Our research is primarily focused on the transmission and evolution of two zoonotic pathogens, enterohemorrhagic Escherichia coli (EHEC) and Salmonella. These pathogens reside in the intestinal tracts of animal hosts where they encounter diverse microbial communities, fluctuating nutrient levels, and myriad host factors. Transmission between hosts requires these pathogens to survive varied environmental conditions. The general stress protection system (regulated by the alternative sigma factor, σs) is known to play a central role in environmental persistence and transmission. Acid and desiccation tolerance are two transmission-associated phenotypes that are dependent upon σs –regulated genes. We are also investigating the role of prophage in fitness. EHEC harbor multiple lambda-like prophage and cryptic phage remnants in their genome that facilitate genomic rearrangements, gene duplications, and deletions by homologous recombination. We are investigating how these phage-mediated genomic rearrangements influence the persistence of EHEC in its bovine host and the environment. The goals of our research are to use results from these fundamental studies in the development of strategies to reduce pathogen transmission.
Microbiology 325: Food Microbiology
Food Research Institute
Professor, Department of Animal Sciences
Molecular and Environmental Toxicology Center
Salmonella enterica is a human pathogen that can produce filamentous cells in response to environmental stress. The molecular mediators and biosynthetic pathways that contribute to the formation of filamentous cells (>10 µm in length) during osmotic stress are mostly unknown. The comparison of filamentous and non-filamentous cells in this study was aided by the use of a filtration step to separate cell types. Osmotic stress caused an efflux of phosphate from cells, and the addition of phosphate and a carbohydrate to Luria broth with 7 % NaCl (LB-7NaCl) significantly increased the proportion of filamentous cells in the population (58 %). In addition to direct measurements of intracellular and extracellular phosphate concentrations, the relative abundance of the iraP transcript that is induced by phosphate limitation was monitored. Non-filamentous cells had a greater relative abundance of iraP transcript than filamentous cells. IraP also affects the stability of RpoS, which regulates the general stress regulon, and was detected in non-filamentous cells but not filamentous cells. Markers of metabolic pathways for the production of acetyl-CoA (pflB, encoding for pyruvate formate lyase) and fatty acids (fabH) that are essential to membrane biosynthesis were found in greater abundance in filamentous cells than non-filamentous cells. There were no differences in the DNA, protein and biomass levels in filamentous and non-filamentous cells after 48 h of incubation, although the filamentous cells produced significantly (P<0.05) more acetate. This study found that phosphate and carbohydrate enhanced the formation of filamentous cells during osmotic stress, and there were differences in key regulatory elements and markers of metabolic pathways in filamentous and non-filamentous S. enterica.
Six major Shiga toxin producing Escherichia coli (STEC) serogroups: O26, O103, O145, O111, O121, and O45 have been declared as adulterants in federally inspected raw beef in the USA effective June 4th, 2012 in addition to the routinely tested STEC O157: H7. This study tests a real-time multiplex PCR assay and pooling of the samples to optimize the detection and quantification (prevalence and contamination) of six major non-O157 STEC, regardless of possessing Shiga toxins. To demonstrate the practicality, one large-scale slaughter plant (Plant LS) and one small-scale slaughter plant (Plant SS) located in the Mid-Western USA were sampled, in 2011, before the establishment of 2013 USDA laboratory protocols. Carcasses were sampled at consecutive intervention stations and beef trimmings were collected at the end of the fabrication process. Plant SS had marginally more contaminated samples than Plant LS (p-value 0.08). The post-hide removal wash, steam pasteurization, and lactic acid (≤5%) spray used in Plant LS seemed to reduce the six serogroups effectively, compared to the hot-water wash and 7-day chilling at Plant SS. Compared to the culture isolation methods, quantification of the non-O157 STEC using real-time PCR may be an efficient way to monitor the efficacy of slaughter line interventions.
A pen infection-transmission experiment was conducted to elucidate the role of pathogen strain and environmental contamination in transmission of Escherichia coli O157:H7 (ECO157) in cattle. Five steers were inoculated with a three-strain mixture of ECO157 and joined with five susceptible steers in each of two experimental replicates. Faecal and environmental samples were monitored for ECO157 presence over 30 days. One ECO157 strain did not spread. Transmission rates for the other two strains were estimated using a generalized linear model developed based on a modified 'Susceptible-Infectious-Susceptible' mathematical model. Transmission rates estimated for the two strains (0·11 and 0·14) were similar. However, the rates significantly (P = 0·0006) increased 1·5 times for every 1-unit increase in the level of environmental contamination measured as log10 c.f.u. Depending on the level of environmental contamination, the estimated basic reproduction numbers varied from <1 to 8. The findings indicate the importance of on-farm measures to reduce environmental contamination for ECO157 control in cattle that should be validated under field conditions.
Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR=16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR=0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR=0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.
Salmonella enterica can survive harsh environmental conditions, including hyperosmotic stress. It is well established that the alternative sigma factor, σ(s) (RpoS), is required for maximal survival of enteric pathogens, including S. enterica. Although RpoS levels are greatest during stationary phase or stress conditions, RpoS can be found in S. enterica during growth. However, its activity during growth is poorly characterized. In this study, the impact of RpoS levels on the growth of S. enterica in LB supplemented with 6% NaCl (LB-NaCl) was examined. Cells in stationary phase prior to inoculation into LB-NaCl had a shorter lag phase than did exponential-phase cells. In addition, the deletion of rpoS from S. enterica Typhimurium M-09 (M-09 ΔrpoS) increased the length of lag phase in LB-NaCl relative to the parental strain. Complementation of M-09 ΔrpoS in trans by an inducible plasmid encoding rpoS reduced the length of lag phase. The length of lag phase in both the rpoS mutant and complemented strain was independent of their growth phase prior to inoculation of LB-NaCl. The results from this study demonstrate that the level of RpoS influences the length of lag phase and the growth of S. enterica in hyperosmotic growth conditions.
Shiga toxin (stx)-producing Escherichia coli O157 : H7 is a prominent food-borne pathogen. Symptoms in human infections range from asymptomatic to haemorrhagic colitis and haemolytic uraemic syndrome, and there is a need for methods that yield information that can be used to better predict clinical and epidemiological outcomes. IS629 is an insertion sequence notable for its prevalence and variable distribution in the chromosome of E. coli O157 : H7, which has been exploited for subtyping and strain characterization. In particular, IS629 distribution is closely aligned with the major phylogenetic lineages that are known to be distinctive in their genome structure and virulence potential. In the present study, a comprehensive subtyping method in which IS629-typing was combined with stx genotyping was developed using a conventional PCR approach. This method consisted of a set of 32 markers based on the unique distribution of IS629 in the three major phylogenetic lineages of E. coli O157 : H7 and six additional markers to determine the stx genotype, a key virulence signature associated with each lineage. The analysis of IS629 loci variation with the 32 markers allowed us to determine the IS629 distribution profile (IDP), phylogenetic lineage and genetic relatedness of the 31 E. coli O157 : H7 strains examined. An association between IDP typing and stx genotype was observed. The use of both IDP and the stx genotype for strain characterization provided confirmative and complementary data in support of lineage placement of closely related strains. In addition, IS629/stx profiles were in agreement with strain segregation based on LSPA-6 (lineage-specific polymorphism assay) and PFGE subtyping, demonstrating its potential as a subtyping and strain tracking method.
Disease outbreaks due to the consumption of legume seedlings contaminated with human enteric bacterial pathogens like Escherichia coli O157:H7 and Salmonella enterica are reported every year. Besides contaminations occurring during food processing, pathogens present on the surface or interior of plant tissues are also responsible for such outbreaks. In the present study, surface and internal colonization of Medicago truncatula, a close relative of alfalfa, by Salmonella enterica and Escherichia coli O157:H7 were observed even with inoculum levels as low as two bacteria per plant. Furthermore, expression analyses revealed that approximately 30% of Medicago truncatula genes were commonly regulated in response to both of these enteric pathogens. This study highlights that very low inoculum doses trigger responses from the host plant and that both of these human enteric pathogens may in part use similar mechanisms to colonize legume seedlings.
Escherichia coli O157:H7 is a human pathogen capable of causing hemorrhagic colitis and in some cases hemolytic uremic syndrome. Cattle are an asymptomatic carrier and a major reservoir of this pathogen that can be transmitted by contaminated foods like beef products and vegetables. To further understand persistence in cattle and on farms, a total of 1716 samples over a two-year period were collected from a Wisconsin dairy farm (Farm R) and 91 were positive for the presence of E. coli O157:H7. Seventy-six of 1373 (4.8%) fecal samples and 10/190 (5.3%) water samples were positive. Genotyping of the 341 E. coli O157 isolates by pulsed-field gel electrophoresis showed nine different restriction enzyme digestion profile (REDP) types, seven of which were 93-98% similar (comprised of serotype O157:H7 isolates) and two that were dissimilar (serotype O157:H-isolates). The REDP 31 strain dominated and was isolated from 59 fecal and 9 water samples; 75% of the positive samples (68/91) contained this strain. Growth studies of representative strains from each the REDP groups in Luria broth at 25 and 39 °C found no significant differences between the strains. In LB supplemented with bile salts (3, 6, and 9%; 39 °C, 48 h), the REDP 30 strain had a longer lag phase and achieved a lower maximum density than the other strains in the presence of 6 and 9% bile salts. Likewise, the survival of the strains in low-pH conditions (HCl, pH 2.0 and acetic acid, pH 3.0) were similar except for the REDP 30 strain which was significantly less acid tolerant at pH 2.0. A screening for differences in carbohydrate utilization found that the dominant strain (REDP 31) utilized the most carbon sources and was the only strain that oxidized amygdalin, citraconic acid, α-ketoglutarate, and γ-cyclodextrin. The inoculation of Holstein calves with a three-strain mixture (REDP 30, 31, and 36 strains) found the REDP 31 strain (FRIK 2455) dominated in fecal and rectal swab samples throughout the durations of shedding. These results suggested that carbohydrate utilization and host factors encountered during animal passage select for persistent and predominant strains on farms.
Escherichia coli O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms, stx(2) subtypes, and the levels of stx(2) transcript and toxin. The E. coli O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of stx(2a) by IS1203v (farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished stx(2c) transcription and induction of the prophage and stx(2a)::IS1203v in Stx2a-encoding prophage generated a truncated stx(2a) mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e., stx(2a)::IS1203v). To our knowledge, this is the first study that details the evolution of E. coli O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative E. coli O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.
This study analysed the growth and survival of 18 strains of the six serotypes of non-O157 Shiga toxin-producing Escherichia coli (STEC) (O26, O45, O103, O111, O121 and O145) most frequently implicated in human illness and compared them with Escherichia coli O157:H7 strain ATCC43895. The data from growth in Luria-Bertani broth (LB)-HCl (pH 4.0, 4.5, 4.8), LB-lactate (pH 4.5 and 4.8) and LB-NaCl (5%, 7%) were fitted to modified Gompertz equations to enable quantitative comparisons across strains and media conditions. Serogroup O45 strains had growth rates that were equal to or significantly greater than the O157:H7 control strain in all growth conditions tested. The growth rate was independent from the maximum growth achieved, but three strains (103A, 121A and 45B) had significantly faster growth and greater maximum cell densities in LB-NaCl 5% (strain 103A), LB-HCl pH 4·0 (strain 121A) and LB-NaCl 7% (strain 45B). Survival in LB-HCl pH 3.0 of four strains (103C, 111B, 26B and 26C) was significantly greater and five strains (26A, 45A, 111A, 121A and 145A) were significantly reduced in comparison with the O157:H7 control strain. None of the STEC strains had greater survival in LB-NaCl 12% than the O157:H7 control strain. A significant association was found between the exponential phase, but not stationary phase, RpoS level and survival of STEC. Some STEC strains had growth or survival properties that exceeded those of the O157:H7 control strain, but none of the non-O157 STEC had both significantly greater growth and survival properties. STEC survival was associated with the exponential-phase RpoS level. Results from this study define the variability in growth and survival of STEC strains that will be useful defining food product formulations and process interventions to control STEC. The presence of exponential phase σ(s) expands the significance of this alternative sigma factor.
The contribution of RecA, Dps, and RpoS to survival of Escherichia coli O157:H7 during desiccation and osmotic stress was determined in Luria-Bertani broth with 12 % NaCl (LB-12) at 30 and 37 °C, on filter disks at 23 and 30 °C, and in sterile bovine feces at 30 °C. RecA did not significantly contribute to survival in any condition or temperature. The contribution of Dps to survival was only significant in LB-12 at 37 °C. RpoS was necessary for survival during desiccation and osmotic stress, and survival of the RpoS mutant was significantly less than the parent in all conditions and temperatures. The RpoS mutant survived up to 21 days in bovine feces, <4 days on filter disks, and >8 and <4 days in LB-12 at 30 and 37 °C, respectively. The parent, ΔrecA, dps, and dps/ΔrecA mutant strains survived >8 days in LB-12, >28 days on filter disks, and >28 days in bovine feces. Increased incubation temperatures were associated with decreased survival. E. coli O157:H7 can persist in desiccating and osmotically challenging environments, especially sterile feces, for an extended period time.
Experimental oral challenge studies with three different genotypes of Escherichia coli O157:H7 were conducted in cattle to determine the genotype-specific variability in shedding frequencies and concentrations and the frequency and extent of contamination of the environment. The results indicated that the E. coli O157:H7 genotype and ecological origin maybe important factors for the occurrence and concentration in the cattle host. Four groups of six young Holstein steers each were orally challenged with 10(6) CFU of one of three E. coli O157:H7 strains: FRIK 47 (groups 1 and 2), FRIK 1641 (group 3), and FRIK 2533 (group 4). Recto-anal mucosal swabs (RAMS) and environmental samples were taken on alternate days over 30 days. The numbers of E. coli O157:H7 cells and generic E. coli cells per sample were determined. Also, the presence and absence of 28 gene targets were determined for 2,411 isolates using high-throughput real-time PCR. Over the study period, strains FRIK 47, FRIK 1641, and FRIK 2533 were detected in 52%, 42%, and 2% of RAMS, respectively. Environmental detection of the challenge strains was found mainly in samples of the hides and pen floors, with strains FRIK 47, FRIK 1641, and FRIK 2533 detected in 22%, 27%, and 0% of environmental samples, respectively. Based on the panel of 28 gene targets, genotypes of enterohemorrhagic E. coli (EHEC) and generic E. coli from the experimental samples were clustered into three subgroups. In conclusion, the results suggested that the type and intensity of measures to control this pathogen at the preharvest level may need to be strain specific.
Salmonella enterica forms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were active in vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F(0)F(1)-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2 × Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.
The objective of this study was to compare the survival of non-O157 Shiga toxin-producing Escherichia coli (STEC) with E. coli O157:H7 during pepperoni production. Pepperoni batter was inoculated with 7 log CFU/g of a seven-strain STEC mixture, including strains of serotypes O26, O45, O103, O111, O121, O145, and O157. Sausages were fermented to pH ≤4.8, heated at 53.3°C for 1 h, and dried for up to 20 days. STEC strains were enumerated at designated intervals on sorbitol MacConkey (SMAC) and Rainbow (RA) agars; enrichments were completed in modified EC (mEC) broth and nonselective tryptic soy broth (TSB). When plated on SMAC, total E. coli populations decreased 2.6 to 3.5 log after the 1-h heating step at 53.3°C, and a 4.9- to 5-log reduction was observed after 7 days of drying. RA was more sensitive in recovering survivors; log reductions on it were 1.9 to 2.6, 3.8 to 4.2, and 4.6 to 5.3 at the end of cook, and at day 7 and day 14 of drying, respectively. When numbers were less than the limit of detection by direct plating on days 14 and 20 of drying (representing a 5-log kill), no more than one of three samples in each experiment was positive by enrichment with mEC broth; however, STEC strains were recovered in TSB enrichment. Freezing the 7-day dried sausage for 2 to 3 weeks generated an additional 1- to 1.5-log kill. Confirmation by PCR revealed that O103 and O157 had the greatest survival during pepperoni productions, but all serotypes except O111 and O121 were occasionally recovered during drying. This study suggests that non-O157 STEC s trains have comparable or less ability than E. coli O157 to survive the processing steps involved in the manufacture of pepperoni. Processes suitable for control of E. coli O157 will similarly inactivate the other STEC strains tested in this study.
Salmonella enterica serovar Enteritidis strain E40 filaments were developed under conditions of a reduced water activity (a(w)) of 0.95 in tryptic soy broth (TSB) or tryptic soy agar (TSA) supplemented with 8% or 7% NaCl, respectively. Filament formation was accompanied by an increase of biomass without an increase in CFU and was affected by incubation temperature and the physical milieu. The greatest amount of filaments was recovered from TSA with 7% NaCl and incubation at 30°C. Within 2 h of transfer to fresh TSB, filaments started to septate into normal-sized cells, resulting in a rapid increase in CFU. S. Enteritidis E40 filaments were not more tolerant of low- or high-temperature stresses than nonfilamented control cells. However, there was greater survival of filaments in 10% bile salts after 24 to 48 h of incubation, during pH 2.0 acid challenge for 10 min, and under desiccation on stainless steel surfaces at 25°C and 75.5% relative humidity for 7 days. S. Enteritidis E40 filaments invaded and multiplied within Caco-2 human intestinal epithelial cells to a similar degree as control cells when a comparable CFU of filaments and control cells was used. S. Enteritidis E40 filaments established a successful infection in mice via intragastric inoculation. The filaments colonized the gastrointestinal tract and disseminated to the spleen and liver at levels comparable to those attained by control cells, even when animals were inoculated with 10- to 100-fold fewer CFU. To our knowledge this is the first demonstration of virulence of stress-induced Salmonella filaments in vitro and in vivo. Formation of filaments by Salmonella in food products and food processing environments is significant to food safety, because detection and quantitation of the pathogen may be compromised. The finding that these filaments are virulent further enhances their potential public health impact.
Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin-producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx(1) and stx(2)), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non-E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100 % specificity. The detection limits of the assays were 10(3) or 10(4) CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 10(0) CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.
The effectiveness of environmental decontamination (ED) as a measure in the control of infectious diseases is controversial. This work quantifies the effectiveness of ED by analysing the transmission of pathogens from the environment to susceptible hosts in a Susceptible-Infected-Susceptible model. Analysis of the model shows that ED can render a population disease-free only when the duration of infection (D) is within a certain range. As host-to-host transmission rate is increased, D falls outside this range and the higher levels of ED have a diminishing return in reducing the number of infected hosts at endemic equilibrium. To avoid this, ED can be combined with other control measures, such as treating infected individuals to push the duration of infection into the specified range. We propose decision criteria and minimum ED efforts required for control policies to be effective.
Enterohemorrhagic Escherichia coli (EHEC), including O157 and non-O157 serotypes are significant foodborne pathogens that require sensitive and discriminatory methods for detection and characterization. There are numerous PCR-based methods for the detection of EHEC virulence factors, but the time and cost involved with large-scale screening efforts and population level analyses have limited the size and scope of studies. Recent technological advancements have combined the high-throughput performance of the microarray with the specificity and sensitivity of real-time qPCR to make large-scale screening efforts both time- and cost-effective. This study identified and evaluated a panel of 28 genetic markers including known virulence and regulatory genes, O-antigen genes, and select prophage regions of O157 and non-O157 EHEC that can be used with high-throughput PCR to virulotype, serotype, and preliminarily subtype large numbers of isolates. The PCR assays for the target genes were shown to be robust using multiple extraction methods and PCR platforms. Preliminary quantitative PCR showed that an EHEC concentration of 10(4) CFU/ml or lower could be detected, with a linear range of detection over five to six orders of magnitude. The panel of 28 target genes has the potential to become an integral tool in outbreak, environmental, and genetic investigations of EHEC.
No abstract available.
Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.
No abstract available.
Acid tolerance in Escherichia coli O157:H7 contributes to persistence in its bovine host and is thought to promote passage through the gastric barrier of humans. Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear. This study investigated the mechanism by which Dps contributes to acid tolerance in E. coli O157:H7. The results from this study showed that acid stress lead to damage of chromosomal DNA, which was accentuated in dps and recA mutants. The use of Bal31, which cleaves DNA at nicks and single-stranded regions, to analyze chromosomal DNA extracted from cells challenged at pH 2.0 provided in vivo evidence of acid damage to DNA. The DNA damage in a recA mutant further corroborated the hypothesis that acid stress leads to DNA strand breaks. Under in vitro assay conditions, Dps was shown to bind plasmid DNA directly and protect it from acid-induced strand breaks. Furthermore, the extraction of DNA from Dps-DNA complexes required a denaturing agent at low pH (2.2 and 3.6) but not at higher pH (>pH4.6). Low pH also restored the DNA-binding activity of heat-denatured Dps. Circular dichroism spectra revealed that at pH 3.6 and pH 2.2 Dps maintains or forms alpha-helices that are important for Dps-DNA complex formation. Results from the present work showed that acid stress results in DNA damage that is more pronounced in dps and recA mutants. The contribution of RecA to acid tolerance indicated that DNA repair was important even when Dps was present. Dps protected DNA from acid damage by binding to DNA. Low pH appeared to strengthen the Dps-DNA association and the secondary structure of Dps retained or formed alpha-helices at low pH. Further investigation into the precise interplay between DNA protection and damage repair pathways during acid stress are underway to gain additional insight.
To determine if Escherichia coli O157:H7 is capable of residing in the gall bladder of cattle, inoculation studies were conducted with O157:H7 strain 86-24 in weaned Holstein calves. Strain 86-24 was isolated from the gall bladders of five calves 36 days after inoculation. Two other calves contained the inoculation strain in the distal colon but the organism was absent in their gall bladders. A second trial in which the calves were euthanized 15 days after inoculation found strain 86-24 in six of seven inoculated calves but only in colon and/or rumen samples. In a third trial that inoculated eight calves with a four-strain cocktail of O157:H7 strains, the gall bladders from all eight animals were positive 9 days after inoculation. The colon and rumen samples from these calves were also positive. E. coli O157:H7 isolates recovered from bile samples and subtyped by pulsed field gel electrophoresis found that three of the four inoculation strains were present in one or more of the calves. Thus, residence in the gall bladder is not restricted to a single strain. Additional evidence of the ability to localize in the gall bladder of cattle was provided by testing the bile from 150 gall bladders (five collection dates, 30 samples each) obtained at an abbatoir and the isolation of E. coli O157:H7 from four samples (2.7%). This study establishes that E. coli O157:H7 can reside transiently or permanently at a low level in the gall bladder of cattle.
H-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in Escherichia coli. There are over 60 genes that are influenced by H-NS many of which are involved in metabolism. To determine the significance of H-NS-regulated genes in metabolism and stress tolerance, an hns mutant of E. coli O157:H7 was generated (hns::nptI, FRIK47001P) and its growth, metabolism, and gastrointestinal passage compared to the parent strain (43895) and strain FRIK47001P harboring pSC0061 which contains a functional hns and 90-bp upstream of the open-reading frame. The hns mutant grew slower and was non-motile in comparison to the parent strain. Carbon and nitrogen metabolism was significantly altered in the hns mutant, which was incapable of utilizing 42 carbon, and 19 nitrogen sources that the parent strain metabolized. Among the non-metabolized substrates were several amino acids, organic acids, and key metabolic intermediates (i.e., pyruvate) that limit carbon acquisition and energy generation. Growth studies determined that the parent strain grew in LB containing 14 to 15% bile or bile salts, while the hns mutant grew in 6.5 and 9% of these compounds, respectively. Conversely, log-phase cells of the hns mutant were significantly (p < 0.05) more acid tolerant than the parent strain and hns mutant complemented with pSC0061. In mouse passage studies, the parent strain was recovered at a higher frequency (p < 0.01) than the hns mutant regardless of whether log- or stationary-phase phase cells were orally administered. These results demonstrate that H-NS is a powerful regulator of carbon and nitrogen metabolism as well as tolerance to bile salts. It is likely that the metabolic impairments and/or the reduced bile tolerance of the E. coli O157:H7 hns mutant decreased its ability to survive passage through mice. Collectively, these results expand the influence of H-NS on carbon and nitrogen metabolism and highlight its role in the ability of O157:H7 strains to respond to changing nutrients and conditions encountered in the environment and its hosts.
A portion of the cbpA gene from Escherichia coli K-12 encoding a 24 amino acid proton-buffering peptide (Pbp) was cloned via the shuttle vector pJB99 into E. coli JM105 and subsequently into Zymomonas mobilis CP4. Expression of Pbp was confirmed in both JM105 and CP4 by HPLC. Z. mobilis CP4 carrying pJB99-2 (Pbp) exhibited increased acid tolerance (p < 0.05) in acidified TSB (HCl [pH 3.0] or acetic acid [pH 3.5]), glycine-HCl buffer (pH 3.0), and sodium acetate-acetic acid buffer (pH 3.5) in comparison to the parent strain (CP4) and CP4 with pJB99 (control plasmid). Although the expression of Pbp influenced survival at a low pH, the minimum growth pH was unaffected. Growth of Z. mobilis in the presence of ampicillin also significantly increased acid tolerance by an unknown mechanism. Results from this study demonstrate that the production of a peptide with a high proportion of basic amino acids can contribute to protection from low pH and weak organic acids such as acetic acid.
When tetracycline was present, tetA(C) reduced acid tolerance, suppressed rpoS expression, and increased the concentration of total soluble proteins in stationary-phase Escherichia coli. The suppression of acid tolerance was reversed by 85 mM sodium, potassium, magnesium, and calcium ions but not by 85 mM sucrose. Implications for using TetA(C) are discussed.
The DNA binding protein from starved cells (Dps) is a general stress protein that provides Escherichia coli protection from osmotic, oxidative, and acid stresses. While Dps production and accumulation is primarily associated with stationary phase, during log phase, this protein protects against oxidative stress in an OxyR-dependent manner. In this study, evidence is provided that expands the role of Dps in acid tolerance to both log- and stationary-phase E. coli O157:H7. The transcription of dps occurred in log-phase cells without OxyR or stress and was upregulated during entry into stationary phase. The expression in log and stationary phase involved sigma70 and sigmas, respectively, with both sigma factors recognizing the same promoter region. Site-directed mutagenesis identified an extended -10 region that was essential to both sigma70 and sigmas transcription of dps. cAMP receptor protein (CRP) was found to repress dps expression as a crp mutant had a significant increase in the dps mRNA level. However, a CRP binding site was not found in the dps promoter and upregulation of dps in the crp mutant was absent in a crp rpoS double mutant. The findings from this study demonstrated that dps was expressed at a basal level during growth, both sigma70- and sigmas-driven transcription required an extended -10, and CRP repression is mediated through the alternative sigma factor sigmas (rpoS).
Campylobacter jejuni undergoes a dramatic morphological transformation from a corkscrew-shaped rod to a coccoid form in response to unfavorable conditions. It has been speculated that the coccoid plays an important role in the survival and dissemination of C. jejuni but questions still remain regarding the viability of coccoid cells. Characterization of the genome of coccoid cells found that newly formed coccoid cells (i.e., 1-3 days) had a SmaI-digestion profile identical to that of spiral-shaped cells; however, there was a progressive degradation of the DNA with continued incubation at 37 degrees C. Concomitant with genome degradation was the detection of DNA in supernatants of coccoid cells. In contrast, cells incubated at 4 degrees C retained a spiral shape and their SmaI-digestion profile for 8 weeks and released little DNA into the medium. Thus, low temperature inhibited both coccoid formation and genome degradation. Collectively, these data support the theory that the coccoid form of C. jejuni is a manifestation of cellular degradation and spiral-shaped cells, or possibly coccoid cells formed at low temperature, are the most probable candidates for a viable but nonculturable form of this pathogen.
While cattle in general have been identified as a reservoir of Escherichia coli O157:H7, there are limited data regarding the prevalence and clonality of this pathogen in downer dairy cattle and the potential impact to human health that may occur following consumption of meat derived from downer dairy cattle. In the present study, conducted at two slaughter facilities in Wisconsin between May and October of 2001, we established a higher prevalence of E. coli O157:H7 in fecal and/or tissue samples obtained aseptically from intact colons of downer dairy cattle (10 of 203, 4.9%) than in those from healthy dairy cattle (3 of 201, 1.5%). Analyses of 57 isolates, representing these 13 positive samples (one to five isolates per sample), by pulsed-field gel electrophoresis, revealed 13 distinct XbaI restriction endonuclease digestion profiles (REDP). Typically, isolates from different animals displayed distinct REDP and isolates from the same fecal or colon sample displayed indistinguishable REDP. However, in one sample, two different, but highly related, REDP were displayed by the isolates recovered. Antimicrobial susceptibility testing indicated that 10 of the 57 isolates, recovered from 2 (1 downer and 1 healthy animal) of the 13 positive samples, were resistant to at least 1 of 18 antimicrobials tested. However, there was no appreciable difference in the frequency of resistance of isolates recovered from downer and healthy dairy cattle, and not all isolates with the same REDP displayed the same antimicrobial susceptibility profile. Lastly, it was not possible to distinguish between isolates recovered from downer and healthy cattle based on their XbaI REDP or antimicrobial susceptibility. These results indicate that downer cattle had a 3.3-fold-higher prevalence of E. coli O157:H7 than healthy cattle within the time frame and geographic scope of this study.
A study of Escherichia coli O157:H7 transmission and shedding was conducted with bull calves housed in individual pens within a confined environment. For comparative purposes, the numbers and duration of E. coli O157:H7 shedding in naturally infected calves were monitored after a single purchased calf (calf 156) tested positive prior to inoculation. During the next 8 days, the calves in adjacent pens and a pen directly across a walkway from calf 156 began to shed this serotype O157:H7 strain. Five of the eight calves in this room shed this O157:H7 strain at some time during the following 8 weeks. The numbers of E. coli O157:H7 isolates shed in these calves varied from 60 to 10(5) CFU/g of feces, and the duration of shedding ranged from 17 to >31 days. The genomic DNAs from isolates recovered from these calves were indistinguishable when compared by using XbaI digestion and pulsed-field gel electrophoresis. Inoculation of calves with 1 liter of water containing ca. 10(3) to 10(4) CFU of E. coli O157:H7/ml resulted in shedding in 10 of 12 calves (trial 1, 4 of 4 calves; trial 2, 6 of 8 calves). The inoculated calves shed the inoculation strain (FRIK 1275) as early as 24 h after administration. The duration of shedding varied from 18 to >43 days at levels from 10(2) to 10(6) CFU/g of feces. The numbers of doses necessary to initiate shedding varied among calves, and two calves in trial 2 never shed FRIK 1275 after four doses (ca. 10(6) CFU per dose). Results from this study confirm previous reports of animal-to-animal and waterborne dissemination of E. coli O157:H7 and highlight the need for an effective water treatment to reduce the spread of this pathogen in cattle.
Beef carcass quarters and fat-covered subprimal cuts were suspended vertically and inoculated with a bovine manure slurry containing a five-strain mixture of Escherichia coli O157:H7 to deliver about 4 to 5 log10 CFU/cm2. To identify treatments that would improve the effectiveness of spraying with lactic acid (LA), the inoculated quarters and cuts were treated as follows: experiment A, (i) not treated (control), (ii) sprayed with 2% (vol/vol) LA, (iii) tempered at 21 degrees C for 4 h, and (iv) tempered and then sprayed with LA; experiment B, (v) sprayed with water, (vi) sprayed with LA, (vii) sprayed with LA containing 0.5% (vol/vol) sodium benzoate (SB), and (viii) sprayed with LA containing SB and 5% (vol/vol) Tween 20 (TW20); and experiment C, (ix) sprayed with water (no prespray), (x) presprayed with TW20 and then sprayed with LA, and (xi) presprayed with TW20 and then sprayed with LA containing SB. In experiment A, spraying carcasses with LA significantly (P < 0.05) reduced the numbers of E. coli Biotype I and serotype O157:H7 after 1 and 3 days of storage, respectively. The tempering process employed did not affect the effectiveness of the LA spray on either type of E. coli. In experiment B, there was no significant difference in the reduction of E. coli O157:H7 on subprimal cuts sprayed with water and that on cuts sprayed with LA alone or with LA in combination with SB and TW20 after 1 or 3 days of storage (total reductions ranged from about 1.6 to 2.8 log10 CFU/cm2). In experiment C, prespraying subprimal cuts with TW20 significantly (P < 0.05) increased the effectiveness of LA (reductions of 2.8 and 3.2 log10 CFU/cm2, respectively) and that of LA with SB (reductions of 2.6 and 3.3 log10 CFU/cm2, respectively) compared with spraying with water alone (reductions of ca. 1.0 and 2.0 log10 CFU/cm2, respectively) after I and 3 days of storage, respectively. In a separate experiment, the incorporation of TW20 (0.1 or 0.25%) into buffered peptone water prior to the maceration of excised carcass surface samples resulted in the recovery of significantly larger numbers (ca. 5.1 to 5.2 log10 CFU/cm2) of E. coli O157:H7 cells than did the control treatment without added TW20 (ca. 3.8 to 4.6 log10 CFU/cm2). These results demonstrate that the treatment of beef carcasses with LA reduces the number of viable E. coli O157:H7 cells and that this inactivation or removal by LA is enhanced by prespraying of the carcass with a 5% solution of TW20.
An Escherichia coli O157:H7 dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log(10)-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log(10)-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log(10) CFU/ml while the parent strain decreased by approximately 2 log(10) CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.
Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator sigma(S) and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10(4) CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P </= 0.05). Thus, sigma(S) appears to play a role in E. coli O157:H7 passage in mice and shedding from calves, possibly by inducing expression of the glucose-repressed RpoS-dependent AR determinant and thus increasing resistance to gastrointestinal stress. These findings may provide clues for future efforts aimed at reducing or eliminating this pathogen from cattle herds.
The survival of Escherichia coli O157:H7 and of a nonpathogenic control strain of E. coli was monitored in raw ground beef that was stored at 2 degrees C for 4 weeks, -2 degrees C for 4 weeks, 15 degrees C for 4 h and then -2 degrees C for 4 weeks, and -20 degrees C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 10(5) CFU/g), formed into patties (30 to 45 g), and stored at the appropriate temperature. The numbers of the E. coli control strain decreased by 1.4 log 10 CFU/g, and pathogen numbers declined 1.9 log 10 CFU/g when patties were stored for 4 weeks at 20 degrees C. When patties were stored at -2 degrees C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log 10 CFU/g, respectively. Patties stored at 15 degrees C for 4 h prior to storage at -2 degrees C for 4 weeks resulted in 1.6 and 2.7 log 10-CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15 degrees C for 4 h (tempering) did not result in increased numbers of colony forming units per gram, as determined with violet red bile, MRS lactobacilli, and plate-count agars. Frozen storage (-20 degrees C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log 10-CFU/g reduction in the numbers of the control strain and individual serotype O157:H7 strains after 1 year. There was no significant difference between the survival of the control strain and the O157:H7 strains, nor was there a difference between O157:H7 strains. These data demonstrate that tempering of ground-beef patties prior to low-temperature storage accelerated the decline in the numbers of E. coli O157:H7.
A by-product of glucose produced during sterilization (121 degrees C, 15 lb/in2, 15 min) at neutral pH and in the presence of phosphate (i.e., phosphate-buffered saline) was bactericidal to Escherichia coli O157:H7 (ATCC 43895). Other six-carbon (fructose and galactose) and five-carbon (arabinose, ribose, and xylose) reducing sugars also produced a toxic by-product under the same conditions. Fructose and the five-carbon sugars yielded the most bactericidal activity. Glucose concentrations of 1% (wt/vol) resulted in a 99.9% decline in the CFU of stationary-phase cells per milliliter in 2 days at 25 degrees C. An rpoS mutant (pRR10::rpoS) of strain 43895 (FRIK 816-3) was significantly (P < 0.001) more sensitive to the glucose-phosphate by-product than the parent strain, as glucose concentrations from 0.05 to 0.25% resulted in a 2- to 3-log10 reduction in CFU per milliliter in 2 days at 25 degrees C. Likewise, log-phase cells of the wild-type strain, 43895, were significantly more sensitive (P < 0.001) to the glucose-phosphate by-product than were stationary-phase cells, which is consistent with the stability of rpoS and the regulation of rpoS-regulated genes. The bactericidal effect of the glucose-phosphate by-product was reduced when strains ATCC 43895 and FRIK 816-3 were incubated at a low temperature (4 degrees C). Also, growth in glucose-free medium (i.e., nutrient broth) did not alleviate the sensitivity to the glucose-phosphate by-product and excludes the possibility of substrate-accelerated death as the cause of the bactericidal effect observed. The glucose-phosphate by-product was also bactericidal to Salmonella typhimurium, Shigella dysenteriae, and a Klebsiella sp. Attempts to identify the glucose-phosphate by-product were unsuccessful. These studies demonstrate the production of a glucose-phosphate by-product bactericidal to E. coli O157:H7 and the protective effects afforded by rpoS-regulated gene products. Additionally, the detection of sublethally injured bacteria may be compromised by the presence of this by-product in recovery media.
This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.
Pepperoni batter was prepared with fat contents of about 15, 20, and 32% (wt/wt) and inoculated with a pediococcal starter culture and > or = 2.0 x 10(7) CFU/g of a five-strain inoculum of Escherichia coli O157:H7. The batter was fermented at 96 degrees F (ca. 36 degrees C and 85% relative humidity (RH) to pH < or = 4.8 and then dried at 55 degrees F (ca. 13 degrees C) and 65% RH to a moisture/protein ratio of < or = 1.6:1. For storage, slices were packaged under air or vacuum and stored at 39 degrees F (ca. 4 degrees C) and 70 degrees F (ca. 21 degrees C). For baking, frozen slices were placed on retail frozen cheese pizzas that were subsequently baked at 275 degrees F (ca. 135 degrees C), 375 degrees F (ca. 191 degrees C), or 475 degrees F (ca. 246 degrees C) for 0 to 20 min. Appreciable differences related to fat levels were observed after drying; pathogen numbers decreased by 1.04, 1.31 and 1.62 log10 units in sticks prepared from batter at initial fat levels of 15, 20, and 32%, respectively. During storage, the temperature rather than the atmosphere had the greater effect on pathogen numbers, with similar viability observed among the three fat levels tested. At 70 degrees F (ca. 21 degrees C), compared to original levels, pathogen numbers decreased by > or = 5.56 and > or = 4.53 log10 units within 14 days in slices stored under air and vacuum, respectively, whereas at 39 degrees F (ca. 4 degrees C) numbers decreased by < or = 2.43 log10 CFU/g after 60 days of storage under either atmosphere. Baking, as expected, resulted in greater reductions in pathogen numbers as the temperature and/or time of baking increased. However, it was still possible to recover the pathogen by enrichment after baking frozen slices on frozen pizza at 475 degrees F (ca. 246 degrees C) for 10 min or at 375 degrees F (ca. 191 degrees C) for 15 min. The calculated D values for all three temperatures tested increased as the fat content of the batter increased from 15 to 20 to 32%. The present study confirmed that fermentation and drying were sufficient to reduce levels of E. coli O157:H7 in pepperoni sticks by < 2.0 log10 CFU/g. Storage of slices for at least 14 days at ambient temperature under air resulted in a > 5.5-log10-unit total reduction of the pathogen. Baking slices on frozen pizza for at least 15 min at 475 degrees F (ca. 246 degrees C) or 20 min at 375 degrees F (ca. 191 degrees C) was necessary to reduce pathogen numbers to below detection by both direct plating and enrichment.
The fate of Escherichia coli O157:H7 was monitored in salami during conditioning of batter, fermentation and drying of sticks, and storage of slices. The raw batter (75% pork: 25% beef, wt/wt, fat content about 20%) was inoculated with a pediococcal starter culture (about 10(8) CFU/g) and a five-strain cocktail of E. coli O157:H7 ( > or = 2 x 10(7) CFU/g) and stuffed into 104-mm diameter fibrous casings. After being refrigerated at 4 degrees C or being tempered at 13 degrees C, frozen at -20 degrees C, and thawed at 4 degrees C, or being frozen at -20 degrees C, and thawed at 4 degrees C, the inoculated batter was fermented at 24 degrees C and 90% relative humidity (RH) to pH < or = 4.8, dried at 13 degrees C and 65% RH to a moisture/protein ratio of < or = 1.9:1, and then stored at 4 or 21 degrees C under air or vacuum. For salami sticks sampled immediately after drying, appreciable differences were evident among the various batter-conditioning treatments; pathogen numbers were reduced from original levels by 2.1, 1.6, or 1.1 log10 units when batter was tempered, frozen, and thawed, frozen and thawed, or refrigerated, respectively. Similarly, regardless of storage temperature or atmosphere, within 7 days salami slices cut from sticks prepared from batter that was tempered, frozen, and thawed (2.7- to 4.9-log10-unit reduction) or frozen and thawed (2.3- to 4.8-log10-unit reduction) displayed a greater impact on pathogen numbers than slices cut from sticks prepared from batter that was refrigerated (1.6- to 3.1-log10-unit reduction). The effects of batter conditioning notwithstanding, a greater reduction in levels of E. coli O157:H7 was observed when slices were stored at 21 degrees C compared to otherwise similar slices stored at 4 degrees C. After storage for 60 days the pathogen was only detected by enrichment in slices stored at 21 degrees C, whereas pathogen levels ranged from 1.4 to 4.5 log10 CFU/g in slices stored at 4 degrees C. Differences related to storage atmosphere were first observed after slices were stored for 21 days. Such differences were more readily demonstrable after 60 and 90 days, with pathogen numbers for treatments that were statistically different ranging from 0.6- to 1.5-log10 units higher on slices stored under vacuum than in air. These data emphasize the need to implement multiple barriers to appreciably reduce numbers of E. coli O157:H7 in salami.
A 14-month longitudinal study was conducted on four dairy farms (C, H, R, and X) in Wisconsin to ascertain the source(s) and dissemination of Escherichia coli O157:H7. A cohort of 15 heifer calves from each farm were sampled weekly by digital rectal retrieval from birth to a minimum of 7 months of age (range, 7 to 13 months). Over the 14 months of the study, the cohort heifers and other randomly selected cattle from farms C and H tested negative. Farm R had two separate periods of E. coli O157:H7 shedding lasting 4 months (November 1995 to February 1996) and 1 month (July to August 1996), while farm X had at least one positive cohort animal for a 5-month period (May to October 1996). Heifers shed O157:H7 strains in feces for 1 to 16 weeks at levels ranging from 2.0 x 10(2) to 8.7 x 10(4) CFU per g. E. coli O157:H7 was also isolated from other noncohort cattle, feed, flies, a pigeon, and water associated with the cohort heifers on farms R and/or X. When present in animal drinking water, E. coli O157:H7 disseminated through the cohort cattle and other cattle that used the water source. E. coli O157:H7 was found in water at < 1 to 23 CFU/ml. Genomic subtyping by pulsed-field gel electrophoresis demonstrated that a single O157:H7 strain comprised a majority of the isolates from cohort and noncohort cattle, water, and other positive samples (i.e., from feed, flies, and a pigeon, etc.) on a farm. The isolates from farm R displayed two predominant XbaI restriction endonuclease digestion profiles (REDP), REDP 3 and REDP 7, during the first and second periods of shedding, respectively. Six additional REDP that were > or = 89% similar to REDP 3 or REDP 7 were identified among the farm R isolates. Additionally, the REDP of an O157:H7 isolate from a heifer on farm R in 1994 was indistinguishable from REDP 3. Farm X had one O157:H7 strain that predominated (96% of positive samples had strains with REDP 9), and the REDP of an isolate from a heifer in 1994 was indistinguishable from REDP 9. These results suggest that E. coli O157:H7 is disseminated from a common source on farms and that strains can persist in a herd for a 2-year period.
Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE) was used to compare Wisconsin isolates of Escherichia coli O157:H7, including 39 isolates from a 1994 day care center outbreak, 28 isolates from 18 individuals from the surrounding geographic area with sporadic cases occurring during the 3 months before the outbreak, and 3 isolates, collected in 1995, from patients with hemolytic-uremic syndrome (HUS) who were from eastern Wisconsin counties other than those inhabited by the day care center and sporadic-case individuals. The technique of CHEF-PFGE using XbaI identified seven highly related restriction endonuclease digestion profiles (REDPs) (93 to 98% similarity) among the 39 day care center isolates and nine XbaI REDPs (63 to 93% similarity) among the 28 isolates from sporadic-case individuals, including REDP 33, which was exhibited by both day care and sporadic-case isolates. PFGE analyses of sequential E. coli O157:H7 isolates from symptomatic day care center attendees revealed that the REDPs of 25 isolates from eight patients were indistinguishable whereas the REDPs of 2 of 6 isolates from two patients differed slightly (93 to 95% similarity). The REDPs of the three isolates from 1995 HUS patients were 78 to 83% similar, with REDP 26 being exhibited by one HUS-associated isolate and an isolate from one day care attendee who did not develop HUS. The genes for both Shiga toxins I and II (stx1 and stx2, respectively) were detected in all but one isolate (sporadic case), and Shiga toxin production by the day care center isolates was not significantly different from that of the other isolates, including the three HUS-associated isolates. Analyses of E. coli O157:H7 isolates from both the day care center outbreak and sporadic cases by CHEF-PFGE permitted us to define the REDP variability of an outbreak and geographic region and demonstrated that the day care center outbreak and a HUS case in 1995 were caused by E. coli O157:H7 strains endemic to eastern Wisconsin.
Escherichia coli was isolated from 58% (11/19) of retail soft and semi-soft cheeses tested, but E. coli O157:H7 was not detected. The presence of E. coli in retail cheeses and the lack of baseline data on the prevalence of serotype O157:H7 strains prompted us to survey ingredients and the environment in 15 cheese and dairy plants. Escherichia coli O157:H7 was not detected in any of the 1104 samples tested, including 42 raw milk samples. These results suggest that serotype O157:H7 is not prevalent within dairy product ingredients and processing environments.
A total of 765 Escherichia coli isolates from point and nonpoint sources were collected from the Apalachicola National Estuarine Research Reserve, and their multiple-antibiotic-resistance (MAR) profiles were determined with 10 antibiotics. E. coli isolates from point sources showed significantly greater resistance (P < 0.05) to antibiotics and higher MAR indices than isolates from nonpoint sources. Specifically, 65 different resistance patterns were observed among point source isolates, compared to 32 among nonpoint source isolates. Examples of this contrast in MAR profiles included percentages of isolates with resistance to chlortetracycline-sulfathiazole of 33.7% and to chlortetracycline-penicillin G-sulfathiazole of 14.5% for point source isolates versus 15.4 and 1.7%, respectively, for nonpoint source isolates. MAR profile homology, based on coefficient similarity, showed that isolates from point sources were markedly more diverse than isolates from nonpoint sources. Seven clusters were observed among point source isolates, with a coefficient value of approximately 1.8. In contrast, only four clusters were observed among nonpoint source isolates. Covariance matrices of data displayed six very distinct foci representing nonpoint source E. coli isolates. Importantly, E. coli isolates obtained directly from human and animal feces also clustered among point and nonpoint sources, respectively. We conclude that E. coli MAR profiles were associated with point and nonpoint sources of pollution within Apalachicola Bay and that this method may be useful in facilitating management of other estuaries.
Forty illness associated phage-type (PT) 4 and PT 8 strains of Salmonella enteritidis were analyzed by the pulsed-field technique of clamped homogeneous electric fields (CHEF) electrophoresis. Using NotI and XbaI, the 40 strains were subdivided by each enzyme into seven restriction endonuclease digestion profiles (REDP). The 35 PT 4 isolates from Austria were subdivided into six NotI and five XbaI REDP, while the five PT 8 isolates from the United States displayed a single NotI and two XbaI REDP. When highly-concentrated, uncleaved genomic DNA was subjected to CHEF electrophoresis, plasmid DNA in the size range of 350 kb relative to a linear DNA standard was discernible in 38 of the 40 strains. Subsequent isolation and restriction analyses of plasmid DNA from one strain (E40) revealed a single plasmid (pE40; ca. 54 kb) with one XbaI and two NotI cleavage sites that was similar in size to the S. enteritidis virulence plasmid pRQ29. Hybridization of the PE40 probe with S. enteritidis genomic DNAs identified a 54 kb fragment within the XbaI REDP and two fragments, 20 and 34 kb, in NotI REDP of plasmid-positive strains. It was not possible to identify plasmid-specific bands in NotI REDP without hybridization due to comigrating chromosomal and plasmid DNA fragments. Regardless of PT, all 40 S. enteritidis strains showed highly related REDP. The similarity between PT 4 and PT 8 strains as further revealed by Dice similarity coefficients was 90% to 95% for NotI REDP and 79% to 93% for XbaI REDP. These results support the hypothesis that the pandemic observed today is the result of the efficient spread of a single clone, or clusters of closely related clones, of S. enteritidis.
Vibrio vulnificus belongs to the autochthonous bacterial flora of warm estuarine waters. It can cause life-threatening extraintestinal disease in persons who have underlying illness and who consume raw shellfish or contact wounds with estuarine water. Currently, very little is known about genetic diversity within this species. In this report, we describe high-level variation in restriction fragment length polymorphism profiles among 53 clinical and 78 environmental isolates, as determined by pulsed-field gel electrophoresis. In contrast, ribotype profiles showed greater similarity. When combined ribotype profiles of clinical and environmental isolates were analyzed, four predominant clusters were observed. Interestingly, a low number (16%) of clinical isolates were found in cluster C, compared with clusters A, B, and D (range, 50 to 83%). In addition, 83% of all Hawaiian isolates were located in a single cluster, indicating a possible relationship between geography and genotype. We also report that spontaneous translucent colonial morphotypes were distinct by both restriction fragment length polymorphism and biochemical profiles, compared with opaque parent strains.
An rpoS mutant (rpoS::pRR10) of Escherichia coli O157:H7 ATCC 43895 was generated. Stationary-phase acid, heat, and salt tolerance was significantly reduced, and starvation-induced acid tolerance did not develop in the mutant. RpoS was also important for survival of E. coli O157:H7 in dry, fermented sausage.
A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.
The genomic fingerprints of 26 Escherichia coli O157:H7 isolates from calves on 20 farms in 16 states were determined by pulsed-field gel electrophoresis (PFGE). Digestion of genomic DNAs with the restriction enzymes SfiI and XbaI yielded 14 and 18 restriction endonuclease digestion profiles (REDP), respectively. Seventeen farms (85%) had E. coli O157:H7 with a unique REDP, and when more than one calf tested positive on a farm, the isolates displayed identical REDP. Isolates from different farms within the same state displayed distinct REDP, as did most isolates from farms in different states. The exceptions were three farms in New York, Ohio, and Washington that had calves harboring E. coli O157:H7 with the same REDP. In addition to REDP, the toxin profiles of all 26 isolates were determined using oligonucleotide probes to Shiga-like toxins (SLT) I and II. Nineteen (73%) of the E. coli O157:H7 isolates harbored the genes for both SLT and II, while the remaining seven isolates (27%) had the gene for SLT II only. Also, isolates with the same REDP had the same toxin profile. The genomic relatedness among the E. coli O157:H7 isolates was also determined by principal component analysis of Dice similarity indices of REDP. Three clusters were identified, but none of these were associated with a geographic region or toxin profile.
Pulsed-field gel electrophoresis established the linkage between recalled chocolate milk and a multistate invasive listeriosis outbreak during a four-product recall period. Listeria monocytogenes isolates from four hospitalized patients and an environmental dairy sample displayed AscI restriction endonuclease digestion profiles identical to that of the chocolate milk isolate.
Stationary phase and the starvation of log-phase cells increased the acid tolerance of Escherichia coli O157:H7 strains. Although the degree of acid tolerance varied, the survival of most O157:H7 strains exceeded that of other, related, pathogens in a synthetic gastric fluid.
Clamped homogeneous electric field gel electrophoresis and a computer program for managing electrophoresis banding patterns (ELBAMAP) were used to analyze genomic DNA of 118 Vibrio vulnificus strains, isolated from three oysters by direct plating. Analysis with SfiI resulted in 60 restriction endonuclease digestion profiles (REDP), while analysis with SrfI produced 53 different REDP. Similarities between REDP ranged from 7 to 93%. Principal-component analysis showed that the strains were heterogeneous.
Sixty strains of Yersinia enterocolitica from five serogroups (O:3; O:9; O:8; O:5; and O:5,27) and eight non-Y. enterocolitica strains, recovered from diverse sources (humans, animals, food, and the environment) in Europe, Argentina, and the United States, were examined by the pulsed-field gel electrophoresis (PFGE) technique of contour clamped homogeneous electric field electrophoresis (CHEF) by using NotI and XbaI as restriction enzymes. NotI and XbaI generated 36 and 33 restriction endonuclease digestion profiles (REDP), respectively. By combining the results of both enzymes, 42 unique genomic groups were differentiated. DNA fragments were transferred to nylon membranes and hybridized with digoxigenin-labelled oligonucleotide probes to the ail gene and virulence plasmid to determine hybridization patterns and the potential virulence of the strains. The strains were tested for the presence of the plasmid by PFGE-CHEF and phenotypic characteristics encoded for by the virulence plasmid. Thirty of the 60 Y. enterocolitica strains tested harbored the virulence plasmid. The specificity of the ail and pYV probes was 100% when tested with 68 Yersinia strains and 19 different non-Yersinia strains. Sixteen selected Y. enterocolitica strains were tested for their virulence by lethality in iron- and desferrioxamine-sensitized mice. No correlation between REDP and the virulence of the strains was observed. The observed REDP and the hybridization patterns were very homogeneous within a serogroup and independent of the source of isolation. In addition, PFGE-CHEF was shown to be valuable in identifying and confirming serogroups. Principal component analysis of Dice similarity indices from REDP was an excellent tool for determining genetic relatedness among strains.
Yersinia enterocolitica gastroenteritis was first recognized in the early 1960s and has since been reported with increasing frequency. To determine if strains of Y. enterocolitica, within a restricted region isolated over 8 years (1985-1993), originated from a single or multiple clones, pulsed-field gel electrophoresis (PFGE) of large chromosomal DNA restriction fragments generated by XbaI or NotI was used. A total of 27 isolates of Y. enterocolitica were analyzed, 24 from Austria (Vienna and Graz) consisting of serogroups 0:3 (17 isolates), 0:9 (6 isolates), 0:5 (1 isolate); 2 from Germany of serogroups 0:3 and 0:9 (1 isolate each); 1 from the U.S.A. of serogroup 0:8. Genomic fingerprints of these strains were compared to those of 8 other Yersinia species to ascertain if their restriction endonuclease digestion profiles (REDP) were serogroup and/or species specific. The 27 Y. enterocolitica strains could be divided into 16 genomic varieties according to their restriction patterns with NotI and XbaI. PFGE was highly discriminatory as strains belonging to the same serogroup could be subdivided into different genomic groups. Furthermore, Y. enterocolitica strains isolated from the same region, over an 8 year period, belonged to a few closely related clones. The genomic fingerprints of Yersinia were found to be species and serogroup specific.
Fresh blue crab (Callinectes sapidus) meat was obtained from retail markets in Florida and sampled for viable Listeria monocytogenes. The pathogen was found in crabmeat in three of four different lots tested by enrichment and at levels of 75 CFU/g in one of the same four lots by direct plating. Next, crabmeat was steam sterilized, inoculated with a three-strain mixture of L. monocytogenes (ca. 5.5 log10 CFU/g), washed with various lactic acid bacterium fermentation products (2,000 to 20,000 arbitrary units [AU]/ml of wash) or food-grade chemicals (0.25 to 4 M), and stored at 4 degrees C. Counts of the pathogen remained relatively constant in control samples during storage for 6 days, whereas in crabmeat washed with Perlac 1911 or MicroGard (10,000 to 20,000 AU), numbers initially decreased (0.5 to 1.0 log10 unit/g) but recovered to original levels within 6 days. Numbers of L. monocytogenes cells decreased 1.5 to 2.7 log10 units/g of crabmeat within 0.04 day when washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083, or Nisin per ml. Thereafter, counts increased 0.5 to 1.6 log10 units within 6 days. After washing with food-grade chemicals, modest reductions (0.4 to 0.8 log10 unit/g) were observed with sodium acetate (4 M), sodium diacetate (0.5 or 1 M), sodium lactate (1 M), or sodium nitrite (1.5 M). However, Listeria counts in crabmeat washed with 2 M sodium diacetate decreased 2.6 log10 units/g within 6 days. In addition, trisodium phosphate reduced L. monocytogenes counts from 1.7 (0.25 M) to > 4.6 (1 M) log10 units/g within 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)
The direct viable count (DVC), a microscopic method for the enumeration of viable bacteria, was modified by replacing nalidixic acid with ciprofloxacin. This modification made it possible to apply this method to a variety of Gram-negative and Gram-positive bacteria which was not previously possible. Of the four antibiotics tested (nalidixic acid, novobiocin, ciprofloxacin and mitomycin C), ciprofloxacin and mitomycin C were the only ones effective for use in the DVC with all of the bacteria tested. In addition, ciprofloxacin could be used at a single concentration (1 microgram/ml) while adjustments were necessary with the other antibiotics when examining bacteria from different genera and, in some instances, from different species. The use of ciprofloxacin in the DVC resulted in viable cells that had elongated by 5-11 times their original length. We conclude that the modified DVC will be useful in growth and survival studies of bacterial pathogens and spoilage organisms in milk and other foods.
Genomic DNAs of Escherichia coli O157:H7 strains isolated from patients and food samples were analyzed by pulsed-field gel electrophoresis. The rare-cutting endonucleases SfiI and XbaI generated 6 and 10 distinct genomic profiles, respectively, for the 22 strains analyzed, indicating that this technique may find application for epidemiologic studies. Summation of XbaI fragments from five E. coli O157:H7 strains estimated the genomic length at ca. 4.7 Mb.
Sterilized seawater was used to assess the effects of temperature and salinity on the survival of Vibrio vulnificus. In the temperature range of 13 to 22 degrees C, numbers of V. vulnificus increased during the 6-day incubation. Temperatures outside this range reduced the time of V. vulnificus survival in sterile 10-ppt seawater. At these restrictive temperatures, V. vulnificus numbers were reduced by 90% after 6 days of incubation. Incubation between 0.5 and 10.5 degrees C demonstrated that V. vulnificus survives poorly below 8.5 degrees C. At salinities between 5 and 25 ppt and at 14 degrees C, V. vulnificus numbers actually increased or remained unchanged after 6 days of incubation. At salinities of 30, 35, and 38 ppt, numbers of V. vulnificus decreased 58, 88, and 83%, respectively. V. vulnificus could not be recovered from deionized water, indicating lysis. When a rifampin-resistant strain of V. vulnificus was used to inoculate sterilized and unsterilized seawater (20 ppt, 20 degrees C), numbers increased in sterile seawater but decreased to undetectable levels in 14 days in the unsterilized seawater, indicating that biological factors may play a role in the survival of V. vulnificus in the environment. Since our studies demonstrated sensitivity to low temperatures, the survival of V. vulnificus in naturally contaminated oysters at temperatures of 0, 2, and 4 degrees C was also determined. Numbers of endogenous V. vulnificus in oyster shellstock increased by more than 100-fold in shellstock stored at 30 degrees C but were reduced approximately 10- and 100-fold after 14 days at 2 to 4 degrees C and 0 degrees C, respectively. We conclude that both biological and physicochemical factors are important to the survival of V. vulnificus in the environment and that temperature is critical to controlling its growth in oyster shellstock.
Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H2O, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was approximately 2,000 V. vulnificus cells per EIA well. Epitope FRBT37 was labile at 70 degrees C for 30 min. Immunoblot and EIA plate formats reduced assay time and facilitated handling large numbers of test samples.
A total of 202 Escherichia coli isolated from urban and rural water were tested with 11 antibiotics to assess the prevalence of antibiotic resistance from each source. Urban waters harbored higher percentages of resistant E. coli strains than rural waters. Antibiotic-resistant E. coli may offer an index of water quality related to source.
A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.
Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.
Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli, and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of 13 non-E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non-E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non-E. coli.
Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.