Causative Agents Of Foodborne Diseases Research Articles

Effects of Rhodomyrtus tomentosa Extract on Killing Activity of Human Neutrophils and Membrane Integrity of Enterohaemorrhagic Escherichia coli O157:H7.

Enterohaemorrhagic Escherichia coli (E. coli) O157:H7 is one of the most virulent causative agents of foodborne disease. Use of antibiotics for the treatment against E. coli O157:H7 infection leads to hemolytic uremic syndrome. The present study evaluated the potential of ethanolic leaf extract of a medicinal plant, Rhodomyrtus tomentosa in enhancing the killing activity of human neutrophils against E. coli O157:H7. In addition, the effects of the extract on membrane permeability of the organisms were studied. In the killing assay, percentage survival of the bacterial cells after being exposed to human neutrophils in the presence of various concentrations of the extract were determined. At 45 min, percentage survival of E. coli O157:H7 and E. coli ATCC 25922 after treated with neutrophils in the presence of the extract at 125–250 µg/mL was 58.48%–50.28% and 69.13%–35.35%, respectively. Furthermore, upon treatment with R. tomentosa at 250 µg/mL uptake of crystal violet by E. coli O157:H7 and E. coli ATCC 25922 was increased to 40.07% and 36.16%, respectively. Therefore, it is suggested that the extract exhibited dual effects as immunostimulant and membrane permeabilizing agent perhaps resulted in enhancing the killing activity of neutrophils against the organisms.

Antimicrobial activities of six essential oils commonly used as condiments in Brazil against Clostridium perfringens

Despite recent advances in food production technology, food-borne diseases (FBD) remain a challenging public health concern. In several countries, including Brazil, Clostridium perfringens is among the five main causative agents of food-borne diseases. The present study determines antimicrobial activities of essential oils of six condiments commonly used in Brazil, viz., Ocimum basilicum L. (basil), Rosmarinus officinalis L. (rosemary), Origanum majorana L. (marjoram), Mentha × piperita L. var. Piperita (peppermint), Thymus vulgaris L. (thyme) and Pimpinella anisum L. (anise) against C. perfringens strain A. Chemical compositions of the oils were determined by GC–MS (gas chromatography–mass spectrometry). The identities of the isolated compounds were established from the respective Kováts indices, and a comparison of mass spectral data was made with those reported earlier. The antibacterial activity was assessed from minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the microdilution method. Minimum inhibitory concentration values were 1.25 mg mL−1 for thyme, 5.0 mg mL−1 for basil and marjoram, and 10 mg mL−1 for rosemary, peppermint and anise. All oils showed bactericidal activity at their minimum inhibitory concentration, except anise oil, which was only bacteriostatic. The use of essential oils from these common spices might serve as an alternative to the use of chemical preservatives in the control and inactivation of pathogens in commercially produced food systems.

The Development of a Novel, Validated, Rapid and Simple Method for the Detection of Sarcocystis fayeri in Horse Meat in the Sanitary Control Setting.

Sarcocystis fayeri (S. fayeri) is a newly identified causative agent of foodborne disease that is associated with the consumption of raw horse meat. The testing methods prescribed by the Ministry of Health, Labour and Welfare of Japan are time consuming and require the use of expensive equipment and a high level of technical expertise. Accordingly, these methods are not suitable for use in the routine sanitary control setting to prevent outbreaks of foodborne disease. In order to solve these problems, we have developed a new, rapid and simple testing method using LAMP, which takes only 1 hour to perform and which does not involve the use of any expensive equipment or expert techniques. For the validation of this method, an inter-laboratory study was performed among 5 institutes using 10 samples infected with various concentrations of S. fayeri. The results of the inter-laboratory study demonstrated that our LAMP method could detect S. fayeri at concentrations greater than 10(4) copies/g. Thus, this new method could be useful in screening for S. fayeri as a routine sanitary control procedure.

Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction (MPF7P.703)

Abstract The NF-κB signaling pathway has long been recognized as a crucial activator and regulator of the innate and adaptive immune responses. More recently, the ribosomal protein, RPS3 has been identified as a non-rel component of NF-κB and functions as a “specifier” protein critical for the induction of selective proinflammatory gene transcription. Attaching/effacing pathogens including enteropathogenic Escherichia coli, enterohemorrhagic E. coli and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. A/E pathogens have evolved many strategies to evade the host immune response and encode various effector proteins that target NF-κB at different stages. Our work here, demonstrates how the A/E encoded metalloprotease NleC, specifically cleaves the p65 molecule at the N-terminus generating a fragment that is capable of selectively blocking the function of RPS3. This selective blockade of RPS3 results in a dampened immune response following infection, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction.

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p651–38 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p651–38 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a ‘specifier’ subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

Complete Genome Sequence of Vibrio alginolyticus ATCC 17749T.

Vibrio alginolyticus is a Gram-negative halophilic bacterium and has been recognized as an opportunistic pathogen in both humans and marine animals. It is the causative agent of food-borne diseases, such as gastroenteritis, and it invades through wounds in predisposed individuals. In this study, we present the completed genome of V. alginolyticus ATCC 17749T through high-throughput sequencing.

A new repertoire of informations about the quorum sensing system in Salmonella enterica serovar Enteritidis PT4

Salmonella spp are among the main causative agents of foodborne diseases. Some phenotypes associated with increased drug resistance and virulence are regulated by quorum sensing (QS). In the present study, the autoinducer (AI)-1- and -2-mediated QS mechanisms were characterized in Salmonella enterica serovar Enteritidis PT4 for the first time. Salmonella Enteritidis did not produce AI-1. Phylogenetic analysis of nucleotides encoding the SdiA protein, the response regulator of AI-1-mediated QS, and comparative alignment of its amino acids showed that the gene and protein are conserved within the same bacterial genus. Thus, bacteria of the same genus respond to the same AIs. However, this finding did not preclude the possibility that Salmonella Enteritidis might respond to AIs released from bacteria of a different genus, which might confer a competitive advantage to this pathogen. We found that the regulation of AI-2-mediated QS in Salmonella Enteritidis is similar to that in serovar Typhimurium. The elucidation of the AI-1- and AI-2-mediated QS mechanisms in Salmonella Enteritidis will contribute to the development of new control strategies for this pathogen by indicating new targets for antimicrobial drugs.

New validated rapid screening methods for identifying Kudoa septempunctata in olive flounder (Paralichthys olivaceus).

Kudoa septempunctata is a newly identified causative agent of foodborne diseases associated with consuming raw olive flounder. Qualitative PCR and quantitative real-time PCR have been used as notification methods to identify K. septempunctata in Japan. However, these methods require expensive equipment and are time-consuming (2-3 h for screening). To address these problems, in this study, we developed new rapid and simple methods using real-time loop-mediated isothermal amplification (LAMP) and nucleic acid sequence based amplification-nucleic acid chromatography (NASBA-NAC). Using these methods, the total procedure required approximately 45 min and did not require any expensive equipment. With regard to validating these new methods in comparison with the notification methods used in Japan, we performed an inter-laboratory study of 5 laboratories using samples that included olive flounders infected with 4 different amounts of K. septempunctata. These results demonstrated that the sensitivity of NASBA-NAC was equivalent to that of qualitative PCR, and that the sensitivity of real-time LAMP was equivalent to that of quantitative real-time PCR, which indicated that these new methods were acceptable screening methods for identifying K. septempunctata.

Salmonella serotypes, resistance patterns, and food vehicles of salmonellosis in southern Brazil between 2007 and 2012.

Previous studies have identified Salmonella as the main causative agent of foodborne diseases in the state of Rio Grande do Sul (RS), southern Brazil, between 1997 and 2006. This study aimed to describe the Salmonella serotypes, antimicrobial patterns, and food vehicles of salmonellosis that occurred in RS between 2007 and 2012. Information about Salmonella isolates and salmonellosis outbreaks registered in the official records of the Central Laboratory of RS (FEEPS/IPB-LACEN/RS) was analyzed. Among the 163 isolates investigated, 138 (84.7%) were identified as S. Enteritidis. The second and third most frequent serovars identified were S. Schwarzengrund (5.5%) and S. Typhimurium (3.7%). Homemade mayonnaise was the food vehicle most frequently identified (17.39%), followed by pastry products (15.94%) and beef (12.32%). Antimicrobial resistance was analyzed; 12 drugs were tested. Higher percentages of resistance were observed to nitrofurantoin (94.2%) and nalidixic acid (89.1%). The resistance to these two drugs was verified in 80.43% of the isolates. Multi-resistance to three and five drugs was verified in four and two isolates, respectively. Comparing the results of the present study with results of previous reports, it was possible to conclude that S. Enteritidis and homemade mayonnaise are still the main serotype and food vehicle of salmonellosis in RS and that antimicrobial resistance has been increasing among S. Enteritidis responsible for foodborne outbreaks in southern Brazil.

Proteome profile of a pandemic Vibrio parahaemolyticus SC192 strain in the planktonic and biofilm condition

Vibrio parahaemolyticus is one of the leading causative agents of foodborne diseases in humans. In this study, the proteome profiles of the pandemic strain V. parahaemolyticus SC192 belonging to the O3:K6 serovar during the planktonic and biofilm stages were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry. This non-gel-based multidimensional protein identification technology approach identified 45.5% of the proteome in the reference genome V. parahaemolyticus RIMD 2210633. This is the largest proteome coverage obtained so far in V. parahaemolyticus and provides evidence for expression of 27% of the hypothetical proteins. Comparison of the planktonic and biofilm proteomes based on their cluster of orthologous groups, gene ontologies and KEGG pathways provides basic information on biofilm specific functions and pathways. To the authors’ knowledge, this is the first study to generate a global proteome profile of the pandemic strain of V. parahaemolyticus and the method reported here could be used to rapidly obtain a snapshot of the proteome of any microorganism at a given condition.

Inactivation of Human Norovirus in Contaminated Oysters and Clams by High Hydrostatic Pressure

Human norovirus (NoV) is the most frequent causative agent of food-borne disease associated with shellfish consumption. In this study, the effect of high hydrostatic pressure (HHP) on inactivation of NoV was determined. Genogroup I.1 (GI.1) or genogroup II.4 (GII.4) NoV was inoculated into oyster homogenates and treated at 300 to 600 MPa at 25, 6, and 1°C for 5 min. After HHP, samples were treated with RNase and viral particles were extracted with porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs). Viral RNA was then quantified by real-time reverse transcription (RT)-PCR. Since PGM contains histo-blood group-like antigens, which can act as receptors for NoV, deficiency for binding to PGM is an indication of loss of infectivity of NoV. After binding to PGM-MBs, RT-PCR-detectable NoV RNA in oysters was reduced by 0.4 to >4 log10 by HHP at 300 to 600 MPa. The GI.1 NoV was more resistant to HHP than the GII.4 NoV (P < 0.05). HHP at lower temperatures significantly enhanced the inactivation of NoV in oysters (P < 0.05). Pressure treatment was also conducted for clam homogenates. Treatment at 450 MPa at 1°C achieved a >4 log10 reduction of GI.1 NoV in both oyster and clam homogenates. It is therefore concluded that HHP could be applied as a potential intervention for inactivating NoV in raw shellfish. The method of pretreatment of samples with RNase, extraction of viral particles using PGM-MB binding, and quantification of viral RNA using RT-PCR can be explored as a practical means of distinguishing between infectious and noninfectious NoV.

Characterization of Amylolysin, a Novel Lantibiotic from Bacillus amyloliquefaciens GA1

BackgroundLantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity.Methodology/Findings Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact in vitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus.Conclusion/SignificanceApart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens.

MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars

Salmonella enterica subsp . enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars.

Detection of Noroviruses in Foods: A Study on Virus Extraction Procedures in Foods Implicated in Outbreaks of Human Gastroenteritis

Disease outbreaks in which foods are epidemiologically implicated as the common source are frequently reported. Noroviruses and enteric hepatitis A viruses are among the most prevalent causative agents of foodborne diseases. However, the detection of these viruses in foods other than shellfish is often time-consuming and unsuccessful. In this study, three virus concentration methods were compared: polyethylene glycol (PEG) plus NaCl, ultracentrifugation, and ultrafiltration. Two RNA extraction methods, TRIzol and RNeasy Mini Kit (Qiagen), were compared for detection of viruses in whipped cream and lettuce (as representatives of the dairy and vegetable-fruit food groups, respectively). A seeding experiment with canine calicivirus was conducted to determine the efficiency of each virus extraction procedure. The PEG-NaCl-TRIzol method was most efficient for the detection of viruses in whipped cream and the ultracentrifugation–RNeasy–Mini Kit procedure was best for detection on lettuce. Based on the seeding experiments, food items implicated in norovirus-associated gastroenteritis outbreaks were subjected to the optimal procedure for a specific composition and matrix. No noroviruses were detected in the implicated food items, possibly because the concentration of virus on the food item was too low or because of the presence of inhibitory factors. For each food group, a specific procedure is optimal. Inhibitory factors should be controlled in these procedures because they influence virus detection in food.

Induction of interleukin‐8 production via nuclear factor‐κB activation in human intestinal epithelial cells infected with Vibrio vulnificus

Vibrio vulnificus, a Gram-negative estuarine bacterium, is a causative agent of food-borne diseases, such as life-threatening septicaemia and wound infection disease. V. vulnificus penetrating into the epithelial barrier stimulates an inflammatory response in the adjacent mucosa. Therefore, interaction between V. vulnificus and epithelial cells is important for understanding of both the immunology of mucosal surfaces and V. vulnificus. In this study, we investigated the effect and action mechanism of V. vulnificus infection on production of interleukin (IL)-8, a proinflammatory cytokine, in human intestinal epithelial INT-407 cells. V. vulnificus infection significantly induced IL-8 production in a time- and multiplicity of infection (MOI)-dependent manner, as determined by human IL-8 enzyme-linked immunosorbent assay (ELISA). In addition, V. vulnificus infection significantly increased IL-8 mRNA levels in INT-407 cells, indicating that the increased IL-8 production by V. vulnificus occurred at the transcriptional level. V. vulnificus infection also enhanced IL-8 gene promoter activity in INT-407 cells transiently transfected with IL-8 promoter constructs, but this effect was impaired in INT-407 cells transfected with IL-8 promoter constructs deleted or mutated of a kappaB site. V. vulnificus infection increased the nuclear factor-kappaB (NF-kappaB) binding activity to a kappaB site and the degradation of IkappaB-alpha protein in a time- and a MOI-dependent manner. Furthermore, BAY11-7082, an inhibitor of NF-kappaB activation, significantly reduced the IL-8 production, NF-kappaB binding activity and IkappaB-alpha degradation induced by V. vulnificus infection. Taken together, these results indicate clearly that V. vulnificus infection significantly induces IL-8 production in human intestinal epithelial cells via NF-kappaB activation.

RpoS involvement and requirement for exogenous nutrient for osmotically induced cross protection in Vibrio vulnificus

Vibrio vulnificus is an opportunistic human pathogen which is the causative agent of food-borne disease and wound infections. V. vulnificus is able to adapt to a variety of potentially stressful environmental changes, such as osmotic, nutrient, and temperature variations in estuarine environments, as well as oxidative, osmotic, and acidity differences following infection of a human host. After exposure to sub-lethal levels of a particular environmental stress, many bacteria become resistant to unrelated stresses, a phenomenon termed cross protection. In this study, we examined the ability of osmotic shock to cross protect V. vulnificus to high temperature as well as oxidative stress. Log phase cells of V. vulnificus strain C7184o were cross protected by prior osmotic shock to both heat and oxidative challenge, but only when exogenous nutrient was present during the osmotic upshift. Further, and unlike other bacteria, nutrient starvation alone did not result in cross protection against either stress. When small amounts of nutrient were present during osmotic shock, cross protection to an otherwise lethal heat challenge developed extremely rapidly, with significant protection seen within 10 min. Cross protection to oxidative stress was slower to develop, requiring several hours. Although stationary phase alone conferred some cross protection to heat and oxidative stress, the alternate sigma factor RpoS was required for complete cross protection of log phase cells to oxidative stress but not for resistance to heat challenge. Together these findings suggest that the cross protective response in V. vulnificus is complex and appears to involve multiple mechanisms.

Unusual Food-Borne Pathogens: Listeria Monocytogenes, Aeromonas, Plesiomonas, and Edwardsiella Species

Although these four groups of organisms are perceived as infrequent food-borne pathogens or of dubious significance, increasing epidemiologic data indicate that L. monocytogenes is an emerging cause of infections, particularly gastroenteritis. Furthermore, if data are ever generated that prove that most fecal isolates of Aeromonas are involved in bacterial diarrhea, then aeromonads will become recognized as important food-borne pathogens. For Plesiomonas and Edwardsiella, recognition of possible involvement in food-borne disease requires detailed medical histories, including foreign travel, contact with pets or animals, and food consumption histories.

Salmonella infections in finishing pigs in The Netherlands: bacteriological herd prevalence, serogroup and antibiotic resistance of isolates and risk factors for infection

Salmonellae are wide spread in man and animals world wide and are of increasing significance as causative agents of foodborne diseases in man. The European Union, national authorities and the pig industry are therefore more and more interested in the Salmonella status of the pig population. The aim of this study was to estimate the bacteriological prevalence of Salmonella in finishing pig herds, the serogroup and the resistance to antibiotics of the isolated Salmonellae and a preliminary risk analysis of factors associated with infection. For this, 317 finishing pig herds were randomly selected from a database containing 1500 herds in the southern part of the Netherlands. In each herd 24 samples of fresh faeces were collected from two compartments with pigs close to market weight. Per compartment 12 samples of faeces were pooled into one pooled sample. Pooled samples were cultured in duplicate. Salmonella spp. were recovered from 71 out of 306 herds (23%) in which two compartments could be sampled. A total of 108 isolated Salmonella’s were serotyped: 71 serogroup B, 3 serogroup C1, 6 serogroup C2, 22 serogroup D1, and 6 isolates neither serogroup B, C or D1. Of a total of 115 Salmonella isolates tested, none were resistant to colistin, enrofloxacin, flumequin or gentamicin. Automated liquid feeding of by-products, and membership of an Integrated Quality Control (IQC) production group were associated with a decreased risk of infection, while use of trough feeding was associated with an increased risk of infection. It is necessary to test these presumed risk factors in intervention studies to evaluate their potency to reduce the Salmonella prevalence in finishing pigs and thereby reduce the risk of Salmonellosis in people consuming pork.