Novel multidrug resistance genomic islands and transposon carrying blaVEB-1 identified in mcr-positive Aeromonas strains from raw meat in China.

OBJECTIVES: To characterize the genetic environments of ESBL gene blaVEB-1 in mcr-positive Aeromonas strains from raw meat in China. METHODS: Whole genomes of Aeromonas strains were sequenced using the Illumina or Nanopore platforms. Genetic environments of blaVEB-1 were analysed using the BLAST program. RESULTS: The blaVEB-1 gene was detected in five Aeromonas strains carrying the mcr-7-like gene. WGS revealed that all blaVEB-1 genes were located on Aeromonas chromosome, and were carried by two novel different genomic islands named Aeromonas veronii genomic islands AveGI1 and AveGI2, as well as one transposon named Tn7690. AveGI1 is a new member of the Salmonella genomic island 1 family, incorporated into the 3'-end of mnmE (trmE). AveGI2 is a novel genomic island that has a size of 23 180 bp and is incorporated into the 3'-end of syd. The MDR regions of AveGI1 and AveGI2 are two different class 1 integrons containing 10 and five resistance genes, respectively. Tn7690 is a Tn1722 derivative containing In4-type integron and Tn5393, which harbours 10 resistance genes and integrates into different positions on the chromosomes of three strains with the capacity for mobility. CONCLUSIONS: We report chromosomally located novel MDR genomic islands and transposon that carry blaVEB-1 in mcr-positive Aeromonas strains. These genetic elements may mediate the spread of blaVEB-1 in Aeromonas, and may also evolve by capturing new antimicrobial resistance genes or other mobile genetic elements.

Evaluation of a novel recombinant strain of infectious bronchitis virus emerged from three attenuated live vaccine strains.

Infectious bronchitis virus (IBV) causes avian infectious bronchitis (IB) and there are multiple serotypes worldwide originating from deletions, insertions, point mutations, and RNA recombination. In this study, a recombinant IBV, named CK/CH/MY/2020, was isolated from southwest China. Sequencing and phylogenetic analysis revealed that CK/CH/MY/2020 consists of 27,614 nucleotides and belongs to the GI-28 genotype. Moreover, the strain is a recombination product originating from three live attenuated vaccine strains (H120, 4/91, and LDT3-A). The recombination is complicated involving at least nine recombination sites; the first 3/5 portion is mainly composed of H120 and 4/91, and the second 2/5 contains LDT3-A. Pathogenicity analysis showed that CK/CH/MY/2020 could cause respiratory and kidney diseases in chickens resulting in moderate mortality. Therefore, the recombinant strain is more virulent than the attenuated vaccine strains. This study shows that even in the absence of wild strains, the recombination and revirulence of multiple attenuated vaccines could occur simultaneously, which also highlights the continuous evolution in IBV.

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.

A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum.

Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55-1.65% and 2.58-6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.

Identification and analysis of long non-coding RNAs and mRNAs in chicken macrophages infected with avian infectious bronchitis coronavirus.

BACKGROUND: Avian infectious bronchitis virus (IBV) is a gamma coronavirus that severely affects the poultry industry worldwide. Long non-coding RNAs (lncRNAs), a subset of non-coding RNAs with a length of more than 200 nucleotides, have been recently recognized as pivotal factors in the pathogenesis of viral infections. However, little is known about the function of lncRNAs in host cultured cells in response to IBV infection. RESULTS: We used next-generation high throughput sequencing to reveal the expression profiles of mRNAs and lncRNAs in IBV-infected HD11 cells. Compared with the uninfected cells, we identified 153 differentially expressed (DE) mRNAs (106 up-regulated mRNAs, 47 down-regulated mRNAs) and 181 DE lncRNAs (59 up-regulated lncRNAs, 122 down-regulated lncRNAs) in IBV-infected HD11 cells. Moreover, gene ontology (GO) and pathway enrichment analyses indicated that DE mRNAs and lncRNAs were mainly involved in cellular innate immunity, amino acid metabolism, and nucleic acid metabolism. In addition, 2640 novel chicken lncRNAs were identified, and a competing endogenous RNA (ceRNAs) network centered on gga-miR-30d and miR-146a-5p was established. CONCLUSIONS: We identified expression profiles of mRNAs and lncRNAs during IBV infection that provided new insights into the pathogenesis of IBV.

Florfenicol Enhances Colonization of a Salmonella enterica Serovar Enteritidis floR Mutant with Major Alterations to the Intestinal Microbiota and Metabolome in Neonatal Chickens.

Florfenicol is an important antibiotic commonly used in poultry production to prevent and treat Salmonella infection. However, oral administration of florfenicol may alter the animals' natural microbiota and metabolome, thereby reducing intestinal colonization resistance and increasing susceptibility to Salmonella infection. In this study, we determined the effect of florfenicol (30 mg/kg of body weight) on gut colonization of neonatal chickens challenged with Salmonella enterica subsp. enterica serovar Enteritidis. We then analyzed the microbial community structure and metabolic profiles of cecal contents using microbial 16S amplicon sequencing and liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics, respectively. We also screened the marker metabolites using a multi-omics technique and assessed the effect of these markers on intestinal colonization by S. Enteritidis. Florfenicol administration significantly increased the loads of S. Enteritidis in cecal contents, spleen, and liver and prolonged the residence of S. Enteritidis. Moreover, florfenicol significantly affected cecal colony structures, with reduced abundances of Lactobacillus and Bacteroidetes and increased levels of Clostridia, Clostridium, and Dorea. The metabolome was greatly influenced by florfenicol administration, and perturbation in metabolic pathways related to linoleic acid metabolism (linoleic acid, conjugated linoleic acid [CLA], 12,13-EpOME, and 12,13-diHOME) was most prominently detected. We screened CLA and 12,13-diHOME as marker metabolites, which were highly associated with Lactobacillus, Clostridium, and Dorea. Supplementation with CLA maintained intestinal integrity, reduced intestinal inflammation, and accelerated Salmonella clearance from the gut and remission of enteropathy, whereas treatment with 12,13-diHOME promoted intestinal inflammation and disrupted intestinal barrier function to sustain Salmonella infection. Thus, these results highlight that florfenicol alters the intestinal microbiota and metabolism of neonatal chickens and promotes Salmonella infection mainly by affecting linoleic acid metabolism. IMPORTANCE Florfenicol is a broad-spectrum fluorine derivative of chloramphenicol frequently used in poultry to prevent/treat Salmonella. However, oral administration of florfenicol may lead to alterations in the microbiota and metabolome in the chicken intestine, thereby reducing colonization resistance to Salmonella infection, and the possible mechanisms linking antibiotics and Salmonella colonization in poultry have not yet been fully elucidated. In the current study, we show that increased colonization by S. Enteritidis in chickens administered florfenicol is associated with large shifts in the gut microbiota and metabolic profiles. The most influential linoleic acid metabolism is highly associated with the abundances of Lactobacillus, Clostridium, and Dorea in the intestine. The screened target metabolites in linoleic acid metabolism affect S. Enteritidis colonization, intestinal inflammation, and intestinal barrier function. Our findings provide a better understanding of the susceptibility of animal species to Salmonella after antibiotic intervention, which may help to elucidate infection mechanisms that are important for both animal and human health.

Identification of Proteus genomic island 2 variants in two clonal Proteus mirabilis isolates with coexistence of a novel genomic resistance island PmGRI1.

OBJECTIVES: To characterize the MDR genomic islands (GIs) in Proteus mirabilis isolates. METHODS: Two P. mirabilis strains (C55 and C74) of chicken origin were subjected to WGS (HiSeq and PacBio) and the MDR GIs were determined. RESULTS: P. mirabilis strains C55 and C74 are clonal strains and harbour different Proteus genomic island 2 (PGI2) variants (PGI2-C55 and PGI2-C74). The MDR region of PGI2-C55 is composed of two class 1 integrons, separated by a region containing seven copies of IS26 and eight resistance genes, including blaCTX-M-3 and fosA3. The region in PGI2-C74 is a complete In4-type class 1 integron, harbouring five gene cassettes (dfrA16, blaCARB-2, aadA2, cmlA1 and aadA1). In addition, C55 and C74 carry an SXT/R391 integrative and conjugative element (ICEPmiJpn1), harbouring blaCMY-2, and a novel 50.46 kb genomic resistance island named PmGRI1-C55. PmGRI1-C55 harbours a tyrosine-type recombinase/integrase that might be responsible for the integration of PmGRI1-C55 at the 3' end of tRNA-Sec. It carries an MDR region derived from Tn2670 that harbours a Tn21 region and carries six resistance genes (catA1, blaTEM-1b, aphA1a, sul2, strA and strB). Blast analysis showed diverse PmGRI1 variants in P. mirabilis and Escherichia coli strains. CONCLUSIONS: The finding of the two new PGI2 variants highlights that the homologous recombination between shared components of class 1 integrons and transposition by IS26 promote the diversity of MDR regions in PGI2. PmGRI1 is a new GI that carries various resistance genes identified in P. mirabilis and E. coli.

Integration of the blaNDM-1 carbapenemase gene into a novel SXT/R391 integrative and conjugative element in Proteus vulgaris.

OBJECTIVES: To characterize the genetic environment of the carbapenem resistance determinant in Proteus vulgaris of swine origin. METHODS: The carbapenem-resistant P. vulgaris strain BC22 was isolated from a faecal swab from a diseased pig with diarrhoea in Sichuan Province of China in 2018. The presence of carbapenemase genes was screened by PCR. WGS and bioinformatics analysis were performed to analyse the genetic environment of the carbapenem resistance determinant. RESULTS: P. vulgaris strain BC22 was found to harbour the carbapenemase gene blaNDM-1. WGS data revealed that blaNDM-1 was located in a truncated ISAba125 composite transposon. The carbapenem resistance gene blaNDM-1 and 20 other resistance genes, including the multiresistance gene cfr and the bifunctional aminoglycoside/quinolone resistance gene aac(6')-lb-cr, were located in a novel SXT/R391 integrative and conjugative element (ICE). This new SXT/R391 ICE of 148.7 kb was chromosomally located, and could be transferred to Escherichia coli. CONCLUSIONS: Here, we report a carbapenemase gene, blaNDM-1, integrated into an SXT/R391 ICE. Our study highlights that this SXT/R391 ICE may facilitate the dissemination of clinically important resistance genes such as blaNDM-1, cfr and aac(6')-lb-cr.

A novel cfr-carrying Tn7 transposon derivative characterized in Morganella morganii of swine origin in China.

OBJECTIVES: To characterize the presence and genetic environment of the multiresistance gene cfr in bacterial isolates from a swine farm. METHODS: A total of 97 bacterial isolates, recovered from 32 faecal swabs obtained on one farm, were tested for the presence of the cfr gene by PCR. Species identification of the one cfr-positive strain was conducted using the BD PhoenixTM 100 Automated Microbiology System. Susceptibility testing was carried out by broth microdilution. The genetic environment of the cfr gene was analysed by WGS. RESULTS: The Morganella morganii isolate BCMM24 was the only cfr-positive strain. The cfr gene, as well as 15 other resistance genes, is located on a novel 111238 bp transposon derived from Tn7, designated as Tn6451, which comprises various genetic materials including a novel class 1 integron with five gene cassettes. The cfr-containing region consists of a novel genetic structure IS26-cfr-ΔTn554 tnpB-ΔTn3 family tnpA-IS26, differing from previous reports. Two-step PCR results show that the structure can be looped out and that Tn6451 cannot be excised from the chromosome. CONCLUSIONS: To the best of our knowledge, we report the cfr gene in M. morganii for the first time. The cfr gene and 15 other resistance genes are located on a novel Tn7 transposon derivative, suggesting that the Tn7 transposon may act as a reservoir for various antimicrobial resistance genes and more Tn7 derivatives carrying multiple resistance genes are likely to be discovered in Gram-negative bacteria of both animal and human origin.

Identification of a novel conjugative plasmid carrying the multiresistance gene cfr in Proteus vulgaris isolated from swine origin in China.

The multiresistance gene cfr has a broad host range encompassing both Gram-positive and Gram-negative bacteria, and can be located on the chromosomes or on plasmids. In this study, a novel conjugative plasmid carrying cfr, designated as pPvSC3, was characterized in a Proteus vulgaris strain isolated from swine in China. Plasmid pPvSC3 is 284,528 bp in size and harbors 10 other antimicrobial resistance genes, making it a novel plasmid that differs from all known plasmids due to its unique backbone and repA gene. BLAST analysis of the plasmid sequence shows no significant homology to any known plasmid backbone, but shows high level homology to Providencia rettgeri strain CCBH11880 Contig_9, a strain isolated from surgical wound in Brazil, 2014. There are two resistance-determining regions in pPvSC3, a cfr-containing region and a multidrug-resistant (MDR) region. The cfr-containing region is flanked by IS26, which could be looped out via IS26-mediated recombination. The MDR region harbors 10 antimicrobial resistance genes carried by various DNA segments that originated from various sources. Plasmid pPvSC3 could be successfully transferred to Escherichia coli by conjugation. In summary, we have characterized a novel conjugative plasmid pPvSC3 carrying the multiresistance gene cfr and 10 other antimicrobial resistance genes, and consider that this novel type of plasmid deserves attention.

A tracing method of airborne bacteria transmission across built environments.

Disease transmission across built environments has been found to be a serious health risk. Airborne transmission is a vital route of disease infection caused by bacteria and virus. However, tracing methods of airborne bacteria in both lab and field research failed to veritably express the transporting process of microorganism in the air. A new tracing method of airborne bacteria used for airborne transmission was put forward and demonstrated its feasibility by conducting a field evaluation on the basis of genetic modification and bioaerosol technology. A specific gene fragment (pFPV-mCherry fluorescent protein plasmid) was introduced into nonpathogenic E. coli DH5α as tracer bacteria by high-voltage electroporation. Gel electrophoresis and DNA sequencing proved the success of the synthesis. Genetic stability, effect of aerosolization on the survival rate of tracer bacteria, and the application of the tracer bacteria to the airborne bacteria transmission were examined in both lab and field. Both the introduced plasmid stability rates of tracer E. coli in pre-aerosolization and post-aerosolization were above 95% in five test days. Survival rate of tracer E. coli at 97.5% ±â€¯1.2% through aerosolization was obtained by an air-atomizer operated at an air pressure of 30 Psi. In the field experiment, the airborne transmission of E. coli between poultry houses was proved and emitted E. coli was more easily transmitted into self-house than adjacent house due to the ventilation design and weather condition. Our results suggested that the tracing method of airborne bacteria was available for the investigation of airborne microbial transmission across built environments.

Characterization of a Novel SXT/R391 Integrative and Conjugative Element Carrying cfr, blaCTX-M-65, fosA3, and aac(6')-Ib-cr in Proteus mirabilis.

A novel 139,487-bp SXT/R391 integrative and conjugative element, ICEPmiChnBCP11, was characterized in Proteus mirabilis of swine origin in China. ICEPmiChnBCP11 harbors 20 different antimicrobial resistance genes, including the clinically important rRNA methyltransferase gene cfr, the extended-spectrum ß-lactamase gene blaCTX-M-65, fosfomycin resistance gene fosA3, and fluoroquinolone resistance gene aac(6')-Ib-cr An ISPpu12-mediated composite transposon containing various resistance genes and 10 copies of IS26 is inserted in hot spot 4. ICEPmiChnBCP11 was successfully transferred to Escherichia coli.

Various Sequence Types of Enterobacteriaceae Isolated from Commercial Chicken Farms in China and Carrying the blaNDM-5 Gene.

A total of 108 meropenem-resistant Enterobacteriaceae isolates were obtained from 1,658 rectal swabs collected from 15 unrelated commercial chicken farms in China between 2014 and 2016. These samples yielded 16 Escherichia coli and 2 Klebsiella pneumoniae isolates of diverse sequence types carrying a blaNDM-5-bearing IncX3 plasmid. K. pneumoniae strain sequence type 709 (ST709) has two blaNDM-5-carrying plasmids that were transferred together to E.coli.

Colocation of the Polymyxin Resistance Gene mcr-1 and a Variant of mcr-3 on a Plasmid in an Escherichia coli Isolate from a Chicken Farm.

A colistin-resistant Escherichia coli isolate from a commercial poultry farm in China carried two colistin resistance genes, mcr-1 and variant of mcr-3, in an IncP plasmid. The variant of the mcr-3 gene, named mcr-3.11, encoded two amino acid substitutions compared with the mcr-3 gene. A novel genetic structure, ISKpn40-mcr-3-dgkA-ISKpn40, might be the key element mediating the translocation of mcr-3 through the formation of a circular form. The mcr-1 and mcr-3 genes, which are colocated on a plasmid, might pose a huge threat to public health.

PGI2 Is a Novel SGI1-Relative Multidrug-Resistant Genomic Island Characterized in Proteus mirabilis.

A novel 61,578-bp genomic island named Proteus genomic island 2 (PGI2) was characterized in Proteus mirabilis of swine origin in China. The 23.85-kb backbone of PGI2 is related to those of Salmonella genomic island 1 and Acinetobacter genomic island 1. The multidrug resistance (MDR) region of PGI2 is a complex class 1 integron containing 14 different resistance genes. PGI2 was conjugally mobilized in trans to Escherichia coli in the presence of a conjugative IncC helper plasmid.

Tn6450, a Novel Multidrug Resistance Transposon Characterized in a Proteus mirabilis Isolate from Chicken in China.

A novel 65.8-kb multidrug resistance transposon, designated Tn6450, was characterized in a Proteus mirabilis isolate from chicken in China. Tn6450 contains 18 different antimicrobial resistance genes, including cephalosporinase gene blaDHA-1 and fluoroquinolone resistance genes qnrA1 and aac(6')-Ib-cr It carries a class 1/2 hybrid integron composed of intI2 and a 3' conserved segment of the class 1 integron. Tn6450 is derived from Tn7 via acquisition of new mobile elements and resistance genes.

Novel plasmid-mediated colistin resistance gene mcr-7.1 in Klebsiella pneumoniae.

Objectives: To identify a novel plasmid-mediated colistin resistance gene in Klebsiella pneumoniae isolated from chickens in China. Methods: WGS was used to identify a novel colistin resistance gene. The transferability of plasmids carrying mcr-7.1 was investigated by conjugation experiments. The expression of the mcr-7.1 gene was examined using an expression vector. Results: A novel plasmid-mediated colistin resistance gene mcr-7.1, sharing 70% amino acid identity with the mcr-3 gene, was identified in three K. pneumoniae strains isolated from chickens in China. The mcr-7.1 gene was found in an IncI2-type plasmid (pSC20141012) that co-harboured the blaCTX-M-55 gene in one isolate. pSC20141012 can be transferred from K. pneumoniae SC20141012 to Escherichia coli J53Azr, exhibiting a ≥8-fold increase in colistin MIC compared with the recipient E. coli J53Azr. Conclusions: We identified a novel plasmid-mediated colistin resistance gene named mcr-7.1 in K. pneumoniae in China. The prevalence of mcr-7.1 in various species of human and animal origin needs to be investigated immediately.

Co-occurrence of biofilm formation and quinolone resistance in Salmonella enterica serotype typhimurium carrying an IncHI2-type oqxAB-positive plasmid.

The objective of this study was to investigate the co-occurrence of biofilms and quinolone resistance in Salmonella enterica serotype Typhimurium mediated by IncHI2-type oqxAB-positive plasmids. Among the 40 Salmonella strains, we found that 27 isolates formed biofilms and displayed identical multidrug-resistance profiles to ciprofloxacin, doxycycline, sulfamethoxazole-trimethoprim, ampicillin and streptomycin, based on biofilm formation assays and antimicrobial susceptibility testing. In particular, a single S. Typhimurium isolate named SC523 produced the thickest biofilms and exhibited the highest-level resistance (MIC = 8 µg/mL) to ciprofloxacin compared to those of the other isolates. The detection of known plasmid-mediated quinolone resistance (PMQR) genes and point mutations in the quinolone resistance-determining region (QRDR) by PCR assay showed that oqxAB genes were present in 27 biofilm-positive isolates. Conjugation experiments, S1-pulse-field gel electrophoresis and biofilm formation assays demonstrated that the conjugative plasmid that encoded biofilms and quinolone resistance in Salmonella SC523 could be transferred to a recipient with a frequency of 4.7 × 10-3 per recipient cell. The results of PCR-based replicon typing (PBRT) showed that the IncHI2-type plasmids accounted for 100% of the biofilm-oqxAB-positive isolates and transconjugants. The sequence analysis of Salmonella SC523 confirmed that the oqxAB cassette and fourteen DNA transfer genes in the IncHI2-type oqxAB-positive conjugative plasmid were genetically responsible for the phenotypic quinolone resistance and biofilm formation. The conclusion is that the IncHI2-type plasmid in S. Typhimurium isolate from chicken farm was identified and sequenced, which contained oqxAB and tra/trh and encoded quinolone resistance and biofilms, and could be transferred to recipients through conjugation. Notably, the prevalence of IncHI2-type biofilm-oqxAB-positive plasmids in animal-origin Salmonella poses a threat to public health, as these Salmonella from poultry farms show a decreased susceptibility to quinolones and could spread to humans.

Distribution of Salmonella Enteritidis in internal organs and variation of cecum microbiota in chicken after oral challenge.

The aim study was to explore the distribution of Salmonella Enteritidis (S. enteritidis) in internal organs and variation of cecum microbiota in newly hatched chicken after oral challenge during a 21-day period. The quantities of S. enteritidis DNA in different internal organs (heart, liver, spleen, stomach, pancreas, small intestine, blood and cecum contents) were determined by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). The result showed that all of the above-mentioned samples were positive at 12 h post inoculation (PI) after oral challenge. The highest copy numbers of S. enteritidis in all tissue were heart and liver, with about 2 × 102 to 6 × 106 copies of DNA target sequences/0.5 g. The copy number of S. enteritidis in the stomach was only lower than the heart and liver. The blood at 8 d PI, the pancreas at 10 d PI, the heart at 14 d PI and the stomach at 17 d PI didn't have a positive result. However, the liver, spleen, cecum contents and small intestine were all positive during the 21-day period. The cecum contents at 0 d PI, 4 d PI and 10 d PI from the control group and experiment group were collected for bacterial 16 S rRNA sequencing targeting the V3-V4 hypervariable region. The result showed that at the 0 d PI, the main cecum microbiota ingredient of the two-day old chicken was Enterobacteriaceae (Proteobacteria) and the other microbiology species were fewer. At the 10 d PI, the microbiota ingredient of cecum became abundant and stable mainly including the families Ruminococcaceae (Firmicutes), Enterobacteriaceae (Proteobacteria), Lachnospiraceae (Firmicutes) and clostridiacaea (Firmicutes) both of the two group, suggesting Salmonella infection with 2-day old chicken might not significantly change cecum microbiota community. The study indicated the major organs, which carried numerous S. enteritidis, providing a significantly guideline for salmonella detection in poultry and revealed the main microbiota ingredient of chicken cecum.

An IncX1 plasmid isolated from Salmonella enterica subsp. enterica serovar Pullorum carrying blaTEM-1B, sul2, arsenic resistant operons.

We have identified an IncX1 plasmid named pQJDSal1 from Salmonella enterica subsp. enterica serovar Pullorum (S. Pullorum). The plasmid is 67,685 bp in size and has 72 putative genes. pQJDSal1 harbors a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin plasmid addiction system. Two regions (A and B) that have not been previously reported in IncX1 plasmids are inserted into the backbone. Region A (10.7 kb), inserted between parA and taxD, consists of a new Tn6168-like transposon containing an arsenic resistant operon arsB2CHR and sulfonamide resistance gene sul2. Region B contains another arsenic resistant operon arsADHR, resistance gene blaTEM-1B and three transposable elements. Conjugation experiments showed that pQJDSal1 could transfer from S. Pullorum to Escherichia coli (E. coli) J53. Statistical analysis of 70 sequenced IncX1 plasmids revealed that IncX1 plasmids harbored various antibiotic resistance genes. The results highlight the importance of IncX1 plasmids in disseminating antibiotic resistance genes.