Hemolytic-Uremic Syndrome and Escherichia coli O121 at a Lake in Connecticut, 1999.

OBJECTIVE: Non-O157 Shiga toxin-producing Escherichia coli (STEC) have emerged as an important public health problem. Outbreaks attributed to non-O157 STEC rarely are reported. In 1999, follow-up of routine surveillance reports of children with hemolytic- uremic syndrome (HUS) identified a small cluster of 3 cases of HUS, all of whom had spent overlapping time in a Connecticut lake community in the week before onset of symptoms. We conducted an investigation to determine the magnitude and source of the outbreak and to determine risk factors associated with the transmission of illness. METHODS: We conducted a cohort study and an environmental investigation. The study population included all people who were at the lake in a defined geographic area during July 16-25, 1999. This time and area were chosen on the basis of interviews with the 3 HUS case-patients. A case was defined as diarrhea (>/=3 loose stools/d for >/=3 days) in a person who was at the lake during July 16-25, 1999. Stool samples were requested from any lake resident with diarrheal illness. Stools were cultured for Salmonella, Shigella, Campylobacter, and E coli O157. Broth cultures of stools were tested for Shiga toxin. Case-patients were asked to submit a serum specimen for antibody testing to lipopolysaccharides of selected STEC. Environmental samples from sediment, drinking water, lake water, and ice were obtained and cultured for E coli and tested for Shiga toxin. An environmental evaluation of the lake was conducted to identify any septic, water supply system, or other environmental condition that could be related to the outbreak. RESULTS: Information was obtained for 436 people from 165 (78%) households. Eleven (2.5%) people had illnesses that met the case definition, including the 3 children with HUS. The attack rate was highest among those who were younger than 10 years and who swam in the lake on July 17 or 18 (12%; relative risk [RR]: 7.3). Illness was associated with swimming (RR = 8.3) and with swallowing water while swimming (RR = 7.0) on these days. No person who swam only after July 18 developed illness. Clinical characteristics of case-patients included fever (27%), bloody diarrhea (27%), and severe abdominal cramping (73%). Only the 3 children with HUS required hospitalization. No bacterial pathogen was isolated from the stool of any case-patient. Among lake residents outside the study area, E coli O121:H19 was obtained from a Shiga toxin-producing isolate from a toddler who swam in the lake. Serum was obtained from 7 of 11 case-patients. Six of 7 case-patients had E coli O121 antibody titers that ranged from 1:320 to >1:20 480. E coli indicative of fecal contamination was identified from sediment and water samples taken from a storm drain that emptied into the beach area and from a stream bed located between 2 houses, but no Shiga toxin-producing strain was identified. CONCLUSIONS: Our findings are consistent with a transient local beach contamination in mid-July, probably with E coli O121:H19, which seems to be able to cause severe illness. Without HUS surveillance, this outbreak may have gone undetected by public health officials. This outbreak might have been detected sooner if Shiga toxin screening had been conducted routinely in HUS cases. Laboratory testing that relies solely on the inability of an isolate to ferment sorbitol will miss non-O157 STEC, such as E coli O121. Serologic testing can be used as an adjunct in the diagnosis of STEC infections. Lake-specific recommendations included education, frequent water sampling, and alternative means for toddlers to use lake facilities.

Growth of Escherichia coli O157:H7 in bruised apple (Malus domestica) tissue as influenced by cultivar, date of harvest, and source.

Four of five apple cultivars (Golden Delicious, Red Delicious, McIntosh, Macoun, and Melrose) inoculated with Escherichia coli O157:H7 promoted growth of the bacterium in bruised tissue independent of the date of harvest (i.e., degree of apple ripening) or the source of the apple (i.e., tree-picked or dropped fruit). Apple harvest for this study began 4 September 1998 and ended 9 October, with weekly sampling. Throughout this study, freshly picked (<2 days after harvest) McIntosh apples usually prevented the growth of E. coli O157:H7 for 2 days. Growth of E. coli O157:H7 did occur following 6 days of incubation in bruised McIntosh apple tissue. However, the maximum total cell number was approximately 80-fold less than the maximum total cell number recovered from Red Delicious apples. When fruit was stored for 1 month at 4 degrees C prior to inoculation with E. coli O157:H7, all five cultivars supported growth of the bacterium. For each apple cultivar, the pH of bruised tissue was significantly higher and degrees Brix was significantly lower than the pH and degrees Brix of undamaged tissue regardless of the source. In freshly picked apples, changes in the pH did not occur over the harvest season. Bruised Golden Delicious, McIntosh, and Melrose apple tissue pHs were not significantly different (tree-picked or dropped), and the degrees Brix values of McIntosh, Macoun, and Melrose apple tissue were not significantly different. Single-cultivar preparations of cider did not support growth of E. coli, and the cell concentration of inoculated cider declined over an 11-day test period. The rate of decline in E. coli cell concentration in the McIntosh cider was greater than those in the other ciders tested. The findings of this study suggested that the presence of some factor besides, or in addition to, pH inhibited E. coli growth in McIntosh apples.

Prevalence of Escherichia coli in apple cider manufactured in Connecticut.

Cider samples obtained from 11 cider mills operating in Connecticut during the 1997 to 1998 production season were tested for the presence of Escherichia coli. Cider production began in mid August and continued through March, with peak production in September and October. Of 314 cider samples tested, 11 (4%) were found to contain E. coli. Of the 11 mills, 6 (55%) tested positive for E. coli in the cider at least once during the production year. E. coli was first observed in cider samples produced in mid to late October and was not detected in samples made after January. A trend was observed for cider to decrease in acidity and increase in Brix (soluble sugars) throughout the production season. No correlation between pH and soluble sugars of cider and the presence of E. coli was detected. Eight mills used both dropped apples and tree-picked apples, whereas three mills used tree-picked apples only. The use of dropped apples in cider production began 5 weeks before the first detection of E. coli in cider. E. coli was isolated from cider samples produced using dropped apples and from samples produced using only tree-picked apples. No direct correlation between the use of dropped apples or tree-picked apples and the presence of E. coli in the cider was observed. An association between the time of apple harvest and the appearance of E. coli in cider was noted. For mills providing adequate records, all contaminated cider was produced from apples harvested between mid October and mid November.

Inhibitory Effects of Turf Pesticides on Bacillus popilliae and the Prevalence of Milky Disease.

Fourteen pesticides (fungicides, herbicides, and insecticides) were tested to determine whether they had deleterious effects on the bioinsecticide Bacillus popilliae, the causal agent of milky disease. All of these pesticides reduced levels of spore viability, spore germination, and/or vegetative cell growth when they were tested over a range of concentrations from 0 to 1,000 ppm of active ingredient, and the fungicides had the greatest detrimental effects. As determined by tests in water, the level of spore viability was significantly reduced by chlorothalonil, iprodione, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and 2-[(4-chloro-o-tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid. In tests performed with iprodione, loss of viability was evident at concentrations less than the concentration calculated to result from recommended use. Tests performed in soil demonstrated that triadimefon, chlorothalonil, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and pendimethalin at concentrations resulting from recommended rates of application reduced spore titers. Spore germination did not occur in the continued presence of 2-[(4-chloro-otolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid, isofenphos, and chlordane, whereas exposure of spores to triadimefon or pendimethalin for 2 days stimulated germination. The tests to determine effects on spore germination were inconclusive for all other pesticides. Triadimefon, chlorothalonil, iprodione, pendimethalin, and chlorpyrifos at concentrations less than the concentrations recommended for use inhibited vegetative cell growth of B. popilliae, and chlordane at a concentration that was twice the concentration expected to result from the recommended rate of application repressed cell growth. My data support the hypothesis that use of synthetic pesticides can contribute to a low incidence of milky disease in white grubs.

Selective Medium for Quantitation of Bacillus popilliae in Soil and in Commercial Spore Powders.

A medium consisting of MYPGP agar supplemented with vancomycin was found to be highly selective for Bacillus popilliae, especially for strains originally isolated from Japanese beetle larvae. The medium has proven to be useful for the quantitation of B. popilliae spores in commercial spore powder and in soil.

Presence of N6-methyladenine in GATC sequences of Bacillus popilliae and Bacillus lentimorbus KLN2.

Nine strains of Bacillus popilliae and Bacillus lentimorbus KLN2 contain N6-methyladenine in GATC sequences, as determined by using the restriction enzymes MboI and DpnI. Among eight other Bacillus species examined, all, except one strain of Bacillus brevis (ATCC 9999), lacked adenine methylation in GATC. A methylase with Escherichia coli dcm site specificity was not present in any of the Bacillus species studied.

Purification of aconitase from Bacillus subtilis and correlation of its N-terminal amino acid sequence with the sequence of the citB gene.

The DNA sequence for the promoter region of the Bacillus subtilis citB gene has been determined. Presumed "-10" and "-35" regions of the promoter have been identified, and transcriptional and translational start points of citB have been located. To correlate the DNA sequence of citB with the amino acid sequence of its presumed product, aconitase, it was necessary to devise a scheme for purification of this labile enzyme. This procedure relies on the ability to restore enzyme activity at each stage of purification by incubation in a reducing buffer containing a source of ferrous ions. B. subtilis aconitase appears to be a monomer with a molecular weight of approximately 120,000. The amino-terminal amino acids of aconitase fit the sequence predicted by analysis of the citB gene. Thus, citB codes for aconitase.

Relationship between aconitase gene expression and sporulation in Bacillus subtilis.

The citB of Bacillus subtilis codes for aconitase (D. W. Dingman and A. L. Sonenshein, J. Bacteriol. 169:3060-3065). By direct measurements of citB mRNA levels and by measurements of beta-galactosidase activity in a strain carrying a citB-lacZ fusion, we have examined the expression of citB during growth and sporulation. When cells were grown in nutrient broth sporulation medium, citB mRNA appeared in mid- to late-exponential phase and disappeared by the second hour of sporulation. This timing corresponded closely to the kinetics of appearance of aconitase enzyme activity. Decoyinine, a compound that induces sporulation in a defined medium, caused a rapid simultaneous increase in aconitase activity and citB transcription. After decoyinine addition, the rate of increase in aconitase activity in a 2-ketoglutarate dehydrogenase (citK) mutant and in a citrate synthase (citA) mutant was significantly less than in an isogenic wild-type strain. This is apparently due to a failure to deplete 2-ketoglutarate and accumulate citrate. These metabolites might act as negative and positive effectors of citB expression, respectively. Mutations known to block sporulation at an early stage (spo0H and spo0B) had no appreciable effect on citB expression or aconitase activity. These results suggest that appearance of aconitase is stimulated by conditions that induce sporulation but is independent of certain gene products thought to act at an early stage of sporulation.

Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine.

The activity of aconitase in Bacillus subtilis is greatly reduced in cells cultured in media containing rapidly metabolized carbon sources (e.g., glucose). Thus, expression of this enzyme appears to be subject to a form of catabolite repression. Since the product of the citB gene of B. subtilis is required for aconitase activity, we cloned the wild-type allele of this gene and used this DNA as a probe for transcription of citB in cells grown in various media. The steady-state level of RNA that hybridized to this probe was about 10-fold higher in B. subtilis cells grown in citrate-glutamine medium than in cells grown in glucose-glutamine medium. This result correlates well with the steady-state levels of aconitase activity. Two transcripts were shown to initiate within the cloned DNA; the steady-state level of one of these transcripts varied in the same way as did aconitase activity when cells were grown in media containing different carbon sources. This is the first demonstration of regulation by the carbon source of the level of a vegatative-cell transcript in B. subtilis.

Isolation of two bacteriophages from Bacillus larvae, PBL1 and PBL0.5, and partial characterization of PBL1.

Two temperate bacteriophages have been isolated from Bacillus larvae: PBL1 and PBL0 .5. Strains lysogenic for either of these phages are immune to lysis by the same phage but are sensitive to the other phage. PBL1 has an oval head, a non-contractile tail, and a base plate with a pin structure but no apparent tail fibres. The genome of PBL1 consists of double-stranded DNA with a molecular weight of 24.1 (+/-0.6) X 10(6), a G + C content (derived from melting temperature) of 41.5%, and cohesive ends. Restriction enzyme analysis permitted construction of a physical map of the genome.

Protection of Bacillus larvae from Oxygen Toxicity with Emphasis on the Role of Catalase.

Sporulation of Bacillus larvae NRRL B-3650 occurred only at aeration rates lower than those used for cultivation of most Bacillus species. One possible explanation for the requirement for a low level of aeration in B. larvae is that toxic forms of oxygen such as H(2)O(2) and superoxide are involved. The superoxide dismutase levels of strain B-3650 were similar to those of Bacillus subtilis 168 during sporulation, and no NADH peroxidase was present. Catalase activity was absent during exponential growth and first appeared near the start of the stationary phase. The catalase activity was 2,700 times less than that in B. subtilis 168 at the same stage of development. Therefore, the relative deficiency of catalase (and NADH peroxidase) might be the cause of the apparent O(2) toxicity. It was postulated that B. larvae might accumulate H(2)O(2) in the medium and exhibit more than normal sensitivity to H(2)O(2). Experimental results did not verify either postulate, but the possibilities of intracellular accumulation of H(2)O(2) and unusual sensitivity to endogenous H(2)O(2) were not excluded. The catalase present in early-stationary-phase cells was soluble, heat labile, and inhibited by cyanide, azide, and hydroxylamine. An increase in catalase activity also occurred at the time of appearance of refractile spores in both B. larvae NRRL B-3650 and B. subtilis 168. The level of catalase activity in strain B-3650 was 5,400 times less than that in B. subtilis 168 at this stage. In B. larvae, this second increase occurred primarily within the developing endospore. The activity in spore extracts was particulate, heat stable, and inhibited by hydroxylamine but not by azide or cyanide. Synthesis of catalase in B. larvae was unaffected by H(2)O(2), O(2), or glucose.

Medium Promoting Sporulation of Bacillus larvae and Metabolism of Medium Components.

A new medium, designated TMYGP broth, was developed that allowed the honeybee pathogen Bacillus larvae NRRL B-3650 to produce up to 5 x 10 spores per ml of culture (microscopic count). This species normally sporulates poorly, if at all, in artificial broth media. An aeration rate lower than that normally used to cultivate other Bacillus species was required for sporulation. During the exponential growth phase, acids were produced by catabolism of yeast extract components, causing a decrease in pH of the medium. Thereafter, the pH began to increase, probably because of derepression of the citric acid cycle and consumption of the acids. Only after this time did usage of glucose from the medium occur. Thus, glucose usage seems to be regulated by catabolite repression. The presence of glucose was needed for one or more of the later events of sporulation. Of many substances tested, only gluconic acid and glucosamine partially substituted for glucose as a requirement for sporulation. Pyruvate was also required for good sporulation. It was metabolized during the late-exponential phase of growth.