo durante todo el período del estudio. La tasa de aislamiento de Campylobacter cambió significativamente según el tipo de muestra (mayor con muestras de contenido cecal en comparación con los cubre botas de arrastre) y la estación/ciclo de producción (mayor en primavera frente a otras estaciones). La mayoría (88%; 2364/2675) de los aislados se identificaron como C. jejuni, y casi todo el resto (11%; 303/2675) fueron Campylobacter coli. La genotipificación mostró una diversidad limitada dentro de una parvada y sugirió la persistencia de algunos clones de C. jejuni durante múltiples ciclos de producción en la misma granja. En conclusión, este estudio indicó que, aunque la prevalencia de Campylobacter fue alta en general, hubo marcadas diferencias en la prevalencia entre las parvadas o granjas de pollos de engorde analizadas. Se justifica la conducción de estudios futuros destinados a identificar posibles factores de riesgo asociados con el estado diferencial de Campylobacter para desarrollar intervenciones efectivas en las granjas.

Membrane vesicles derived from Enterococcus faecalis promote the co-transfer of important antibiotic resistance genes located on both plasmids and chromosomes.

BACKGROUND: Bacterial membrane vesicles (BMVs) are novel vehicles of antibiotic resistance gene (ARG) transfer in Gram-negative bacteria, but their role in the spread of ARGs in Gram-positive bacteria has not been defined. The purpose of this study was to evaluate the role of MVs in the transmission of antimicrobial resistance in Gram-positive bacteria. METHODS: A linezolid-resistant Enterococcus faecalis CQ20 of swine origin was selected as the donor strain. Linezolid-susceptible E. faecalis SC032 of human origin, Enterococcus faecium BM4105 and Escherichia coli were selected as recipient strains. The presence of plasmids (pCQ20-1 and pCQ20-2) and an optrA-carrying transposon Tn6674 in CQ20, MVs and vesiculants was verified by WGS or PCR. MVs were isolated with density gradient centrifugation, and MV-mediated transformation was performed to assess the horizontal transferability of MVs. The MICs for CQ20 and its vesiculants were determined by the broth microdilution method. RESULTS: CQ20-derived MVs (CQ20-MV) were isolated, and PCR identified the presence of two plasmids and the optrA gene in the CQ20-MVs. MV-mediated transformation to E. faecalis SC032 and E. faecium BM4105 was successfully performed, and the WGS data also showed that both plasmids pCQ20-1 and pCQ20-2 and optrA-carrying transposon Tn6674 were transferred to E. faecalis SC032 and E. faecium BM4105, but failed for E. coli. Additionally, vesiculants that had acquired ARGs still had the ability to spread these genes via MVs. CONCLUSIONS: To our knowledge, this is the first report of MV-mediated co-transfer of ARG-carrying plasmids and transposons in the Gram-positive bacterium E. faecium.

Field Investigation of the Dynamic Response of Culvert-Embankment-Culvert Transitions in a High-Speed Railway.

The stiffnesses of embankments and culverts differ in the transition sections of high-speed railways (HSRs) due to their different supporting conditions. The dynamic irregularity caused by the different stiffnesses makes this transition area the weakest part of high-speed railways. Graded crushed stone combined with 5% cement is typically used to fill the subgrade in these transition areas. Thus, three different particle size ratios of crushed stone were matched and tested regarding the construction parameters to explore the most suitable materials to fill the roadbed in a transition section. Then, field dynamic tests were carried out on the culvert-embankment-culvert transition area where trains run at speeds of 5-360 km/h. A time-domain analysis of the test data was performed to obtain the laws of variation that cause the dynamic characteristics to change with the railway line and roadbed layer and the changes induced by a train's running speed, operating direction, and axle weight. The results indicate that (i) it is feasible to fill transition section roadbeds with well-graded crushed stone combined with 5% cement with optimal water contents; (ii) extreme dynamic responses in some special sections are observed, suggesting the value of taking special measures at the transition section. For example, the sections 14.5 m and 30 m from the 679 culvert and the bed layer should be specially stabilized; (iii) the train's axle load and driving direction show a great effect on corresponding sections and layers but present a small effect on the sections and layers nearby; and (iv) 260 km/h is a critical speed.

Insights on genetic characterization and pathogenesis of a GI-19 (QX-like) infectious bronchitis virus isolated in China.

Infectious bronchitis virus (IBV) causes respiratory diseases in chickens, incurring great losses to the poultry industry worldwide. In this study, we isolated an IBV strain, designated as AH-2020, from the chickens vaccinated with H120 and 4/91 in Anhui, China. The sequence homology analysis based on the S1 gene revealed that AH-2020 shares low similarities with the 3 vaccine strains, namely, H120, LDT3-A, and 4/91 (78.19, 80.84, and 81.6%, respectively). Phylogenetic analysis based on the S1 gene revealed that AH-2020 clustered with the GI-19 type. Furthermore, protein modeling revealed that the mutations in the amino acids in AH-2020 were mainly located in the N-terminal domain of S1 (S1-NTD), and the pattern of deletion and insertion mutations in the S1 protein may have influenced the structural changes on the surface of S1. Further, approximately 7-day-old SPF chickens were inoculated with AH-2020 at 106.0 EID50. These chickens exhibited clinical signs of the infection such as listlessness, huddling, and head-shaking, accompanied by depression and 40% mortality. Serum antibody test demonstrated that in response to the AH-2020 infection, the antibody level increased the fastest at 7 dpi, with virus shedding rate of cloaca being 100% at 14 dpi. The viral titer in various tissues was detected using hematoxylin and eosin staining and immunohistochemistry, which revealed that AH-2020 infection can damage the kidney, trachea, lung, cecal tonsil, and bursa of Fabricius. Our study provided evidence that the GI-19-type IBV is undergoing more complex mutations, and effective measures are urgently needed to prevent the spread of these variant strains.

Sensitive detection of viable salmonella bacteria based on tertiary cascade signal amplification via splintR ligase ligation-PCR amplification-CRISPR/Cas12a cleavage.

Several viable Salmonella bacteria are capable of causing severe human diseases and huge economic losses. In this regard, viable Salmonella bacteria detection techniques that can identify small numbers of microbial cells are highly valuable. Here, we present a detection method (referred to as SPC) based on the amplification of tertiary signals using splintR ligase ligation, PCR amplification and CRISPR/Cas12a cleavage. The detection limit of the SPC assay was 6 copies (HilA RNA) and 10 CFU (cell). Based on Intracellular HilA RNA detection, this assay can be used to distinguish between viable and dead Salmonella. In addition, it is able to detect multiple serotypes of Salmonella and has been successfully used to detect Salmonella in milk or isolated from farms. Overall, this assay is a promising test for viable pathogens detection and biosafety control.

Chicken interferon-induced transmembrane protein 1 promotes replication of coronavirus infectious bronchitis virus in a cell-specific manner.

Interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit numerous virus infections by impeding viral entry into target cells. However, increasing evidence suggests diverse functions of IFITMs in virus infection, especially with the coronavirus. We analyzed the effect of chicken interferon-induced transmembrane proteins (chIFITMs) on coronavirus infectious bronchitis virus (IBV) infection in vitro. We demonstrated that the antiviral effects of IFITMs are dependent on cell and virus types. The overexpression of chIFITM1 dramatically promoted the replication of IBV Beaudette strain in the chicken hepatocellular carcinoma cell line, LMH. Mechanistically, chIFITMs share roughly the same subcellular localization in different host cells, and overexpressed of chIFITM1 have no effect of viral attachment and entry. Further studies revealed that mutations of amino acids at key positions (60KSRD63, 68KDFV71) in the intracellular loop domain (CIL) caused loss of the promoted function. Interaction with downstream proteins in co-response to viral infection could be the primary reason behind variable functions of chIFITM1 in different cells. In all, our study explored the functions of chIFITMs in viral infection from a new perspective.

Comparative Analysis of Phylogenetic Relationships and Virulence Factor Characteristics between Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates Derived from Clinical Sites and Chicken Farms.

Antimicrobial resistance in bacteria is the most urgent global threat to public health, with extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-E. coli) being one of the most documented examples. Nonetheless, the ESBL-E. coli transmission relationship among clinical sites and chicken farms remains unclear. Here, 408 ESBL-E. coli strains were isolated from hospitals and chicken farms in Sichuan Province and Yunnan Province in 2021. We detected blaCTX-M genes in 337 (82.62%) ESBL-E. coli strains. Although the isolation rate, prevalent sequence type (ST) subtypes, and blaCTX-M gene subtypes of ESBL-E. coli varied based on regions and sources, a few strains of CTX-ESBL-E. coli derived from clinical sites and chicken farms in Sichuan Province displayed high genetic similarity. This indicates a risk of ESBL-E. coli transmission from chickens to humans. Moreover, we found that the high-risk clonal strains ST131 and ST1193 primarily carried blaCTX-M-27. This indicates that drug-resistant E. coli from animal and human sources should be monitored. As well, the overuse of ß-lactam antibiotics should be avoided in poultry farms to ensure public health and build an effective regulatory mechanism of "farm to fork" under a One Health perspective. IMPORTANCE Bacterial drug resistance has become one of the most significant threats to human health worldwide, especially for extended-spectrum ß-lactamase-producing E. coli (ESBL-E. coli). Timely and accurate epidemiological surveys can provide scientific guidance for the adoption of treatments in different regions and also reduce the formation of drug-resistant bacteria. Our study showed that the subtypes of ESBL-E. coli strains prevalent in different provinces are somewhat different, so it is necessary to individualize treatment regimens in different regions, and it is especially important to limit and reduce antibiotic use in poultry farming since chicken-derived ESBL-E. coli serves as an important reservoir of drug resistance genes and has the potential to spread to humans, thus posing a threat to human health. The use of antibiotics in poultry farming should be particularly limited and reduced.

Withdrawal of antibiotic growth promoters in China and its impact on the foodborne pathogen Campylobacter coli of swine origin.

Antibiotic growth promoters (AGPs) have been used as feed additives to improve feed efficiency in food animals for more than six decades. However, the wide use of AGPs has led to the emergence of antibiotic-resistant pathogens of animal origin, posing a significant threat to food safety and public health. China prohibited the addition of AGPs to animal feed from July 2020. The impacts caused by the withdrawal of AGPs on the prevalence and antibiotic resistance of foodborne pathogens have not been illustrated. Here, a total of 471 strains of Campylobacter were isolated from pigs from three pig farms and two slaughterhouses in Sichuan Province for 4 consecutive years (2018-2021), including 2 years before and 2 years after the ban on AGPs in China. The isolation rate of Campylobacter had a slight increase after prohibiting the addition of AGPs to the feed. Contrary to what we expected, the antibiotic susceptibility test and WGS data showed that the antibiotic resistance to gentamicin and florfenicol and the abundance of virulence genes increased significantly after the ban of AGPs. Comparison of the isolates of swine origin with isolates of human origin indicated the potential of antibiotic-resistant Campylobacter transmission from pigs to humans. These data suggested that phasing out AGPs may lead to increased use of therapeutic antimicrobials, promoting the prevalence and transmission of both antibiotic resistance and virulence genes.

Pathogen-Specific Bactericidal Method Mediated by Conjugative Delivery of CRISPR-Cas13a Targeting Bacterial Endogenous Transcripts.

The emergence of antibiotic-resistant bacteria threatens public health, and the use of broad-spectrum antibiotics often leads to unintended consequences, including disturbing the beneficial gut microbiota and resulting in secondary diseases. Therefore, developing a novel strategy that specifically kills pathogens without affecting the residential microbiota is desirable and urgently needed. Here, we report the development of a precise bactericidal system by taking advantage of CRISPR-Cas13a targeting endogenous transcripts of Salmonella enterica serovar Typhimurium delivered through a conjugative vehicle. In vitro, the CRISPR-Cas13a system exhibited specific killing, growth inhibition, and clearance of S. Typhimurium in mixed microbial flora. In a mouse infection model, the CRISPR-Cas13a system, when delivered by a donor Escherichia coli strain, significantly reduced S. Typhimurium colonization in the intestinal tract. Overall, the results demonstrate the feasibility and efficacy of the designed CRISPR-Cas13a system in selective killing of pathogens and broaden the utility of conjugation-based delivery of bactericidal approaches. IMPORTANCE Antibiotics with broad-spectrum activities are known to disturb both pathogens and beneficial gut microbiota and cause many undesired side effects, prompting increased interest in developing therapies that specifically eliminate pathogenic bacteria without damaging gut resident flora. To achieve this goal, we developed a strategy utilizing bacterial conjugation to deliver CRISPR-Cas13a programmed to specifically kill S. Typhimurium. This system produced pathogen-specific killing based on CRISPR RNA (crRNAs) targeting endogenous transcripts in pathogens and was shown to be effective in both in vitro and in vivo experiments. Additionally, the system can be readily delivered by conjugation and is adaptable for targeting different pathogens. With further optimization and improvement, the system has the potential to be used for biotherapy and microbial community modification.

Outer Membrane Vesicles of Avian PathogenicEscherichia coli Mediate the Horizontal Transmission of blaCTX-M-55.

The CTX-M-55 type extended-spectrum ß-lactamase (ESBL) producing Enterobacteriaceae is increasing in prevalence worldwide without the transmission mechanism being fully clarified, which threatens public and livestock health. Outer membrane vesicles (OMVs) have been shown to mediate the gene horizontal transmission in some species. However, whether blaCTX-M-55 can be transmitted horizontally through OMVs in avian pathogenic Escherichia coli (APEC) has not been reported yet. To test this hypothesis, an ESBL-producing APEC was isolated and whole-genome sequencing (WGS) was performed to analyze the location of blaCTX-M-55. Ultracentrifugation and size exclusion chromatography was used to isolate and purify OMVs, and the transfer experiment of blaCTX-M-55 via OMVs was performed finally. Our results showed that the blaCTX-M-55 was located on an IncI2 plasmid. The number and diameter of OMVs secreted by ESBL-producing APEC treated with different antibiotics were significantly varied. The transfer experiment showed that the OMVs could mediate the horizontal transfer of blaCTX-M-55, and the frequency of gene transfer ranged from 10-5 to 10-6 CFU/mL with the highest frequency observed in the Enrofloxacin treatment group. These findings contribute to a better understanding of the antibiotics in promoting and disseminating resistance in the poultry industry and support the restrictions on the use of antibiotics in the poultry industry.

Development of nanobody-horseradish peroxidase-based sandwich ELISA to detect Salmonella Enteritidis in milk and in vivo colonization in chicken.

BACKGROUND: Salmonella Enteritidis (S. Enteritidis) being one of the most prevalent foodborne pathogens worldwide poses a serious threat to public safety. Prevention of zoonotic infectious disease and controlling the risk of transmission of S. Enteriditidis critically requires the evolution of rapid and sensitive detection methods. The detection methods based on nucleic acid and conventional antibodies are fraught with limitations. Many of these limitations of the conventional antibodies can be circumvented using natural nanobodies which are endowed with characteristics, such as high affinity, thermal stability, easy production, especially higher diversity. This study aimed to select the special nanobodies against S. Enteriditidis for developing an improved nanobody-horseradish peroxidase-based sandwich ELISA to detect S. Enteritidis in the practical sample. The nanobody-horseradish peroxidase fusions can help in eliminating the use of secondary antibodies labeled with horseradish peroxidase, which can reduce the time of the experiment. Moreover, the novel sandwich ELISA developed in this study can be used to detect S. Enteriditidis specifically and rapidly with improved sensitivity. RESULTS: This study screened four nanobodies from an immunized nanobody library, after four rounds of screening, using the phage display technology. Subsequently, the screened nanobodies were successfully expressed with the prokaryotic and eukaryotic expression systems, respectively. A sandwich ELISA employing the SE-Nb9 and horseradish peroxidase-Nb1 pair to capture and to detect S. Enteritidis, respectively, was developed and found to possess a detection limit of 5 × 104 colony forming units (CFU)/mL. In the established immunoassay, the 8 h-enrichment enabled the detection of up to approximately 10 CFU/mL of S. Enteriditidis in milk samples. Furthermore, we investigated the colonization distribution of S. Enteriditidis in infected chicken using the established assay, showing that the S. Enteriditidis could subsist in almost all parts of the intestinal tract. These results were in agreement with the results obtained from the real-time PCR and plate culture. The liver was specifically identified to be colonized with quite a several S. Enteriditidis, indicating the risk of S. Enteriditidis infection outside of intestinal tract. CONCLUSIONS: This newly developed a sandwich ELISA that used the SE-Nb9 as capture antibody and horseradish peroxidase-Nb1 to detect S. Enteriditidis in the spike milk sample and to analyze the colonization distribution of S. Enteriditidis in the infected chicken. These results demonstrated that the developed assay is to be applicable for detecting S. Enteriditidis in the spiked milk in the rapid, specific, and sensitive way. Meanwhile, the developed assay can analyze the colonization distribution of S. Enteriditidis in the challenged chicken to indicate it as a promising tool for monitoring S. Enteriditidis in poultry products. Importantly, the SE-Nb1-vHRP as detection antibody can directly bind S. Enteritidis captured by SE-Nb9, reducing the use of commercial secondary antibodies and shortening the detection time. In short, the developed sandwich ELISA ushers great prospects for monitoring S. Enteritidis in food safety control and further commercial production.

Immunocapture Magnetic Beads Enhanced the LAMP-CRISPR/Cas12a Method for the Sensitive, Specific, and Visual Detection of Campylobacter jejuni.

Campylobacter jejuni is one of the most important causes of food-borne infectious disease, and poses challenges to food safety and public health. Establishing a rapid, accurate, sensitive, and simple detection method for C. jejuni enables early diagnosis, early intervention, and prevention of pathogen transmission. In this study, an immunocapture magnetic bead (ICB)-enhanced loop-mediated isothermal amplification (LAMP) CRISPR/Cas12a method (ICB-LAMP-CRISPR/Cas12a) was developed for the rapid and visual detection of C. jejuni. Using the ICB-LAMP-CRISPR/Cas12a method, C. jejuni was first captured by ICB, and the bacterial genomic DNA was then released by heating and used in the LAMP reaction. After the LAMP reaction, LAMP products were mixed and detected by the CRISPR/Cas12a cleavage mixture. This ICB-LAMP-CRISPR/Cas12a method could detect a minimum of 8 CFU/mL of C. jejuni within 70 min. Additionally, the method was performed in a closed tube in addition to ICB capture, which eliminates the need to separate preamplification and transfer of amplified products to avoid aerosol pollution. The ICB-LAMP-CRISPR/Cas12a method was further validated by testing 31 C. jejuni-positive fecal samples from different layer farms. This method is an all-in-one, simple, rapid, ultrasensitive, ultraspecific, visual detection method for instrument-free diagnosis of C. jejuni, and has wide application potential in future work.

Identification of Three Novel PmGRI1 Genomic Resistance Islands and One Multidrug Resistant Hybrid Structure of Tn7-like Transposon and PmGRI1 in Proteus mirabilis.

The widespread use of antibiotics in large-scale livestock production has led to serious antibiotic resistance. Proteus mirabilis is an important pathogenic bacterium on large-scale farms. Chromosomally localized mobilizable genetic elements (genomic islands) and mobile genetic elements (Tn7-like transposons) play an important role in the acquisition and transmission of resistance genes by P. mirabilis. To study the prevalence and resistance characteristics of antibiotic-resistant genomic islands in P. mirabilis of animal origin in China, we performed whole genome sequencing of P. mirabilis isolated from large-scale pig and chicken farms. Three new variants of PmGRI1 (HN31, YN8, and YN9), and a hybrid structure (HN2p) formed by the multidrug-resistant Tn7-like-HN2p transposon and a genomic island PmGRI1-HN2p, were identified from P. mirabilis. All variants underwent homologous recombination mediated by insertion sequence IS26. A genomic rearrangement in the chromosome between the Tn7-like-HN2p transposon and PmGRI1-HN2p occurred in HN2p. The heterozygous structure contained various antimicrobial resistance genes, including three copies of fluoroquinolone resistance gene qnrA1 and 16S rRNA methylase gene rmtB, which are rarely found in P. mirabilis. Our results highlight the structural genetic diversity of genomic islands by characterizing the novel variants of PmGRI1 and enrich the research base of multidrug resistance genomic islands.

Tracking Salmonella enterica by whole genome sequencing of isolates recovered from broiler chickens in a poultry production system.

Salmonella enterica is a major cause of foodborne diseases, and is also an important pathogenic bacterium in poultry industry. Whole genome sequencing (WGS) has become a crucial molecular typing technology used for the surveillance of the pathogenic bacteria. In the present study, we adopted WGS for tracking transmission of S. enterica in the production chain of broiler chickens. A total of 74 S. enterica strains were isolated from the different steps of breeding and slaughtering in a large production enterprise in Sichuan Province, China. The isolation rate of Salmonella was the highest in procedure of defeathering (50.0%) and evisceration (36.7%). Serotype identification showed that 74 Salmonella isolates included 7 serotypes, among which Mbandaka accounted for the highest proportions (35.1%). WGS revealed that 74 strains belonged to 7 different sequence types (STs), as well as 7 different ribosomal STs and 35 core genome STs. cgMLST-based Minimum Spanning Trees and phylogenetic tree based on the SNPs indicated that three serotypes, Mbandaka, Indiana and Kentucky, could be clonally transmitted between broiler farm and slaughterhouse. Heterogeneous resistant phenotypes and genotypes were found in two serotypes, Indiana and Kentucky. Our study indicated WGS in an accurate tool for molecular typing of S. enterica. Routine surveillance of S. enterica in the production chain of broiler chickens is needed.

Novel insertion sequence ISChh1-like mediating acquisition of optrA gene in foodborne pathogen Campylobacter coli of swine origin.

The optrA gene encodes an ABC-F protein which confers cross-resistance to oxazolidinones and phenicols, and its transmission has so far been associated with multiple transposable elements, including IS1216E, prophages, ICEs and Tn558. Here, we identified an optrA gene flanked by two copies of a novel insertion sequence ISChh1-like in the same direction in Campylobacter coli. Seven optrA-positive C. coli were identified from 263 Campylobacter isolates obtained from one swine farm and two slaughterhouses in Sichuan province of China. The optrA genes in 6 isolates were directly flanked by two copies of ISChh1-like elements in the same orientation, in the remaining one isolate, optrA was co-located with fexA and flanked by two copies of IS1216E and inserted in the downstream of the aadE-sat4-aphA3 gene cluster on chromosome. Cloning of optrA into C. jejuni NCTC 11,168 confirmed its role in elevated MICs to oxazolidinones and phenicols. Translocatable units (TUs) and natural transformants were only detected from the isolate with optrA bracketed by IS1216E, not from these with optrA bracketed by ISChh1-like. ISChh1-like in all isolates inserts specifically either next to or between adjacent GG nucleotides, neither have terminal inverted repeats nor generate target site duplications. Interestingly, ISChh1-like were also found mediating integration of other antibiotic resistance genes in Campylobacter, including tet(O), aphA3 and aadE-sat4-aphA3 gene cluster. Taken together, these results identify ISChh1-like as a novel transposon mediating acquisition of multiple antibiotic resistance genes in Campylobacter, including the very important optrA gene, suggesting that it plays an essential role in the transmission of antibiotic resistance genes to Campylobacter.

Genetic environments and related transposable elements of novel cfr(C) variants in Campylobacter coli isolates of swine origin.

The cfr(C) is a cfr-like gene that confers cross-resistance to antibiotics targeting the 23S rRNA through methylation of nucleotide A2503. Here, we identified 7 C. coli isolates containing 4 novel cfr(C) variants from swine farm and slaughterhouses samples. Of the 7 cfr(C)-carrying isolates, one had a frame-shift mutation, while the other 6 had intact genes. However, one of the 6 intact genes did not show a PhLOPSA phenotype in the original isolate, but was fully functional when cloned into C. jejuni NCTC 11168. Cloning of cfr(C) variants into C. jejuni NCTC 11168 and conjugative transfer of the two cfr(C)-containing plasmids further confirmed their role in conferring resistance to PhLOPSA antimicrobials, and resulted in an 8-128-fold increase in their MICs. In all cfr(C)-carrying isolates, cfr(C) genes were located in the downstream of the kanamycin resistant gene aphA3. IS607* and IS1595-like were located immediately upstream of aphA3 gene and seemed to play a role in its recombination. A novel transposable element named ISCco7, which located immediately downstream of cfr(C) in two isolates, was probably associated with the integration of cfr(C). However, neither insertion sequence nor other transposable elements were identified near cfr(C) in the remaining five cfr(C)-positive isolates, indicating the mechanism underlying the integration of cfr(C) into plasmids or chromosomal DNA requires further investigation. These results reveal novel cfr(C) variants and their associated genetic environments in C. coli isolates and indicate the flexibility of C. coli in acquiring new antibiotic resistance genes.

Emergence of fexA in Mediating Resistance to Florfenicols in Campylobacter.

Florfenicol belongs to a class of phenicol antimicrobials widely used as feed additives and for the treatment of respiratory infections. In recent years, increasing resistance to florfenicol has been reported in Campylobacter spp., the leading foodborne enteric pathogens causing diarrheal diseases worldwide. Here, we reported the identification of fexA, a novel mobile florfenicol resistance gene in Campylobacter Of the 100 Campylobacter jejuni strains isolated from poultry in Zhejiang, China, 9 were shown to be fexA positive, and their whole-genome sequences were further determined by integration of Illumina short-read and MinION long-read sequencing. The fexA gene was found in the plasmid of one strain and chromosomes of eight strains, and its location was verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting. Based on comparative analysis, the fexA gene was located within a region with the tet(L)-fexA-catA-tet(O) gene arrangement, demonstrated to be successfully transferable among C. jejuni strains. Functional cloning indicated that acquisition of the single fexA gene significantly increased resistance to florfenicol, whereas its inactivation resulted in increased susceptibility to florfenicol in Campylobacter Taken together, these results indicated that the emerging fexA resistance is horizontally transferable, which might greatly facilitate the adaptation of Campylobacter in food production environments where florfenicols are frequently used.