A Single Nucleotide Polymorphism in lptG Increases Tolerance to Bile Salts, Acid, and Staining of Calcofluor-Binding Polysaccharides in Salmonella enterica Serovar Typhimurium E40.

The outer membrane of Salmonella enterica plays an important role in combating stress encountered in the environment and hosts. The transport and insertion of lipopolysaccharides (LPS) into the outer membrane involves lipopolysaccharide transport proteins (LptA-F) and mutations in the genes encoding for these proteins are often lethal or result in the transport of atypical LPS that can alter stress tolerance in bacteria. During studies of heterogeneity in bile salts tolerance, S. enterica serovar Typhimurium E40 was segregated into bile salts tolerant and sensitive cells by screening for growth in TSB with 10% bile salts. An isolate (E40V) with a bile salts MIC >20% was selected for further characterization. Whole-genome sequencing of E40 and E40V using Illumina and PacBio SMRT technologies revealed a non-synonymous single nucleotide polymorphism (SNP) in lptG. Leucine at residue 26 in E40 was substituted with proline in E40V. In addition to growth in the presence of 10% bile salts, E40V was susceptible to novobiocin while E40 was not. Transcriptional analysis of E40 and E40V, in the absence of bile salts, revealed significantly greater (p < 0.05) levels of transcript in three genes in E40V; yjbE (encoding for an extracellular polymeric substance production protein), yciE (encoding for a putative stress response protein), and an uncharacterized gene annotated as an acid shock protein precursor (ASPP). No transcripts of genes were present at a greater level in E40 compared to E40V. Corresponding with the greater level of these transcripts, E40V had greater survival at pH 3.35 and staining of Calcofluor-binding polysaccharide (CBPS). To confirm the SNP in lptG was associated with these phenotypes, strain E40E was engineered from E40 to encode for the variant form of LptG (L26P). E40E exhibited the same differences in gene transcripts and phenotypes as E40V, including susceptibility to novobiocin, confirming the SNP was responsible for these differences.

Phosphate and carbohydrate facilitate the formation of filamentous Salmonella enterica during osmotic stress.

Salmonella enterica is a human pathogen that can produce filamentous cells in response to environmental stress. The molecular mediators and biosynthetic pathways that contribute to the formation of filamentous cells (>10 µm in length) during osmotic stress are mostly unknown. The comparison of filamentous and non-filamentous cells in this study was aided by the use of a filtration step to separate cell types. Osmotic stress caused an efflux of phosphate from cells, and the addition of phosphate and a carbohydrate to Luria broth with 7 % NaCl (LB-7NaCl) significantly increased the proportion of filamentous cells in the population (58 %). In addition to direct measurements of intracellular and extracellular phosphate concentrations, the relative abundance of the iraP transcript that is induced by phosphate limitation was monitored. Non-filamentous cells had a greater relative abundance of iraP transcript than filamentous cells. IraP also affects the stability of RpoS, which regulates the general stress regulon, and was detected in non-filamentous cells but not filamentous cells. Markers of metabolic pathways for the production of acetyl-CoA (pflB, encoding for pyruvate formate lyase) and fatty acids (fabH) that are essential to membrane biosynthesis were found in greater abundance in filamentous cells than non-filamentous cells. There were no differences in the DNA, protein and biomass levels in filamentous and non-filamentous cells after 48 h of incubation, although the filamentous cells produced significantly (P<0.05) more acetate. This study found that phosphate and carbohydrate enhanced the formation of filamentous cells during osmotic stress, and there were differences in key regulatory elements and markers of metabolic pathways in filamentous and non-filamentous S. enterica.

RpoS impacts the lag phase of Salmonella enterica during osmotic stress.

Salmonella enterica can survive harsh environmental conditions, including hyperosmotic stress. It is well established that the alternative sigma factor, σ(s) (RpoS), is required for maximal survival of enteric pathogens, including S. enterica. Although RpoS levels are greatest during stationary phase or stress conditions, RpoS can be found in S. enterica during growth. However, its activity during growth is poorly characterized. In this study, the impact of RpoS levels on the growth of S. enterica in LB supplemented with 6% NaCl (LB-NaCl) was examined. Cells in stationary phase prior to inoculation into LB-NaCl had a shorter lag phase than did exponential-phase cells. In addition, the deletion of rpoS from S. enterica Typhimurium M-09 (M-09 ΔrpoS) increased the length of lag phase in LB-NaCl relative to the parental strain. Complementation of M-09 ΔrpoS in trans by an inducible plasmid encoding rpoS reduced the length of lag phase. The length of lag phase in both the rpoS mutant and complemented strain was independent of their growth phase prior to inoculation of LB-NaCl. The results from this study demonstrate that the level of RpoS influences the length of lag phase and the growth of S. enterica in hyperosmotic growth conditions.

DNA polymorphisms and biocontrol of Bacillus antagonistic to citrus bacterial canker with indication of the interference of phyllosphere biofilms.

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a devastating disease resulting in significant crop losses in various citrus cultivars worldwide. A biocontrol agent has not been recommended for this disease. To explore the potential of bacilli native to Taiwan to control this disease, Bacillus species with a broad spectrum of antagonistic activity against various phytopathogens were isolated from plant potting mixes, organic compost and the rhizosphere soil. Seven strains TKS1-1, OF3-16, SP4-17, HSP1, WG6-14, TLB7-7, and WP8-12 showing superior antagonistic activity were chosen for biopesticide development. The genetic identity based on 16S rDNA sequences indicated that all seven native strains were close relatives of the B. subtilis group and appeared to be discrete from the B. cereus group. DNA polymorphisms in strains WG6-14, SP4-17, TKS1-1, and WP8-12, as revealed by repetitive sequence-based PCR with the BOXA1R primers were similar to each other, but different from those of the respective Bacillus type strains. However, molecular typing of the strains using either tDNA-intergenic spacer regions or 16S-23S intergenic transcribed spacer regions was unable to differentiate the strains at the species level. Strains TKS1-1 and WG6-14 attenuated symptom development of citrus bacterial canker, which was found to be correlated with a reduction in colonization and biofilm formation by X. axonopodis pv. citri on leaf surfaces. The application of a Bacillus strain TKS1-1 endospore formulation to the leaf surfaces of citrus reduced the incidence of citrus bacterial canker and could prevent development of the disease.

Characterization of osmotically induced filaments of Salmonella enterica.

Salmonella enterica forms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were active in vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F(0)F(1)-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2 × Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.

Swarming behavior of and hemolysin BL secretion by Bacillus cereus.

An association between swarming and hemolysin BL secretion was observed in a collection of 42 Bacillus cereus isolates (P=0.029). The highest levels of toxin were detected in swarmers along with swarm cell differentiation (P=0.021), suggesting that swarming B. cereus strains may have a higher virulence potential than nonswarming strains.

Requirement of flhA for swarming differentiation, flagellin export, and secretion of virulence-associated proteins in Bacillus thuringiensis.

Bacillus thuringiensis is being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen. While phospholipases, hemolysins, and enterotoxins are claimed to be responsible for B. thuringiensis virulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions. This report describes the characterization of a mini-Tn10 mutant of B. thuringiensis that is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C. The mutant strain was determined to carry the transposon insertion in flhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus. Interestingly, the flhA mutant of B. thuringiensis synthesized flagellin but was impaired in flagellin export. Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable in B. thuringiensis, thus suggesting that the flagellar gene expression hierarchy of B. thuringiensis differs from that described for Bacillus subtilis. The flhA mutant of B. thuringiensis was also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant. Since complementation of the mutant with a plasmid harboring the flhA gene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence in B. thuringiensis may be coordinately regulated by flhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.

Swarming motility in Bacillus cereus and characterization of a fliY mutant impaired in swarm cell differentiation.

This report describes a new behavioural response of Bacillus cereus that consists of a surface-induced differentiation of elongated and hyperflagellated swarm cells exhibiting the ability to move collectively across the surface of the medium. The discovery of swarming motility in B. cereus paralleled the isolation of a spontaneous non-swarming mutant that was found to carry a deletion of fliY, the homologue of which, in Bacillus subtilis, encodes an essential component of the flagellar motor-switch complex. However, in contrast to B. subtilis, the fliY mutant of B. cereus was flagellated and motile, thus suggesting a different role for FliY in this organism. The B. cereus mutant was completely deficient in chemotaxis and in the secretion of the L2 component of the tripartite pore-forming necrotizing toxin, haemolysin BL, which was produced exclusively by the wild-type strain during swarm-cell differentiation. All the defects in the fliY mutant of B. cereus could be complemented by a plasmid harbouring the B. cereus fliY gene. These results demonstrate that the activity of fliY is required for swarming and chemotaxis in B. cereus, and suggest that swarm-cell differentiation is coupled with virulence in this organism.

Cooperative, synergistic and antagonistic haemolytic interactions between haemolysin BL, phosphatidylcholine phospholipase C and sphingomyelinase from Bacillus cereus.

Haemolysis of erythrocytes from different species (sheep, bovine, swine and human), caused by various combinations of phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC), sphingomyelinase (SMase) and the three-component, pore-forming toxin haemolysin BL (HBL) from Bacillus cereus was analysed. The lytic potency of HBL did not correlate with phospholipid (PL) content, but lysis by the individual or combined enzymes did. SMase alone lysed ruminant erythrocytes, which contain 46-53% sphingomyelin (SM). The cooperative action of PC-PLC and SMase was needed to lyse swine and human erythrocytes (22-31% PC and 28-25% SM). SMase synergistically enhanced haemolysis caused by HBL for all erythrocytes tested, which all contained >25% SM. PC-PLC enhanced HBL haemolysis only in cells containing significant amounts of PC (swine, 22% PC; human, 31% PC). Unexpectedly, PC-PLC inhibited HBL lysis of sheep erythrocytes (<2% PC) and enhanced the discontinuous haemolysis pattern that is characteristic of HBL in sheep blood agar. Inhibition and pattern enhancement was abolished by washing PC-PLC-treated erythrocytes or by adding EDTA, suggesting that enzymic alteration of the membrane is not involved, but that zinc in the active site is required, perhaps to facilitate binding. These observations highlight the potential for cooperative and synergistic interactions among virulence factors in B. cereus infections and dependence of these effects on tissue composition.

Tripartite haemolysin BL: isolation and characterization of two distinct homologous sets of components from a single Bacillus cereus isolate.

Haemolysin BL (HBL), a three-component enterotoxic/necrotizing/vascular permeability toxin, is a likely virulence factor of Bacillus cereus diarrhoeal food poisoning and necrotic infections. This paper describes the isolation of two distinct homologous sets of all three HBL components from a single B. cereus isolate, MGBC 145. The proteins of one set (designated HBL, consisting of B, L(1) and L(2)), were about 87-100% identical in N-terminal amino acid sequences to their respective prototype components from strain F837/76, and the proteins of the homologous set (HBL(a), consisting of B(a), L(1a) and L(2a)) were all about 62-65% identical. Only the latter homologues differed immunochemically and physicochemically from the prototypes. HBL and HBL(a) exhibited similar haemolytic and vascular permeability potencies, and the homologues could be interchanged freely. There were no notable differences in activity between the L component homologues. However, components B and B(a) were significantly different. Both were secreted as monomers, but unlike B, B(a) was isolated as a relatively inactive complex that could be reactivated with urea. When B(a) was substituted for B in gel-diffusion assays the distinct discontinuous haemolysis pattern typical of the presence of B did not occur. In suspension assays, excess B inhibited the haemolysis of B-primed cells by L(1) (as previously described), but not that of B(a)-primed cells. Excess B(a) had the opposite effect and enhanced lysis of B(a)-primed cells, but not that of B-primed cells. These differences reveal details about how the toxin components interact on target cell membranes. The authors' observations indicate that HBL represents a new family of multicomponent toxins that was generated by a process of gene and operon duplication that occurred either intracellularly or by horizontal transfer, and raise the possibility of the existence of other related toxins in the genetically diverse B. cereus taxonomic group.

Survival and Growth Characteristics of Listeria monocytogenes and Salmonella typhimurium on Stainless Steel and Buna-N Rubber.

Microorganisms harbored on food-contact surfaces are part of a complex ecosystem. The interactions of temperature, relative humidity (RH), soil and surface on the survival of Listeria monocytogenes and Salmonella typhimurium were studied. Survival and growth were monitored at 25°C and 6°C and 32.5% RH and 75.5% RH. Survival in phosphate-buffered saline and dilute pasteurized whole milk on both stainless steel and buna-n was highest at 6°C and 75.5% RH. Both organisms were recoverable on the two surfaces after 10 days storage at 6°C and 75.5% RH. Survival of L. monocytogenes and S. typhimurium at 25°C and 75.5% RH was increased in dilute pasteurized whole milk on stainless steel, but not on buna-n. Organisms grew in pasteurized whole milk on stainless steel at 25°C and 75.5% RH, but failed to grow on buna-n. At 25°C and 75.5% RH, S. typhimurium was not recoverable on buna-n after 10 days in whole milk; however, L. monocytogenes remained close to initial levels. The survival and growth of both organisms in raw milk soil was similar to that in pasteurized whole milk soil. Buna-n was not bacteriostatic towards all organisms, as the total viable count in raw milk increased by more than a factor of 10 after 1 day storage at 25°C and 75.5% RH. Unlike other soils tested, survival of S. typhimurium at in conditions and L. monocytogenes at 25°C and both RHs in whey was higher on buna-n than on stainless steel. At 6°C and both RHs, L. monocytogenes levels remained constant on both surfaces in whey. The bacteriostatic effect of buna-n was not affected significantly by exposure to 20 cycles of a simulated clean-in-place (CIP) process.

Attachment of Listeria monocytogenes and Salmonella typhimurium to Stainless Steel and Buna-N in the Presence of Milk and Individual Milk Components.

The effects of milk and individual milk components on the attachment of Listeria monocytogenes and Salmonella typhimurium to two commonly used materials in the dairy industry were studied. Attachment of both organisms to stainless steel and Buna-N was significantly inhibited by the presence of skim, 2%, whole, or chocolate 2% milk compared to the phosphate-buffered saline (PBS) control. The addition of individual milk components, casein, α-lactalbumin, and ß-lactoglobulin to the attachment menstruum significantly reduced attachment. Pretreating surfaces with milk and milk components for 1 h prior to attachment in PBS gave similar results. The presence of lactose did not affect attachment of either organism; however, attachment of S. typhimurium was significantly decreased on pretreated Buna-N. Cells of either organism pretreated with skim milk or ß-lactoglobulin prior to attachment in PBS showed significantly less attachment than untreated cells. Pretreating S. typhimurium cells with casein had no effect on attachment to stainless steel. Pretreatment of S. typhimurium with lactose increased attachment to both surfaces while pretreatment had no effect on L. monocytogenes . Attachment of both organisms was significantly reduced in diluted whole milk. Both organisms attached significantly less to surfaces soiled with one or more layers of whole milk.

Optimal Condition for the Production of Unidentified Staphylococcal Enterotoxins.

The sac culture method in combination with 6% NZ-Amine A plus 1% yeast extract was found to be the optimal condition for staphylococcal enterotoxins A (SEA) and D (SED) production. This growth condition was tested for the production of unidentified staphylococcal enterotoxins (SEs). Twenty-one Staphylococcus aureus strains that previously showed an emetic response in monkeys but were negative for any of the identified SEs when cultured by the membrane-over-agar method were grown by the sac culture method. All 21 strains produced at least one known SE. One strain produced only enterotoxin C (SEC). Nine strains produced only SED. One strain produced both SEA and SED. Four produced both SEC and SED. One produced SEA, enterotoxin B (SEB), and SED. Two produced SEA, SEC, and SED. Three produced SEA, SEB, SEC, and SED. One of these strains, FRI-569, that produced only SED at a low level (10 ng/ml) was selected to confirm the production of an unidentified SE by the monkey feeding test. Emesis was induced in five out of six monkeys. The total amount of SED in the supernatant fluids fed to monkeys was only 0.5 µg, or 2.5% of the 50% emetic dose (20 µg). Production of an unidentified SE by strain FRI-569 was therefore confirmed. This optimal condition can be used to produce increased amounts of unidentified SEs for purification.

Biofilm Development and Sanitizer Inactivation of Listeria monocytogenes and Salmonella typhimurium on Stainless Steel and Buna-n Rubber.

Biofilm formation by seven strains of Listeria monocytogenes and one strain of Salmonella typhimurium on stainless steel and Buna-n rubber was examined under two nutrient conditions. The type of surface, nutrient level, and organism influenced biofilm development and production of extracellular materials. Buna-n had a strong bacteriostatic effect on L. monocytogenes , and biofilm formation on Buna-n under low nutrient conditions was reduced for four of the seven strains tested. Buna-n was less bacteriostatic toward S. typhimurium . It inhibited the growth of several other pathogens to varying degrees. An ethylene propylene diamine monomer rubber was less inhibitory than Buna-n, and Viton rubber had no effect. The effectiveness of sanitizers on biofilm bacteria was examined. Biofilms were challenged with four types of detergent and nondetergent sanitizers. Resistance to sanitizers was strongly influenced by the type of surface. Bacterial biofilm populations on stainless steel were reduced 3-5 log by all the sanitizers, but those on Buna-n were resistant to these sanitizers and were reduced less than 1-2 log. In contrast, planktonic (suspended) bacteria were reduced 7-8 log by these sanitizers. Chlorine and anionic acid sanitizers generally removed extracellular materials from biofilms better than iodine and quaternary ammonium detergent sanitizers. Scanning electron microscopy demonstrated that biofilm cells and extracellular matrices could remain on sanitized biofilm cells and extracellular matrices could remain surfaces from which no viable cells were recovered.