Shlomo Sela

Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs

Despite washing and decontamination, outbreaks linked to consumption of fresh or minimally-processed leafy greens have been increasingly reported in recent years. In order to assure the safety of produce it is necessary to gain knowledge regarding the exact routes of contamination. Leaf internalization through stomata was previously reported as a potential route of contamination, which renders food-borne pathogens protected from washing and disinfection by sanitizers. In the present study we have examined the incidence (percentage of microscopic fields harboring ≥ 1 GFP-tagged bacteria) of Salmonella Typhimurium on the surface and underneath the epidermis in detached leaves of seven vegetables and fresh herbs. The incidence of internalized Salmonella varied considerably among the different plants. The highest incidence was observed in iceberg lettuce (81 ± 16%) and arugula leaves (88 ± 16%), while romaine (16 ± 16%) and red-lettuce (20 ± 15%), showed significantly lower incidence (P < 0.05). Internalization incidence in fresh basil was 46 ± 12%, while parsley and tomato leaves demonstrated only marginal internalization (1.9 ± 3.3% and 0.56 ± 1.36%, respectively). Internalization of Salmonella in iceberg lettuce largely varied (0-100%) through a 2 year survey, with a higher incidence occurring mainly in the summer. These results imply that Salmonella internalization occurs in several leafy vegetables and fresh herbs, other than iceberg lettuce, yet the level of internalization largely varies among plants and within the same crop. Since internalized bacteria may evade disinfection, it is of great interest to identify plants which are more susceptible to bacterial internalization, as well as plant and environmental factors that affect internalization.

Interactions of Salmonella enterica with lettuce leaves.

Aims:  To investigate the interactions of Salmonella enterica with abiotic and plant surfaces and their effect on the tolerance of the pathogen to various stressors.

Effect of Desiccation on Tolerance of Salmonella enterica to Multiple Stresses

ABSTRACT Reducing the available water in food is a long-established method for controlling bacterial growth in the food industry. Nevertheless, food-borne outbreaks of salmonellosis due to consumption of dry foods have been continuously reported. Previous studies showed that dried Salmonella cells acquire high tolerance to heat and ethanol. In order to examine if dehydration also induces tolerance to other stressors, dried Salmonella enterica serotype Typhimurium cells were exposed to multiple stresses, and their viability was assessed. Indeed, desiccated S. Typhimurium acquired higher tolerance to multiple stressors than nondesiccated cells. The dried cells were significantly more resistant to most stressors, including ethanol (10 to 30%, 5 min), sodium hypochlorite (10 to 100 ppm, 10 min), didecyl dimethyl ammonium chloride (0.05 to 0.25%, 5 min), hydrogen peroxide (0.5 to 2.0%, 30 min), NaCl (0.1 to 1 M, 2 h), bile salts (1 to 10%, 2 h), dry heat (100°C, 1 h), and UV irradiation (125 μW/cm2, 25 min). In contrast, exposure of Salmonella to acetic and citric acids reduced the survival of the dried cells (1.5 log) compared to that of nondesiccated cells (0.5 log). Three other S. enterica serotypes, S. Enteritidis, S. Newport, and S. Infantis, had similar stress responses as S. Typhimurium, while S. Hadar was much more susceptible and gained tolerance to only a few stressors. Our findings indicate that dehydration induces cross-tolerance to multiple stresses in S. enterica, demonstrating the limitations of current chemical and physical treatments utilized by the food industry to inactivate food-borne pathogens.

Distribution of Salmonella typhimurium in romaine lettuce leaves

Leafy greens are occasionally involved in outbreaks of enteric pathogens. In order to control the plant contamination it is necessary to understand the factors that influence enteric pathogen-plant interactions. Attachment of Salmonella enterica serovar typhimurium to lettuce leaves has been demonstrated before; however, only limited information is available regarding the localization and distribution of immigrant Salmonella on the leaf surface. To extend our knowledge regarding initial pathogen-leaf interactions, the distribution of green-fluorescent protein-labeled Salmonella typhimurium on artificially contaminated romaine lettuce leaves was analyzed. We demonstrate that attachment of Salmonella to different leaf regions is highly variable; yet a higher attachment level was observed on leaf regions localized close to the petiole (7.7 log CFU g(-1)) compared to surfaces at the far-end region of the leaf blade (6.2 log CFU g(-1)). Attachment to surfaces located at a central leaf region demonstrated intermediate attachment level (7.0 log CFU g(-1)). Salmonella displayed higher affinity toward the abaxial side compared to the adaxial side of the same leaf region. Rarely, Salmonella cells were also visualized underneath stomata within the parenchymal tissue, supporting the notion that this pathogen can also internalize romaine lettuce leaves. Comparison of attachment to leaves of different ages showed that Salmonella displayed higher affinity to older compared to younger leaves (1.5 log). Scanning electron microscopy revealed a more complex topography on the surface of older leaves, as well as on the abaxial side of the examined leaf tissue supporting the notion that a higher attachment level might be correlated with a more composite leaf landscape. Our findings indicate that initial attachment of Salmonella to romaine lettuce leaf depends on multiple plant factors pertaining to the specific localization on the leaf tissue and to the developmental stage of the leaf.

Biofilm Formation by and Multicellular Behavior of Escherichia coli O55:H7, an Atypical Enteropathogenic Strain

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an important causal agent of diarrheal illness throughout the world. Nevertheless, researchers have only recently begun to explore its capacity to form biofilms. Strain O55:H7 (DMS9) is a clinical isolate belonging to the atypical EPEC (aEPEC) group, which displays a high degree of genetic relatedness to enterohemorrhagic E. coli. Strain DMS9 formed a robust biofilm on an abiotic surface at 26°C, but not at 37°C. It also formed a dense pellicle at the air-liquid interface and developed a red, rough, and dry (RDAR) morphotype on Congo red agar. Unlike a previously described E. coli O157:H7 strain, the aEPEC strain seems to express cellulose. Transposon mutagenesis was used to identify biofilm-deficient mutants. One of the mutants was inactivated in the csgFG genes, required for assembly and secretion of curli fimbriae, while a second mutant had a mutation in crl, a thermosensitive global regulator that modulates σS activity and downstream expression of curli and cellulose. The two mutants were deficient in their biofilm formation capabilities and did not form a pellicle at the air-liquid interface. Unlike in Salmonella, the csgFG mutant in aEPEC completely lost the RDAR phenotype, while the crl mutant displayed a unique RDAR “pizza”-like morphotype. Genetic complementation of the two mutants resulted in restoration of the wild-type phenotype. This report is the first to describe and analyze a multicellular behavior in aEPEC and support a major role for curli and the crl regulator in biofilm development at low temperatures corresponding to the nonmammalian host environment.

Identification of a bovine mastitis Escherichia coli subset.

Eleven Escherichia coli isolates from clinical bovine mastitis cases (mastitic strains) and 11 from the cowshed environment (environmental strains) were compared, to determine if the former were a subset of the latter. The mastitic and environmental strains could not be distinguished according to O antigen and antibiotic sensitivity. All mastitic isolates showed significantly (P<0.0001) faster growth in milk and faster lactose fermentation than most (approximately 64%) environmental strains, but growth rates in nutrient broth did not differ. The rates of lactose fermentation and growth in milk were positively correlated. Adhesion and phagocytosis of mastitic strains by bovine PMN were significantly (P<0.0001) lower than those of environmental strains, and correlated negatively with growth in milk and lactose fermentation. The average percentages of killing by bovine leukocytes in the two sources were not statistically different. All mastitic strains were serum sensitive, whereas most ( approximately 72%) environmental ones were resistant. Finally, pulse-field gel electrophoresis revealed two main pulse type clusters, sharing a similarity coefficient of 79%. Cluster 1 comprised only environmental strains, whereas cluster 2 comprised mostly mastitic strains and only three environmental ones. Four mastitic strains shared a similarity coefficient of less than 74% with the other strains and were not included in the clusters. Our results suggest that clinical bovine mastitis E. coli isolates may form a subset of the general environmental E. coli population; they seem better able to multiply in the udder medium and to evade the host cellular innate immune response, and are genetically distinct from most environmental strains.

Persistence of Salmonella enterica during dehydration and subsequent cold storage

Despite the fact that Salmonella enterica serotype Typhimurium SL 1344 has served as a model pathogen in many studies, information regarding its desiccation response is still scarce. In this study, we investigated environmental conditions that affect Salmonella survival following dehydration and subsequent cold storage, using a 96-well polystyrene plate model. The SL 1344 strain exhibited high survival compared with other Typhimurium isolates and S. enterica serotypes. Further characterization of desiccation tolerance in this strain revealed that temperature, stationary-phase of growth, solid medium, and the presence of increasing NaCl concentrations (0.5-5.0%) in the growth medium enhanced desiccation tolerance. Dehydration at basic pHs (8-10), or in trehalose, sucrose, but not in glycine-betaine, improved bacterial persistence. Dehydrated Salmonella survived over 100 weeks at 4 °C with a ∼5-log reduction in numbers. However, viability staining revealed only a ∼50% reduction in viable cells, suggesting bacterial transition into a viable-but-not-cultivable state (VBNC). Addition of chloramphenicol reduced bacterial survival implying that adaptation to desiccation stress requires de-novo protein synthesis. Consistent with this finding, shortening the dehydration time resulted in lower survival. This study emphasizes the impact of environmental conditions on the fate of dried Salmonella in the food chain and highlights the potential transition of the pathogen to the VBNC state.

Efficiency of phenol biodegradation by planktonic Pseudomonas pseudoalcaligenes (a constructed wetland isolate) vs. root and gravel biofilm.

In the last two decades, constructed wetland systems gained increasing interest in wastewater treatment and as such have been intensively studied around the world. While most of the studies showed excellent removal of various pollutants, the exact contribution, in kinetic terms, of its particular components (such as: root, gravel and water) combined with bacteria is almost nonexistent. In the present study, a phenol degrader bacterium identified as Pseudomonas pseudoalcaligenes was isolated from a constructed wetland, and used in an experimental set-up containing: plants and gravel. Phenol removal rate by planktonic and biofilm bacteria (on sterile Zea mays roots and gravel surfaces) was studied. Specific phenol removal rates revealed significant advantage of planktonic cells (1.04 × 10(-9) mg phenol/CFU/h) compared to root and gravel biofilms: 4.59 × 10(-11)-2.04 × 10(-10) and 8.04 × 10(-11)-4.39 × 10(-10) (mg phenol/CFU/h), respectively. In batch cultures, phenol biodegradation kinetic parameters were determined by biomass growth rates and phenol removal as a function of time. Based on Haldane equation, kinetic constants such as μ(max) = 1.15/h, K(s) = 35.4 mg/L and K(i) = 198.6 mg/L fit well phenol removal by P. pseudoalcaligenes. Although P. pseudoalcaligenes planktonic cells showed the highest phenol removal rate, in constructed wetland systems and especially in those with sub-surface flow, it is expected that surface associated microorganisms (biofilms) will provide a much higher contribution in phenol and other organics removal, due to greater bacterial biomass. Factors affecting the performance of planktonic vs. biofilm bacteria in sub-surface flow constructed wetlands are further discussed.

The response of foodborne pathogens to osmotic and desiccation stresses in the food chain.

In combination with other strategies, hyperosmolarity and desiccation are frequently used by the food processing industry as a means to prevent bacterial proliferation, and particularly that of foodborne pathogens, in food products. However, it is increasingly observed that bacteria, including human pathogens, encode mechanisms to survive and withstand these stresses. This review provides an overview of the mechanisms employed by Salmonella spp., Shiga toxin producing E. coli, Cronobacter spp., Listeria monocytogenes and Campylobacter spp. to tolerate osmotic and desiccation stresses and identifies gaps in knowledge which need to be addressed to ensure the safety of low water activity and desiccated food products.

Phenotypic and genotypic characterization of Pseudomonas aeruginosa strains isolated from mastitis outbreaks in dairy herds

During 1998-2002 outbreaks of Pseudomonas sp. mastitis among more than 15 Israeli sheep and goat dairy herds were observed. The animals presented a wide spectrum of clinical signs ranging from subclinical to gangrenous udder. Ninety-five isolates of Pseudomonas sp. were isolated from clinical and subclinical mastitis of 47 sheep, 17 goats and 31 cows from 34 different farms. Biochemical and genetic analyses revealed that the all-causative organism was Ps. aeruginosa. Selections of isolates were further analysed on the bases of colony morphology, biochemical traits and capacity to form biofilm. All the strains displayed a wide heterogeneity in all the tested traits. No association between bacterial isolates, farm of origin and type of animal was found. Pulsed-field gel electrophoresis and cluster analysis showed no clonality among the tested strains. The present study revealed that a large variety of Ps. aeruginosa strains may cause mastitis outbreaks in sheep, goat and cattle in Israel.