A large nontypical outbreak of Norwalk virus. Gastroenteritis associated with exposing celery to nonpotable water and with Citrobacter freundii.

The US Air Force Academy experienced a point-source outbreak of gastroenteritis originally believed to be caused by Salmonella. The overall attack rate was 48% among approximately 3000 cadets and staff. Food-specific attack rates implicated chicken salad. The odds ratio for chicken salad consumption in ill cadets was 10.7 (95% confidence interval: 8.2; 13.8). The celery component had been exposed to nonpotable water. Citrobacter freundii were statistically associated with consumption of the suspected vehicle and subsequent illness. Most aspects were consistent with the epidemiology of Norwalk gastroenteritis. However, the clinical presentation was not typical of reported outbreaks. One hundred five cadets required intravenous rehydration. Serum samples implicated Norwalk virus as the most probable cause of this outbreak. The Centers for Disease Control (Atlanta, Ga) recently began national surveillance for viral gastroenteritis. All outbreaks of gastroenteritis associated with nonpotable water should be investigated for evidence of viral cause.

Ureaplasma urealyticum demonstrated by open lung biopsy in newborns with chronic lung disease.

Lung biopsy tissue from eight infants with chronic lung disease was evaluated for the presence of Ureaplasma urealyticum. Specimens from four infants grew the organism. Pleural fluid cultures matched lung tissue but tracheal cultures were negative in two babies with positive lung tissue. There were no distinguishing pathologic findings in the four culture-positive infants which could be used to identify them vs. the culture-negative infants. Three culture-positive infants improved clinically after therapy directed at Ureaplasma even though two remained culture-positive. Ureaplasma grows in lung tissue of infants with chronic lung disease, it does not demonstrate any specific standard pathologic findings and tissue cultures do not match endotracheal cultures.

Non-01 Vibrio cholerae bacteremia--complication of a LeVeen shunt.

Vibrio cholerae bacteremia occurred in a patient with cirrhosis after placement of a LeVeen shunt. At the time of bacteremia, cultures of peritoneal fluid were negative and fluid dynamics did not suggest spontaneous bacterial peritonitis. Despite apparent successful treatment of the bacteremia, relapse and death occurred with culture positivity of peritoneal fluid for V. cholerae. Simultaneously, blood cultures were positive for Klebsiella pneumoniae. Agglutination studies demonstrated the V. cholerae to be a non-01 strain. Insertion of a LeVeen shunt, which bypasses the hepatic clearance mechanisms, appeared to have allowed bacteremia to occur with this bacterium that is rarely isolated from blood. In patients with LeVeen shunts, bacteremia with noninvasive pathogens may occur, and in coastal areas, Vibrios should be considered when bacteremia occurs.

Isolation of oxidase-negative Pseudomonas aeruginosa from urine culture.

An isolate of Pseudomonas aeruginosa lacking characteristic indophenol oxidase was recovered from a catheterized urine specimen.

The bacteriology of recurrent otitis media and the effect of sulfisoxazole chemoprophylaxis.

Middle ear effusion specimens were obtained from 31 children with recurrent episodes of acute otitis media. Of 75 total specimens 28 were obtained from children during chemoprophylaxis with sulfisoxazole. A single organism was isolated in 65 of 70 instances. Beta-lactamase was produced from Gram-negative organisms in 11 instances, and penicillin resistance from Streptococcus pneumoniae occurred in one instance. Haemophilus influenzae predominated during prophylaxis; S. pneumoniae predominated without it. Serotyping and biotyping were performed on 28 isolates from 8 children with consecutive episodes. In 17 instances the infecting organism was the same species but seven of these strains differed in serotype or biotype. The average number of weeks between onset of recurrence in children with homologous strains was shorter (2.6 weeks) than in the children from whom heterologous strains were found (5.7 weeks). Three media were evaluated for efficacy in 32 episodes, and direct plating resulted in the highest rate of recovery.

Pneumonia due to Haemophilus influenzae (H. aegyptius) biotype 3.

Haemophilus influenzae (H. aegyptius) biotype 3 was isolated from eye, nasopharyngeal, and sputum cultures of a 23-month-old male and from sputum and transtracheal aspirate cultures of his 39-year-old mother, both with diffuse bronchopneumonia.

Bacteremia associated with Haemophilus influenzae type e biotype 4.

Haemophilus influenzae type e biotype 4 was isolated from a single antemortem blood culture obtained from a 60-year-old, white male with abdominal carcinomatosis.

Development of a DNA probe for Ureaplasma urealyticum.

Ureaplasma urealyticum has been associated with a variety of disease conditions in humans. However, its exact etiologic role has not been well established because of the difficulties encountered in cultural diagnosis and the time needed for positive identifications. A DNA probe which is specific for a target DNA sequence unique to this suspected pathogen offers a rapid, sensitive and specific means of diagnosis. This study details the development of a polymerase chain reaction system for U. urealyticum. Using conventional hybridization techniques, a cloned genomic fragment was found to be specific for this organism. Sequencing of part of this probe DNA permitted the assignment of oligonucleotide primers which amplified a 186 bp target segment. This PCR system is specific for U. urealyticum but not for other closely related species of mycoplasma. This highly sensitive diagnostic technique will aid in determining the etiologic role, tissue tropism and dynamics of pathogenesis of this organism, and thereby result in better patient care.

Efficacies of rapid agglutination tests for identification of methicillin-resistant staphylococcal strains as Staphylococcus aureus.

Four commercially available rapid agglutination tests for the identification of Staphylococcus aureus were compared with the tube coagulase test for the identification of 300 methicillin-resistant isolates of staphylococci. Isolates tested included 207 methicillin-resistant S. aureus and 93 coagulase-negative staphylococci, collected from five medical centers. Strain variability was documented by phage typing and antimicrobial susceptibility patterns. Results of rapid identification tests ranged between 82 and 86% sensitivity, significantly poorer than the 98% sensitivity which the tube coagulase test provided.

Septic arthritis involving Capnocytophaga ochracea.

Septic arthritis of the knee developed in a 21-month-old child. The causative organism, isolated from two separate arthrocenteses, was identified as Capnocytophaga ochracea morphologically and by biochemical reactions. Previous human infections (bacteremias) have occurred in granulocytopenic hosts with concomitant oral pathology including periodontitis and gingivitis. No abnormalities of oral hygiene were present in this patient, and granulocyte numbers were normal or elevated. Eradication of the infection was accomplished with 8 weeks of antibiotic therapy combined with surgical drainage. Septic arthritis expands the spectrum of infections reported to be caused by Capnocytophaga spp.

Sterilization of complete dentures with sodium hypochlorite.

It is important to give the patient a denture that is clean and free from cross-contamination. This study was made to determine if Clorox could be used as a rapid, safe, and clinically effective way to sterilize complete dentures. The data obtained from this study indicate that a 5-minute immersion of dentures in undiluted Clorox accomplished sterilization against a variety of microorganisms, including a spore-forming bacteria and C. albicans.

Development of a diagnostic polymerase chain reaction assay for detection of Mycoplasma hominis.

A polymerase chain reaction (PCR)-based assay was developed for the detection of Mycoplasma hominis. This assay generates a 152-bp PCR product which was part of an initial 471-bp M. hominis genomic DNA fragment. The 471-bp DNA fragment was shown by hybridization analysis to be unique for M. hominis. The PCR assay can amplify as few as 18 molecules of target DNA. This diagnostic assay offers potential for wide clinical application as it is rapid and can be successfully performed on crude sample preparations from a variety of media or biopsies. The use of this assay should aid in defining the aetiologic and pathologic roles played by M. hominis and thereby benefit patients.

DNA fingerprinting of Candida rugosa via repetitive sequence-based PCR.

A repetitive sequence-based PCR (rep-PCR) technique was developed to characterize the genotypic relatedness among Candida rugosa isolates. Two repetitive sequences, viz., Care-2 and Com29 from Candida albicans, were used to design primers Ca-21, Ca-22, and Com-21, respectively. When used alone or in combination, these primers generated discriminatory fingerprints by amplifying the adjacent variable regions of the genome. Twenty-three isolates from burn patients, eight from other human sources, and four C. rugosa isolates pathogenic in animals were placed into nine fingerprinting groups. Different primers placed these isolates into identical groups, indicating that rep-PCR is a specific and reproducible technique for molecular characterization of C. rugosa. Moreover, these primers unequivocally discriminated among other important Candida species such as C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, and C. lusitaniae. These data confirm the conservation of repetitive sequences in Candida species. Because of its ease and sensitivity, rep-PCR offers a relatively rapid and discriminatory method for molecular typing of C. rugosa in outbreaks.

Identification of bacteria from a non-healing diabetic foot wound by 16 S rDNA sequencing.

Approximately 10-20% of diabetic foot wounds fail initial antibiotic treatment. It is generally believed that several bacterial species may be present in these types of wounds. Because some of these organisms cannot be easily cultured, proper identification is problematic and thus, appropriate treatment modalities cannot be applied. This report examined the bacterial flora present in a chronic diabetic foot wound that failed antibiotic treatment. A tissue sample was collected from the base of the wound and used for standard microbiological culturing. DNA from the sample was used to amplify bacterial 16 S rDNA gene sequences and a library of these sequences was made. The clones were placed into two major groups on the basis of their melting temperatures. Representatives of these groups were sequenced, and information was used to identify the bacteria present in the wound. The culture-based method identified a single anaerobic species, Bacteroides fragilis. The method employing rDNA sequencing identified B. fragilis as a dominant organism and Pseudomonas (Janthinobacterium) mephitica as a minor component. The results indicate that rDNA sequencing approach can be an important tool in the identification of bacteria from wounds.

Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore-enhanced repetitive-sequence PCR.

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.

An outbreak of gastroenteritis due to a heat-labile enterotoxin-producing strain of Escherichia coli.

In April 1981, an outbreak of gastroenteritis, characterized by diarrhea and abdominal cramps, occurred in 282 of approximately 3,000 personnel at a large metropolitan hospital in San Antonio, Tex. There was a significant association between illness and eating at the hospital cafeteria (P = 0.0008), but no specific food could be incriminated. Stools or rectal swabs from 54 ill individuals produced almost pure cultures of Escherichia coli. Cultures from 51 of these subjects had identical antibiotic sensitivity patterns, and 38 had the same biotype. Isolates from 45 persons were tested for production of heat-stable and heat-labile enterotoxins, using the suckling mouse and Y-1 adrenal cell assays, respectively. Of 45 isolates, 41 produced heat-labile enterotoxins, while 0 of 45 produced heat-stabile enterotoxins. Two isolates were rough, and 34 of the remaining 43 were serotype O25:H-. Two strains were O25:H+. None of the 45 strains possessed hemagglutination patterns typical of colonization factor antigens I or II. Six of seven O25:H- heat-labile enterotoxin-positive strains selected at random were piliated as seen by electron microscopy but did not agglutinate with anti-colonization factor antigens I or II antisera.

Cloning of insertion sequence IS1485 from Enterococcus species.

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.

A PCR assay for identification of Enterococcus faecium.

Enterococcus faecium has recently emerged as a serious nosocomial pathogen. The prevalence and severity of enterococcal infections, the mortality rate from such infections, and the antibiotic resistance of enterococci are often species dependent. Since conventional biochemical methods fail to differentiate E. faecium from certain newly described enterococcal species, a PCR-based assay was developed for the rapid identification of E. faecium.

Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR.

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia, Streptococcus pneumoniae was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of S. pneumoniae, verified by latex agglutination, were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the lytA gene, correctly detected all S. pneumoniae genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.