Virulence, antimicrobial susceptibility and phylogenetic analysis of Cronobacter sakazakii isolates of food origins from Jordan.

AIMS: The aim was to characterize a collection of Cronobacter sakazakii isolates collected from various origins in Jordan. METHODS AND RESULTS: The isolates were characterized using 16S rRNA sequencing, DNA microarray, multi-locus sequence typing (MLST), O-serotyping, virulence gene identification and antibiotic susceptibility testing. The identities and phylogenetic relatedness revealed that C. sakazakii sequence type 4 (ST4) and Csak O:1 serotype were the most prevalent STs and serovars amongst these C. sakazakii strains. PCR screening of putative virulence genes showed that the siderophore-interacting protein gene (sip) and iron acquisition gene clusters (eitCBAD and iucABCD/iutA) were the most detected genes with noticeable variability in the type 6 secretion system (T6SS) and filamentous hemagglutinin/adhesion (FHA) gene loci. The antibiotic resistance profiles revealed that the majority of the isolates were susceptible to all antibiotics used despite harbouring a class C ß-lactamase resistance gene. CONCLUSIONS: The results described in this report provide additional insights about the considerable genotypic and phenotypic heterogeneity within C. sakazakii. SIGNIFICANCE AND IMPACT OF THE STUDY: The information reported in this study might be of great value in understanding the origins of C. sakazakii isolates, in addition to their diversity and variability, which might be helpful in preventing future outbreaks of this pathogen.

Predictors of growth of Escherichia coli on lab coats as part of hospital-acquired infection transmission through healthcare personnel attire.

OBJECTIVES: Previous research has documented the presence of microbes on healthcare personnel (HCP) attire. This study aimed to explore the bacterial contamination and predictors of Escherichia coli (E coli) growth, as well as, hygiene and handling practices of HCP attire that could influence growth of E coli. METHODS: Descriptive, cross-sectional study was used in this study. Convenience sampling of the 188 HCP was recruited from a main comprehensive hospital in the northern part of Jordan. Three swab samples were collected from three different parts of lab coats used by each participant. The generalised mixed linear model was used for the categorical variables and to identify the predictors of E coli growth on HCP attire. RESULTS: Enterococcus faecalis was the most common species of bacteria found on lab coat. The HCP attire coming from the emergency department (ED) was highlighted with slightly higher contamination of E coli compared with other departments, such as critical care units. Factors associated with significant E coli growth on HCP attire were lab coat use over scrubs and borrowing of lab coats. The predictors of positive E coli growth were working in the ED, storing HCP attire in hospital lockers, believing the transmission of pathogens by HCP attire and carrying attire wrapped around arms. IMPLICATIONS: Hygiene practices and policies, including a washing facility on the hospital premises, are a must to keep the lab coats clean. CONCLUSION: HCP should be cautious about the method of use and storage of lab coats they wear.

Antagonistic effects of Lactobacillus reuteri against Escherichia coli O157:H7 in white-brined cheese under different storage conditions.

This study aimed to investigate the survival of the foodborne pathogen Escherichia coli O157:H7 in white-brined cheeses as influenced by the presence of Lactobacillus reuteri. The white cheeses were made from pasteurized bovine milk inoculated with E. coli O157:H7 (cocktail of 3 strains) to achieve ∼5 log10 cfu/g with absence or presence of Lb. reuteri (∼6 log10 cfu/g). Cheese samples were brined in 10% or 15% NaCl solution and stored at 10°C and 25°C for 28 d. The white-brined cheeses were assessed for salt content, pH, water activity (Aw), and numbers of E. coli O157:H7, Lb. reuteri, nonstarter lactic acid bacteria (NSLAB), yeasts, and molds. Results showed that E. coli O157:H7 survived in cheese stored in both brine solutions at 10°C and 25°C regardless of the presence of Lb. reuteri. A substantial reduction was observed in cheese stored in 10% NaCl brine at 25°C, followed by cheese stored in 15% NaCl brine at 10°C by 2.64 and 2.16 log10 cfu/g, respectively, in the presence of Lb. reuteri and by 1.02 and 1.87 log10 cfu/g, respectively, in the absence of Lb. reuteri under the same conditions. The pathogen in brine solutions survived but at a lower rate. Furthermore, the growth of Lb. reuteri and NSLAB were enhanced or slightly decreased in cheese and brine by 28 d, respectively. The salt concentrations of cheese ranged from 4 to 6% and 5 to 7% (wt/wt), during 28-d ripening in 10 and 15% brine, respectively. Values of pH and Aw slightly increased at d 1 after exposure to brine and reached 4.69 to 6.08 and 0.91 to 0.95, respectively, in all treatments. Therefore, the addition of Lb. reuteri can be used as a biopreservation method to inhibit the survival of E. coli O157:H7 in white-brined cheese when combined with the appropriate temperature, NaCl level, and storage time.

Inactivation of Salmonella spp. in tahini using plant essential oil extracts.

Tahini is a popular food product in the Middle East region and is used as a major ingredient in several ready-to-eat food products. Tahini and its products have been linked to foodborne illness outbreaks and product recalls worldwide as a result of Salmonella spp. contamination. The objectives of the current study were to investigate: i) the effectiveness of 10 plant essential oil extracts on the viability of Salmonella spp. using disc diffusion ii) the antimicrobial activity of the most effective oils against Salmonella spp. in commercial or 10% w/v hydrated tahini (tahini-based product model) stored at 37, 25 and 10 °C for 28 d and iii) the effect of the addition of essential oil extracts on the sensory acceptability of tahini and hydrated tahini. Among the tested essential oils, thyme (TO) and cinnamon oil (CO) showed the highest antimicrobial activity against tested Salmonella spp. at 37 and 10 °C using a disc diffusion assay method. In tahini, the addition of 2.0% CO reduced the numbers of Salmonella spp. by 2.87, 2.64 or 2.35 log10 CFU/ml at 37, 25 or 10 °C, respectively, by 28 d. However, the antimicrobial activity of CO was more pronounced at all storage temperatures in hydrated tahini where no viable cells were detected after 3 d storage at 25 and 37 °C, or after 7 d at 10 °C. However, at 25 and 37 °C, the antimicrobial activity of CO was more evident since no viable cells were detected after 14 d when 0.5% was used. The numbers of Salmonella spp. were reduced by 3.29, 3.03 or 2.17 log10 CFU/ml at 37, 25 or 10 °C, respectively, after 28 d when 2.0% TO was added to tahini. Salmonella spp. were not detected in the hydrated tahini treated with 2.0% TO after 28 d at 37 °C or 25 °C, while at 10 °C, the numbers of Salmonella spp. were not significantly reduced after 28 d in hydrated tahini compared to the initial numbers at zero time. Therefore, the addition of TO and CO could be used to preclude the post process contamination of tahini with foodborne pathogens, yet, the addition of TO and CO to tahini reduced its consumer acceptability compared untreated tahini.

Force degradation of orthodontic latex elastics: An in-vivo study.

INTRODUCTION: Our objectives were to assess the force degradation of orthodontic latex elastics over 48 hours in vivo and to study the relationship between the amount of mouth opening and the degree of force decay. METHODS: Fifty-two orthodontic patients wearing fixed appliances using Class II elastics were asked to wear premeasured-force 3/16-in heavy and medium intermaxillary elastics. The force amounts were measured and compared at different time intervals. RESULTS: Fifty percent of the force was lost after 3.9 hours for the medium elastics and after 4.9 hours for the heavy elastics. A continuous significant force drop in all elastics was seen at all time intervals (P <0.05, P <0.001). There was greater force loss in the heavy elastics compared with the medium elastics in vivo at all time intervals (P <0.001); the rates of force loss, however, were similar. CONCLUSIONS: Fifty percent of force degradation occurred in the first 4 to 5 hours. Because of breakage and for oral hygiene purposes, orthodontic elastics should be changed daily; otherwise, elastics can be used for 48 hours. Force decay of the elastics was correlated to the lateral distance between the maxillary canine and the mandibular first molar in occlusion.

Behavior of Escherichia coli O157:H7 and Listeria monocytogenes during fermentation and storage of camel yogurt.

In addition to its nutritional and therapeutic properties, camel milk has the ability to suppress the growth of a wide range of foodborne pathogens, but there is a lack of information regarding the behavior of these pathogens in products such as yogurt produced from camel milk. The objective of the current study was to investigate the behavior of Listeria monocytogenes and Escherichia coli O157:H7 during manufacture and storage of camel yogurt. Camel milk inoculated with L. monocytogenes and E. coli O157:H7 was fermented at 43° C for 5h using freeze-dried lactic acid bacteria (LAB) starter cultures (Streptococcus thermophilus and Lactobacillus bulgaricus) and stored at 4 or 10 °C for 14 d. Camel milk inoculated with L. monocytogenes and E. coli O157:H7 without starter culture was also prepared. During fermentation, the numbers of L. monocytogenes and E. coli O157:H7 increased 0.3 and 1.6 log cfu/mL, respectively, in the presence of LAB, and by 0.3 and 2.7 log cfu/mL in the absence of LAB. During storage at 4 or 10 °C, L. monocytogenes increased 0.8 to 1.2 log cfu/mL by 14 d in camel milk without LAB, but in the presence of LAB, the numbers of L. monocytogenes were reduced by 1.2 to 1.7 log cfu/mL by 14 d. Further, E. coli O157:H7 numbers in camel milk were reduced by 3.4 to 3.5 log cfu/mL in the absence of LAB, but E. coli O157:H7 was not detected (6.3 log cfu/mL reduction) by 7d in camel yogurt made with LAB and stored at either temperature. Although camel milk contains high concentrations of natural antimicrobials, L. monocytogenes was able to tolerate these compounds in camel yogurt stored at refrigerator temperatures. Therefore, appropriate care should be taken during production of yogurt from camel milk to minimize the potential for postprocess contamination by this and other foodborne pathogens.

Effects of osmotic pressure, acid, or cold stresses on antibiotic susceptibility of Listeria monocytogenes.

Prevalence of antibiotic resistance of Listeria monocytogenes isolated from a variety of foods has increased in many countries. L. monocytogenes has many physiological adaptations that enable survival under a wide range of environmental stresses. The objective of this study was to evaluate effects of osmotic (2, 4, 6, 12% NaC), pH (6, 5.5, 5.0) and cold (4 °C) stresses on susceptibility of three isolates of L. monocytogenes towards different antibiotics. The minimal inhibitory concentrations (MICs) of tested antibiotics against unstressed (control), stressed or post-stressed L. monocytogenes isolates (an ATCC strain and a meat and dairy isolate) were determined using the broth microdilution method. Unstressed cells of L. monocytogenes were sensitive to all tested antibiotics. In general, when L. monocytogenes cells were exposed to salt, cold and pH stresses, their antibiotic resistance increased as salt concentration increased to 6 or 12%, as pH was reduced to pH 5 or as temperature was decreased to 10 °C. Results showed that both meat and dairy isolates were more resistant than the ATCC reference strain. Use of sub-lethal stresses in food preservation systems may stimulate antibiotic resistance responses in L. monocytogenes strains.

Use of acetic and citric acids to control Salmonella Typhimurium in tahini (sesame paste).

Since tahini and its products have been linked to Salmonella illness outbreaks and product recalls in recent years, this study assessed the ability of Salmonella Typhimurium to survive or grow in commercial tahini and when hydrated (10% w/v in water), treated with 0.1%-0.5% acetic or citric acids, and stored at 37, 21 and 10 °C for 28 d. S. Typhimurium survived in commercial tahini up to 28 d but was reduced in numbers from 1.7 to 3.3 log10 CFU/ml. However, in the moist or hydrated tahini, significant growth of S. Typhimurium occurred at the tested temperatures. Acetic and citric acids at ≤0.5% reduced S. Typhimurium by 2.7-4.8 log10 CFU/ml and 2.5-3.8 log10 CFU/ml, respectively, in commercial tahini at 28 d. In hydrated tahini the organic acids were more effective. S. Typhimurium cells were not detected in the presence of 0.5% acetic acid after 7 d or with 0.5% citric acid after 21 d at the tested temperatures. The ability of S. Typhimurium to grow or survive in commercial tahini and products containing hydrated tahini may contribute to salmonellosis outbreaks; however, use of acetic and citric acids in ready-to-eat foods prepared from tahini can significantly minimize the risk associated with this pathogen.

Inactivation of stressed Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in hummus using low dose gamma irradiation.

Hummus is a popular dip in the Middle East region prepared by mixing the boiled chickpeas with tahini and other ingredients, and because its consumption has increased world-wide some notoriety has developed following an increase in the incidence of hummus-related illness outbreaks and recalls. The objectives of the current research were (i) to study the efficiency of low dose gamma irradiation to inhibit Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in hummus, and (ii) to assess the effect of environmental stresses namely cold, heat, and desiccation on the resistance of these pathogens to gamma irradiation. The samples of hummus were prepared and then individually inoculated with approximately 7.0 log CFU/g of unstressed or cold-, heat-, or desiccated-stressed cocktail cultures of each of E. coli O157:H7, S. enterica, and L. monocytogenes. The inoculated samples were then exposed to gamma irradiation at doses of 0.1 to 0.6 kGy. The numbers of unstressed E. coli O157:H7, S. enterica, and L. monocytogenes were decreased by 0.6-3.9, 0.7-2.9, and 1.0-3.0 log CFU/g, respectively, by irradiation treatment. The resistance of E. coli O157:H7 to gamma irradiation was not affected by desiccation, heat, and cold stresses. However, the pre-exposure of S. enterica and L. monocytogenes cells to these stresses reduced their resistance toward gamma irradiation. PRACTICAL APPLICATION: Gamma irradiation is a non-thermal treatment that can be used in food processing to ensure food safety and quality. The current study proved that low levels (≤0.6 kGy) of gamma irradiation can effectively decrease the risk of unstressed and cold-, heat-, or desiccation-stressed Salmonella enterica, Listeria monocytogenes, or Escherichia coli O157:H7 in hummus.

Characterization of surface proteins of Cronobacter muytjensii using monoclonal antibodies and MALDI-TOF Mass spectrometry.

BACKGROUND: Cronobacter spp. is a newly emerging pathogen that causes meningitis in infants and other diseases in elderly and immunocompromised individuals. This study was undertaken to investigate surface antigenic determinants in Cronobacter spp. using monoclonal antibodies (MAbs) and MALDI-TOF Mass spectrometry. RESULTS: Spleenocytes from mice that were immunized with heat-killed (20 min, 80°C) Cronobacter cells were fused with SP2 myeloma cells. Five desirable MAbs (A1, B5, 2C2, C5 and A4) were selected. MAbs A1, B5, 2C2 and C5 were of IgG2a isotype while A4 was an IgM. Specificity of the MAbs was determined by using immunoblotting with outer membrane protein preparations (OMPs) extracted from 12 Cronobacter and 6 non-Cronobacter bacteria. All MAbs recognized proteins with molecular weight ranging between 36 and 49 kDa except for one isolate (44) in which no OMPs were detected. In addition, MAbs recognized two bands (38-41 kDa) in four of the non-Cronobacter bacteria. Most of the proteins recognized by the MAbs were identified by MALDI-TOF peptide sequencing and appeared to be heterogeneous with the identities of some of them are still unknown. All MAbs recognized the same epitope as determined by an additive Index ELISA with their epitopes appeared to be conformational rather than sequential. Further, none of the MAbs recognized purified LPS from Cronobacter spp. Specificity of the MAbs toward OMPs was further confirmed by transmission electron microscopy. CONCLUSIONS: Results obtained in this study highlight the immunological cross-reactivity among Cronobacter OMPs and their Enterobacteriaceae counterparts. Nevertheless, the identity of the identified proteins appeared to be different as inferred from the MALDI-TOF sequencing and identification.

Occurrence, distribution and pattern analysis of methicillin resistant (MRSA) and methicillin sensitive (MSSA) Staphylococcus aureus on fomites in public facilities.

Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen incriminated as a causative agent of hospital nosocomial infections as well as a wide range of diseases in communities. This study was conducted to assess the occurrence and distribution of MRSA and methicillin-sensitive (MSSA) on different fomites in public facilities in northern Jordan and to determine their antibiograms, toxin genes profiles, as well as identify their genetic relatedness. A total of 2600 swabs were collected from 14 fomite surfaces in a variety of public facilities including hospitals, universities, schools, transportation sites, and market places. The identity of the 380 S. aureus isolates was confirmed. Among them, 158 (41.6%) were MRSA while the rest of the isolates, 222 (58.4%) were MSSA. MRSA isolates were recovered from all fomites sites. However, among the total collected samples, the percentages of MRSA in public facilities were significantly higher in hospitals and transportation fomites, while percentages of MRSA among fomites sites were higher in public reception sites, chairs, and toilet seats. Antibiotic resistance profiles indicated that 24.5% of the isolates were resistant to cefoxitin, oxacillin, and oxytetracycline. In contrast, only 3.95% were resistant to trimethoprim-sulfamethoxazole, and 15.3% were resistant to ciprofloxacin. Multidrug-resistant patterns were higher in MRSA than in MSSA isolates. There was no apparent difference in toxin gene profiles between MRSA and MSSA. Molecular analysis revealed 85 patterns and 16 clusters at a 9% mean similarity level. In conclusion, this study provides evidence for the potential of MRSA transmission via inanimate surfaces.

Conductometric Immunosensor for Escherichia coli O157:H7 Detection Based on Polyaniline/Zinc Oxide (PANI/ZnO) Nanocomposite.

A conductometric immunosensor was developed for the detection of one of the most common foodborne pathogens, Escherichia coli O157:H7 (E. coli O157:H7), by conductometric sensing. The sensor was built based on a polyaniline/zinc oxide (PANI/ZnO) nanocomposite film spin-coated on a gold electrode. Then, it was modified with a monoclonal anti-E. coli O157:H7 antibody as a biorecognition element. The fabricated nanostructured sensor was able to quantify the pathogens under optimal detection conditions, within 30 min, and showed a good detection range from 101 to 104 CFU/mL for E. coli O157:H7 and a minimum detection limit of 4.8 CFU/mL in 0.1% peptone water. The sensor efficiency for detecting bacteria in food matrices was tested in ultra-heat-treated (UHT) skim milk. E. coli O157:H7 was detected at concentrations of 101 to 104 CFU/mL with a minimum detection limit of 13.9 CFU/mL. The novel sensor was simple, fast, highly sensitive with excellent specificity, and it had the potential for rapid sample processing. Moreover, this unique technique for bacterial detection could be applicable for food safety and quality control in the food sector as it offers highly reliable results and is able to quantify the target bacterium.

Methicillin Resistant Staphylococcus aureus and public fomites: a review.

Staphylococcus genus is a Gram-positive coccus normally associated with skin and mucous membranes of warm-blooded animals. It is part of the commensal human microflora, or found in animals, or contaminating surfaces in the community and hospital settings. Staphylococcus aureus is the most pathogenic species belonging to this genus, as it possesses a collection of virulence factors that are expressed solely to evade the immune system. The increase in the misuse of antimicrobial agents predisposed S. aureus to develop antibiotic resistance, including the resistance to methicillin which led to the emergence of Methicillin-Resistant S. aureus (MRSA). MRSA is considered one of the most dangerous nosocomial pathogens causing many hard to treat infections in hospitals and was named as Hospital Associated MRSA (HA-MRSA). Over the past 20-25 years, MRSA was isolated from community settings and thus Community Associated MRSA (CA-MRSA) has emerged. Inside hospitals, MRSA has been isolated from fomites in contact with patients, as well as staff's protective and personal items. This review highlights the worldwide prevalence of MRSA on fomites within the contexts of hospital and community settings.

Isolation of Cronobacter spp. (formerly Enterobacter sakazakii) from infant food, herbs and environmental samples and the subsequent identification and confirmation of the isolates using biochemical, chromogenic assays, PCR and 16S rRNA sequencing.

BACKGROUND: Cronobacter spp. (formerly Enterobacter sakazakii), are a group of Gram-negative pathogens that have been implicated as causative agents of meningitis and necrotizing enterocolitis in infants. The pathogens are linked to infant formula; however, they have also been isolated from a wide range of foods and environmental samples. RESULTS: In this study, 233 samples of food, infant formula and environment were screened for the presence of Cronobacter spp. in an attempt to find its source. Twenty nine strains were isolated from samples of spices, herbs, infant foods, and dust obtained from household vacuum cleaners. Among the 76 samples of infant food, infant formula, milk powder and non-milk dairy products tested, only one sample of infant food contained Cronobacter spp. (1.4%). The other Cronobacter spp. isolates recovered include two from household vacuum dust, and 26 from 67 samples of herbs and spices. Among the food categories analyzed, herbs and spices harbored the highest number of isolates, indicating plants as a possible reservoir of this pathogen. Initial screening with API 20E test strips yielded 42 presumptive isolates. Further characterization using 3 chromogenic media (alpha-MUG, DFI and EsPM) and 8 sets of PCR primers detecting ITS (internal transcribed spacer sequences), 16S rRNA, zpx, gluA, gluB, OmpA genes followed by nucleotide sequencing of some PCR amplicons did not confirm the identity of all the isolates as none of the methods proved to be free of both false positives or false negatives. The final confirmation step was done by 16S rRNA sequence analysis identifying only 29 of the 42 isolates as Cronobacter spp. CONCLUSION: Our studies showed that Cronobacter spp. are highly diverse and share many phenotypic traits with other Enterobacteriaceae members highlighting the need to use several methods to confirm the identity of this pathogen. None of the biochemical, chromogenic or PCR primers proved to be a reliable method for confirmation of the identity of the isolates as all of them gave either false positives or false negatives or both. It is therefore concluded that 16S rRNA sequencing is pivotal to confirm the identity of the isolates.

Draft genomes of Cronobacter sakazakii strains isolated from dried spices bring unique insights into the diversity of plant-associated strains.

Cronobacter sakazakii is a Gram-negative opportunistic pathogen that causes life- threatening infantile infections, such as meningitis, septicemia, and necrotizing enterocolitis, as well as pneumonia, septicemia, and urinary tract and wound infections in adults. Here, we report 26 draft genome sequences of C. sakazakii, which were obtained from dried spices from the USA, the Middle East, China, and the Republic of Korea. The average genome size of the C. sakazakii genomes was 4393 kb, with an average of 4055 protein coding genes, and an average genome G + C content of 56.9%. The genomes contained genes related to carbohydrate transport and metabolism, amino acid transport and metabolism, and cell wall/membrane biogenesis. In addition, we identified genes encoding proteins involved in osmotic responses such as DnaJ, Aquaproin Z, ProQ, and TreF, as well as virulence-related and heat shock-related proteins. Interestingly, a metabolic island comprised of a variably-sized xylose utilization operon was found within the spice-associated C. sakazakii genomes, which supports the hypothesis that plants may serve as transmission vectors or alternative hosts for Cronobacter species. The presence of the genes identified in this study can support the remarkable phenotypic traits of C. sakazakii such as the organism's capabilities of adaptation and survival in response to adverse growth environmental conditions (e.g. osmotic and desiccative stresses). Accordingly, the genome analyses provided insights into many aspects of physiology and evolutionary history of this important foodborne pathogen.

Antibiograms, toxin profiling and molecular typing of Staphylococcus aureus isolates from two tertiary hospitals in Jordan.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infections. This study was undertaken to investigate toxin profiles as well as antibiotic resistance patterns of S. aureus isolates form two tertiary hospitals in Jordan. METHODOLOGY: A total of 250 S. aureus isolates from clinical samples of two tertiary hospitals were analyzed for the presence of the mecA, vanA, vanB, and 16 Staphylococcus toxin encoding genes using PCR. The isolates were further tested for antimicrobial sensitivities using the disc diffusion method. DNA from all the isolates were fingerprinted by coa gene Restriction Fragment Length Polymorphism (RFLP) to study relationships between isolates from the two hospitals. RESULTS: 73.2% of the isolates contained the mecA gene and thus were designated MRSA. All MRSA isolates showed high levels of resistance to many of the antibiotics compared to those of MSSA. All MRSA isolates were susceptible to vancomycin and teicoplanin while all MSSA isolates were susceptible to nitrofurantoin, teicoplanin, vancomycin, cefoxitin, clindamycin, erythromycin and gentamycin. The isolates exhibited high prevalence of the toxin genes and none of the isolates contained less than 4 genes with one isolate contained 14 genes with no apparent differences in gene profiles among MRSA and MSSA. About 60% of the isolates contained 12 to 13 toxin genes and were isolated either from pus or blood. CONCLUSION: Antibiograms of the MRSA isolates were significantly different from MSSA antibiograms while there were no apparent differences in the toxin genes profiles. Further, coagulase gene RFLP of the isolates showed that the isolates are very heterogenic.

Adhesion characteristics of Listeria adhesion protein (LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor.

Listeria adhesion protein (LAP) is an important adhesion factor in Listeria monocytogenes and interacts with its cognate receptor, mammalian heat shock protein 60 (Hsp60). The genetic identity of LAP was determined to be alcohol acetaldehyde dehydrogenase (Aad). A recombinant Escherichia coli strain expressing aad confirmed the involvement of Aad in adhesion to Caco-2 cells. Binding kinetics (ka) of recombinant LAP (rLAP) to Hsp60 was examined in a surface plasmon resonance sensor and was determined to be 5.35 x 10(8) M(-1) s(-1) and it was equivalent to the binding of anti-Hsp60 antibody (ka = 2.15 x 10(9) M(-1) s(-1)) to Hsp60. In contrast, Internalin B, an adhesion/invasion protein from L. monocytogenes, used as a control, had binding kinetics (ka) of only 2.9 x 10(6) M(-1) s(-1). The KD value of rLAP was 1.68 x 10(-8) M, which was significantly lower than Internalin B (KD = 6.5 x 10(-4) M). These results suggest that Hsp60 has significantly higher avidity for anti-Hsp60 antibody and LAP than Internalin B. In summary, LAP is identified as an alcohol acetaldehyde dehydrogenase and binding of recombinant E. coli to Caco-2 cells or rLAP to Hsp60 protein was found to be highly specific.

Adverse effects of T-2 toxin on chicken lymphocytes blastogenesis and its protection with Vitamin E.

T-2 toxin, a trichothecene mycotoxin that is produced by fusarium species, is prevalent mainly in cereal crops and poultry feed. One of the major effects of this toxin is immunomodulation. The effect of T-2 toxin on chicken lymphocyte proliferation in the presence of mitogens and the subsequent protection with Vitamin E in both fat and water soluble forms was studied using an MTT colorimetric assay. T-2 toxin was administered in concentrations ranging from 0 to 10ng/mL of lymphocytes in the presence of either concanavalin A (ConA) or phytohemagglutinine (PHA-M) at optimum concentration of 333ng/mL and a dilution of 1:160 for ConA and PHA-M, respectively. Lymphocyte proliferation in response to ConA and PHA-M mitogens was depressed at T-2 doses of 1ng/mL or higher (p<0.05). The proliferation was completely abolished at 10ng/mL when the toxin was added at 0 time, while it was decreased by 80% when the toxin was added to the lymphocytes after 24h. The addition of Vitamin E in the fat soluble form (alpha-tocopheryl acetate) did not exert any protection effect against the toxin when it was added at either 25 or 100microg. However, when the water soluble form (Trolox) was added at a concentration of (200microg) (equivalent to 100microM of alpha-tocopherol), it provided considerable protection (p<0.05) against T-2 toxin inhibition of lymphocyte proliferation. The difference in the effect between the two forms of Vitamin E might be related to their relative solubility in the culture media which in turn may affect their availability for protection.

Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9 in a resonant mirror biosensor.

Typical detection of Listeria monocytogenes involves selective enrichment, isolation and biochemical testing. Development of antibodies to Listeria species has improved detection; however, most antibodies detect all species of Listeria. A previously developed monoclonal antibody (MAb)-C11E9 was examined for its reaction to 13 L. innocua and 40 L. monocytogenes strains representing all 13 serotypes by ELISA. Absorbance values for L. monocytogenes strains were 0.44-3.58 and for L. innocua 0.22-1.44. ELISA reactions were divided into three arbitrary groups of high (Abs 1.0 or higher), intermediate (0.6-0.99) and low (0.18-0.59). Most L. monocytogenes strains (32/41, 78%) were in the high group while only 23% (3/13) of L. innocua were in the same group. In the Western blot assay, antibody reacted with phosphate-buffered saline (PBS) extracted protein preparations of 52, 66 and 97 kDa. Ribopattern of all strains was analyzed and no clear relationship was observed for antibody reaction and ribotype of a given strain. MAb C11E9 was used in a resonant mirror biosensor (IAsys sensor), but failed to detect any viable intact L. monocytogenes cells at levels as high as 10(8) cells/ml; however, it showed binding (85-150 arc/s) with the surface protein preparations containing the 97-, 66- and 52-kDa proteins at 208 mug/ml. Binding kinetics of L. monocytogenes and L. innocua surface protein extracts showed significantly (p<0.05) higher responses than the three other Listeria species (L. ivanovii, L. welshimeri and L. grayi), which could be detected in 10-20 min. These data corroborate with ELISA results. In summary, this study suggest that MAb-C11E9 is suitable for detection of all serotypes of L. monocytogenes despite cross-reaction with L. innocua and could be used for detection of soluble protein extracts in the resonant mirror (IAsys) biosensor.

Cronobacter spp.--opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits.

The genus Cronobacter consists of a diverse group of Gram-negative bacilli and comprises seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti. Cronobacter are regarded as opportunistic pathogens, and have been implicated in newborn and infant infections, causing meningitis, necrotizing enterocolitis and bacteraemia or sepsis. Cronobacter virulence is believed to be due to multiple factors. Some strains were found to produce diarrhoea or cause significant fluid accumulation in suckling mice. Two iron acquisition systems (eitCBAD and iucABCD/iutA), Cronobacter plasminogen activator gene (cpa), a 17 kb type VI secretion system (T6SS), and a 27 kb filamentous haemagglutinin gene (fhaBC) and associated putative adhesins locus are harboured on a family of RepFIB-related plasmids (pESA3 and pCTU1), suggesting that these are common virulence plasmids; 98% of 229 tested Cronobacter strains possessed these plasmids. Even though pESA3 and pCTU1 share a common backbone composed of the repA gene and eitCBAD and iucABCD/iutA gene clusters, the presence of cpa, T6SS and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type and species. Other factors were observed, in that Cronobacter form biofilms, and show unusual resistance to heat, dry and acid stress growth conditions. The outer-membrane protein A is probably one of the best-characterized virulence markers of Cronobacter. Furthermore, it was reported that Cronobacter employ phosphatidylinositide 3-kinase/Akt signalling, which activates protein kinase C-α and impairs the host cell's mitogen-activated protein kinase pathway, in order to invade cells. Cronobacter can also use immature dendritic cells and macrophages to escape the immune response. This review addresses the various virulence and environmental-adaptive characteristics possessed by members of the genus Cronobacter.