Differential effects of magnesium, calcium, and sodium on Listeria monocytogenes biofilm formation.

Listeria monocytogenes is a gram-positive foodborne pathogen that causes outbreaks of listeriosis associated with a diverse range of foods. L. monocytogenes forms biofilms as a strategy to enhance its survival in the environment. These biofilms then provide a source of contamination in processing plant environments. Cations like magnesium, calcium, and sodium are commonly found in the environment and are important to bacteria to maintain their homeostasis. It is, therefore, valuable to understand the relationship between these cations and biofilm formation. In this study, four isolates of L. monocytogenes from seafood processing environments were used to investigate the influence of magnesium, calcium, and sodium (1, 10, and 50 mM) on biofilms. The isolates selected were defined as being either a low biofilm former, a high biofilm former, an outbreak isolate, and a persistent isolate from the seafood industry. The study showed that the divalent cations magnesium and calcium increased biofilm formation compared with the monovalent cation, sodium. Fifty mM concentrations of the divalent cations significantly enhanced biofilm formation. The cations did not have a significant effect on the initial stages of biofilm formation but appeared to influence the later stages of biofilm development.

Culture and genome-based analysis of four soil Clostridium isolates reveal their potential for antimicrobial production.

BACKGROUND: Soil bacteria are a major source of specialized metabolites including antimicrobial compounds. Yet, one of the most diverse genera of bacteria ubiquitously present in soil, Clostridium, has been largely overlooked in bioactive compound discovery. As Clostridium spp. thrive in extreme environments with their metabolic mechanisms adapted to the harsh conditions, they are likely to synthesize molecules with unknown structures, properties, and functions. Therefore, their potential to synthesize small molecules with biological activities should be of great interest in the search for novel antimicrobial compounds. The current study focused on investigating the antimicrobial potential of four soil Clostridium isolates, FS01, FS2.2 FS03, and FS04, using a genome-led approach, validated by culture-based methods. RESULTS: Conditioned/spent media from all four Clostridium isolates showed varying levels of antimicrobial activity against indicator microorganism; all four isolates significantly inhibited the growth of Pseudomonas aeruginosa. FS01, FS2.2, and FS04 were active against Bacillus mycoides and FS03 reduced the growth of Bacillus cereus. Phylogenetic analysis together with DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and functional genome distribution (FGD) analyses confirmed that FS01, FS2.2, and FS04 belong to the species Paraclostridium bifermentans, Clostridium cadaveris, and Clostridium senegalense respectively, while FS03 may represent a novel species of the genus Clostridium. Bioinformatics analysis using antiSMASH 5.0 predicted the presence of eight biosynthetic gene clusters (BGCs) encoding for the synthesis of ribosomally synthesized post-translationally modified peptides (RiPPs) and non-ribosomal peptides (NRPs) in four genomes. All predicted BGCs showed no similarity with any known BGCs suggesting novelty of the molecules from those predicted gene clusters. In addition, the analysis of genomes for putative virulence factors revealed the presence of four putative Clostridium toxin related genes in FS01 and FS2.2 genomes. No genes associated with the main Clostridium toxins were identified in the FS03 and FS04 genomes. CONCLUSIONS: The presence of BGCs encoding for uncharacterized RiPPs and NRPSs in the genomes of antagonistic Clostridium spp. isolated from farm soil indicated their potential to produce novel secondary metabolites. This study serves as a basis for the identification and characterization of potent antimicrobials from these soil Clostridium spp. and expands the current knowledge base, encouraging future research into bioactive compound production in members of the genus Clostridium.

Influence of the incubation temperature and total dissolved solids concentration on the biofilm and spore formation of dairy isolates of Geobacillus stearothermophilus.

Geobacillus species are an important contaminant in the dairy industry and their presence is often considered as an indicator of poor plant hygiene with the potential to cause spoilage. They can form heat resistant spores that adhere to surfaces of processing equipment and germinate to form biofilms. Therefore, strategies aimed towards preventing or controlling biofilm formation in the dairy industry are desirable. In this study we demonstrate that the preferred temperature for biofilm and spore formation among Geobacillus stearothermophilus A1, D1, P3 and ATCC 12980 was 65°C. Increasing the total dissolved milk solids concentration to 20% (w/v) caused an apparent delay in the onset of biofilm and spore formation to detectable concentrations among all the strains at 55°C. Compared to the onset time of the biofilm formation of A1 in 10% (w/v) reconstituted skim milk, addition of milk protein (whey protein and sodium caseinate) caused an apparent delay in the onset of biofilm formation to detectable concentrations by an average of 10 h at 55°C. This study proposes that temperature and total dissolved solids concentration have a cumulative effect on the biofilm and spore formation of G. stearothermophilus A1, D1, P3 and ATCC 12980. In addition, the findings from this study may indicate that preconditioning of stainless-steel surface with adsorbed milk proteins may delay the onset of biofilm and spore formation of thermophilic bacteria during milk powder manufacture.IMPORTANCE The thermophilic bacilli, Geobacillus stearothermophilus is a predominant spoilage bacterium in milk powder manufacturing plants. If their numbers exceed the accepted levels, this may incur financial loses by lowering the price of the end product. Furthermore, they can form heat resistant spores which adhere to processing surfaces and can germinate to form biofilms. Previously conducted research had highlighted the variation in the spore and biofilm formation among three specific strains of G. stearothermophilus isolated from a milk powder manufacturing plant in New Zealand. The significance of our research is demonstrating the effect of two abiotic factors namely temperature and total dissolved solids concentration on the biofilm and spore formation of these three dairy isolates, leading to modifications in the thermal processing steps aimed towards controlling the biofilm and spore formation of G. stearothermophilus in the dairy industry.

Biofilm Formation by Listeria monocytogenes 15G01, a Persistent Isolate from a Seafood-Processing Plant, Is Influenced by Inactivation of Multiple Genes Belonging to Different Functional Groups.

Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafood-processing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants.IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.

In silico analysis revealing CsrA roles in motility-sessility switching and tuning VBNC cells in Vibrio parahaemolyticus.

The formation of biofilms is a survival strategy employed by bacteria to help protect them from changing or unfavourable environments. In this research, 319 genes which govern biofilm formation in V. parahaemolyticus, as reported in 1,625 publications, were analysed using protein-protein-interaction (PPI) network analysis. CsrA was identified as a motility-sessility switch and biofilm formation regulator. Through robust rank aggregation (RRA) analysis of GSE65340, the generation of viable but non-culturable (VBNC) cells that may enhance cell tolerance to stress, was found to be associated with the TCA cycle and carbon metabolism biological pathways. The finding that CsrA is likely to play a role in the development of VBNC cells improves understanding of the molecular mechanisms of VBNC formation in V. parahaemolyticus and contributes to on-going efforts to reduce the hazard posed by this foodborne pathogen.

Bacillus cereus spores and toxins - The potential role of biofilms.

Bacillus cereus is a well-known foodborne pathogen capable of causing two types of gastrointestinal diseases, diarrhoea and emesis. It is of particular concern for the food industry causing food safety issues, due to the formation of spores, biofilms and diarrhoea and/or emetic toxins. This review reveals the possible link between two food safety issues - toxins and spores - and the role of biofilms. The review highlights genetic determinants that are involved in sporulation, toxin production and biofilm formation based on current research, and evidence showing the possible correlation of spore, toxin and biofilm formation of B. cereus. This is the first review highlighting the potential relationship between toxin production and biofilm formation in B. cereus.

Magnesium and calcium ions: roles in bacterial cell attachment and biofilm structure maturation.

The ubiquitous divalent cations magnesium and calcium are important nutrients required by bacteria for growth and cell maintenance. Multi-faceted roles are shown both in bacterial initial attachment and biofilm maturation. The effects of calcium and magnesium can be highlighted in physio-chemical interactions, gene regulation and bio-macromolecular structural modification, which lead to either promotion or inhibition of biofilms. This review outlines recent research addressing phenotypic changes and mechanisms undertaken by calcium and magnesium in affecting bacterial biofilm formation.

Update on the management of neonatal sepsis in horses.

Despite advances in neonatal intensive care sepsis, severe sepsis and septic shock remain the biggest killers of neonatal foals. Management of this severe syndrome remains difficult, requiring intensive intervention. Key aspects of management include infection control, hemodynamic support, immunomodulatory interventions, and metabolic/endocrine support. Infection control largely consists of early antimicrobial therapy, plasma transfusions, and local therapy for the infected focus. In cases with severe sepsis or septic shock, hemodynamic support with fluids, vasoactive agents, and respiratory support insuring oxygen delivery to vital organs is important. Nutritional support is important, but close monitoring is needed to avoid hyperglycemia and hypoglycemia.

Preconditioning with cations increases the attachment of Anoxybacillus flavithermus and Geobacillus species to stainless steel.

Preconditioning of Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 with cations prior to attachment often significantly increased (P ≤ 0.05) the number of viable cells that attached to stainless steel (by up to 1.5 log CFU/cm(2)) compared with unconditioned bacteria. It is proposed that the transition of A. flavithermus and Geobacillus spp. from milk formulations to stainless steel product contact surfaces in milk powder manufacturing plants is mediated predominantly by bacterial physiological factors (e.g., surface-exposed adhesins) rather than the concentrations of cations in milk formulations surrounding bacteria.

An improved MTT colorimetric method for rapid viable bacteria counting.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay has been employed in the analysis of bacterial growth. In comparison to experiments conducted on mammalian cells, the MTT bacterial assay encounters a greater number of interfering factors and obstacles that impact the accuracy of results. In this study, we have elucidated an improved MTT assay protocol and put forth an equation that establishes a correlation between colony-forming units (CFU) and the amount of formazan converted by the bacteria, drawing upon the fundamental principle of the MTT assay. This equation is represented as CFU=kF. Furthermore, we have explicated a methodology to determine the scale factor "k" by employing S. aureus and E. coli as illustrative examples. The findings indicate that S. aureus and E. coli reduce MTT by a cyclic process, from which the optimal reduction time at room temperature was determined to be approximately 30 mins. Furthermore, individual E. coli exhibits an MTT reduction capacity approximately four times greater than that of S. aureus. HPLC analysis proves to be the most accurate method for mitigating interferences during the dissolution and quantification of formazan. Additionally, this study has identified a new constraint related to the narrow linear range (0-125 µg/mL) of formazan concentration-absorbance and has presented strategies to circumvent this limitation.

Non-Targeted Metabolomic Profiling Identifies Metabolites with Potential Antimicrobial Activity from an Anaerobic Bacterium Closely Related to Terrisporobacter Species.

This work focused on the metabolomic profiling of the conditioned medium (FS03CM) produced by an anaerobic bacterium closely related to Terrisporobacter spp. to identify potential antimicrobial metabolites. The metabolome of the conditioned medium was profiled by two-channel Chemical Isotope Labelling (CIL) LC-MS. The detected metabolites were identified or matched by conducting a library search using different confidence levels. Forty-eight significantly changed metabolites were identified with high confidence after the growth of isolate FS03 in cooked meat glucose starch (CMGS) medium. Some of the secondary metabolites identified with known antimicrobial activities were 4-hydroxyphenyllactate, 3-hydroxyphenylacetic acid, acetic acid, isobutyric acid, valeric acid, and tryptamine. Our findings revealed the presence of different secondary metabolites with previously reported antimicrobial activities and suggested the capability of producing antimicrobial metabolites by the anaerobic bacterium FS03.

Efficacy of commercial peroxyacetic acid on Vibrio parahaemolyticus planktonic cells and biofilms on stainless steel and Greenshell™ mussel (Perna canaliculus) surfaces.

The potential of using commercial peroxyacetic acid (PAA) for Vibrio parahaemolyticus sanitization was evaluated. Commercial PAA of 0.005 % (v/v, PAA: 2.24 mg/L, hydrogen peroxide: 11.79 mg/L) resulted in a planktonic cell reduction of >7.00 log10 CFU/mL when initial V. parahaemolyticus cells averaged 7.64 log10 CFU/mL. For cells on stainless steel coupons, treatment of 0.02 % PAA (v/v, PAA: 8.96 mg/L, hydrogen peroxide: 47.16 mg/L) achieved >5.00 log10 CFU/cm2 reductions in biofilm cells for eight strains but not for the two strongest biofilm formers. PAA of 0.05 % (v/v, PAA: 22.39 mg/L, hydrogen peroxide: 117.91 mg/L) was required to inactivate >5.00 log10 CFU/cm2 biofilm cells from mussel shell surfaces. The detection of PAA residues after biofilm treatment demonstrated that higher biofilm production resulted in higher PAA residues (p < 0.05), suggesting biofilm is acting as a barrier interfering with PAA diffusing into the matrices. Based on the comparative analysis of genomes, robust biofilm formation and metabolic heterogeneity within niches might have contributed to the variations in PAA resistance of V. parahaemolyticus biofilms.

Biofilm formation, sodium hypochlorite susceptibility and genetic diversity of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine oriented pathogen; and biofilm formation enables its survival and persistence on seafood processing plant, complicating the hygienic practice. The objectives of this study are to assess the ability of V. parahaemolyticus isolated from seafood related environments to form biofilms, to determine the effective sodium hypochlorite concentrations required to inactivate planktonic and biofilm cells, and to evaluate the genetic diversity required for strong biofilm formation. Among nine isolates, PFR30J09 and PFR34B02 isolates were identified as strong biofilm forming strains, with biofilm cell counts of 7.20, 7.08 log10 CFU/cm2, respectively, on stainless steel coupons after incubation at 25 °C. Free available chlorine of 1176 mg/L and 4704 mg/L was required to eliminate biofilm cells of 1.74-2.28 log10 CFU/cm2 and > 7 log10 CFU/cm2, respectively, whereas 63 mg/L for planktonic cells, indicating the ineffectiveness of sodium hypochlorite in eliminating V. parahaemolyticus biofilm cells at recommended concentration in the food industry. These strong biofilm-forming isolates produced more polysaccharides and were less susceptible to sodium hypochlorite, implying a possible correlation between polysaccharide production and sodium hypochlorite susceptibility. Genetic diversity in mshA, mshC and mshD contributed to the observed variation in biofilm formation between isolates. This study identified strong biofilm-forming V. parahaemolyticus strains of new multilocus sequence typing (MLST) types, showed a relationship between polysaccharide production and sodium hypochlorite resistance.

Comparative genome identification of accessory genes associated with strong biofilm formation in Vibrio parahaemolyticus.

Vibrio parahaemolyticus biofilms on the seafood processing plant surfaces are a potential source of seafood contamination and subsequent food poisoning. Strains differ in their ability to form biofilm, but little is known about the genetic characteristics responsible for biofilm development. In this study, pangenome and comparative genome analysis of V. parahaemolyticus strains reveals genetic attributes and gene repertoire that contribute to robust biofilm formation. The study identified 136 accessory genes that were exclusively present in strong biofilm forming strains and these were functionally assigned to the Gene Ontology (GO) pathways of cellulose biosynthesis, rhamnose metabolic and catabolic processes, UDP-glucose processes and O antigen biosynthesis (p < 0.05). Strategies of CRISPR-Cas defence and MSHA pilus-led attachment were implicated via Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Higher levels of horizontal gene transfer (HGT) were inferred to confer more putatively novel properties on biofilm-forming V. parahaemolyticus. Furthermore, cellulose biosynthesis, a neglected potential virulence factor, was identified as being acquired from within the order Vibrionales. The cellulose synthase operons in V. parahaemolyticus were examined for their prevalence (22/138, 15.94 %) and were found to consist of the genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC. This study provides insights into robust biofilm formation of V. parahaemolyticus at the genomic level and facilitates: identification of key attributes for robust biofilm formation, elucidation of biofilm formation mechanisms and development of potential targets for novel control strategies of persistent V. parahaemolyticus.

Influence of cations on growth of thermophilic Geobacillus spp. and Anoxybacillus flavithermus in planktonic culture.

Free ions of Na(+), K(+), Ca(2+), and Mg(2+) influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca(2+) and Mg(2+) were associated with increases in optical density more so than Na(+) and K(+). Overall, it appeared that Ca(2+) and/or Mg(2+) was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.

Coconut Cream Agar as a simple and rapid semiquantitative method to screen citrinin-producing Monascus spp. isolates isolated from red fermented rice.

Red fermented rice (RFR) is produced using Monascus spp. This product has some health benefits. However, RFR can also contain the mycotoxin, citrinin (CIT) and that has adverse effects on human health. The objective of the study was to develop a simple and rapid screening method for the detection of Monascus spp. isolates that can produce CIT by using Coconut Cream Agar (CCA). RFR was spread onto CCA and other media and incubated at 30 °C for 7 days. All the media were observed daily under ultraviolet (UV) light and any Monascus spp. colony that produced light blue fluorescence was recorded as a CIT-producer. Two different isolates (MF1 and MS1) isolated from CCA were selected for further analysis. All (100%; 10/10 plates) of CCA inoculated with MF1 produced light blue fluorescence after incubation for 4 days, meanwhile 30% (3/10 plates) of MS1 produced weak fluorescence on CCA after incubation for 7 days. Isolates MF1 and MS1 were identified as M. purpureus with the ability to produce CIT by having polyketide synthase (pksCT) and transcriptional regulator (ctnA) genes. CIT was quantified by high-performance liquid chromatography (HPLC). CCA is a simple and rapid method to detect CIT-producers of Monascus spp.

Monascus spp. and citrinin: Identification, selection of Monascus spp. isolates, occurrence, detection and reduction of citrinin during the fermentation of red fermented rice.

Red fermented rice (RFR) is rice fermented using Monascus spp. This product contains monacolin K, providing health benefits including mitigation of diarrhoea and improving blood circulation. RFR can produce pigments that can act as natural colour and flavouring agents. However, Monascus spp. (a fungal starter to ferment RFR) can also produce the mycotoxin, citrinin (CIT) which is believed to have adverse effects on human health. CIT in RFR has been reported worldwide by using different methods of detection. This review focuses on the production of RFR by solid-state fermentation (SSF) and submerged fermentation (SmF), the occurrence of CIT in RFR, CIT quantification, the factors affecting the growth of Monascus spp., pigments and CIT production in RFR, and possible methods to reduce CIT in RFR. This review will help the food industries, researchers, and consumers understand the risk of consuming RFR, and the possibility of controlling CIT in RFR.

Patient reported outcomes after definitive open tibial fracture management.

AIMS: Open tibial fractures are often life-changing injuries and patient outcomes remain poor despite the introduction of national management guidelines. The longer-term impact to the patient can be considerable but this is often overlooked in the literature. This study aims to establish the functional, physical, and psychosocial impact of sustaining an open tibial fracture. METHODS: We reviewed 69 consecutive Gustilo-Anderson grade IIIB and IIIC open tibial fractures that presented to our Major Trauma Centre (MTC) between September 2012 and April 2018. Each participant was interviewed and sent patient-reported outcome questionnaires, a minimum of 12 months following injury. Our primary outcome was the Lower Extremity Functional Scale (LEFS). Secondary outcomes included the Short-Form 36 Healthy Survey (SF-36), Sickness Impact Profile 128 (SIP) and return to occupation. Subgroups were analysed according to age, Injury Severity Score (ISS) and limb amputation. RESULTS: The mean follow up was 43 months. 96% were grade IIIB and 4% grade IIIC. The response rate for our study was 72%. The mean LEFS was 42 (IQR 21.5-58.5). All total and sub-domain scores within both the SF-36 and SIP questionnaires were reduced when compared to normative population data. Only 48% of patients returned to full time employment. Subgroup analysis revealed significantly reduced LEFS, SIP and SF-36 subdomain scores for those with a presenting ISS >14 and those undergoing limb amputation. CONCLUSION: Patients are at significant risk of longer-term functional, physical and psychosocial harm after suffering an open tibial fracture. Those sustaining major polytrauma or amputation demonstrated to have the greatest risk of poor outcome. Early identification of these individuals likely to suffer most from their injury would help direct appropriate resources to those with greatest need at the earliest opportunity.

Investigation of UV light treatment (254 nm) on the reduction of aflatoxin M1 in skim milk and degradation products after treatment.

This study investigates the reduction of aflatoxin M1 (AFM1) in skim milk by using ultraviolet light at 254 nm and the effects of influencing factors on the efficacy including treatment time (min), depth of samples (mm), contamination level (µg L-1), stirring, temperature, and fat content in milk. The colour and pH of milk samples were measured to evaluate the influence of the treatment on these values. It was found that short-wave ultraviolet radiation (UVC) reduced up to 50% of AFM1 in milk after 20 min of treatment regardless of the initial AFM1 contamination level. Treatment time, depth of samples, and stirring were all found to significantly (P < 0.05) enhance the reduction of AFM1. The milk colour was affected but there was no influence on the pH of milk samples at any duration of UV exposure. It is concluded that UVC light treatment has the potential to reduce AFM1 in milk.

Control of aflatoxin M1 in skim milk by high voltage atmospheric cold plasma.

Cold plasma has potential for the degradation of aflatoxins in corn and hazelnuts; however, this has not been demonstrated for aflatoxin in milk. In this study, the efficacy of high voltage atmospheric cold plasma (HVACP) on the reduction of aflatoxin M1 (AFM1) in skim milk improved with increasing treatment times (1-20 min), using gas containing 65% oxygen (MA65) rather than air, increasing voltage (60-80 kV) and reducing sample volume (30 mL-10 mL). Direct treatment was more effective than indirect treatment. AFM1 in milk was degraded by 65.0 % and 78.9 % by air and MA65 respectively in 20 min with no change in milk colour. The toxicity of AFM1 after treatment was assessed using a brine shrimp model. A five-minute HVACP treatment reduced the toxicity of AFM1 by 83.9 % based on the increase in brine shrimp survival. HVACP is a promising method to reduce AFM1 in milk.