RpoS acts as a global repressor of virulence gene expression in E. coli O104:H4 and enteroaggregative E. coli.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.

AefR, a TetR Family Transcriptional Repressor, Regulates Several Auxin Responses in Pseudomonas syringae Strain PtoDC3000.

The plant hormone indole-3-acetic acid (IAA), also known as auxin, plays important roles in plant growth and development, as well as in several plant-microbe interactions. IAA also acts as a microbial signal and in many bacteria regulates metabolism, stress responses, and virulence. In the bacterial plant pathogen Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000), exposure to IAA results in large-scale transcriptional reprogramming, including the differential expression of several known virulence genes. However, how PtoDC3000 senses and responds to IAA and what aspects of its biology are regulated by IAA is not understood. To investigate the mechanisms involved in perceiving and responding to IAA, we carried out a genetic screen for mutants with altered responses to IAA. One group of mutants of particular interest carried disruptions in the aefR gene encoding a TetR family transcriptional regulator. Gene expression analysis confirmed that the aefR mutants have altered responses to IAA. Thus, AefR is the first demonstrated auxin response regulator in PtoDC3000. We also investigated several aspects of PtoDC3000 biology that are regulated by both AefR and IAA, including antibiotic resistance, motility, and virulence. The observation that the aefR mutant has altered virulence on Arabidopsis, suggests that the sector of the IAA response regulated by aefR is important during pathogenesis. Our findings also provide evidence that AefR plays a role in coordinating changes in gene expression during the transition from early to late stages of infection. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of an antivirulence agent targeting the master regulator of virulence genes in Staphylococcus aureus.

The emergence of bactericidal antibiotic-resistant strains has increased the demand for alternative therapeutic agents, such as antivirulence agents targeting the virulence regulators of pathogens. Staphylococcus aureus exoprotein expression (sae) locus, the master regulator of virulence gene expression in multiple drug-resistant S. aureus, is a promising therapeutic target. In this study, we screened a small-molecule library using a SaeRS green fluorescent protein (GFP)-reporter that responded to transcription controlled by the sae locus. We identified the compound, N-(2-methylcyclohexyl)-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SKKUCS), as an efficient repressor of sae-regulated GFP activity. SKKUCS inhibited hemolysin production and reduced α-hemolysin-mediated cell lysis. Moreover, SKKUCS substantially reduced the expression levels of various virulence genes controlled by the master regulators, sae, and the accessory gene regulator (agr), demonstrating its potential as an antivirulence reagent targeting the key virulence regulators. Furthermore, autokinase inhibition assay and molecular docking suggest that SKKUCS inhibits the kinase activity of SaeS and potentially targets the active site of SaeS kinase, possibly inhibiting ATP binding. Next, we evaluated the efficacy and toxicity of SKKUCS in vivo using murine models of staphylococcal intraperitoneal and skin infections. Treatment with SKKUCS markedly increased animal survival and significantly decreased the bacterial burden in organs and skin lesion sizes. These findings highlight SKKUCS as a potential antivirulence drug for drug-resistant staphylococcal infections.

Susceptibility of emetic and enterotoxigenic Bacillus cereus grown at high temperature to disinfectants.

The prevalence of emetic strains in food products is rare; however, infection with these may be fatal to the vulnerable population. Bacterial control of the emetic Bacillus cereus strains is still unclear. This study aimed to assess the influence of high temperature on the disinfection of emetic and enterotoxigenic B. cereus. Emetic B. cereus strains survived up to 50 °C; the lag time and maximum growth rate were higher at 42 °C than those at 30 °C. Compared to enterotoxigenic B. cereus, all emetic food strains showed higher minimum inhibitory concentrations and minimum bactericidal concentrations for sodium hypochlorite and citric acid. The disinfectant susceptibility of the emetic B. cereus OS-05 strain incubated at a higher temperature did not increase and was maintained at the highest MBC value. In all emetic B. cereus strains, enterotoxin gene expression was upregulated at 42 °C and 45 °C. Increased ces gene expression was also found in emetic B. cereus strains GP-15 and OS-05, with upregulation of 128- and 820-fold at 42 °C. Thus, emetic B. cereus grown at high temperatures may resist common disinfectants of the food industry. The findings may help control B. cereus in food or the food processing industry.

Effect of in-water administration of quorum system inhibitors in broilers' productive performance and intestinal microbiome in a mild necrotic enteritis challenge.

The poultry industry has been facing the impact of necrotic enteritis (NE), a disease caused by the bacterium Clostridium perfringens producing the haemolytic toxin NetB. NE severity may vary from mild clinical to prominent enteric signs causing reduced growth rates and affecting feed conversion ratio. NetB production is controlled by the Agr-like quorum-sensing (QS) system, which coordinates virulence gene expression in response to bacterial cell density. In this study, the peptide-containing cell-free spent media (CFSM) from Enterococcus faecium was tested in NE challenged broilers in two battery cage and one floor pen studies. Results showed a significant reduction of NE mortality. Metagenomic sequencing of the jejunum microbiome revealed no impact of the CFSM on the microbial community, and growth of C. perfringens was unaffected by CFSM in vitro. The expression of QS-controlled virulence genes netB, plc and pfoA was found to be significantly repressed by CFSM during the mid-logarithmic stage of C. perfringens growth and this corresponded with a significant decrease in haemolytic activity. Purified fractions of CFSM containing bioactive peptides were found to cause reduced haemolysis. These results showed that bioactive peptides reduce NE mortality in broilers by interfering with the QS system of C. perfringens and reducing bacterial virulence. Furthermore, the microbiome of C. perfringens-challenged broilers is not affected by quorum sensing inhibitor containing CFSM.

Exposure to sublethal concentrations of chlorine enhances the cytotoxicity of Acanthamoeba castellanii.

Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections in humans and animals. Many studies are being conducted to find effective compounds against amoebae, but their sublethal concentration effects on surviving amoebae seem to have been overlooked. Chlorine is a common disinfection agent commonly added to public water facilities and supplies. In this study, the cytopathic and phagocytic properties of Acanthamoeba castellanii trophozoites following exposure to sublethal concentrations of chlorine were examined. Two hours of exposure to 5 ppm hypochlorite calcium was considered the sublethal concentration for A. castellanii trophozoites. To compare the pathogenic potential of treated and untreated Acanthamoeba trophozoites, cytotoxicity, adhesion assays in RAW 264.7 macrophages, osmo, and thermotolerance tests were carried out. Bacterial uptake was assessed in treated cells to evaluate their phagocytic characteristics. Oxidative stress biomarkers and antioxidant activities were compared in treated and untreated trophozoites. Finally, the mRNA expression of the mannose-binding protein (MBP), cysteine protease 3 (CP3), and serine endopeptidase (SEP) genes was determined in cells. In all the experiments, untreated trophozoites were considered the control. In comparison to untreated trophozoites, in chlorine-treated trophozoites, cytopathic effects were more extensive and resulted in the detachment of macrophage monolayers. Treated trophozoites could not grow at high temperatures (43 °C). Besides, they showed osmotolerance to 0.5 M D-mannitol but not to 1 M. Results demonstrated a higher bacterial uptake rate by chlorine-treated trophozoites than untreated cells. The treated and untreated cells had significantly different glutathione and glutathione/glutathione disulfide ratios. Antioxidant enzyme activities, total antioxidant capacity, and malondialdehyde levels were increased significantly in chlorine-treated cells. Quantifying mRNA expression in chlorine-treated trophozoites revealed that virulence genes were upregulated. Chlorine can form resistance and virulent amoebae if it is not used at a proper concentration and exposure time. Identification of stress responses, their mechanisms in Acanthamoeba, and their relation to amoeba virulence would give us a better perception of their pathophysiology.

Effects of BlueM® against Streptococcus mutans biofilm and its virulence gene expression

Abstract This study evaluated the antimicrobial capacity of BlueM® mouthwash against the bacterium Streptococcus mutans and its influence on gbpA gene expression as well as its cytotoxic effect on fibroblast cells. BlueM® showed antimicrobial activity, with MIC and MBC values of 0.005% and 0.01%, respectively. The MBIC was 6.25% for S. mutans. CFU count and confocal microscopy revealed significant effect of BlueM® on S. mutans biofilm pre-formed on dentin surfaces. Interestingly, the analysis of gbpA gene expression indicated a decrease in gene expression after 15 min of treatment with BlueM® at a concentration of 25%. Moreover, BlueM® exhibited low levels of cytotoxicity. In conclusion, our results showed the antimicrobial effectiveness of BlueM® against S. mutans, its ability to modulate the expression of the gbpA gene and its low cytotoxicity. This study supports the therapeutic potential of BlueM® as an alternative agent for the control of oral biofilm.

Resumo Este estudo avaliou a capacidade antimicrobiana do enxaguatório BlueM® contra a bactéria Streptococcus mutans e sua influência na expressão do gene gbpA, bem como seu efeito citotóxico em células de fibroblastos. BlueM® apresentou atividade antimicrobiana, com valores de CIM e CBM de 0,005% e 0,01%, respectivamente. O MBIC foi de 6,25% para S. mutans. A contagem de UFC e a microscopia confocal revelaram efeito significativo do BlueM® no biofilme de S. mutans pré-formado nas superfícies de dentinas. Curiosamente, a análise da expressão do gene gbpA, indicou uma diminuição na expressão do gene após 15 min de tratamento com BlueM® na concentração de 25%. Além disso, BlueM® exibiu baixos níveis de citotoxicidade. Em conclusão, nossos resultados mostraram a eficácia antimicrobiana do BlueM® contra S. mutans, sua capacidade de modular a expressão do gene gbpA e sua baixa citotoxicidade. Este estudo apoia o potencial terapêutico do BlueM® como agente alternativo para o controle do biofilme oral.

Dietary Eugenol Nanoemulsion Potentiated Performance of Broiler Chickens: Orchestration of Digestive Enzymes, Intestinal Barrier Functions and Cytokines Related Gene Expression With a Consequence of Attenuating the Severity of E. coli O78 Infection.

Recently, the use of essential oils (EOs) or their bioactive compounds encapsulated by nanoparticles as alternative supplements for in-feed antimicrobials is gaining attention, especially in organic poultry production. Focusing on eugenol, its incorporation into the nanoformulation is a novel strategy to improve its stability and bioavailability and thus augment its growth-boosting and antimicrobial activities. Therefore, we explored eugenol nanoemulsion activities in modulating growth, digestive and gut barrier functions, immunity, cecal microbiota, and broilers response to avian pathogenic E. coli challenge (APEC) O78. A total of 1,000 one-day-old broiler chicks were allocated into five groups; negative control (NC, fed basal diet), positive control (PC), and 100, 250, and 400 mg/kg eugenol nanoemulsion supplemented groups. All groups except NC were challenged with APEC O78 at 14 days of age. The results showed that birds fed eugenol nanoemulsion displayed higher BWG, FI, and survivability and most improved FCR over the whole rearing period. Birds fed 400 mg/kg of eugenol nanoemulsion sustained a higher growth rate (24% vs. PC) after infection. Likely, the expression of digestive enzymes' genes (AMY2A, CCK, CELA1, and PNLIP) was more prominently upregulated and unaffected by APEC O78 challenge in the group fed eugenol nanoemulsion at the level of 400 mg/kg. Enhanced gut barrier integrity was sustained post-challenge in the group supplemented with higher levels of eugenol nanoemulsion as evidenced by the overexpression of cathelicidins-2, ß-defensin-1, MUC-2, JAM-2, occludin, CLDN-1, and FABP-2 genes. A distinct modulatory effect of dietary eugenol nanoemulsion was observed on cytokine genes (IL-1ß, TNF-α, IL-6, IL-8, and IL-10) expression with a prominent reduction in the excessive inflammatory reactions post-challenge. Supplementing eugenol nanoemulsion increased the relative cecal abundance of Lactobacillus species and reduced Enterobacteriaceae and Bacteriods counts. Notably, a prominent reduction in APEC O78 loads with downregulation of papC, iroN, iutA, and iss virulence genes and detrimental modifications in E. coli morphological features were noticed in the 400 mg/kg eugenol nanoemulsion group at the 3rd-week post-challenge. Collectively, we recommend the use of eugenol nanoemulsion as a prospective targeted delivery approach for achieving maximum broilers growth and protection against APEC O78 infection.

Escherichia coli O157:H7 is challenged by the presence of Pseudomonas, but successfully co-existed in dual-species microbial communities.

Escherichia coli O157:H7 and Pseudomonas were considered as common colonizers of fresh and spoilage meat, where they tended to live in the proximity. In this study, we tested the interplay between different isolates of E. coli O157:H7 and Pseudomonas in random two-by-two combinations grown as dual-species consortia. Results showed that the growth fitness of E. coli was not facilitated by the presence of all tested Pseudomonas strains, and vice versa. Representative combinations were further selected to investigate the property changes following the time course of biofilms formation as compared to single species. Cell counting confirmed that the growth of E. coli O157:H7 was challenged by the presence of Pseudomonas strains as previously described. Our findings shed new light on the evidence that the pathogenicity of E. coli O157:H7 was negatively affected by the presence of Pseudomonas according to the evaluation of spatial organization and genetic expression of virulence factors, which might be a naturally existing biological phenomenon constraining the safety risk of former strains in meat processing and preservation. Intriguingly, we observed that E. coli managed to stably co-exist at low cellular abundance in the progress of dual-species consortia, indicating successful adaptive mechanisms that need further investigations to uncover.

Research Note: Virulence gene downregulation and reduced intestinal colonization of Salmonella enterica serovar Typhimurium PHL2020 isolate in broilers by a natural antimicrobial (NeutraPath™).

The reduction in antibiotic growth promoter use in poultry, due to antibiotic resistance concerns, has created a need for natural solutions that control enteric pathogens like Salmonella. One of these natural feed additives, a select blend of essential oils, fatty acids, and an enterosorbent mineral (NeutraPath), was assessed for its effects on the intestinal colonization of Salmonella enterica serovar Typhimurium PHL2020 isolate (ST-PHL2020) in broiler chickens and ST-PHL2020 virulence gene expression. An in vitro digestion model simulating the pH and enzymatic conditions of 3 gastrointestinal compartments (crop, proventriculus, and intestine) was first used to evaluate the antibacterial effects of NeutraPath on ST-PHL2020. For the in vivo study, day-old male broilers (n = 90) were randomly allocated to 1 of 3 groups: control or NeutraPath supplemented at 0.25 or 0.5%. The dose rates were chosen to enable observable statistical effects during high Salmonella challenge. All groups were challenged with ST-PHL2020 (106 cfu/bird) via oral gavage on day 9. Bacterial load and prevalence of ST-PHL2020 were examined in ceca-cecal tonsils, and intestinal permeability was assessed via serum recovery of fluorescein isothiocyanate dextran (FITC-d) 24 h postchallenge. NeutraPath inhibited (P < 0.05) ST-PHL2020 growth in the in vitro digestion model compared to the control at all concentrations and in all compartments other than NeutraPath 0.25% in the crop. In vivo, NeutraPath 0.25 and 0.5% reduced (P < 0.05) the total cfu recovered and total prevalence of ST-PHL2020 in the ceca. The serum FITC-d levels were also reduced (P < 0.05) by NeutraPath. Further, NeutraPath's effects on ST-PHL2020's Salmonella pathogenicity island-1 virulence network development were explored via treating ST-PHL2020 at subinhibitory concentration (1 mg/mL) of NeutraPath in vitro. Compared to the control, NeutraPath downregulated ST-PHL2020 hilA and invF mRNA expression, which further blocked expression of key downstream effectors involved in ST-PHL2020 invasion. Collectively, NeutraPath has the potential to reduce ST-PHL2020 intestinal colonization in broilers and preserve intestinal barrier integrity.

Identification, Characterization, and Virulence Gene Expression of Marine Enterobacteria in the Upper Gulf of Thailand.

Enterobacteria that commonly inhabit marine environments have a great impact on human health. In this study, enterobacteria isolated from seawater in the Upper Gulf of Thailand were identified and characterized. Seawater from nine sampling sites along the Upper Gulf of Thailand contained presumptive enterobacteria that ranged from 0.22 ± 0.44 to 17.00 ± 3.97 CFU/mL. The 101 strains belonged to seven species in which Klebsiella pneumoniae was the majority (47.5% of strains). The highest prevalence was resistant to ampicillin (76.2%) and ticarcillin (72.3%), respectively, whereas none was resistant to imipenem. Forty-five antibiotic resistance patterns were observed and 33.7% exhibited multidrug resistance, emphasizing the concern about public health. Three ß-lactamase genes, including ampC, blaSHV, and blaTEM, were detected at the frequencies of 47.5%, 21.8%, and 11.9%, respectively. Six virulence genes, including csgD, uge, kfu, eaeA, magA, and LTI, were detected at the frequencies of 37.6%, 31.7%, 19.8%, 16.8%, 12.9%, and 5.9%, respectively. The condition of 4% NaCl downregulated the expression of the kfu and uge genes. The 67.3% and 63.4% of strains synthesized silver nanoparticles ranging between 3.04 ± 0.64 and 20.64 ± 0.95 µg/mL and gold nanoparticles ranging between 7.77 ± 0.45 and 57.57 ± 8.00 µg/mL, respectively.

Growth and Expression of Virulence Genes of Listeria monocytogenes during the Processing of Dry-Cured Fermented "Salchichón" Manufactured with a Selected Lactilactobacillus sakei.

The effect of the dry-cured fermented processing of "salchichón" inoculated with a selected strain of Lactilactobacillus sakei (205) on the growth and transcriptional response of three virulence genes (plcA, hly, and iap) of Listeria monocytogenes was evaluated. For this, three different batches of "salchichón" were analyzed: batch B (inoculated only with L. sakei), batch L (inoculated only with L. monocytogenes), and batch L + B (inoculated with both microorganisms). Sausages were ripened for 90 days according to a traditional industrial process. The processing of "salchichón" provoked a reduction in L. monocytogenes counts of around 2 log CFU/g. The downregulation of the expression of the three genes was found at the end of ripening when the water activity (aw) of "salchichón" was <0.85 aw. The combined effect on the reduction in L. monocytogenes counts together with the downregulation in the expression of the virulence genes throughout the "salchichón" processing could be of great interest to control the hazard caused by the presence of this pathogenic bacterium.

Chitosan propolis nanocomposite alone or in combination with apramycin: an alternative therapy for multidrug-resistant Salmonella Typhimurium in rabbits: in vitro and in vivo study.

Introduction. The emergence of multidrug-resistant Salmonella Typhimurium strains has increased the need for safe, alternative therapies from natural sources with antibacterial properties.Hypothesis/Gap Statement. There are no published data regarding the use of chitosan propolis nanocomposite (CPNP) either alone or in combination with antibiotics as antimicrobials against S. Typhimurium, especially in Egypt.Aim. This study evaluated the antibacterial activities of five antimicrobials [apramycin, propolis, chitosan nanoparticles (CNPs), chitosan propolis nanocomposite (CPNP) and CPNP +apramycin] against ten virulent and multidrug-resistant (MDR) S. Typhimurium field strains recovered from diarrheic rabbits through in vitro and in vivo study.Methodology. The expression levels of three virulence genes of S. Typhimurium strains were determined by quantitative reverse-transcription PCR (RT-qPCR) after exposure to sub-inhibitory concentrations of apramycin, propolis, CNPs, CPNP alone, and CPNP +apramycin. Additionally, 90 New Zealand rabbits were divided into control and experimentally S. Typhimurium-infected groups. The infected rabbits were orally administered saline solution (infected-untreated); 10 mg apramycin/kg (infected-apramycin-treated); 50 mg propolis/kg (infected-propolis-treated); 15 mg CPNP/kg (infected-CPNP-treated) and 15 mg CPNP +10 mg apramycin/kg (infected-CPNP +apramycin-treated) for 5 days.Results. The RT-qPCR analysis revealed different degrees of downregulation of all screened genes. Furthermore, the treatment of infected rabbits with CPNP or CPNP +apramycin significantly improved performance parameters, and total bacterial and Salmonella species counts, while also modulating both oxidative stress and altered liver and kidney parameters.Conclusion. This work demonstrates the use of CPNP alone or in combination with apramycin in the treatment of S. Typhimurium in rabbits.

Five major two components systems of Staphylococcus aureus for adaptation in diverse hostile environment.

Staphylococcus aureus is an eminent and opportunistic human pathogen that can colonize in the intestines, skin tissue and perineal regions of the host and cause severe infectious diseases. The presence of complex regulatory network and existence of virulent gene expression along with tuning metabolism enables the S. aureus to adopt the diversity of environments. Two component system (TCS) is a widely distributed mechanism in S. aureus that permit it for changing gene expression profile in response of environment stimuli. TCS usually consist of transmembrane histidine kinase (HK) and cytosolic response regulator. S. aureus contains totally 16 conserved pairs of two component systems, involving in different signaling mechanisms. There is a connection among these regulatory circuits and they can easily have effect on each other's expression. This review has discussed five major types of TCS in S. aureus and covers the recent knowledge of their virulence gene expression. We can get more understanding towards staphylococcal pathogenicity by getting insights about gene regulatory pathways via TCS, which can further provide implications in vaccine formation and new ways for drug design to combat serious infections caused by S. aureus in humans.

Effect of Bifidobacterium crudilactis and 3'-sialyllactose on the toddler microbiota using the SHIME® model.

The important changes in diet during the first years of life strongly modulate the intestinal microbiota of young children. Among in vitro digestive models, the simulator of human intestinal microbial ecosystem (SHIME®) model, seems particularly adapted to study the effects of prebiotics and/or probiotics on the dynamic microbiota of toddlers. The main purpose of this study was to investigate different formulations with prebiotic (3'-sialyllactose: 3'SL) and probiotic (Bifidobacterium crudilactis FR/62/b/3) effects on young child microbiota using the SHIME® model. The ascending (AC), transverse (TC) and descending (DC) colons of the SHIME® model were inoculated with feces from 3 donors aged between 1 and 2 years, in three separate vessels. After two weeks of microbiota stabilization, four treatments of one week (prebiotic, probiotic, synbiotic and cell-free spent media from the synbiotic) were administered. In all the colon vessels, the short chain fatty acid analyses, determined using high-performance liquid chromatography highlighted a ratio acetate/propionate/butyrate proportion of 5:19:6, situated between infant and adult normal values. As already observed in other validated studies focusing on the SHIME® model, the 16S rDNA sequencing highlighted a low richness and diversity in the AC, while the microbial communities in the TC and the DC remained similar to each other. Although some bacteria involved in biofilm development have been identified (Stenotrophomonas, Megasphera and Enterobacter), specific bacterial populations, proper to each colon were developed. Some bacteria associated to the upper intestinal tract, such as Lactobacillus and Veillonella genera, seemed to grow easily in the AC. The quantitative polymerase chain reaction (qPCR) targeting the hsp60 gene confirmed the ability of bifidobacteria to survive in this toddler model. In addition, the synbiotic treatment tended to a bifidogenic effect (P < 0.1). On the other hand, the feces of the donors and the content of the three colon vessels were filtered and placed in contact with Escherichia coli O157:H7 ATCC 43890 to evaluate the modulation of virulence gene expression using reverse transcription PCR. Finally, filtered supernatants from donor feces significantly up-regulated the expression of the luxS gene of E. coli O157:H7 (P = 0.013). In conclusion, despite the presence of biofilms, the toddler SHIME® model used in his study shared characteristics found both in adults and infants. Although additional investigations should be performed, combining 3'SL and B. crudilactis FR/62/b/3 could lead to a beneficial effect on infant microbiota by favoring bifidobacterial presence. Finally, the filtrated supernatant from young child feces could be able to modulate the quorum sensing mechanism for E. coli O157:H7.

Exploring the Pathogenic Potential of Vibrio vulnificus Isolated from Seafood Harvested along the Mangaluru Coast, India.

It has been observed that not all strains of Vibrio vulnificus are virulent. Determining the virulence of strains that are frequently present in seafood is of significance for ensuring seafood safety. This study is an attempt to predict the virulence of seafood-borne V. vulnificus isolated along the Mangaluru Coast, India. The isolates tested possessed a vcgC gene sequence with high similarity to that in the clinical strain. Transcriptional analysis of core virulence genes in seafood isolate E4010 showed the phenomenon of contact-mediated expression of rtxA1 which correlated well with the actin disintegration and cytotoxicity. These results suggest that the seafood isolates tested in this study possess a functional RtxA1 which could help in initiating the infection. However, other putative virulence genes such as vvpE encoding an extracellular protease, vvhA encoding hemolysin, flp encoding tad pilin and ompU encoding fibronectin-binding protein were also constitutively expressed. Virulence-associated attributes such as cytotoxicity and adherence matched the response of the clinical strain (p > 0.05). On the other hand, the environmental strains showed higher serum sensitivity compared with the clinical strain. These findings show that the part of virulence attributes required for the disease process might be intact in these isolates.

Different Impacts of MucR Binding to the babR and virB Promoters on Gene Expression in Brucella abortus 2308.

The protein MucR from Brucella abortus has been described as a transcriptional regulator of many virulence genes. It is a member of the Ros/MucR family comprising proteins that control the expression of genes important for the successful interaction of α-proteobacteria with their eukaryotic hosts. Despite clear evidence of the role of MucR in repressing virulence genes, no study has been carried out so far demonstrating the direct interaction of this protein with the promoter of its target gene babR encoding a LuxR-like regulator repressing virB genes. In this study, we show for the first time the ability of MucR to bind the promoter of babR in electrophoretic mobility shift assays demonstrating a direct role of MucR in repressing this gene. Furthermore, we demonstrate that MucR can bind the virB gene promoter. Analyses by RT-qPCR showed no significant differences in the expression level of virB genes in Brucella abortus CC092 lacking MucR compared to the wild-type Brucella abortus strain, indicating that MucR binding to the virB promoter has little impact on virB gene expression in B. abortus 2308. The MucR modality to bind the two promoters analyzed supports our previous hypothesis that this is a histone-like protein never found before in Brucella.

Dual Role of Auxin in Regulating Plant Defense and Bacterial Virulence Gene Expression During Pseudomonas syringae PtoDC3000 Pathogenesis.

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen strain Pseudomonas syringae DC3000 (PtoDC3000) produces the plant hormone auxin (indole-3-acetic acid [IAA]) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth and that this phenotype was suppressed by introducing the sid2-2 mutation, which impairs SA synthesis. Thus, host auxin signaling is required for normal susceptibility to PtoDC3000 and is involved in suppressing SA-mediated defenses. Unexpectedly, tir1 afb1 afb4 afb5 quadruple-mutant plants lacking four of the six known auxin coreceptors that exhibit decreased auxin perception, supported increased levels of bacterial growth. This mutant exhibited elevated IAA levels and reduced SA-mediated defenses, providing additional evidence that auxin promotes disease by suppressing host defense. We also investigated the hypothesis that IAA promotes PtoDC3000 virulence through a direct effect on the pathogen and found that IAA modulates expression of virulence genes, both in culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

The MexE/MexF/AmeC Efflux Pump of Agrobacterium tumefaciens and Its Role in Ti Plasmid Virulence Gene Expression.

Expression of the tumor-inducing (Ti) plasmid virulence genes of Agrobacterium tumefaciens is required for the transfer of DNA from the bacterium into plant cells, ultimately resulting in the initiation of plant tumors. The vir genes are induced as a result of exposure to certain phenol derivatives, monosaccharides, and low pH in the extracellular milieu. The soil, as well as wound sites on a plant-the usual site of the virulence activity of this bacterium-can contain these signals, but vir gene expression in the soil would be a wasteful utilization of energy. This suggests that mechanisms may exist to ensure that vir gene expression occurs only at the higher concentrations of inducers typically found at a plant wound site. In a search for transposon-mediated mutations that affect sensitivity for the virulence gene-inducing activity of the phenol, 3,5-dimethoxy-4-hydroxyacetophenone (acetosyringone [AS]), an RND-type efflux pump homologous to the MexE/MexF/OprN pump of Pseudomonas aeruginosa was identified. Phenotypes of mutants carrying an insertion or deletion of pump components included hypersensitivity to the vir-inducing effects of AS, hypervirulence in the tobacco leaf explant virulence assay, and hypersensitivity to the toxic effects of chloramphenicol. Furthermore, the methoxy substituents on the phenol ring of AS appear to be critical for recognition as a pump substrate. These results support the hypothesis that the regulation of virulence gene expression is integrated with cellular activities that elevate the level of plant-derived inducers required for induction so that this occurs preferentially, if not exclusively, in a plant environment.IMPORTANCE Expression of genes controlling the virulence activities of a bacterial pathogen is expected to occur preferentially at host sites vulnerable to that pathogen. Host-derived molecules that induce such activities in the plant pathogen Agrobacterium tumefaciens are found in the soil, as well as in the plant. Here, we tested the hypothesis that mechanisms exist to suppress the sensitivity of Agrobacterium species to a virulence gene-inducing molecule by selecting for mutant bacteria that are hypersensitive to its inducing activity. The mutant genes identified encode an efflux pump whose proposed activity increases the concentration of the inducer necessary for vir gene expression; this pump is also involved in antibiotic resistance, demonstrating a relationship between cellular defense activities and the control of virulence in Agrobacterium.

Effect of fermented milk from Lactococcus lactis ssp. cremoris strain JFR1 on Salmonella invasion of intestinal epithelial cells.

The process of fermentation contributes to the organoleptic properties, preservation, and nutritional benefits of food. Fermented food may interfere with pathogen infections through a variety of mechanisms, including competitive exclusion or improving intestinal barrier integrity. In this study, the effect of milk fermented with Lactococcus lactis ssp. cremoris JFR1 on Salmonella invasion of intestinal epithelial cell cultures was investigated. Epithelial cells (HT29-MTX, Caco-2, and cocultures of the 2) were treated for 1 h with Lactococcus lactis ssp. cremoris JFR1 fermented milk before infection with Salmonella enterica ssp. enterica Typhimurium. Treatment with fermented milk resulted in increased transepithelial electrical resistance, which remained constant for the duration of infection (up to 3 h), illustrating a protective effect. After gentamicin treatment to remove adhered bacterial cells, enumeration revealed a reduction in numbers of intracellular Salmonella. Quantitative reverse-transcription PCR data indicated a downregulation of Salmonella virulence genes hilA, invA, and sopD after treatment with fermented milk. Fermented milk treatment of epithelial cells also exhibited an immunomodulatory effect reducing the production of proinflammatory IL-8. In contrast, chemically acidified milk (glucono delta-lactone) failed to show the same effect on monolayer integrity, Salmonella Typhimurium invasion, and gene expression as well as immune modulation. Furthermore, an oppA knockout mutant of Salmonella Typhimurium infecting treated epithelial cells did not show suppressed virulence gene expression. Collectively, these results suggest that milk fermented with Lactococcus lactis ssp. cremoris JFR1 is effective in vitro in the reduction of Salmonella invasion into intestinal epithelial cells. A functional OppA permease in Salmonella is required to obtain the antivirulence effect of fermented milk.