Growth In Laboratory Media Research Articles

A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia.

Ventilator-associated pneumonia is defined as pneumonia that develops in a patient who has been on mechanical ventilation for more than 48 hours through an endotracheal tube. It is caused by biofilm formation on the indwelling tube, which introduces pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans into the patient's lower airways. Currently, there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates the biofilm formation of these pathogens in a host-mimicking environment and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilated airway mucus medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-scanning electron microscopy were employed to characterize the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved the biofilm eradication of all pathogens. Our in vitro endotracheal tube model informs on fundamental microbiology in the ventilator-associated pneumonia context and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants.

Correlation of gene content in selected bifidobacteria with folate supplier or scavenger status during growth in laboratory media

Five different bifidobacterial cultures were screened to investigate if their de novo folate production ability could be predicated based on their folate biosynthetic gene content. As previously developed folate free media could not adequately support the growth of these five strains, even with added folate, an alternative medium (YNB+) was developed. Consistent with its genome prediction, Bifidobacterium animalis subsp. lactis strain Bb-12 required folate for its growth even in the presence of p-aminobenzoic acid (PABA). Although B. longum DJO10A and B. breve ATCC 15701 lack an alkaline phosphatase gene for the formation of one pathway intermediate (DHPPP), both were found to be able to grow independent from folate and PABA suggesting that lack of these genes in genome annotations cannot be used to predict folate dependency in bifidobacteria. Similarly, having all required genes cannot always predict folate production, as B. longum subsp. infantis RECb4 contained a full set of pathway genes, but required folate or PABA for growth. While many bifidobacteria can produce folate, this also cannot be used to predicate their ability to supply folate as some can act as scavengers when folate is readily available in the environment, such as B. breve ATCC 15701 in this study. As bifidobacterial cultures are sometimes considered for use in foods based on their folate production abilities, it is important to consider whether they function as folate scavengers or suppliers when growing in folate containing media.

Correlation of Gene Content in Selected Bifidobacteria with Folate Supplier or Scavenger Status During Growth in Laboratory Media

Correlation of Gene Content in Selected Bifidobacteria with Folate Supplier or Scavenger Status During Growth in Laboratory Media

Genome-wide analysis of fitness-factors in uropathogenic Escherichia coli during growth in laboratory media and during urinary tract infections.

Uropathogenic Escherichia coli (UPEC) UTI89 is a well-characterized strain, which has mainly been used to study UPEC virulence during urinary tract infection (UTI). However, little is known on UTI89 key fitness-factors during growth in lab media and during UTI. Here, we used a transposon-insertion-sequencing approach (TraDIS) to reveal the UTI89 essential-genes for in vitro growth and fitness-gene-sets for growth in Luria broth (LB) and EZ-MOPS medium without glucose, as well as for human bacteriuria and mouse cystitis. A total of 293 essential genes for growth were identified and the set of fitness-genes was shown to differ depending on the growth media. A modified, previously validated UTI murine model, with administration of glucose prior to infection was applied. Selected fitness-genes for growth in urine and mouse-bladder colonization were validated using deletion-mutants. Novel fitness-genes, such as tusA, corA and rfaG; involved in sulphur-acquisition, magnesium-uptake, and LPS-biosynthesis, were proved to be important during UTI. Moreover, rfaG was confirmed as relevant in both niches, and therefore it may represent a target for novel UTI-treatment/prevention strategies.

Salmonella enterica serovar Typhimurium genetic variants isolated after lethal treatment with Thymbra capitata essential oil (TCO) showed increased resistance to TCO in milk

The high prevalence of Salmonella enterica in milk poses a risk of considerable concern in the preservation of certain dairy products, mainly those elaborated from raw milk. Essential oils (EOs) have been proposed as a promising food preservative for such products due to their strong antimicrobial properties. Additionally, these natural antimicrobials have been shown to be effective against multi-drug resistant strains. They can thus also be utilized to prevent the dissemination of antimicrobial resistances (AMR). However, recent evidence of the development of bacterial resistance under EO treatments may call their use into question. This study sought to assess the emergence of antimicrobial resistant genetic variants of S. enterica serovar Typhimurium from survivors after cyclic exposure to lethal doses (>5 log10 cycles of inactivation) of Thymbra capitata EO (TCO), in order to evaluate the impact that it could have on milk preservation, to ascertain whether cross-resistance to antibiotics occurs, and to identify the genomic changes responsible for their phenotype. Isolated strains by TCO (SeTCO) showed a two-fold increase in minimum inhibitory and bactericide concentrations (MIC and MBC) of TCO compared to Salmonella enterica serovar Typhimurium wild-type strain (SeWT) in laboratory growth medium, as well as a greater adaptation and growth rate in the presence of the EOs and a higher survival to TCO treatments in buffers of pH 4.0 and 7.0. The increased resistance of SeTCO was confirmed in skimmed milk: 300 μL/L TCO reduced only 1 log10 cycle of SeTCO population, whereas it inactivated more than 5 log10 cycles in SeWT. Moreover, SeTCO showed an increased cross-resistance against aminoglycosides, quinolones and tetracyclines. Whole genome sequencing revealed 5 mutations in SeTCO: 2 in genes involved in O-antigens synthesis (rfbV and rfbX), 2 in genes related to adaptation to the growing medium (trkA and glpK), and 1 in a redox-sensitive transcriptional regulator (soxR). The phenotypic characterization of a constructed SeWT strain with mutant soxRSeTCO demonstrated that the mutation of soxR was the main cause of the increased resistance and tolerance observed in SeTCO against TCO and antibiotics. The emergence of resistant strains against EOs might jeopardize their use as food preservatives. Further studies will thus be required to determine under which conditions such resistant strains might occur, and to assess the food risk they may pose, as well as to ascertain their impact on the spread of AMR.

The gigA/gigB Genes Regulate the Growth, Stress Response, and Virulence of Acinetobacter baumannii ATCC 17978 Strain.

Acinetobacter baumannii is an important pathogen of nosocomial infection. Recently, a group of genes, named “gig” (for Growth in Galleria), have been identified in a contemporary multi-drug resistant clinical isolate of A. baumannii—strain AB5075. Among these so-called gig genes, gigA and gigB were found to promote antibiotic resistance, stress survival, and virulence of AB5075 by interacting with the nitrogen phosphotransferase system (PTSNtr). This study aimed to investigate the roles of gigA/gigB, which appear to comprise a stress-signaling pathway (encoding for an atypical two-component system response regulator and a predicted anti-anti-sigma factor, respectively), and the involvement of ptsP (encoding the Enzyme I component of the PTSNtr) in the growth, stress resistance, and virulence of the widely studied A. baumannii strain ATCC 17978. Genetic analyses of strains harboring mutations of gigA and gigB were performed to investigate the roles of these genes in bacterial growth, stress resistance, evading macrophage defense, and killing of Galleria mellonella larva. In contrast with findings from strain AB5075 where gigA and gigB contribute to aminoglycoside resistance, the data presented herein indicate that the loss of gigA/gigB does not impact antibiotic resistance of strain ATCC 17978. Interestingly, however, we found that deletion of gigA/gigB in the ATCC 17978 background imparts a general growth in laboratory medium and also conferred growth and replication defects within murine macrophages and an inability to kill G. mellonella larvae. Importantly, studies as well as the loss of ptsP restored the phenotypes of the gigA/gigB mutant to that of the wild-type. The data presented herein indicate that in A. baumannii ATCC 17978, the gigA/gigB genes play a key role in both growth and virulence traits, but are dispensable for other stress-resistance survival phenotypes, including aminoglycoside resistance. Our findings thus highlight several similarities and also important differences between the gigA/gigB stress-signaling pathway in two commonly studied isolates of this troublesome pathogen.

Investigating the Involvement of Cytoskeletal Proteins MreB and FtsZ in the Origin of Legume-Rhizobial Symbiosis

Rhizobia are rod-shaped bacteria that form nitrogen-fixing root nodules on leguminous plants, but they don't carry MreB, a key determinant of rod-like cell shape. Here, we introduced an actin-like mreB homologue from pseudomonad into Mesorhizobium huakuii 7653R (a microsymbiont of Astragalus sinicus L.) and examined the molecular, cellular and symbiotic phenotypes of the resultant mutant. Exogenous mreB caused an enlarged cell size and slower growth in laboratory medium. However, the mutant formed small ineffective nodules on A. sinicus (Nod+ Fix-), and rhizobial cells in the infection zone were unable to differentiate into bacteroids. RNA-seq analysis also revealed minor effects of mreB on global gene expression in free-living cells but larger effects for cells grown in planta. Differentially expressed nodule-specific genes include cell cycle regulators such as the tubulin-like ftsZ1 and ftsZ2. Unlike the ubiquitous FtsZ1, FtsZ2 was commonly found in Rhizobium, Sinorhizobium and Mesorhizobium, but not in closely related non-symbiotic species. Bacterial Two-Hybrid analysis revealed that MreB interacts with FtsZ1 and FtsZ2, which are targeted by the host-derived NCR peptides. Significantly, MreB mutation D283A disrupted the protein-protein interactions and restored the aforementioned phenotypic defects caused by MreB in M. huakuii. Together, our data indicate that MreB is detrimental for modern rhizobia and its interaction with FtsZ1/FtsZ2 cause the symbiotic process to cease at the late stage of bacteroid differentiation. These findings led to a hypothesis that loss of mreB in the common ancestor of Rhizobiales and subsequent acquisition of ftsZ2 are critical evolutionary steps leading to legume-rhizobial symbiosis.

A mycobacterial DivIVA domain-containing protein involved in cell length and septation

Mycobacterial cells elongate via polar deposition of cell wall material, similar to the filamentous Streptomyces species, which contain a tip-organizing centre. Coiled-coiled proteins such as DivIVA play an important role in this process. The genome of Mycobacterium tuberculosis , the causative agent of tuberculosis, encodes many coiled-coil proteins that are homologous to DivIVA with a potential role in mycobacterial cell elongation. Here we describe studies on Mycobacterium smegmatis MSMEG_2416, a homologue of M. tuberculosis Rv2927c. Two previous independent studies showed that MSMEG_2416 was involved in septation (subsequently referred to as sepIVA). Contrary to these previous reports, we found sepIVA to be dispensable for growth in laboratory media by generating a viable null mutant. The mutant strain did, however, show a number of differences, including a change in colony morphology and biofilm formation that could be reversed on complementation with sepIVA as well as Rv2927c, the sepIVA homologue from M. tuberculosis . However, analysis of cell wall lipids did not reveal any alterations in lipid profiles of the mutant strain. Microscopic examination of the mutant revealed longer cells with more septa, which occurred at irregular intervals, often generating mini-compartments, a profile similar to that observed in the previous studies following conditional depletion, highlighting a role for sepIVA in mycobacterial growth.

Saccharomyces cerevisiae nutrient signaling pathways show an unexpected early activation pattern during winemaking

BackgroundSaccharomyces cerevisiae wine strains can develop stuck or sluggish fermentations when nutrients are scarce or suboptimal. Nutrient sensing and signaling pathways, such as PKA, TORC1 and Snf1, work coordinately to adapt growth and metabolism to the amount and balance of the different nutrients in the medium. This has been exhaustively studied in laboratory strains of S. cerevisiae and laboratory media, but much less under industrial conditions.ResultsInhibitors of such pathways, like rapamycin or 2-deoxyglucose, failed to discriminate between commercial wine yeast strains with different nutritional requirements, but evidenced genetic variability among industrial isolates, and between laboratory and commercial strains. Most signaling pathways involve events of protein phosphorylation that can be followed as markers of their activity. The main pathway to promote growth in the presence of nitrogen, the TORC1 pathway, measured by the phosphorylation of Rps6 and Par32, proved active at the very start of fermentation, mainly on day 1, and ceased soon afterward, even before cellular growth stopped. Transcription factor Gln3, which activates genes subject to nitrogen catabolite repression, was also active for the first hours, even when ammonium and amino acids were still present in media. Snf1 kinase was activated only when glucose was exhausted under laboratory conditions, but was active from early fermentation stages. The same results were generally obtained when nitrogen was limiting, which indicates a unique pathway activation pattern in winemaking. As PKA remained active throughout fermentation, it could be the central pathway that controls others, provided sugars are present.ConclusionsWine fermentation is a distinct environmental situation from growth in laboratory media in molecular terms. The mechanisms involved in glucose and nitrogen repression respond differently under winemaking conditions.

Transposon Insertion Site Sequencing of Providencia stuartii: Essential Genes, Fitness Factors for Catheter-Associated Urinary Tract Infection, and the Impact of Polymicrobial Infection on Fitness Requirements.

Providencia stuartii is a common cause of polymicrobial catheter-associated urinary tract infection (CAUTI), and yet literature describing the molecular mechanisms of its pathogenesis is limited. To identify factors important for colonization during single-species infection and during polymicrobial infection with a common cocolonizer, Proteus mirabilis, we created a saturating library of ∼50,000 transposon mutants and conducted transposon insertion site sequencing (Tn-Seq) in a murine model of CAUTI. P. stuartii strain BE2467 carries 4,398 genes, 521 of which were identified as essential for growth in laboratory medium and therefore could not be assessed for contribution to infection. Using an input/output fold change cutoff value of 20 and P values of <0.05, 340 genes were identified as important for establishing single-species infection only and 63 genes as uniquely important for polymicrobial infection with P. mirabilis, and 168 genes contributed to both single-species and coinfection. Seven mutants were constructed for experimental validation of the primary screen that corresponded to flagella (fliC mutant), twin arginine translocation (tatC), an ATP-dependent protease (clpP), d-alanine-d-alanine ligase (ddlA), type 3 secretion (yscI and sopB), and type VI secretion (impJ). Infection-specific phenotypes validated 6/7 (86%) mutants during direct cochallenge with wild-type P. stuartii and 3/5 (60%) mutants during coinfection with P. mirabilis, for a combined validation rate of 9/12 (75%). Tn-Seq therefore successfully identified genes that contribute to fitness of P. stuartii within the urinary tract, determined the impact of coinfection on fitness requirements, and added to the identification of a collection of genes that may contribute to fitness of multiple urinary tract pathogens.IMPORTANCEProvidencia stuartii is a common cause of polymicrobial catheter-associated urinary tract infections (CAUTIs), particularly during long-term catheterization. However, little is known regarding the pathogenesis of this organism. Using transposon insertion site sequencing (Tn-Seq), we performed a global assessment of P. stuartii fitness factors for CAUTI while simultaneously determining how coinfection with another pathogen alters fitness requirements. This approach provides four important contributions to the field: (i) the first global estimation of P. stuartii genes essential for growth in laboratory medium, (ii) identification of novel fitness factors for P. stuartii colonization of the catheterized urinary tract, (iii) identification of core fitness factors for both single-species and polymicrobial CAUTI, and (iv) assessment of conservation of fitness factors between common uropathogens. Genomewide assessment of the fitness requirements for common uropathogens during single-species and polymicrobial CAUTI thus elucidates complex interactions that contribute to disease severity and will uncover conserved targets for therapeutic intervention.

Casamino acids slow motility and stimulate surface growth in an extreme oligotroph.

Environmental cues that regulate motility are poorly understood, but specific carbon and nitrogen sources, such as casamino acids (CAA), are known to stimulate motility in model organisms. However, natural environments are commonly more nutrient-limited than laboratory growth media, and the effect of energy-rich CAA on the motility of oligotrophic microorganisms is unknown. In this study, an extreme oligocarbotroph, Variovorax paradoxus YC1, was isolated from weathered shale rock within a disused mine level in North Yorkshire, UK. The addition of 0.1% CAA to minimal media significantly reduced the motility of YC1 after 72 h and inhibited swimming motility resulting in enhanced surface growth. We propose this response to CAA is a physiological adaptation to oligotrophy, facilitating the colonization of nutrient-rich environments.

Relevant Aspects of Clostridium estertheticum as a Specific Spoilage Organism of Vacuum-Packed Meat.

Clostridium estertheticum is a psychrotolerant, gram-positive, motile, anaerobic, spore-forming, rod-shaped bacteria that causes blown pack spoilage (BPS). Spoilage occurs in vacuum-packed meat without temperature abuse. Having been reported in the last 30 years in several countries, BPS by Cl. estertheticum is a major issue around the world and presents a huge economic impact on the meat industry. Despite being an important spoilage microorganism, studies on Cl. estertheticum are challenged by numerous aspects. These include, lack or poor growth in laboratory media, long culturing periods, and unpredictable isolation on the media. These factors hamper the detection of Cl. estertheticum before occurrence of BPS, which further undermines efforts to prevent the occurrence of BPS. Nevertheless, considerable developments have taken place with regard to culture-independent methods. Although information on Cl. estertheticum is available, it is limited and remains highly fragmented. Therefore, this review collates the available information and discusses relevant aspects of Cl. estertheticum as a specific spoilage organism of BPS in vacuum-packed meat.

Effect of Food Residues in Biofilm Formation on Stainless Steel and Polystyrene Surfaces by Salmonella enterica Strains Isolated from Poultry Houses.

Salmonella spp. is a major food-borne pathogen around the world. The ability of Salmonella to produce biofilm is one of the main obstacles in reducing the prevalence of these bacteria in the food chain. Most of Salmonella biofilm studies found in the literature used laboratory growth media. However, in the food chain, food residues are the principal source of nutrients of Salmonella. In this study, the biofilm formation, morphotype, and motility of 13 Salmonella strains belonging to three different subspecies and isolated from poultry houses was evaluated. To simulate food chain conditions, four different growth media (Tryptic Soy Broth at 1/20 dilution, milk at 1/20 dilution, tomato juice, and chicken meat juice), two different surfaces (stainless steel and polystyrene) and two temperatures (6 °C and 22 °C) were used to evaluate the biofilm formation. The morphotype, motility, and biofilm formation of Salmonella was temperature-dependent. Biofilm formation was significantly higher with 1/20 Tryptic Soy Broth in all the surfaces and temperatures tested, in comparison with the other growth media. The laboratory growth medium 1/20 Tryptic Soy Broth enhanced biofilm formation in Salmonella. This could explain the great differences in biofilm formation found between this growth medium and food residues. However, Salmonella strains were able to produce biofilm on the presence of food residues in all the conditions tested. Therefore, the Salmonella strain can use food residues to produce biofilm on common surfaces of the food chain. More studies combining more strains and food residues are necessary to fully understand the mechanism used by Salmonella to produce biofilm on the presence of these sources of nutrients.

Assessing biofilm formation by Salmonella enterica serovar Typhimurium on abiotic substrata in the presence of quorum sensing signals produced by Hafnia alvei

This study aimed to evaluate the possible impact of N-acyl-homoserine lactones (AHLs) produced by Hafnia alvei on the biofilm-forming ability of Salmonella enterica ser. Typhimurium. To do this, S. Typhimurium was left to form biofilms on two abiotic substrata, stainless steel (SS) and polystyrene (PS), through their static incubation for up to 48 h, at 20 °C, in laboratory growth media, also containing crude or ethyl acetate extracted cell-free culture supernatant of either the AHL-producing H. alvei 718 strain or its AHL-lacking isogenic mutant. Apart from the presence of signals, two nutritional conditions, a rich and a poor one, were also evaluated in those experiments involving the ethyl acetate extracted supernatants. The presence of AHLs in all media was tested using Agrobacterium tumefaciens biosensor-based bioassay, while the quantities of biofilms formed were in parallel estimated through three different quantification methods, namely agar plating, conductance, and absorbance measurements. Results revealed a significant correlation in biofilm formation among both tested surfaces, with the conductance detection times to be also inversely well correlated to the biofilm counts on SS. Interestingly, S. Typhimurium in general presented a higher capability to form biofilms under the nutrient poor compared to rich conditions. However, all hereby employed biofilm setup and quantification methods did not reveal any significant influence of H. alvei AHLs on Salmonella biofilm-forming behavior. In agreement, further experiments conducted using a synthetic AHL (i.e.3OC6-HSL), simulating a major such signal produced by H. alvei, did not show any effect. To sum up, these results expand our knowledge on the potential role of interspecies signaling on biofilm formation by S. Typhimurium.

INFLUENCE OF RECONSTITUTION TEMPERATURE ON SURVIVAL OF CRONOBACTER SAKAZAKII IN POWDERED INFANT FORMULA

Cronobacter spp. are emerging opportunistic pathogens commonly associated with the feeding of powdered infant formula (PIF). This study aimed to investigate the ability of C.sakazakii (ATCC 29544) to survive heat injury in laboratory growth medium and powdered infant formula (PIF) in comparison to Escherichia coli (ATCC 25922) and other selected Cronobacter strains. As part of this work the thin agar layer (TAL) method for the recovery of heat-injured foodborne pathogens was evaluated for use with C.sakazakii (ATCC 29544) and E.coli (ATCC 25922). We report that heat treatment at 55°C for 10 minutes had no significant effect (P &gt; 0.05) on C.sakazakii (ATCC 29544) viability in peptone or PIF. Significant differences in survival of Cronobacter strains after this heat treatment were identified indicating that heat tolerance in this genus may be strain dependent. PIF did not enhance survival of this organism in comparison to other media. Results indicate that a decrease in temperature of 5°C from the recommended 70°C for the reconstitution of PIF was sufficient to produce a significant increase in survival of C.sakazakii (ATCC 29544). Reconstituted PIF stored at room temperature was shown to support the survival and proliferation of this pathogen indicating that temperature abuse during PIF preparation and storage may significantly increase the risk of disease caused by these opportunistic pathogens.

Effect of tea tree oil on Staphylococcus aureus growth and enterotoxin production

Staphylococcus aureus (S. aureus), a major food-borne pathogen, causes disease in mammalian hosts by producing a wide variety of exoproteins, such as α-hemolysin and staphylococcal enterotoxins. Tea tree oil (TTO), an essential oil, has broad-spectrum antimicrobial activity. The objectives of this study were to evaluate the inhibitory effects of TTO on S. aureus growth and on α-hemolysin, enterotoxins A and B production. In this study, the effect of TTO on S. aureus growth in laboratory medium and pasteurized milk was determined by time-kill assays. Treatment with half of minimal inhibitory concentration (MIC) of TTO demonstrated very little or no reduction in numbers of viable ATCC 29213 cells; however, 1 × MIC of TTO reduced the viable cell count more noticeably, and 2 × MIC of TTO demonstrated an even greater reduction in the viable cell count, both in TSB and milk. The influence of TTO on enterotoxins was determined by real-time reverse transcriptase-PCR (real-time RT-PCR), a hemolysis assay, Western blot and a tumor necrosis factor alpha (TNF-α) assay. The real-time RT-PCR results revealed that the transcription of genes encoding α-hemolysin, staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) were down regulated after S. aureus was exposed to TTO (0.0625 mg/mL-0.5 mg/mL). The hemolytic assay showed that S. aureus hemolytic activity was inhibited by subinhibitory concentrations of TTO (0.0625 mg/mL-0.5 mg/mL). The Western blot assay identified that the production of the three virulence factors was inhibited by TTO (0.0625 mg/mL-0.5 mg/mL). Moreover, the enzyme linked immunosorbent assay (ELISA) demonstrated that TNF-α production was suppressed by TTO treatment in RAW264.7 cells stimulated by S. aureus supernatant containing staphylococcal enterotoxins (SEs).

Effect of growth media on gene expression levels in Salmonella Typhimurium biofilm formed on stainless steel surface

Salmonella biofilm is recognized as a frequent source of cross-contamination and has been associated with food-borne outbreaks. It is more meaningful for evaluating biofilm formation in food-based medium than in laboratory-growth medium. In the present study, the expression of functional genes during biofilm formation of Salmonella Typhimurium grown in meat-based and standard growth substrates were determined by RT-qPCR assays. The number of viable cells and the expression of tested genes during biofilm formation displayed a specific behavior according to the growth conditions. When biofilm was grown for 7 days in a laboratory trypticase soy broth (TSB), the expression of the tested genes increased, but when grown in a meat thawing loss broth (MTLB), gene expression was inhibited as compared to corresponding planktonic cells. Relative gene expression of biofilm in MTLB was lower than that in TSB. Positive relationships were found between each gene tested in biofilm cells grown in TSB and MTLB at 3, 5 and 7 days incubation, their correlation did not changed with the variation of growth media of biofilm. A preliminary positive relationship was also observed between growth days and the expression levels of some tested genes. Our results suggested that biofilm formation was coordinately governed by a highly complex regulatory network, where the genes tested in this study (adrA, bapA, csgB, csgD, csrA, rpoS, invA, sipB-C, sdiA and luxS) played important roles.

Effect of sodium chloride on the survival of Shigella flexneri in acidified laboratory media and cucumber puree.

This study investigated the effects of sodium chloride (NaCl) and various acids, alone or in combination, on Shigella flexneri growth in laboratory medium and cucumber puree. Shigella flexneri was treated with various acids (acetic, citric, malic, tartaric, propionic, lactic and phosphoric acid) alone or with 3, 6 or 9% NaCl. Pronounced antagonistic effects were observed in Sh.flexneri treated with acetic or lactic acid in combination with 3% NaCl. Next, Sh.flexneri was pre-exposed to 3% NaCl and then treated with various acids; acid-stressed cells were then inoculated onto agar plates containing 3% NaCl. There was no significant difference in the reduction of Sh.flexneri, regardless of treatment (P>0·05). Finally, Sh.flexneri was inoculated into cucumber puree to which various concentrations of acetic acid had been added with and without 3% NaCl. Antagonistic effects were observed with a treatment of either 0·5 or 1% acetic acid combined with 3% NaCl. Antagonistic effects were observed when Sh.flexneri was exposed to acetic or lactic acid with NaCl. This study shows that depending on the type of acid, the addition of NaCl can increase the resistance of Sh.flexneri to acid treatments. This may provide useful information for developing methods of preserving acidified foods.

Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

Benzopyrone Coumarin Leads to an Inhibition of Ochratoxin Biosynthesis in Representatives of Aspergillus and Penicillium spp. via a Type of Feedback Response Mechanism

Growth and mycotoxin biosynthesis of the ochratoxin-producing fungal strains Aspergillus carbonarius, Aspergillus steynii, Penicillium verrucosum, and Penicillium nordium were analyzed on standard laboratory growth medium supplemented with different amounts of coumarin, an organic compound of the benzopyrone class. Neither the growth nor the phenotypic morphology of the filamentous fungi analyzed was affected by using coumarin concentrations equivalent to 2.5 to 25 μg/ml of medium. In contrast, the ochratoxin biosynthesis was strongly inhibited in both strains of the Aspergillus species and nearly completely inhibited in both Penicillium strains at coumarin concentrations above 8.75 μg/ml. Analyzing the transcriptional activity of the otapksPN polyketide synthase gene in P. nordicum using real-time PCR revealed a strong concentration-dependent decrease in gene expression. Taken together, the data show that ochratoxin biosynthesis in representative strains of the genera Aspergillus and Penicillium could be effectively inhibited by coumarin in a concentration-dependent manner. It could be suggested that the molecular background behind this inhibition is some kind of feedback response mechanism, based on the structural similarity of coumarin to the benzopyrone moiety of the ochratoxin molecule.