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Farm products are considered important vehicles for the transmission of Listeria monocytogenes (L. monocytogenes). The typing of L. monocytogenes from farm products contributes to the surveillance and source tracing of the pathogen. In this study, 77 L. monocytogenes strains from seven farm product categories in Shanghai were analyzed by serological typing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and whole-genome nucleotide polymorphism (wgSNP). The results showed that the 77 isolates were classified into four serovars (1/2a, 1/2c, 1/2b, and 4b), and of these, 1/2a (n=47) was the most prevalent. Seventeen sequence types (STs) were generated by MLST with two novel STs (ST1402 and ST1403), and 20 of 77 L. monocytogenes isolates had high genetic identity with previously documented outbreak isolates according to the minimum spanning tree from the MLST results. Moreover, 34 PFGE patterns (PF1–PF34) were differentiated, and based on a similarity value higher than 80% by the unweighted pair group method dendrogram, the discriminatory index was relatively low (equal to 0.775). Furthermore, 14 isolates were chosen and further analyzed by wgSNP based on the previous typing results, which demonstrated that wgSNP and MLST yielded mostly consistent typing results but higher resolution than PFGE. In conclusion, 77 L. monocytogenes isolates from farm products collected in nine districts in Shanghai were highly genetically diverse, and 20 of these isolates had high relatedness with previously documented outbreak strains worldwide. The results indicate a possible cross-contamination risk of L. monocytogenes and a potential public health concern resulting from farm products during the supply chain in Shanghai, China.

Disease caused by antibiotic-resistant Salmonella is a serious clinical problem that poses a great threat to public health. The present study is aimed at assessing differences in bacterial kinetics with different antibiotic resistance profiles under environmental stress and at developing microbial tolerance models in lettuce during storage from 4 to 36°C. The drug-resistance phenotypes of 10 Salmonella Typhimurium (S. Typhimurium) isolates were examined using the broth microdilution method. The results of 10 S. Typhimurium isolates in the suspensions showed that a slow trend towards reduction of drug-sensitive (DS) isolates in relation to the others though without statistical difference. Compared to DS S. Typhimurium SA62, greater bacterial reduction was observed in multidrug-resistant (MDR) S. Typhimurium HZC3 during lettuce storage at 4°C ( P < 0.05 ). It was likely that a cross-response between antibiotic resistance and food-associated stress tolerance. The greater growth in lettuce at 12°C was observed for DS S. Typhimurium SA62 compared to MDR S. Typhimurium HZC3 and was even statistically different ( P < 0.05 ), while no significant difference was observed for bacterial growth between MDR S. Typhimurium HZC3 and DS S. Typhimurium SA62 strains in lettuce storage from 16 to 36°C ( P > 0.05 ). The goodness-of-fit indices indicated the Log-linear primary model provided a satisfactory fit to describe the MDR S. Typhimurium HZC3 and DS S. Typhimurium SA62 survival at 4°C. A square root secondary model could be used to describe the effect of temperature (12, 16, 28, and 36°C) on the growth rates of S. Typhimurium HZC3 ( a d j − R 2 = 0.91 , RMSE = 0.06 ) and S. Typhimurium SA62 ( a d j − R 2 = 0.99 , RMSE = 0.01 ) derived from the Huang primary model. It was necessary to pay attention to the tolerance of antibiotic resistant bacteria under environmental stress, and the generated models could provide parts of the input data for microbial risk assessment of Salmonella with different antibiotic resistance profile in lettuce.

Listeria monocytogenes is an important foodborne pathogen that can cause listeriosis with high mortality rates. Sodium lactate has been used as an antimicrobial in meat products to inhibit the growth of foodborne pathogens. However, the adaptation mechanism of L. monocytogenes to sodium lactate remains unclear. In this paper, the growth/survival of L. monocytogenes was determined in ready-to-eat (RTE) meats with 4% sodium lactate treatment. RNA-Seq was conducted to compare the transcriptional profiles between cells cultured in Brain Heart Infusion broth with and without 4% sodium lactate to identify differentially expressed genes (DEGs). DEGs (766) including 329 up-regulated and 437 down-regulated genes were identified from RNA-Seq analysis in response to sodium lactate treatment. RNA-Seq data were validated using quantitative reverse transcription PCR (qRT-PCR) assays. The DEGs include genes involved in pathways including signal transduction, ABC transporter and PTS systems. Interestingly, the DEGs involved in bacterial chemotaxis and flagellar assembly were exclusively down-regulated, indicating that flagellar synthesis was inhibited by sodium lactate treatment. Most importantly, virulence-related genes were induced, suggesting that sodium lactate may increase the virulence potential of L. monocytogenes. Our study provides insight on the adaptation mechanism of L. monocytogenes with treatment of sodium lactate, and may aid in searching for strategies that may have a synergistic effect in combination with sodium lactate to inhibit L. monocytogenes in RTE meat.

Among 13 serotypes of Listeria monocytogenes, serotypes 4b and 1/2a account for most of the sporadic listeriosis cases and environmental isolations. To understand the mechanism of stress response in these serotypes, six strains of each serotype were selected in this study to differentiate the physiological and genetic response of these L. monocytogenes under oxidative stress. Oxidative stress tolerance was examined by plate count after hydrogen peroxide exposure during exponential phase, long-term-survival phase and biofilm state. The biofilm forming capability of the two serotypes was also compared using crystal violet assay. Three oxidation resistance genes (kat, sod and fri), two stress-regulator encoding genes (sigB and perR), and one DNA repair gene (recA) were selected for transcription analysis by quantitative RT-PCR. Most serotype 4b strains exhibited a stronger resistance to 0.6% H2O2 than serotype 1/2a during exponential phase and long-term-survival phase, while the latter demonstrated stronger biofilm forming capacity in BHI broth at 37 °C. At the genetic level, there were significant differences in the expression of three genes (sod, fri and perR) under oxidative stress between two serotypes, which suggests a role in the observed variation towards stress resistance. In addition, the expression of recA was induced under oxidative stress in L. monocytogenes strains of both serotypes.

Listeria monocytogenes is a major foodborne pathogen that is well known for its high mortality rate in humans upon infection. This study aimed to investigate the prevalence and serotype diversity of L. monocytogenes isolates in various foods. From July 2013 to June 2014, a total of 648 food samples, originating from raw milk, quick-frozen food, vegetables, raw meats, cooked meats, aquatic products, dairy products and bean products, were collected from open-air markets, large supermarkets, delis and dairy farms, in Shanghai, China. Forty samples (6.2%) tested positive for L. monocytogenes , confirmed by both bacteriological and molecular methods. Among the food categories surveyed, the highest prevalence was observed in raw meat samples at 16.0% (15/94), followed by 9.9% (10/101) in quick-frozen food samples and 7.3% (4/55) in cooked meat samples. Serotyping showed that majority of the isolates (67.5%) belonged to serotype 1/2a, followed by 1/2b (22.5%), 1/2c (7.5%) and 3c (2.5%). Isolates were assessed for their ability to form biofilms. Significant differences in biofilm forming abilities were observed between storage temperatures and serotypes. Anti-biofilm efficacy of industrial disinfectants, chlorine dioxide (ClO 2 ) and hydrogen peroxide (H 2 O 2 ), was examined by measuring minimal effective concentration (MEC) on L. monocytogenes in biofilm. A positive correlation was observed among the biofilm’s resistance to H 2 O 2 and the biomass of biofilms. ClO 2 was found to be more effective against biofilm and inactivated all the listerial cells in the biofilm state, whereas H 2 O 2 was not able to kill all the cells in biofilms at the recommended concentrations according to National Food Safety Standard of China. Our study suggests that there is potential public health risk of L. monocytogenes infection in Shanghai. Efficacy against L. monocytogenes biofilm should be considered when evaluating disinfectants.

Corynespora cassiicola as a necrotrophic plant pathogenic ascomycetes fungus can infect hundreds of species of plants, and also rarely cause human disease. The pathogen infects cucumber and causes cucumber target spot, which has given rise to great yield loss of cucumber in China recently. Genome sequence and spore germination associated transcriptome analysis will contribute to the understanding of the molecular mechanism of pathogenicity and spore germination of C. cassiicola .Results Firstly, we reported the draft genome sequences of a cucumber-sampled C. cassiicola isolate HGCC with high virulence. Although being conspecific, HGCC had distinct difference with a rubber-sampled isolate (CCP) and a human-sampled isolate (UM591) in genome sequences. The proportion of secreted proteins was 7.4% in HGCC. 28.6% of HGCC predicted genes were highly homologous to experimentally proven virulence-associated genes, which was close to that in CCP, UM591 and some plant fungal pathogens, but far more than 21.9% in Phaeosphaeria nodorum and 19.6% in Botrytis cinerea . Thousands of putative virulence-associated genes in various pathways or families were identified in HGCC. Secondly, a global view of the transcriptome of C. cassiicola spores during germination was evaluated using RNA sequencing (RNA-seq). A total of 3,288 differentially expressed genes (DEGs) were identified. The majority of KEGG annotated DEGs were involved in metabolism, genetic information processing, cellular processes, organismal system, human diseases and environmental information processing.Conclusions These results not only facilitated the exploration of the molecular pathogenic mechanism of C. cassiicola to cucumber and the understanding of molecular and cellular processes during spore germination, but also laid the foundation for the disease control.

The aim of this study was to determine whether sublethal concentrations of ceftriaxone could alter antibiotic resistance patterns in Salmonella strains. Three multiple antibiotic-resistant Salmonella isolates and the control strain ATCC 13076 were subjected to induction experiments by stepwise increases in sublethal concentrations of ceftriaxone. Sublethal levels of ceftriaxone induced antibiotic resistance but not control Salmonella isolates to ceftriaxone and to other antibiotics. After 100 generations in 2 months when the antibiotic stress was removed, only one isolate (Salmonella Typhimurium 11202) maintained the induction changes in antibiotic resistance phenotype (tetracycline from resistance to sensitive and ampicillin from sensitive to resistance). Consistent with its stable phenotypic resistance changes, expression of the tetracycline and β-lactam resistance-related genes tetA and blaTEM were >10-fold down- and upregulated, respectively. Moreover, this strain had increased mRNA levels of efflux pump associated genes acrB and tolC and the SOS response regulator lexA and downregulation of the porin gene ompC. We found no overt changes in plasmid profiles before and after resistance induction. In all, sublethal concentrations of ceftriaxone induced alterations in Salmonella isolates to multiple antibiotics and some of them kept stable maintenance. The increased blaTEM expression may pose a potential danger for new generation β-lactam antibiotics.

Magnetic separation is an efficient method for target enrichment and elimination of inhibitors in the molecular detection systems for foodborne pathogens. In this study, we prepared magnetic capture probes by modifying oligonucleotides complementary to target sequences on the surface of amino-modified silica-coated magnetic nanoparticles and optimized the conditions and parameters of probe synthesis and hybridization. We innovatively put the complexes of magnetic capture probes and target sequences into qPCR without any need for denaturation and purification steps. This strategy can reduce manual steps and save time. We used the magnetic capture probes to separate invA mRNA from Salmonella in artificially contaminated milk samples. The detection sensitivity was 104 CFU/ml, which could be increased to 10 CFU/ml after a 12 h enrichment step. The developed method is robust enough to detect live bacteria in a complex environmental matrix.

Salmonella enterica serovar Enteritidis is the leading global cause of salmonellosis. A total of 146 Salmonella Enteritidis isolates obtained from retail chicken products in Shanghai, China were characterized for their antimicrobial susceptibilities, virulence and antibiotic resistance gene profiles, and molecular subtypes using pulsed-field gel electrophoresis (PFGE). Approximately 42% (61/146) of the isolates were susceptible to all 13 antimicrobials tested. More than half of the isolates (50.70%) were resistant to ampicillin, 49.32% to sulfisoxazole, 17.12% to tetracycline, and 15.75% to doxycycline. Thirty (20.55%) isolates were resistant to three or more antimicrobials. The avrA, mgtC, and sopE virulence genes were identified in all isolates, while 97.2% and 92.4% were positive for bcfC and spvC genes, respectively. Genes associated with resistance to streptomycin (aadA), β-lactams (blaTEM, blaCMY, blaSHV, and blaCTX), tetracycline (tetA and tetB), and sulfonamides (sulI, sulII, and sulIII) were detected among corresponding resistant isolates. A total of 41 PFGE patterns were identified from 77 antimicrobial resistance (AMR) isolates and were primarily grouped into seven clusters (A-G), each with 90% similarity. The majority of Salmonella Enteritidis isolates (63.63%, 49/77) shared the same PFGE cluster, indicating potential cross contamination during processing and cutting or working during retailing and marketing. A significantly (p 0.05) lower percentage (25%) of isolates belonging to clusters D and E were resistant to sulfisoxazole compared with those belonging to clusters A, B, C, F, and G (80%), indicating that sulfisoxazole resistance might be associated with genetic content (PFGE profiles) of Salmonella Enteritidis. This study provides important and updated information about the baseline antimicrobial-resistant data for food safety risk assessment of Salmonella Enteritidis from retailed chicken in Shanghai, which is the first step for the development and implementation of China's AMR National Action Plan, and can be helpful for future surveillance activities to ensure the safety of the chicken supply.

Staphyloxanthin (STX), a golden carotenoid pigment produced by Staphylococcus aureus, is suggested to act as an important virulence factor due to its antioxidant properties. Restraining biosynthesis of STX was considered as an indicator of virulence decline in pigmented S. aureus isolates. However, it is not clear whether natural non-pigmented S. aureus isolates have less virulence than pigmented ones. In this study, it is aimed to compare the pigmented and non-pigmented S. aureus isolates to clarify the genetic and virulent differences between the two groups. Here, 132 S. aureus isolates were divided into two phenotype groups depending on the absorbance (OD450) of the extracted carotenoids. Then, all isolates were subjected to spa typing and multilocus sequence typing (MLST), and then the detection of presence of 30 virulence factors and the gene integrity of crtN and crtM. Furthermore, 24 typical S. aureus isolates and 4 S. argenteus strains were selected for the murine infection assay of in vivo virulence, in which the histological observation and enumeration of CFUs were carried out. These isolates were distributed in 26 sequence types (STs) and 49 spa types. The pigmented isolates were scattered in 25 STs, while the non-pigmented isolates were more centralized, which mainly belonged to ST20 (59%) and ST25 (13%). Among the 54 non-pigmented isolates, about 20% carried intact crtN and crtM genes. The in vivo assay suggested that comparing with pigmented S. aureus, non-pigmented S. aureus and S. argenteus strains did not show a reduced virulence in murine sepsis models. Therefore, it suggested that there were no significant genetic and virulent differences between pigmented and non-pigmented S. aureus.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, which is a major public health concern due to the high fatality rate. LMOf2365_0442, 0443, and 0444 encode for fructose-specific EIIABC components of phosphotransferase transport system (PTS) permease that is responsible for sugar transport. In previous studies, in-frame deletion mutants of a putative fructose-specific PTS permease (LMOf2365_0442, 0443, and 0444) were constructed and analyzed. However, the virulence potential of these deletion mutants has not been studied. In this study, two in vitro methods were used to analyze the virulence potential of these L. monocytogenes deletion mutants. First, invasion assays were used to measure the invasion efficiencies to host cells using the human HT-29 cell line. Second, plaque forming assays were used to measure cell-to-cell spread in host cells. Our results showed that the deletion mutant ΔLMOf2365_0442 had reduced invasion and cell-to-cell spread efficiencies in human cell line compared to the parental strain LMOf2365, indicating that LMOf2365_0442 encoding for a fructose specific PTS permease IIA may be required for virulence in L. monocytogenes strain F2365. In addition, the gene expression levels of 15 virulence and stress-related genes were analyzed in the stationary phase cells of the deletion mutants using RT-PCR assays. Virulence-related gene expression levels were elevated in the deletion mutants ΔLMOf2365_0442-0444 compared to the wild type parental strain LMOf2365, indicating the down-regulation of virulence genes by this PTS permease in L. monocytogenes. Finally, stress-related gene clpC expression levels were also increased in all of the deletion mutants, suggesting the involvement of this PTS permease in stress response. Furthermore, these deletion mutants displayed the same pressure tolerance and the same capacity for biofilm formation compared to the wild-type parental strain LMOf2365. In summary, our findings suggest that the LMOf2365_0442 gene can be used as a potential target to develop inhibitors for new therapeutic and pathogen control strategies for public health.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis. Paraquat can generate reactive oxygen species (ROS) in cells, which results in oxidative stress. It was first shown that 1 mM of paraquat inhibited the growth rate of a superoxide dismutase (sod)-deletion mutant (∆sod) generated from L. monocytogenes 4b G but not in the wild-type, and induced the expression of other resistance genes (kat, fri, perR, sigB, and recA) as well as sod in the wild type. Interestingly, without paraquat treatment the expression of all the 5 genes were repressed in ∆sod compared to the wild type, while the expression of recA triggering SOS response, a global response to DNA damage, was increased in ∆sod in the presence of 1 mM paraquat. Taken together, these results suggest that SOD plays a central role in oxidant defense of L. monocytogenes 4b G, and SOS probably significantly impacts ∆sod survival under oxidative stress.

Background Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China. Genome sequencing of the pathogen will provide important information for globally understanding its virulence mechanism. Results We report the genome sequences of a highly virulent C. lunata strain. Phylogenomic analysis indicates that C. lunata was evolved from Bipolaris maydis (Cochliobolus heterostrophus). The highly virulent strain has a high potential to evolve into other pathogenic stains based on analyses on transposases and repeat-induced point mutations. C. lunata has a smaller proportion of secreted proteins as well as B. maydis than entomopathogenic fungi. C. lunata and B. maydis have a similar proportion of protein-encoding genes highly homologous to experimentally proven pathogenic genes from pathogen-host interaction database. However, relative to B. maydis, C. lunata possesses not only many expanded protein families including MFS transporters, G-protein coupled receptors, protein kinases and proteases for transport, signal transduction or degradation, but also many contracted families including cytochrome P450, lipases, glycoside hydrolases and polyketide synthases for detoxification, hydrolysis or secondary metabolites biosynthesis, which are expected to be crucial for the fungal survival in varied stress environments. Comparative transcriptome analysis between a lowly virulent C. lunata strain and its virulence-increased variant induced by resistant host selection reveals that the virulence increase of the pathogen is related to pathways of toxin and melanin biosynthesis in stress environments, and that the two pathways probably have some overlaps. Conclusions The data will facilitate a full revelation of pathogenic mechanism and a better understanding of virulence differentiation of C. lunata. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-627) contains supplementary material, which is available to authorized users.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis. Paraquat can generate reactive oxygen species (ROS) in cells, which results in oxidative stress. It was first shown that 1 mM of paraquat inhibited the growth rate of a superoxide dismutase (sod)-deletion mutant (∆sod) generated from L. monocytogenes 4b G but not in the wild-type, and induced the expression of other resistance genes (kat, fri, perR, sigB, and recA) as well as sod in the wild type. Interestingly, without paraquat treatment the expression of all the 5 genes were repressed in ∆sod compared to the wild type, while the expression of recA triggering SOS response, a global response to DNA damage, was increased in ∆sod in the presence of 1 mM paraquat. Taken together, these results suggest that SOD plays a central role in oxidant defense of L. monocytogenes 4b G, and SOS probably significantly impacts ∆sod survival under oxidative stress.

Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant Δsod, and double deletion mutant of the sod and lm. G_1771 genes Δ1771Δsod were used to investigate the role of SOD and its relationship to the expression of the putative ABC transporter permease in biofilm formation. Our results showed that the ability to form a biofilm was significantly reduced in the Δsod mutant and the Δ1771Δsod double mutant. Both Δsod and Δ1771Δsod mutants exhibited slow growth phenotypes and produced more reactive oxygen species (ROS). The growth was inhibited in the mutants by methyl viologen (MV, internal oxygen radical generator) treatment. In addition, the expression of one oxidation resistance gene (kat), two stress regulators encoding genes (perR and sigB), and one DNA repair gene (recA) were analyzed in both the wild-type L. monocytogenes 4b G and the deletion mutants by RT-qPCR. The expression levels of the four genes were increased in the deletion mutants when biofilms were formed. Taken together, our data indicated that SOD played an important role in biofilm formation through coping with the oxidant burden in deficient antioxidant defenses.