The largest reported outbreak of waterborne Escherichia coli O157:H7 in the United States occurred in upstate New York following a county fair in August 1999. Culture methods were used to isolate E. coli O157:H7 from specimens from 128 of 775 patients with suspected infections. Campylobacter jejuni was also isolated from stools of 44 persons who developed diarrheal illness after attending this fair. There was one case of a confirmed coinfection with E. coli O157:H7 and C. jejuni. Molecular detection of stx 1 and stx 2 Shiga toxin genes, immunomagnetic separation (IMS), and selective culture enrichment were utilized to detect and isolate E. coli O157:H7 from an unchlorinated well and its distribution points, a dry well, and a nearby septic tank. PCR for stx 1 and stx 2 was shown to provide a useful screen for toxin-producing E. coli O157:H7, and IMS subculture improved recovery. Pulsed-field gel electrophoresis (PFGE) was used to compare patient and environmental E. coli O157:H7 isolates. Among patient isolates, 117 of 128 (91.5%) were type 1 or 1a (three or fewer bands different). Among the water distribution system isolates, 13 of 19 (68%) were type 1 or 1a. Additionally, PFGE of C. jejuni isolates revealed that 29 of 35 (83%) had indistinguishable PFGE patterns. The PFGE results implicated the water distribution system as the main source of the E. coli O157:H7 outbreak. This investigation demonstrates the potential for outbreaks involving more than one pathogen and the importance of analyzing isolates from multiple patients and environmental samples to develop a better understanding of bacterial transmission during an outbreak.

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A genomics-based PCR method was developed and used to test specimens from patients involved in a large outbreak of Mycoplasma pneumoniae in a closed religious community in New York State. New P1 adhesin gene primers were designed to bind to 9 of 10 target sequences in the repetitive-element sequences obtained from the whole genome sequence of M. pneumoniae. This PCR method had a sensitivity of 0.006 CFU and a specificity of 100% for M. pneumoniae. The PCR was validated by testing a subset of patient samples by culture and comparing the results to those obtained by PCR. Of the initial 280 samples tested, 73 were positive by PCR and 22 were positive by culture. All samples positive by culture were also positive by PCR. Follow-up testing of selected patients 3 to 6 weeks after antibiotic treatment revealed that eight samples remained positive by PCR and that three samples remained positive by culture. Additionally, no nonspecific PCR inhibition was detected as a result of the specimen type, transport medium, or sample preparation methodology. The study demonstrates that the PCR described here is a rapid, sensitive, and specific method for the identification of M. pneumoniae and was helpful for the detection and monitoring of the outbreak.Mycoplasma pneumoniae is primarily a respiratory pathogen that is responsible for approximately 15 to 20% of all community-acquired pneumonias (8). The clinical presentation of patients with M. pneumoniae infection is not significantly different from that of patients with infections caused by other respiratory pathogens such as Chlamydia pneumoniae (22), so diagnosis of infection relies primarily on laboratory testing. M. pneumoniae is, however, a fastidious organism, requiring a laborious effort and 21 days or more for growth in culture. Likewise, serology is lacking in sensitivity, has questionable specificity (22), and is dependent on specific collection times relative to the onset of illness (5). DNA probes have also been used, but cross-reactivity between M. pneumoniae and Mycoplasma genitalium has been observed, and methods that use these probes also lack sensitivity (4).Numerous PCR approaches have been developed to provide a rapid, sensitive method for the detection of M. pneumoniae. PCR targets have included the P1 adhesin gene (3,4,5,12,17,21), the ATPase operon (11), genomic clones (2, 7), or a combination of these (1). The P1 adhesin gene is responsible for cytadherence, which is necessary for colonization and infection of host cells by M. pneumoniae (22). This gene is also an intriguing target for PCR because of its repetitive nature within the M. pneumoniae genome. Approximately 8% of this genome is composed of repetitive DNA elements with regions homologous to the P1 adhesin gene (10). Use of the entire genomic DNA sequence enabled us to design primers that would theoretically amplify the maximum number of target molecules, resulting in an improved sensitivity of the PCR for M. pneumoniae.A large outbreak of respiratory illness occurred in a closed religious communit...

Haemophilus ducreyi ATCC 33922, a virulent, well-characterized strain, was used to construct a genomic library in a bacteriophage expression vector. Three DNA fragments were selected for use as probes on the basis of their ability to encode H. ducreyi-specific proteins, as demonstrated by reactivity with rabbit polyclonal antiserum. With DNA-DNA hybridization, the three probes, labeled with 32p, reacted strongly with 16 strains of H. ducreyi obtained from a variety of sources. Thirty-seven other bacterial isolates, representing 33 different species and including organisms likely to be encountered in the urogenital tract, were also tested with the three probes. Twenty-eight of these isolates, including the genital pathogen Neisseria gonorrhoeae, showed no hybridization with the probes. In addition, herpes simplex virus-infected tissue culture cells and Treponema pallidum-infected rabbit testicular fluid were also completely nonreactive. Nine isolates, six belonging to other Haemophilus species and three belonging to Pasteurella species, reacted weakly with the probes when approximately 3.0 x 107 to 6.0 x 107 CFU was tested. When 105 to 106 CFU of these organisms was tested, the weak reactions could no longer be seen. Yet this number of H. ducreyi still reacted strongly. In fact, the three probes consistently detected 104 CFU of H. ducreyi in pure and mixed cultures and even produced a weak signal when only 103 CFU was present. It is clear from our results that use of these probes will greatly facilitate the laboratory diagnosis of this genital pathogen.

Chancroid is a sexually transmitted genital ulcer disease caused by Haemophilus ducreyi. Previously, we developed diagnostic DNA probes for H. ducreyi (L.

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Staphylococcus epidermidis was isolated from the blood of a 25-year-old pregnant woman following the administration of eight units of platelets. She had developed chills and a fever of 41.4°C soon after the transfusions were completed. S. epidermidis was also obtained from all eight platelet units, as well as from the packed-erythrocyte unit associated with the first unit of platelets. The isolation of the same organism from these epidemiologically related sources provided us with the opportunity to phenotypically and genetically characterize the isolates. Several typing methods, including four molecular techniques, were used to increase our chances of finding any differences between the isolates under investigation. Phenotypic analyses demonstrated that S. epidermidis isolates from the patient, platelet units, and erythrocyte unit reacted in exactly the same manner in 15 biochemical tests, exhibited slime production, and had the same antibiotic susceptibility pattern. Genetic analyses, which included plasmid profiles, plasmid cross-hybridization, field inversion gel electrophoresis, and ribotyping, substantiated the relationships between the S. epidermidis isolates from the patient, platelet units, and erythrocyte unit. Eight S. epidermidis control strains unrelated to the case were found to differ significantly from the platelet-related strain.