Suresh D. Pillai

Suppression of bacterial cell–cell signalling, biofilm formation and type III secretion system by citrus flavonoids

Aim:  This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids.

Antimicrobial Resistance Markers of Class 1 and Class 2 Integron- bearing Escherichia coli from Irrigation Water and Sediments

Municipal and agricultural pollution affects the Rio Grande, a river that separates the United States from Mexico. Three hundred and twenty-two Escherichia coli isolates were examined for multiple antibiotic resistance phenotypes and the prevalence of class 1 and class 2 integron sequences. Thirty-two (10%) of the isolates were resistant to multiple antibiotics. Four (13%) of these isolates contained class 1–specific integron sequences; one isolate contained class 2 integron–specific sequences. Sequencing showed that the class 1 integron–bearing strain contained two distinct gene cassettes, sat-1 and aadA. Although three of the four class 1 integron–bearing strains harbored the aadA sequence, none of the strains was phenotypically resistant to streptomycin. These results suggest that integron-bearing E. coli strains can be present in contaminated irrigation canals and that these isolates may not express these resistance markers.

Grapefruit juice and its furocoumarins inhibits autoinducer signaling and biofilm formation in bacteria.

Cell-to-cell communications in bacteria mediated by small diffusible molecules termed as autoinducers (AI) are known to influence gene expression and pathogenicity. Oligopeptides and N-acylhomoserine lactones (AHL) are major AI molecules involved in intra-specific communication in gram-positive and gram-negative bacteria respectively, whereas boronated-diester molecules (AI-2) are involved in inter-specific communication among both gram-positive and gram-negative bacteria. Naturally occurring furocoumarins from grapefruit showed >95% inhibition of AI-1 and AI-2 activities based on the Vibrio harveyi based autoinducer bioassay. Grapefruit juice and furocoumarins also inhibited biofilm formation by Escherichia coli O157:H7, Salmonella typhimurium and Pseudomonas aeruginosa. These results suggest that grape fruit juice and furocoumarins could serve as a source to develop bacterial intervention strategies targeting microbial cell signaling processes.

Autoinducer-2-like activity associated with foods and its interaction with food additives.

The autoinducer-2 (AI-2) molecule produced by bacteria as part of quorum sensing is considered to be a universal inducer signal in bacteria because it reportedly influences gene expression in a variety of both gram-negative and gram-positive bacteria. The objective of this study was to determine whether selected fresh produce and processed foods have AI-2-like activity and whether specific food additives can act as AI-2 mimics and result in AI-2-like activity. The luminescence-based response of the reporter strain Vibrio harveyi BB170 was used as the basis for determining AI-2 activity in the selected foods and food ingredients. Maximum AI-2 activity was seen on the frozen fish sample (203-fold, compared with the negative control) followed by tomato, cantaloupe, carrots, tofu, and milk samples. Interestingly, some samples were capable of inhibiting AI-2 activity. Turkey patties showed the highest inhibition (99.8% compared with the positive control) followed by chicken breast (97.5%), homemade cheeses (93.7%), beef steak (90.6%), and beef patties (84.4%). AI-2 activity was almost totally inhibited by sodium propionate, whereas sodium benzoate caused 93.3% inhibition, compared with 75% inhibition by sodium acetate. Sodium nitrate did not have any appreciable effect, even at 200 ppm. Understanding the relationships that exist between AI-2 activity on foods and the ecology of pathogens and food spoilage bacteria on foods could yield clues about factors controlling food spoilage and pathogen virulence.

Bioaerosol transport modeling and risk assessment in relation to biosolid placement.

Abstract A field study was performed in which bioaerosols were sampled at a field site undergoing land placement of anaerobically digested, de‐watered biosolid material. The data from these field studies were then used to generate microbial release rates from the biosolids for use in modeling bioaerosol transport. Continuous‐point sources represented by large biosolid piles (temporary storage before placement) in the field, and continuous‐area sources represented by large fields upon which biosolids were placed by spraying, were modeled using microbial transport models; and downwind microbial concentrations were generated. These quantified transport data were then entered into microbial dose‐response models in an attempt to characterize the risk of pathogenic bacteria and viruses infecting workers and nearby population centers. The risk of viral and bacterial infection to workers at biosolid land application sites is 3:100 and 2:100, respectively, under 2‐m/s wind conditions and 1 hr of exposure. The route of exposure proposed in this model is the transport, inhalation, deposition, and swallowing of bacterial or viral pathogens. Note that these risk models by nature would tend to overestimate the actual risk to populations (wastewater workers) consisting primarily of immunocompetent individuals. Under these low‐wind conditions, nearby population centers where such immunocompetent populations may exist (here considered to be 10 000 m from the land application sites) are predicted to be at little risk (1.95 × 10−:100) of infection from aerosolized bacteria and at no risk from aerosolized viruses.

Zeta Potential of Selected Bacteria in Drinking Water When Dead, Starved, or Exposed to Minimal and Rich Culture Media

The zeta potentials of E. coli, GFP (green fluorescence protein)-labeled E. coli, Salmonella Newport, and Pseudomonas sp. in different states (nutrient-starved and dead) and grown in rich and minimal media were measured. Capillary electrophoresis experiments were conducted to measure the zeta potential of the different cells suspended in a drinking water sample. Salmonella Newport strain showed a lower zeta potential compared to E. coli, GFP-labeled E. coli, and Pseudomonas sp. Starved E. coli cells had a lower zeta potential compared to E. coli cells grown under rich media conditions. Salmonella Newport cells grown in minimal media also had a lower zeta potential compared to rich, starved, and dead cells. The different bacterial cell types exhibited differences in size as well. These results suggest that when bacterial cells are present in drinking water they can exhibit significant heterogeneity in the size and zeta potential, depending on their physiological state.

Temporal regulation of enterohemorrhagic Escherichia coli virulence mediated by autoinducer-2

The autoinducer-2 (AI-2) molecule is produced by many bacterial species, including various human gastrointestinal (GI) tract commensal bacteria, and has been proposed to be involved in interspecies communication. Because pathogens are likely to encounter AI-2 in the GI tract, we studied the effects of AI-2 on various phenotypes associated with enterohemorrhagic Escherichia coli (EHEC) infections. AI-2 attracted EHEC in agarose plug chemotaxis assays and also increased swimming motility, as well as increased EHEC attachment to HeLa cells. The molecular basis underlying the stimulation of EHEC chemotaxis, motility, and colonization by AI-2 was investigated at the transcriptome level using DNA microarrays. We found that exposure to AI-2 altered the expression of 23 locus of enterocyte effacement (LEE) genes directly involved in the production of virulence determinants, as well as other genes associated with virulence (e.g., 46 flagellar/fimbrial genes, 24 iron-related genes), in a temporally defined manner. To our knowledge, this is the first study to report AI-2-mediated regulation of EHEC chemotaxis and colonization, as well as temporal regulation of EHEC transcriptome by AI-2. Our results suggest that AI-2 is an important signal in EHEC infections of the human GI tract.

Variability of virus attachment patterns to butterhead lettuce.

Enteric viruses account for most foodborne illness in the United States. The objective of this study was to determine whether the isoelectric point (pI) of viruses such as feline calicivirus (FCV), echovirus 11, and bacteriophages phiX174 and MS2 had any effect on their attachment to butterhead lettuce. The adsorption of virus particles to the lettuce was variable. Bacteriophage MS2 was the only virus that fit the current Derjaguin-Landau-Verway-Overbeek model of virus attachment. Echovirus 11 had the highest affinity to lettuce surface. Echovirus 11 appeared to exhibit reversible attachment above its pI, whereas below its pI strong adsorption was observed. Adsorption of FCV was at its maximum above its pI. Bacteriophage phiX174 exhibited the most complex adsorption pattern, with attachment occurring only at the pH extremes (pH 3.0 and 8.0). These results suggest the current model for virus adsorption to sediment does not adequately explain the attachment of virus to lettuce. Importantly, the results indirectly suggest that current sample processing methods to recover viruses from lettuce may differentially select for the recovery of only certain virus types.

Citrus limonoids interfere with Vibrio harveyi cell–cell signalling and biofilm formation by modulating the response regulator LuxO

Citrus limonoids are unique secondary metabolites, characterized by a triterpenoid skeleton with a furan ring. Studies have demonstrated beneficial health properties of limonoids. In addition, certain citrus limonoids play a role in plant defence against insect pests. In the present study, five limonoids were purified from sour orange and evaluated for their ability to inhibit cell-cell signalling. The purified limonoids were tested for their ability to interfere with cell-cell signalling and biofilm formation in Vibrio harveyi. Isolimonic acid, deacetylnomilinic acid glucoside and ichangin demonstrated significant inhibition of autoinducer-mediated cell-cell signalling and biofilm formation. Furthermore, isolimonic acid and ichangin treatment resulted in induced expression of the response regulator gene luxO. In addition, luxR promoter activity was not affected by isolimonic acid or ichangin. Therefore, the ability of isolimonic acid and ichangin to interfere with cell-cell signalling and biofilm formation seems to stem from the modulation of luxO expression. The results suggest that isolimonic acid and ichangin are potent modulators of bacterial cell-cell signalling.

Male-specific coliphages as an additional fecal contamination indicator for screening fresh carrots.

The objective of this study was to evaluate the efficacy of male-specific (F+) coliphages as a fecal-contamination indicator for fresh carrots. The prevalence of specific pathogens and indicator organisms on the surface of carrots obtained from a farm, truck, and processing shed was studied. Twenty-five carrot samples collected from each of these locations were washed, and aliquots of the wash were analyzed for the presence of F+ coliphages, Escherichia coli, Salmonella, and Shigella. Additionally, the Salmonella isolates were genotyped using pulsed-field gel electrophoresis (PFGE). Our studies detected the presence of F+ coliphages, E. coli, and Salmonella on carrots. All samples, however, tested negative for Shigella. Although none of the carrot samples from the field were positive for E. coli, one sample was positive for Salmonella, and another was positive for F+ coliphages. From the truck, two carrot samples (8%) were positive for Salmonella, four (16%) were positive for F+ coliphages, and four (16%) were positive for E. coli. None of the carrot samples from the processing shed were positive for Salmonella. However, 2 carrot samples (8%) were positive for E. coli, and 14 carrot samples (56%) were positive for F+ coliphages. The PFGE results suggest that there were three distinct Salmonella genotypes among the carrot samples from the truck and that the Salmonella isolates identified on carrot samples from the field and truck locations were different. Microbiological screening of fresh produce such as carrots (which can be exposed to fecal contaminants in soils and water) should ensure the detection of both viral and bacterial contaminants. Overall, in this study, F+ coliphages were detected in 25% of the carrot samples, compared to E. coli (8%), Salmonella (4%), and Shigella (0%). The results suggest F+ coliphages can serve as a conservative indicator of fecally associated viruses on carrots. This suggests that in addition to E. coli screening, F+ coliphages should be included when produce such as carrots that are vulnerable to fecal contaminants are screened. Since the detection of specific enteric viral pathogens is expensive, screening for viral indicators of fecal contamination using F+ coliphages can be an economical approach to providing an additional level of assurance about the microbiological quality of fresh carrots.