Ziad W. Jaradat

International survey of Cronobacter sakazakii and other Cronobacter spp. in follow up formulas and infant foods

A coordinated survey for Cronobacter and related organisms in powdered infant formula, follow up formula and infant foods was undertaken by 8 laboratories in 7 countries in recognition of and in response to the data needs identified in an FAO/WHO call for data in order to develop global risk management guidance for these products. The products (domestic and imported) were purchased from the local market and were categorised according to their principle ingredients. A total of 290 products were analysed using a standardised procedure of pre-enrichment in 225 ml Buffered Peptone Water (BPW), followed by enrichment in Enterobacteriaceae Enrichment (EE) broth, plating on the chromogenic Cronobacter Druggan-Forsythe-Iversen (DFI) agar and presumptive identification with ID 32 E. Presumptive Cronobacter isolates were identified using 16S rRNA gene sequence analysis. Aerobic plate counts (APC) of the products were also determined on nutrient agar. Fourteen samples had APC>10(5) cfu/g, 3 of which contained probiotic cultures. C. sakazakii was isolated from 27 products; 3/91 (3%) follow up formulas (as defined by Codex Alimentarius Commission), and 24/199 (12%) infant foods and drinks. Hence C. sakazakii was less prevalent in follow up formula than other foods given to infants over the same age range. A range of other bacteria were also isolated from follow up formulas, including Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, Citrobacter freundii, and Serratia ficaria. There was significant variation in the reconstitution instructions for follow up formulas. These included using water at temperatures which would enable bacterial growth. Additionally, the definition of follow up formula varied between countries.

Genetic homogeneity among Listeria monocytogenes strains from infected patients and meat products from two geographic locations determined by phenotyping, ribotyping and PCR analysis of virulence genes

Thirty Listeria monocytogenes isolates from human patients and foods originated from two different geographic locations without any epidemiological relations were analyzed for their genotypic and phenotypic virulence gene expressions and genetic relatedness. All strains contained virulence genes, inlA, inlB, actA, hlyA, plcA and plcB, with expected product size in PCR assay except for the actA gene. Some strains produced actA gene product of 268 and others 385 bp. Phenotypically, all were hemolytic but showed variable expressions of phospholipase activity. Ribotyping classified isolates into 12 different groups based on the similarity to DuPont Identification numbers (DID), which consisted primarily of clinical or food isolates or both. Cluster analysis also indicated possible existence of clones of L. monocytogenes that are found in food or human hosts or are evenly distributed between these two. Two isolates (F1 from food and CHL1250 from patient) had unique ribotype patterns that were not previously reported in the RiboPrinter database. This study indicates distribution of diverse L. monocytogenes strains in clinical and food environments. The isolates showed 92-99% genetic homogeneity, in spite of their origins from two different geographic locations and environments.

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.

BackgroundCronobacter 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.ResultsIn 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 (α-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.ConclusionOur 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.

Heat Shock Protein 60 Acts as a Receptor for the Listeria Adhesion Protein in Caco-2 Cells

ABSTRACT The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

Effect of T-2 toxin on in vivo lipid peroxidation and vitamin E status in mice

The effects of an acute administration of T-2 toxin on vitamin E status and the corresponding degree of lipid peroxidation, as determined by the plasma and organ content of malondialdehyde (MDA), was studied in mice. The effects of T-2 toxin administration on the body weight and weights of liver, spleen and thymus were also assessed. T-2 toxin was administered in doses ranging from 1 to 6.25 mg/kg body weight, depending on the experiment, while the dietary content of vitamin E ranged from near 0 to 5000 IU/kg. There was a significant decrease in vitamin E content of plasma after the administration of the toxin with the concentrations remaining low for periods as long as 48-72 h. MDA content of liver increased significantly after 24-48 h of toxin administration in contrast to the controls. However, MDA levels returned to the control range after 72 h. The concentrations of MDA in liver were inversely related to the vitamin E content of the diet, and were always higher for the toxin-treated animals (significant linear regression between MDA content of liver and the log10 of vitamin E content of the diet). Weights of spleen and thymus decreased after T-2 toxin administration; however, the weight of liver either increased or did not change in the different experiments. In conclusion, T-2 toxin treatment of mice increased lipid peroxidation in the liver as measured by MDA production. This process was maximal after 48 h of T-2 challenge, and decreased thereafter. Plasma alpha-tocopherol levels decreased as soon as 6 h after the toxin challenge, while MDA did not increase until there was a severe depletion of vitamin E. These changes were accompanied by decrease in weight of spleen and thymus.

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 cells 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.

Glucose and Nutrient Concentrations Affect the Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

ABSTRACT Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K2HPO4 reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.

Influence of Culture Conditions on Pectinase Production by Streptomyces sp. (Strain J9)

The purpose of this study was to determine the influence of growth conditions and medium composition on the cellulase enzyme production by Streptomyces sp. Production of cellulase enzyme by a Streptomyces strain (J2) was detected on cellulose agar (CA) medium after 4 days of incubation at 28 °C that exhibited a clear zone of 22 mm around the colony. Cellulase production was assayed by measuring the amount of glucose liberated in μmol/ml/min by using the dinitrosalicylic acid assay method. The highest crude enzyme activity (432 U/l) was observed after 3 days of incubation at pH 7 and 60 °C in a medium that was supplemented with 0.5% glucose, 0.2% starch, and 0.2% NH4CL. However, enzyme production and activity were strongly decreased at 45°C and acidic pH. Enzyme production and activity were also inhibited when Streptomyces strain (J2) was grown in CMC broth supplemented with arabinose and yeast extract as a sole carbon and nitrogen source, respectively. ﻂﺳﻮﻟا ﺐﻴآﺮﺗو ﻮﻤﻨﻟا فوﺮﻇ ﺮﻴﺛﺄﺗ ﺔﺳارد ﺚﺤﺒﻟا اﺬه ﻦﻣ فﺪﻬﻟا نﺎآ ـﻟا ﺎﻳﺮﻴﺘﻜﺑ ﻦﻣ ﺰﻴﻠﻴﻠﺴﻟا ﻢﻳﺰﻧإ جﺎﺘﻧإ ﻰﻠﻋ ﻲﺋاﺬﻐﻟا Streptomyces . ﻢﺗ

A Listeria adhesion protein-deficient Listeria monocytogenes strain shows reduced adhesion primarily to intestinal cell lines

Abstract.Listeria monocytogenes adheres and penetrates intestinal cell linings for systemic infection. A 104-kDa Listeria adhesion protein (LAP) from L. monocytogenes was previously demonstrated to be responsible for adhesion to intestinal enterocyte-like Caco-2 cells. We investigated the adhesion and invasion characteristics of a LAP-deficient mutant L. monocytogenes strain (A572) to various human intestinal and non-intestinal cell lines to assess the possible target host cells. Among the intestinal cell lines, A572 showed significantly reduced adhesion than the wild type (WT) strain to the cells of ileum-cecum (HCT-8) and colon (Caco-2 and HT-29), whereas A572 and WT did not show any significant differences in adhesion to other intestinal cell lines from duodenum (HuTu-80) or jejunum (Int-407). Differences in adhesion between A572 and WT were little or none in non-intestinal cell lines from liver, kidney, bladder, ovary, cervix, breast, larynx, or skin. Invasion data showed that A572 was invasive but the invasion efficiency was proportional to its adhesion characteristics to respective cell lines. In mouse bioassay, A572 was not found in liver following oral administration, suggesting that LAP mutant was possibly unable to pass through intestinal cell linings. Immuno-electron microscopy revealed that the LAP is localized in the bacterial surface as well as the cytoplasm. In summary, this study indicated that the LAP-mediated adhesion is associated with the intestinal cells originating from the lower part of small intestine and from the upper part of large intestine, and possibly plays an important role during the intestinal phase of infection.

Impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii.

Cronobacter sakazakii is an emerging foodborne pathogen that has been implicated in severe forms of meningitis, septicemia or necrotizing colitis in pre-term neonates. Although illness outbreaks (primarily associated with powdered infant formula, PIF) caused by this pathogen are rare, the case-fatality rate may reach 50%. Successful treatment of C. sakazakii infection is reliant upon clinical use of antibiotics (AB) such as ampicillin. Recent reports showed increased resistance of C. sakazakii to broad-spectrum antibiotics. The objective of this study was to evaluate the effect of extreme pH (3.5 for 30 min or 11.25 for 5 min), cold (4°C for 24h), heat (55°C for 5 min), and desiccation (cells were dried at 40°C for 2h and held at 21°C for 4 d) stresses on susceptibility of five isolated strains of C. sakazakii to streptomycin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, tilmicosin, florfenicol, ampicillin, amoxicillin, vancomycin, ciprofloxacin and enrofloxacin. All unstressed strains of C. sakazakii were sensitive to streptomycin, gentamycin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and amoxicillin, but were moderately resistant or resistant to the rest. Exposing cells to alkaline or acidic stress did not change their sensitivity toward streptomycin, gentamycin, kanamycin or ciprofloxacin, but their resistance toward the other AB was increased. Cells stressed by desiccation showed increased sensitivity toward streptomycin, gentamicin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and doxycycline, but showed resistance toward the others. Cold-stressed cells were more sensitive to streptomycin, gentamicin, kanamycin, and ciprofloxacin compared with heat-stressed cells, but both heat and cold-stressed cells showed increased resistance toward all the other AB. Results obtained will help in understanding the effect of environmental stresses during processing on C. sakazakii susceptibility to AB.