Baseline and storm event monitoring of Bacteroidales marker concentrations and enteric pathogen presence in a rural Canadian watershed.

Bacteroidales 16S rRNA gene markers were evaluated for their use as a microbial source tracking tool in a well characterized 750 ha agricultural watershed in Nova Scotia, Canada. Water quality monitoring was conducted following the validation of host-specific and universal Bacteroidales (AllBac) markers for their proficiency in this particular geographic region, which provided further evidence that these markers are geographically stable. Increasing Escherichia coli concentrations were positively correlated (p < 0.01) with concentrations of the AllBac marker in water samples, suggesting that this universal marker is more suited as a positive DNA control rather than as an indicator of recent fecal contamination. Ruminant (BacR) and bovine (CowM2) specific marker detection was associated with increased runoff due to precipitation in sub-watersheds putatively impacted by cattle farming, demonstrating that the BacR and CowM2 markers can be used to detect the recent introduction of fecal matter from cattle farming activities during rainfall events. However, the human associated marker (BacH) was only detected once in spite of numerous on-site residential wastewater treatment systems in the watershed, suggesting that this assay is not sensitive enough to detect this type of human sewage source. E. coli O157:H7 and Salmonella spp. DNA was not detected in any of the 149 watershed samples; however, 114 (76.5%) of those samples tested positive for Campylobacter spp. No significant correlation (p > 0.05) was found between Campylobacter spp. presence and either E. coli or AllBac marker levels. Further studies should be conducted to assess the origins of Campylobacter spp. in these types of watersheds, and to quantify pathogen cell numbers to allow for a human health risk assessment.

Population dynamics of Escherichia coli inoculated by irrigation into the phyllosphere of spinach grown under commercial production conditions.

Recent outbreaks of food-borne illnesses associated with the consumption of fresh produce have increased attention on irrigation water as a potential source of pathogen contamination. A better understanding of the behaviour of enteric pathogens introduced into agricultural systems during irrigation will aid in risk assessments and support the development of appropriate farm-level water management practices. For this reason, the survival dynamics of two nalidixic acid resistant strains of Escherichia coli after their spray inoculation into the phyllosphere and soil of field spinach were examined over two growing seasons. E. coli strains NAR, an environmental isolate, and DM3n, a non-pathogenic serotype O157:H7, were applied at rates of 104 to 107 cfu/100ml to the fully developed spinach plants that arose subsequent to the harvesting of their upper leafy portions for commercial purposes (secondary-growth plants). After 72 h, E. coli on spinach were reduced by 3-5 logs. Culturable E. coli were recovered from plants up to 6 days post-inoculation. Survival in soil was greater than in the phyllosphere. Under ambient conditions, the mean 72 h first order decay constant computed by Chick's Law was 0.1 h⁻¹. Although light reduction studies indicated UV irradiation negatively influenced the persistence of E. coli, a simple relationship between UV exposure and phyllosphere E. coli densities could not be established. E. coli introduced to the leafy portions of spinach via spray irrigation displayed rapid declines in their culturability under the open environmental conditions experienced during this study. A 6 day period between the last irrigation and harvest would minimize the risks of E. coli survival in the spinach phyllosphere. E. coli NAR was identified as a possible surrogate for the O157:H7 strain, DM3n.

Presence and potential for horizontal transfer of antibiotic resistance in oxidase-positive bacteria populating raw salad vegetables.

To assess whether domestically grown fresh salad vegetables constitute a possible reservoir of antibiotic resistance for Canadian consumers, aerobic bacteria capable of forming colonies at 30 degrees C on nutrient-limited media were recovered from a single sampling of Romaine lettuce, Savoy spinach and alfalfa sprouts, then examined for their susceptibility to ten antibiotics and the carriage of potentially mobile R-plasmids and integrons. Of the 140 isolates resistant to one or more antibiotic, 93.5 and 90.0% were resistant to ampicillin and cephalothin; 35.7% to chloramphenicol, 10.0% to streptomycin, 4.2% to nalidixic acid, 4.2% to kanamycin, and 2.8% to gentamicin. Gram-positive isolates accounted for less than 4% of the antibiotic resistant strains. A small portion (23.1%) of the predominant oxidase-positive, gram-negative isolates was resistant to two or more antimicrobials. Members of the Pseudomonas fluorescens/putida complex were most prevalent among the 34 resistant strains identified. Sphingobacterium spp. and Acinetobacter baumanni also were detected. Ten of 52 resistant strains carried plasmids, 3 of which were self-transmissible and bore resistance to ampicillin and kanamycin. Eighteen of 48 gave PCR evidence for integron DNA. Class 2 type integrons were the most prevalent, followed by class 1. We conclude that the foods examined here carry antibiotic resistant bacteria at the retail level. Further, our determination that resistant strains contain integron-specific DNA sequences and self-transmissible R-plasmids indicates their potential to influence the pool of antibiotic resistance in humans via lateral gene transfer subsequent to ingestion.

Helicobacter pylori: primary susceptibility to clarithromycin in vitro in Nova Scotia.

Resistance to antimicrobial agents is a major determinant of the efficacy of regimens to eradicate Helicobacter pylori. Clarithromycin (CLA) has become one of the most commonly used antibiotics for treatment of H pylori infection. In this study, the rate of primary resistance to CLA in H pylori isolated from patients was determined. One hundred sixty-two strains were recovered from patients before treatment. Strains were grown and inoculated onto Mueller-Hinton agar with 7% sheep blood. CLA epsilometer gradient agar diffusion test (E test) strips were used to test for susceptibility. Appropriate control organisms were tested to validate the assay. Plates were incubated at 37 degrees C in a microaerophilic atmosphere for up to five days. E test results were easy to interpret. Strains were considered resistant if the minimum inhibitory concentration (MIC) was 2 micrograms/mL or greater. Three strains were resistant (two strains with MIC 8 micrograms/mL and one strain with MIC 12 micrograms/mL) and 159 strains were sensitive (MICs ranged from less than 0.016 to 0.38 micrograms/mL). Ninety per cent of the strains had MICs of 0.023 micrograms/mL. Primary resistance was 1.8%. These susceptibility data support the use of CLA for the treatment of H pylori in the Nova Scotia population.

Characterization and cloning of a 37.6-kb plasmid carried by Legionella pneumophila recovered from patients and hospital water over a 12-year period.

For 12 years, strains of Legionella pneumophila serogroup 1 harbouring a 37.6-kb (23 MDa) plasmid have predominated among patient and potable water isolates at the Victoria General Hospital, Halifax, N.S. Plasmid DNA recovered from 24 strains isolated between 1983 and 1995 was digested with the restriction endonucleases EcoRI, HindIII, KpnI, PvuII, XbaI, and BamHI. The distribution of cutting sites indicated that the 23-MDa size group had remained essentially unchanged during this period, suggesting the persistence of a single plasmid type. Further fragmentation pattern analysis permitted the construction of a physical map of the prototype 23-MDA plasmid, pLp4269. Double digestion with BamHI-HindIII enabled the cloning of 94.4% of pLp4269 into pBluescript vector. A 2.1-kb fragment was not clonable. Plasmid pLp4269 is the first of the smaller Legionella extrachromosomal DNAs to be characterized in this way.

Genomic stability of Legionella pneumophila isolates recovered from two cardiac transplant patients with nosocomial Legionnaires' disease.

Pulsed-field gel electrophoresis revealed that multiple consecutive isolates of Legionella pneumophila from two cardiac transplant patients remained genomically stable, despite exposure to host defenses and antimicrobial agents.

Serological detection of Helicobacter pylori antibodies in children and their parents.

The antibody response to Helicobacter pylori was examined in 56 children (ages 5 to 18) to determine whether serological tests can be used for diagnosis. Twenty-four children (43%) were H. pylori positive and 32 children (57%) were H. pylori negative by culture and histological examination of endoscopic biopsy specimens. The immune response was also examined in 39 nonendoscoped parents of the children. H. pylori-specific immunoglobulin G (IgG) and IgA antibodies were detected by the flow microsphere immunofluorescent assay (FMIA). IgG was also detected by using the Pyloristat enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity, and positive and negative predictive values for the FMIA for IgG were 100, 97, 96, and 100%, respectively. The respective values for the Pyloristat ELISA for IgG were 96, 94, 92, and 97%. The respective values for the FMIA for IgA were 50, 100, 100, and 73%. Both assays identified the same 19 parents as IgG positive, while FMIA identified 17 of the 19 parents as IgA positive.

Increasing prevalence of Helicobacter pylori infection with age: continuous risk of infection in adults rather than cohort effect.

It remains unclear whether acquisition of Helicobacter pylori is due to a continuous risk of acquiring the infection or a cohort effect. In this prospective 3-year cohort study, the seroprevalence, conversion, and reversion of H. pylori infection as determined by IgG antibodies was examined. The cohort consisted of 316 randomly selected, nonpatient subjects aged 18-72 years who each provided at least 2 suitable samples. Seroprevalence of H. pylori increased from 21% in the third decade to 50% in the eighth decade. Crude annual seroconversion rate was 1% and the "spontaneous" seroreversion rate was 1.6%. Age was the only identified risk factor for H. pylori infection. A continuous risk of acquisition of 1%/year rather than a cohort effect best explains the pattern of H. pylori infection in this Canadian population. Seroconversion continues in adult life, and spontaneous reversions do occur, especially in the later decades.

Serological detection of Helicobacter pylori by a flow microsphere immunofluorescence assay.

A flow cytometric immunofluorescence assay (FMIA) for the detection of immunoglobulin G antibodies to Helicobacter pylori was developed. A multicomponent antigen was prepared and used to coat carboxylated polystyrene microspheres for reaction with patient sera followed by fluorescein isothiocyanate-labelled goat anti-human immunoglobulin G. The reacted microspheres were collected with a flow cytometer, and fluorescence was quantitated relative to the cutoff value provided by pooled sera from patients in whom H. pylori could not be demonstrated by culture or histology. Serum samples from 28 H. pylori-positive patients and 27 H. pylori-negative patients were tested by FMIA. Additionally, an in-house enzyme-linked immunosorbent assay (ELISA) employing the same antigen preparation and a commercially available ELISA were used to assay the patient population. Both the FMIA and in-house ELISA were 100% sensitive and 89% specific with positive and negative predictive values of 90 and 100% and no equivocal results. The commercial ELISA was 96% sensitive and 89% specific with positive and negative predictive values of 90 and 96% and five equivocal results. FMIA provides a rapid, inexpensive, and easily performed means for serodiagnosis of H. pylori.

Characterization of Escherichia coli serotype O157:H7.

A total of 174 strains of Escherichia coli serotype O157:H7 representing human isolates obtained from outbreaks and sporadic cases of hemorrhagic colitis, hemolytic-uremic syndrome, and nonbloody diarrheal illnesses as well as from asymptomatic carriers across Canada and the United States were examined. E. coli serotype O157:H7 possessed distinct biochemical markers, a 100% negative reaction for beta-glucuronidase and sorbitol, and a 100% positive reaction for raffinose and dulcitol; all strains otherwise were biochemically typical of E. coli. The vast majority (97%) of the strains were susceptible to commonly used antimicrobial agents. All strains produced readily detectable levels of Verotoxin; however, with polymyxin extraction, nearly 50% of the strains showed up to a 10-fold increase in the toxin level. None were found to mediate hemagglutination of human group A erythrocytes with or without D-mannose. The majority (approximately 70%) of the strains showed localized and diffuse adherence to HEp-2 cells and Henle 407 cells, and the adherence patterns were not very different from those observed among other E. coli strains. Twenty phage types were recognized, with phage types 1 and 2 accounting for 65% of the test strains. Plasmid analysis indicated three basic plasmid profiles: profile I was characterized by 68.7- and 4.2-megadalton (MDa) plasmids (62% of strains), profile II was characterized by 66.2- and 1.8-MDa plasmids (20% of strains), and profile III was characterized by a 62.5-MDa plasmid (18% of strains). A small number (19%) of the strains carried at least one additional plasmid over the basic complements, and these could be considered to constitute a miscellaneous category. None of the above-described characteristics of E. coli serotype O157:H7 could be directly correlated with one another, with the nature of infection, or with the geographical distribution of strains.

Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak.

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.

Plasmid profiles of value in differentiating Salmonella muenster isolates.

Strains of Salmonella muenster displaying identical antibiotic susceptibility and bacteriophage reaction patterns were found to differ in their complements of plasmid DNA. Plasmid profiles were used to separate 40 isolates from diverse sources into four distinct groups, one of which contained 30 strains without plasmids.

Antibiotic resistance and R-plasmids in food chain Salmonella: evidence of plasmid relatedness.

A large number of strains (1,783) belonging to 15 Salmonella serovars isolated, in Canada, from the three major links of the human food chain were screened for multiple antibiotic resistance and the presence of R-plasmids. Multiresistant strains occurred among animal feed, livestock, and human isolates at frequencies of 4, 22, and 14%, respectively. Conjugation analysis revealed that 58% of the isolates from feeds, 87% of those from livestock, and 89% of the human strains carried all or part of their resistance determinants extrachromosomally on R-plasmids. Conjugative plasmids representing nine different incompatibility groups were detected, with the Inc I alpha group being predominant. Within the limits of the parameters measured, certain of these plasmids show a degree of relatedness suggestive of a common ancestry.

In vitro system for the replication of the mini R1 factor Rsc11.

The in vitro synthesis of the mini R1-factor, Rsc11, was achieved using a soluble Escherichia coli cell-extract system. Triton X-100 lysates of the K12 strain 1101 (Rsc11) fractionated by Sephadex G25 chromatography supported the incorporation of labeled deoxyribonucleotides into covalently-closed circular (30S) and open-circular (25S) plasmid DNA as well as other molecules of various sizes. DNA synthesis required the presence of all four ribonucleotides and was rifampicin sensitive. Pulse-chase experiments indicated that this reaction is discontinuous. A dependence on ATP and sensitivity to nalidixic acid suggested this system capable of the replicative synthesis of Rsc11 DNA. Density-shift analysis confirmed this. In addition to hybrid, fully-heavy plasmid supercoils were synthesized indicating that more than one round of replication was completed. Approximately one-third of the molecules available to the system participate in this reaction.

dnaB gene of Escherichia coli K-12 affects superinfection inhibition between F' plasmids.

F' Escherichia coli K-12 strains bearing the chromosomal mutation dnaB43 offer significantly less resistance to the conjugational introduction of a second F' plasmid than do nonmutant strains. Both the entry exclusion and incompatibility components of superinfection inhibition are altered. This action of dnaB43 occurs regardless of the presence of a recA-minus mutation in matings in liquid cultures and on membrane filters and is not limited to a particular set of F' plasmids. These effects are co-transducible by phage P1 with the temperature sensitivity conferred by dnaB43. The effects also occur with a strain carrying dnaB107. In the double F' strains that arise, the two plasmids exist as units autonomous of one another and the chromosome.

Restoration of F' superinfection inhibition in a DnaB mutant of Escherichia coli upon construction of heterozygous DnaB merodiploids or P1 lysogens carrying a dnaB analogue.

F-prime derivatives of the Escherichia coli strain CR34 bearing the thermosensitivity mutation dnaB43 display low levels of plasmid-determined superinfection inhibition in conjugational crosses at 30 C. Salt-mediated phenotypic suppression of this temperature sensitivity fails to restore normal levels of inhibition, indicating its alteration is not a secondary effect of dnaB43 a-tion on growth or deoxyribonucleic acid syntheiss. Superinfection inhibition is fully restored in mutant cells made merodiploid for the dnaB region by introduction of the F' dnaB-+ plasmid F134-1. dnaB43-bearing strains lysogenized with P1 phage contribution dnaB-analogue protein show eight to nine times more superinfection inhibition than do the same cells carrying P1 prophage repressed dnaB-analogue protein production. Taken together, this evidence suggests a direct causal relationship between dnaB43 and the altered superinfection inhibition phenotype.