Sequence analysis and plasmid mobilization of a 6.6-kb kanamycin resistance plasmid, pSNC3-Kan, from a Salmonella enterica serotype Newport isolate.

Research on the transfer of antibiotic resistance plasmids has been mainly focused on the large multi-drug resistance conjugative plasmids, while the transmission of small mobilizable plasmids remains under-investigated. A series of diverse ColE-like kanamycin resistance plasmids ("KanR plasmids") from Salmonella enterica were characterized previously. In this study, the 6.6-kb pSNC3-Kan from a Salmonella enterica serotype Newport isolate was investigated. It possessed highly conserved RNA I/II and Tn602 (IS903-aph-IS903) regions to two other KanR plasmids pSe-Kan and pSBardo-Kan, but carried a mobC-mobA/BD operon. The mobilization proteins encoded by the mob operon of pSNC3-Kan showed high sequence identity (~95%) to those of an E. coli plasmid pEC34B, except that MobE was not present; and were much less conserved to those of another KanR plasmid pSN11/00Kan (43% - 86% identity). Four structurally different KanR plasmids were investigated for their ability to be mobilized by the conjugal transfer (tra) genes from F and IncP plasmids. Transfer genes derived from IncP plasmids can efficiently mobilize KanR plasmids possessing the mob operons (mobC-mobA/BD), such as pSNC3-Kan and pSN11/00Kan, in bi-parental mating experiments. On the other hand, F tra genes were able to mobilize pU302S, pSNC3-Kan and pSe-Kan, but not pSN11/00Kan. A plasmid-borne mob operon was not required for mobilization of the oriT(F)-bearing pSe-Kan by the F tra genes. This study underscores the complexity of plasmid interaction and the importance of how small mobilizable plasmids may contribute to the spread of antibiotic resistance genes.

Complete Genome Sequence and Annotation of Campylobacter jejuni YH003, Isolated from Retail Chicken.

The complete genome sequence of Campylobacter jejuni YH003, isolated from retail chicken, was determined using PacBio and Illumina technologies. The assembled genome is 1,743,985 bp (G+C content of 30.3%). Genome annotation revealed several genes encoding virulence and antibiotic resistance factors, including a type VI secretion system, cytolethal distending toxins, and a multidrug efflux system.

Detection of Shiga toxin-producing Escherichia coli (STEC) in beef products using droplet digital PCR.

Many of the current accredited methods for the molecular detection of Shiga toxin-producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods can inaccurately conclude the presence of E.coli containing both stx and eae because of the inability of the methods to determine if the two genes originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate if a droplet digital PCR (ddPCR)-based method that does not require DNA isolation could reliably identify the presence of an STEC containing eae in beef samples by confirming that both genes reside within the same cell, even when present in a mixed culture. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works without the need for DNA isolation by partitioning intact cells into emulsion droplets, where they are lysed, and subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparisons were made between the dd-Check STEC Solution, the BAX System Real-Time PCR STEC assay suite (Hygiena), and the iQ-Check STEC PCR detection kit (Bio-Rad) using 37 unique simulations of E. coli contamination in ground beef. While no single platform was consistently superior at detecting eae and stx across all pathogens tested, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of inaccurately identified samples when screening for E. coli with a stx+, eae+ genotype because it can identify the co-existence of multiple virulence genes within a cell even when in the presence of a mixed microbial population containing identical genes. Ultimately, incorporation of this system could result in substantial cost savings by reducing the expenses incurred when product samples are incorrectly classified as containing E. coli with a stx+, eae+ genotype.

Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA.

Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect ≥0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1× and 10× diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.

Whole-genome sequence data and analysis of a Staphylococcus aureus strain SJTUF_J27 isolated from seaweed.

The complete genome sequence data of S. aureus SJTUF_J27 isolated from seaweed in China is reported here. The size of the genome is 2.8 Mbp with 32.9% G + C content, consisting of 2614 coding sequences and 77 RNAs. A number of virulence factors, including antimicrobial resistance genes (fluoroquinolone, beta-lactams, fosfomycin, mupirocin, trimethoprim, and aminocoumarin) and the egc enterotoxin cluster, were found in the genome. In addition, the genes encoding metal-binding proteins and associated heavy metal resistance were identified. Phylogenetic data analysis, based upon genome-wide single nucleotide polymorphisms (SNPs), and comparative genomic evaluation with BLAST Ring Image Generator (BRIG) were performed for SJTUF_J27 and four S. aureus strains isolated from food. The completed genome data was deposited in NCBI׳s GenBank under the accession number CP019117, https://www.ncbi.nlm.nih.gov/nuccore/CP019117.

Isolation and characterization of two novel groups of kanamycin-resistance ColE1-like plasmids in Salmonella enterica serotypes from food animals.

While antimicrobial resistance in Salmonella enterica is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic samples of human or animals were reported. In this study, over 200 KanR S. enterica isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three KanR ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that KanR ColE1-like plasmids are widely distributed among different S. enterica serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes.

Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens.

BACKGROUND: Magnesium oxide nanoparticles (MgO nanoparticles, with average size of 20 nm) have considerable potential as antimicrobial agents in food safety applications due to their structure, surface properties, and stability. The aim of this work was to investigate the antibacterial effects and mechanism of action of MgO nanoparticles against several important foodborne pathogens. RESULTS: Resazurin (a redox sensitive dye) microplate assay was used for measuring growth inhibition of bacteria treated with MgO nanoparticles. The minimal inhibitory concentrations of MgO nanoparticles to 10(4) colony-forming unit/ml (CFU/ml) of Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella Enteritidis were determined to be 0.5, 1 and 1 mg/ml, respectively. To completely inactivate 10(8-9) CFU/ml bacterial cells in 4 h, a minimal concentration of 2 mg/ml MgO nanoparticles was required for C. jejuni whereas E. coli O157:H7 and Salmonella Enteritidis required at least 8 mg/ml nanoparticles. Scanning electron microscopy examination revealed clear morphological changes and membrane structural damage in the cells treated with MgO nanoparticles. A quantitative real-time PCR combined with ethidium monoazide pretreatment confirmed cell membrane permeability was increased after exposure to the nanoparticles. In a cell free assay, a low level (1.1 µM) of H2O2 was detected in the nanoparticle suspensions. Consistently, MgO nanoparticles greatly induced the gene expression of KatA, a sole catalase in C. jejuni for breaking down H2O2 to H2O and O2. CONCLUSIONS: MgO nanoparticles have strong antibacterial activity against three important foodborne pathogens. The interaction of nanoparticles with bacterial cells causes cell membrane leakage, induces oxidative stress, and ultimately leads to cell death.

Phage insertion in mlrA and variations in rpoS limit curli expression and biofilm formation in Escherichia coli serotype O157: H7.

Biofilm formation in Escherichia coli is a tightly controlled process requiring the expression of adhesive curli fibres and certain polysaccharides such as cellulose. The transcriptional regulator CsgD is central to biofilm formation, controlling the expression of the curli structural and export proteins and the diguanylate cyclase adrA, which indirectly activates cellulose production. CsgD itself is highly regulated by two sigma factors (RpoS and RpoD), multiple DNA-binding proteins, small regulatory RNAs and several GGDEF/EAL proteins acting through c-di-GMP. One such transcription factor MlrA binds the csgD promoter to enhance the RpoS-dependent transcription of csgD. Bacteriophage, often carrying the stx1 gene, utilize an insertion site in the proximal mlrA coding region of E. coli serotype O157 : H7 strains, and the loss of mlrA function would be expected to be the major factor contributing to poor curli and biofilm expression in that serotype. Using a bank of 55 strains of serotype O157 : H7, we investigated the consequences of bacteriophage insertion. Although curli/biofilm expression was restored in many of the prophage-bearing strains by a wild-type copy of mlrA on a multi-copy plasmid, more than half of the strains showed only partial or no complementation. Moreover, the two strains carrying an intact mlrA were found to be deficient in biofilm formation. However, RpoS mutations that attenuated or inactivated RpoS-dependent functions such as biofilm formation were found in >70 % of the strains, including the two strains with an intact mlrA. We conclude that bacteriophage interruption of mlrA and RpoS mutations provide major obstacles limiting curli expression and biofilm formation in most serotype O157 : H7 strains.

Phenotypic and genotypic characterization of biofilm forming capabilities in non-O157 Shiga toxin-producing Escherichia coli strains.

The biofilm life style helps bacteria resist oxidative stress, desiccation, antibiotic treatment, and starvation. Biofilm formation involves a complex regulatory gene network controlled by various environmental signals. It was previously shown that prophage insertions in mlrA and heterogeneous mutations in rpoS constituted major obstacles limiting biofilm formation and the expression of extracellular curli fibers in strains of Escherichia coli serotype O157:H7. The purpose of this study was to test strains from other important serotypes of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O113, O121, and O145) for similar regulatory restrictions. In a small but diverse collection of biofilm-forming and non-forming strains, mlrA prophage insertions were identified in only 4 of the 19 strains (serotypes O103, O113, and O145). Only the STEC O103 and O113 strains could be complemented by a trans-copy of mlrA to restore curli production and Congo red (CR) dye affinity. RpoS mutations were found in 5 strains (4 serotypes), each with low CR affinity, and the defects were moderately restored by a wild-type copy of rpoS in 2 of the 3 strains attempted. Fourteen strains in this study showed no or weak biofilm formation, of which 9 could be explained by prophage insertions or rpoS mutations. However, each of the remaining five biofilm-deficient strains, as well as the two O145 strains that could not be complemented by mlrA, showed complete or nearly complete lack of motility. This study indicates that mlrA prophage insertions and rpoS mutations do limit biofilm and curli expression in the non-serotype O157:H7 STEC but prophage insertions may not be as common as in serotype O157:H7 strains. The results also suggest that lack of motility provides a third major factor limiting biofilm formation in the non-O157:H7 STEC. Understanding biofilm regulatory mechanisms will prove beneficial in reducing pathogen survival and enhancing food safety.

Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars.

A group of low molecular-weight ColE1-like plasmids carrying the aph sequence type aph(ii) from three different Salmonella serovars were sequenced. These plasmids carry two or more copies of IS903 elements, with up to 21bp sequence differences to one another, two of which flank the aph gene. This group of plasmids did not appear to carry any known mobilization genes and instead carry three open reading frames encoding hypothetical proteins of unknown function possibly organized in an operon. The plasmid replication region (RNA I/II--rom) of this plasmid group showed extensive homology to that of pKPN2 plasmid of Klebsiella pneumoniae and pCol-let plasmid of Escherichia coli. Three of the four plasmids had identical sequences, and the fourth had an extra copy of IS903 with target duplication, suggesting a recent divergence in the different Salmonella serovars from a common ancestor.

Prevalence of ColE1-like plasmids and kanamycin resistance genes in Salmonella enterica serovars.

Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kan(r)) phenotypes, 102 Kan(r) Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kan(r) Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3')-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group.

Characterization of small ColE1-like plasmids conferring kanamycin resistance in Salmonella enterica subsp. enterica serovars Typhimurium and Newport.

Multi-antibiotic resistant (MR) Salmonella enterica serovars Typhimurium and Newport are an increasing concern in human and animal health. Many strains are known to carry antibiotic resistance determinants on multiple plasmids, yet detailed information has been scarce. Three plasmids conferring kanamycin (Kan) resistance were isolated and nucleotide sequences were determined. Two Kan(R) plasmids from Salmonella Newport strains, pSN11/00Kan and pSN02/01Kan, were found to be identical and were 5698bp in size. Plasmid pG7601Kan from Salmonella Typhimurium phage type U302 strain G7601 was 3208bp, and was the same as the previously reported pU302S from another U302 strain G8430. All three plasmids carried identical aph(3')-I genes. The plasmids were ColE1-like, containing RNA I/RNA II and the rom gene. Plasmids pSN11/00Kan and pSN02/01Kan also carried mobilization genes mobC and mobABD, similar to those of the pColK-K235 and pColD-157 plasmids from the colicinogenic Escherichia coli strains. All three plasmids were stable without kanamycin selection for approximately 100 generations.

Analysis of AI-2/LuxS-dependent transcription in Campylobacter jejuni strain 81-176.

Autoinducer-2 (AI-2) is a quorum-sensing signal molecule that controls a variety of cellular activities in response to cell density in both gram-negative and gram-positive bacteria. The production of AI-2 is dependent upon LuxS, the last enzyme in the AI-2 biosynthesis pathway. For this study, we constructed a luxS null mutation (Delta luxS) in Campylobacter jejuni strain 81-176, and showed that it abolished AI-2 production. The Delta luxS mutant had a longer doubling time in Mueller-Hinton (MH) broth and reduced swarming on MH soft agar at 37 degrees C compared to the wild type (wt), whereas growth rate or swarming at 42 degrees C was not affected. The Delta luxS mutant was also more sensitive to hydrogen peroxide (H(2)O(2)) and cumene hydroperoxide than the wt by disc inhibition assays at 42 degrees C, though minimum inhibitory concentration comparisons were inconclusive. Differences in genome-wide gene expression between wt and Delta luxS mutant with and without H(2)O(2) treatments were compared using DNA microarrays. The genes that showed differential expressions (wt/Delta luxS) include operons/pathways involved in AI-2 synthesis and S-adenosylmethionine (SAM) metabolism (metE, metF, and pfs), flagellar assembly/regulation, stress response (ahpC, tpx, and groES), ABC transporters/efflux systems, and two genes of unknown function located downstream of luxS (Cj1199 and Cj1200). The wt/Delta luxS expression ratios of ahpC (encoding alkyl hydroperoxide reductase) and tpx (encoding thiol peroxidase) were increased only with H(2)O(2) treatment, consistent with our finding that the Delta luxS mutant exhibits higher sensitivity to oxidative stress than wt. Our microarray results agreed with the Delta luxS mutant phenotypes, and suggested that LuxS plays a role in central metabolism involving SAM metabolism, but it is uncertain whether AI-2 functions as a true quorum-sensing signal in C. jejuni.

DNA sequence of the Escherichia coli O103 O antigen gene cluster and detection of enterohemorrhagic E. coli O103 by PCR amplification of the wzx and wzy genes.

Escherichia coli serogroup O103 has been associated with gastrointestinal illness and hemolytic uremic syndrome. To develop PCR-based methods for detection and identification of this serogroup, the DNA sequence of the 12,033-bp region containing the O antigen gene cluster of Escherichia coli O103 was determined. Of the 12 open reading frames identified, the E. coli O103 wzx (O antigen flippase) and wzy (O antigen polymerase) genes were selected as targets for development of both conventional and real-time PCR assays specific for this serogroup. In addition, a multiplex PCR targeting the Shiga toxin (Stx) 1 (stx1), Shiga toxin 2 (stx2), wzx, and wzy genes was developed to differentiate Stx-producing E. coli O103 from non-toxigenic strains. The PCR assays can be employed to identify E. coli serogroup O103, replacing antigen-based serotyping, and to potentially detect the organism in food, fecal, or environmental samples.

Construction and Characterization of Escherichia coli O157:H7 Strains Expressing Firefly Luciferase and Green Fluorescent Protein and Their Use in Survival Studies ‡.

The firefly ( Photinus pyralis ) luciferase (luc) gene on plasmid vector pBESTluc and the Aequorea victoria green fluorescent protein (gfp) gene on plasmid vector pGFP were introduced into strains of Escherichia coli O157:H7. The recombinant E. coli strains were indistinguishable from their parent strains in biochemical and immunological assays and in a multiplex PCR reaction. There was no significant difference in the growth kinetics of the luc-bearing recombinants and the parent strains. At 37°C all of the recombinant strains maintained the vectors and expressed luciferase and the green fluorescent protein when grown both with and without antibiotic selection. Individual colonies of luc-bearing E. coli strains were readily luminescent in the dark after being sprayed with a solution of 1 mM beetle luciferin. The recombinants containing pGFP emitted bright green fluorescence when excited with UV light and the addition of any other proteins, substrates, or cofactors was not required. The green fluorescent protein-expressing E. coli O157:H7 strains were used in studies examining the survival of the organism in apple cider and in orange juice. In apple cider the organism declined to undetectable levels in 24 days at refrigeration temperature while in orange juice the strains survived with only small decreases in number during the 24-day sampling period. These recombinant E. coli O157:H7 strains, containing readily identifiable and stable markers, could be useful as positive controls in microbial assays as well as in studies monitoring bacterial survival and the behavior of E. coli O157:H7 in foods and in a food processing environment.