Identification of Bartonella species directly in clinical specimens by PCR-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment.

It is now established that two species of Bartonella, namely, Bartonella henselae and B. quintana, cause bacillary angiomatosis in human immunodeficiency virus-infected patients. In addition, B. henselae causes cat scratch disease and B. quintana, B. henselae, and B. elizabethae can cause bacteremia and endocarditis in immunocompetent persons. We have developed a PCR-restriction fragment length polymorphism-based assay for direct detection and identification to species level of Bartonella in clinical specimens. This is accomplished by PCR amplification of Bartonella DNA using primers derived from conserved regions of the gene carrying the 16S ribosomal DNA, followed by restriction analysis using DdeI and MseI restriction endonucleases. We amplified a Bartonella genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from digestion of B. henselae and B. quintana amplicons with DdeI, whereas a different unique pattern was seen by using the same enzyme with B. vinsonii and B. elizabethae. With MseI digestion, B. henselae and B. vinsonii gave nearly identical patterns while B. quintana and B. elizabethae gave a different pattern. By combining the restriction analysis data generated with MseI and DdeI, unique "signature" restriction patterns characteristic for each species were obtained. These patterns were useful in identifying the Bartonella species associated with each tissue specimen.

A multistate outbreak of Escherichia coli O157:H7 infections associated with consumption of mesclun lettuce.

BACKGROUND: An outbreak of Escherichia coli O157:H7 infections in Connecticut and Illinois during May 28 to June 27, 1996, was investigated to determine the source of infections. METHODS: Independent case-control studies were performed in both states. Pulsed-field gel electrophoresis (PFGE) was performed on E. coli O157:H7 isolates. A case-patient was defined as a Connecticut or northern Illinois resident with diarrhea whose stool culture yielded E. coli O157:H7 of the outbreak-associated PFGE subtype. Controls were town-, age-, and sex-matched to case-patients. We traced implicated lettuce to the farm level and performed environmental investigations to identify unsafe lettuce production practices. RESULTS: In Connecticut and Illinois, infection was associated with consumption of mesclun lettuce (Connecticut matched odds ratio [MOR], undefined; 95% confidence interval [CI], 3.4 to infinity; and Illinois MOR, undefined; 95% CI, 1.4 to infinity). We traced implicated lettuce to a single grower-processor. Cattle, a known E. coli O157:H7 reservoir, were found near the lettuce fields. Escherichia coli (an indicator of fecal contamination) was cultured from wash water and finished lettuce. A trace-forward investigation identified 3 additional states that received implicated lettuce; E. coli O157:H7 isolates from patients in 1 of these states matched the outbreak-associated PFGE subtype. CONCLUSIONS: This multistate outbreak of E. coli O157:H7 infections was associated with consumption of mesclun lettuce from a single producer. Molecular subtyping facilitated the epidemiological investigation. This investigation increased the knowledge about current production practices that may contribute to the contamination of lettuce by microbial pathogens. Lettuce production practices should be monitored for microbiological safety.

An international outbreak of Salmonella infections caused by alfalfa sprouts grown from contaminated seeds.

An outbreak of Salmonella serotype stanley infections occurred in the United States and Finland in 1995. The outbreak was investigated through case-control studies in Arizona, Michigan, and Finland; by isolate subtyping; and by tracing and culturing of the implicated food. Alfalfa sprout consumption was the only exposure associated with S. stanley infections in Arizona (matched odds ratio [MOR] = 11.1; 95% confidence interval [CI], 1.4-513), Michigan (MOR = 5.5; CI, 1.6-23), and Finland (MOR undefined; CI, 4.9-infinity). US and Finnish patient isolates were a unique outbreak strain distinct from S. stanley isolates not linked to the outbreak. Alfalfa sprouts eaten by patients in 6 US states and Finland were traced to seed shipped by a Dutch shipper. Thus, it was concluded that alfalfa sprouts grown from contaminated seed caused an international outbreak of > or =242 S. stanley infections in > or =17 US states and Finland. This outbreak illustrates a new mechanism through which contamination of fresh produce can cause large, widely dispersed outbreaks.

Risk factors for sporadic infection with Escherichia coli O157:H7.

BACKGROUND: Little is known about risk factors for sporadic infection with Escherichia coli O157:H7. In response to a sharp increase in reported cases in New Jersey during July 1994, we conducted a case-control study to identify principal sources of infection and contributing practices. METHODS: Standardized questionnaires were used to evaluate (1) potential exposures of case patients and matched controls and (2) knowledge, attitudes, and practices of food preparers in case and control households. Patient isolates were subtyped by pulsed-field gel electrophoresis. RESULTS: Patients with E coli O157:H7 infection (N = 23; median age, 9 years; 55% female) were more likely than healthy controls to have eaten a hamburger in the week preceding illness (matched odds ratio, undefined; P < .001); 80% of the hamburgers eaten by ill persons were prepared at home. Food preparers in case households were less likely than those in control households to report washing their hands (odds ratio, 8.5; P < .005) and work surfaces (odds ratio, 10.5; P < .05) after handling raw ground beef. Pulsed-field gel electrophoresis yielded 17 unique subtypes among the 23 patient isolates, indicating multiple sources of infection. CONCLUSIONS: Hamburgers prepared at home are an important source of sporadic E coli O157:H7 infections. We estimate that adequate hand washing by food preparers could have prevented 34% of E coli O157:H7 infections in the study population.

A new route of transmission for Escherichia coli: infection from dry fermented salami.

OBJECTIVES: This study evaluated the production of dry fermented salami associated with an outbreak of Escherichia coli O157.H7 infection in Washington State and California. METHODS: Facility inspections, review of plant monitoring data, food handler interviews, and microbiological testing of salami products were conducted. RESULTS: Production methods complied with federal requirements and industry-developed good manufacturing practices. No evidence suggested that postprocessing contamination occurred. Calculations suggested that the infectious dose was smaller than 50 E. coli O157:H7 bacteria. CONCLUSIONS: Dry fermented salami can serve as a vehicle of transmission for O157:H7 strains. Our investigation and prior laboratory studies suggest that E. coli O157:H7 can survive currently accepted processing methods.

Meat grinders and molecular epidemiology: two supermarket outbreaks of Escherichia coli O157:H7 infection.

Between 23 June and 15 July 1994, 21 cases (19 primary and 2 secondary) of Escherichia coli O157:H7 infection were identified in the Bethel, Connecticut, area. Three pulsed-field gel electrophoresis (PFGE) patterns from 15 isolates (I, n = 13; II, n = 2; and III, n = 1) were observed. A case-control study that excluded secondary cases and patients with PFGE II and III patterns (n = 16) demonstrated that consumption of food from one supermarket was associated with illness (15/16 cases vs. 31/47 geographically matched controls, odds ratio [OR] undefined, lower 95% confidence interval OR = 1.45, P = .018). No one food was associated with illness. Inspection of the supermarket revealed deficiencies in hygiene and meat handling practices. The 2 cases with PFGE II ate raw beef and raw lamb from a second supermarket. These outbreaks demonstrate the value of PFGE in supporting epidemiologic investigations and the potential for outbreaks arising from retail outlets.

Reevaluation of the cellular fatty acid composition of Legionella micdadei Bari 2/158.

The cellular fatty acid composition of Legionella micdadei Bari 2/158 was reevaluated because of its purported differences from other L. micdadei strains and its similarity to L. bozemanii. We found the fatty acid content of this strain to be consistent with that of 11 other strains of L. micdadei, including the presence of an anteiso branched-chain, monounsaturated, 17-carbon acid (Ca17:1) which is characteristic of this species. The double-bond position of Ca17:1 was established at the omega 7 (or delta 9) position by combined gas chromatographic-mass spectrometric analysis of dimethyl disulfide derivatives. The Ca17:1 omega 7 acid was absent in each of 14 strains of L. bozemanii.

Isoprenoid quinones of Campylobacter cryaerophila, C. cinaedi, C. fennelliae, C. hyointestinalis, C. pylori, and "C. upsaliensis".

The isoprenoid quinone contents of Campylobacter cryaerophila, C. cinaedi, C. fennelliae, C. hyointestinalis, C. pylori, and "C. upsaliensis" were determined by reverse-phase thin-layer and high-performance liquid chromatography. All six of these recently named Campylobacter species contained menaquinone-6 (MK-6), but only C. hyointestinalis and "C. upsaliensis" contained 2,[5 or 8]-dimethyl-3-farnesyl-farnesyl-1,4-naphthoquinone (*MK-6), a previously described novel menaquinone of the Campylobacter genus. C. cryaerophila, C. cinaedi, C. fennelliae, and C. pylori contained an unidentified quinone (Un-MK-6) with a molecular weight of 580 and a base peak ion of m/e = 225 by mass spectrometry but with chromatographic properties different from those of MK-6. *MK-6 and Un-MK-6 are important chemotaxonomic markers of Campylobacter and Campylobacter-like organisms.

Location of double bonds in monounsaturated fatty acids of Campylobacter cryaerophila with dimethyl disulfide derivatives and combined gas chromatography-mass spectrometry.

Location of the double-bond position of monounsaturated fatty acids in Campylobacter cryaerophila was accomplished with combined gas chromatography-mass spectrometry analysis of dimethyl disulfide (DMDS) derivatives. The monoenoic fatty acids from whole bacterial cells were converted to methyl esters and then to DMDS adducts and analyzed by capillary gas chromatography-mass spectrometry. The mass spectra of DMDS adducts gave an easily recognizable molecular ion (M+) and two major diagnostic ions attributable to fragmentation between the two CH3S groups located at the original site of unsaturation. Two previously unidentified acids that distinguish C. cryaerophila from other bacteria were identified as C14:1 omega 7 and C16:1 omega 7 from their mass spectral fragmentation patterns. Resolution of cis and trans isomers by capillary column gas chromatography permitted assignment of the trans isomer to the C16:1 omega 7 acid.