Isolation of toxigenic Hafnia alvei from a probable case of hemolytic uremic syndrome.

An 11-year-old girl presented to a central California children's hospital with a 3-day history of erythematous lesions on her forehead, neck, and trunk, abdominal pain, persistent emesis, and decreased urinary output. One day prior to admission she had a mild bout of diarrhea with a small amount of blood in her stool. Upon admission her condition rapidly worsened with acute renal failure, anemia, and thrombocytopenia. One of the possible causes of this condition included hemolytic uremic syndrome. Stool cultures of this patient tested at the children's hospital and at a state reference laboratory were repeatedly negative for Escherichia coli O157:H7. However, the state reference laboratory detected a toxigenic strain of Hafnia alvei active on Vero cells from two consecutive stool cultures during the acute phase of her illness.

Non-invasive Providencia alcalifaciens strains fail to attach to HEp-2 cells.

We investigated the adherence properties of six P. alcalifaciens strains with previously characterized differential invasive capabilities in HEp-2 cells. Highly invasive strains were found to attach to HEp-2 cell monolayers within 2 h post-infection and in large numbers on the eukaryotic cell surfaces within 3 h post-infection. In contrast, weakly or non-invasive P. alcalifaciens strains were non-adherent to HEp-2 cells even at 3 h post-infection. Highly invasive isolates were found to weakly bind F-actin using the fluorescent actin staining assay although these strains were negative for Escherichia coli attachment and effacing gene (eaeA) of enteropathogenic E. coli (EPEC). These results suggest that the strain variation in the ability of P. alcalifaciens to invade HEp-2 cells previously noted by several investigators may be linked to expression of key adhesin(s) on the cell surface of invasive isolates.

Biochemical identification and characterization of DNA groups within the Proteus vulgaris complex.

We placed 43 isolates belonging to the Proteus vulgaris complex into proposed DNA groups (genomovars) using five previously recommended tests (tests for esculin hydrolysis, production of acid from salicin, L-rhamnose fermentation, and elaboration of DNase and lipase). On the basis of the results of these five tests, 49% of the isolates fell into DNA groups 5 and 6, 37% fell into DNA group 2, and the remaining 14% fell into DNA groups 3 and 4. Sequencing of the 16S rRNA genes of 11 members of DNA groups 5 and 6 indicated that 10 of these isolates (91%) could be unambiguously assigned to one of these two genomospecies. Overall expression of selected enzymatic and virulence-associated characteristics did not differ significantly among DNA groups.

A new diagnostic problem: isolation of Escherichia coli O157:H7 strains with aberrant biochemical properties.

Over a five-year period (1995-1999) the Microbial Diseases Laboratory received 34 strains of E. coli O157:H7 each with a single aberrant biochemical property. In addition, 27 O157 strains with negative or delayed motility were noted during the same time period. These observations suggest that there may be an increased likelihood to misdiagnose O157:H7 infections using commercial systems in the future due to increasing phenotypic variability.

High incidence of extra-intestinal infections in a Salmonella Havana outbreak associated with alfalfa sprouts.

OBJECTIVE: To determine a vehicle and point source for an outbreak of Salmonella Havana. METHODS: The authors conducted a case-control study and traceback investigation of 14 residents of California and four from Arizona with onsets of illness from Apr 15, 1998, to June 15, 1998, and Salmonella Havana infections with identical PFGE patterns. RESULTS: Seventeen of 18 patients were women. Seventeen were adults 20-89 years of age. Nine (50%) had diarrheal illness, 6 (33%) had urinary tract infections, 2 (11%) had sepsis, and one had an infected surgical wound after appendectomy. Four patients were hospitalized, and one died. Eating alfalfa sprouts was associated with S. Havana infection (OR = 10.0; 95% confidence interval 1.2, 83.1; P = 0.01). CONCLUSIONS: This outbreak resulted in a high incidence of extra-intestinal infections, especially urinary tract infections, and high morbidity. Raw alfalfa sprouts, often considered a safe "heath food," can be a source of serious foodborne disease outbreaks.

Aeromonas salmonicida subsp. pectinolytica subsp. nov., a new pectinase-positive subspecies isolated from a heavily polluted river.

Aeromonas strains which phenotypically and genetically belong to the Aeromonas salmonicida species but that according to their phenotypic properties constitute a new subspecies have been isolated from the water of a heavily polluted river, the Matanza river, situated near the central district of Buenos Aires city. These strains were ascribed to the A. salmonicida species by using 65 biochemical tests and by DNA-DNA hybridization. They produce acid from -sorbitol, an unusual biochemical property found in a few members of the A. salmonicida species. They also utilize urocanic acid and do not ferment L-rhamnose or utilize LD-lactate, and are elastase- and gluconate-negative. The DNA relatedness was over 70%, the current limit accepted for the phylogenetic definition of a species, to the described A. salmonicida subspecies and nearly 100% within the new group of Aeromonas strains. Phenotypic differentiation from other A. salmonicida subspecies was readily achieved using the following characteristics: growth at 37 degrees C, melanin production, indole and Voges-Proskauer assays, growth on KCN broth, mannitol and sucrose fermentation and gas from glucose. A remarkable property of the strains of the new group was their ability to degrade polypectate, an unusual feature among Aeromonas species in general. The complete 16S rRNA gene of one strain of the new group was sequenced. Comparison with rDNA sequences of Aeromonas members available in databases revealed a close relationship between this strain and strains belonging to A. salmonicida subsp. salmonicida, masoucida and achromogenes, in agreement with the biochemical data. Since the new A. salmonicida strains constitute a tight genomic group that can be identified by phenotypic properties it was concluded that they represent a new subspecies for which the name Aeromonas salmonicida subsp. pectinolytica is proposed. The type strain of A. salmonicida subsp. pectinolytica is 34melT (= DSM 12609T).

Urinary tract infections associated with nontyphoidal Salmonella serogroups.

In an analysis of over 23,000 nontyphoidal strains of Salmonella submitted to the Microbial Diseases Laboratory between 1992 and 1996, two groups (C(1) and E) were significantly recovered more often from the urinary tract than stool compared to more common groups such as B and D. An analysis of >60 urine isolates from 1996 suggests that most of these represent true urinary tract infections, as opposed to colonization or fecal contamination, by virtue of being isolated in pure culture and in high concentrations (>100,000 CFU/ml).

Unusual food-borne pathogens. Listeria monocytogenes, Aeromonas, Plesiomonas, and Edwardsiella species.

Although these four groups of organisms are perceived as infrequent food-borne pathogens or of dubious significance, increasing epidemiologic data indicate that L. monocytogenes is an emerging cause of infections, particularly gastroenteritis. Furthermore, if data are ever generated that prove that most fecal isolates of Aeromonas are involved in bacterial diarrhea, then aeromonads will become recognized as important food-borne pathogens. For Plesiomonas and Edwardsiella, recognition of possible involvement in food-borne disease requires detailed medical histories, including foreign travel, contact with pets or animals, and food consumption histories.

Prototypal diarrheagenic strains of Hafnia alvei are actually members of the genus Escherichia.

We analyzed five bacterial strains, designated 19982, 9194, 10457, 10790, and 12502, that were isolated from stool specimens of individuals with diarrheal illness by the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh (M. J. Albert, S. M. Faruque, M. Ansaruzzaman, M. M. Islam, K. Haider, K. Alam, I. Kabir, and R. Robins-Browne, J. Med. Microbiol. 37:310-314, 1992). The strains were initially identified as Hafnia alvei with a commercial identification system and were reported to contain the eae gene of enteropathogenic Escherichia coli. Results of conventional biochemical analyses, testing of susceptibility to cephalothin, lysis by a Hafnia-specific phage, and amplification of the outer membrane protein gene phoE with species-specific primers support the identification of these strains as members of the genus Escherichia rather than Hafnia alvei. These strains varied from typical E. coli strains by their inability to produce acid from lactose or D-sorbitol and failure to elaborate the enzyme beta-D-glucuronidase. PCR analysis confirmed previous findings that the strains were positive for the eae gene and negative for other virulence markers present among recognized categories of diarrheagenic E. coli. Our findings support the hypothesis that these strains are a new category of diarrheagenic isolates belonging to the genus Escherichia and illustrate the importance of using multiple methodologies when identifying new bacterial agents of diarrheal disease.

Biochemical identification of Citrobacter species defined by DNA hybridization and description of Citrobacter gillenii sp. nov. (formerly Citrobacter genomospecies 10) and Citrobacter murliniae sp. nov. (formerly Citrobacter genomospecies 11).

Recent work describing six named species and two unnamed genomospecies within Citrobacter has enlarged the genus to 11 species. DNA relatedness and phenotypic tests were used to determine how well these species can be identified. One hundred thirty-six strains were identified to species level by DNA relatedness and then identified phenotypically in a blinded fashion. By using conventional tests, 119 of the 136 strains (88%) were correctly identified to species level. Three additional strains (2%) were identified as citrobacteria but were not identified to species level, and 14 strains (10%) were misidentified as other Citrobacter species. Carbon source utilization tests were used to identify 86 of the strains. Eighty-four strains (98%) were correctly identified, and two strains (2%) were misidentified as other Citrobacter species. Additional strains of Citrobacter genomospecies 10 and Citrobacter genomospecies 11 were identified, allowing these species to be formally named as Citrobacter gillenii sp. nov. and Citrobacter murliniae sp. nov., respectively.

Identification and initial characterization of elastase activity associated with Vibrio cholerae.

Strains of Vibrio cholerae O1 (Ogawa, Inaba) and non-O1 serogroups have been found to produce an elastolytic protease that can be detected on 0.3% elastin agar plates or in broth cultures. The elastase enzyme appears to be maximally expressed in late log phase (14-18 h postinoculation) and has optimum activity at a pH range between 7 and 8. Comparative studies indicate that more than 60% of V. cholerae strains analyzed quantitatively produce more elastase in broth (two- to fourfold higher) than other elastase-positive Vibrio species such as Vibrio vulnificus. The V. cholerae elastase enzyme was not inhibited by trypsin, serine-protease, or thiol-protease inhibitors, but was inhibited by phosphoramidon. Ultrafiltration studies indicate the V. cholerae elastase enzyme has a molecular weight >30,000, and a 34K protein with possible elastase activity has been detected by SDS-PAGE for one non-O1 isolate (strain 2396). Cumulative results suggest that the V. cholerae elastase is probably a member of the N-type metalloprotease family and shares similar properties with other elastase enzymes described for pathogenic and nonpathogenic species in this genus.

An untypeable Shigella flexneri strain associated with an outbreak in California.

Eleven Shigella flexneri (group B) isolates were recovered from epidemiologically linked patrons and food handlers from a restaurant-associated outbreak of shigellosis. Six isolates available for pulsed-field gel electrophoresis were identical. All strains agglutinated in group B and subgroup factor 6 sera but not in group 1 through group 6 sera.

Two outbreaks of multidrug-resistant Salmonella serotype typhimurium DT104 infections linked to raw-milk cheese in Northern California.

CONTEXT: Salmonella serotype Typhimurium definitive type 104 (DT104), with resistance to 5 drugs (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline), has emerged as the most common multidrug-resistant Salmonella strain in the United States. However, illnesses resulting from this strain have not been associated definitively with a source in this country. OBJECTIVE: To determine the source of 2 outbreaks of Salmonella Typhimurium DT104. DESIGN: Matched case-control study conducted between March 24 and April 5, 1997 (outbreak 1), enhanced surveillance for new cases dating from February 1, 1997 (outbreak 2), and environmental and laboratory investigations. SETTING AND PARTICIPANTS: The case-control study included residents of 2 adjacent counties in northern California infected with the outbreak strain of Salmonella Typhimurium var Copenhagen and age-matched controls. For enhanced surveillance, a case was defined as Salmonella Typhimurium infection in a person exposed to fresh Mexican-style cheese. MAIN OUTCOME MEASURES: Risk factors for infection and source of implicated food. RESULTS: Outbreak 1 peaked in February 1997; 31 patients were confirmed by culture as having Salmonella Typhimurium var Copenhagen infection, isolates of which showed indistinguishable pulsed-field gel electrophoresis (PFGE) patterns. The outbreak strain was phage type DT104 with the 5-drug resistance pattern. Sixteen cases and 25 controls were enrolled in the case-control study; 15 of 16 Salmonella Typhimurium var Copenhagen cases compared with 14 of 24 matched controls reported eating unpasteurized Mexican-style cheese, (matched odds ratio, 7.9; 95% confidence interval, 1.1-354.9). Enhanced surveillance uncovered outbreak 2, which peaked in April 1997 and was caused by a non-Copenhagen variant of Salmonella Typhimurium. During outbreak 2, Salmonella Typhimurium was isolated from 79 persons who ate fresh Mexican-style cheese from street vendors and from cheese samples and raw milk. The PFGE pattern of the milk isolate matched 1 of the 3 patterns recovered from patients; all strains were phage type DT104b with the 5-drug resistance pattern. CONCLUSION: Raw-milk products pose a risk for multidrug-resistant Salmonella Typhimurium DT104 infections.

Invasion of HEp-2 and other eukaryotic cell lines by Providenciae: further evidence supporting the role of Providencia alcalifaciens in bacterial gastroenteritis.

Wild-type strains of Providencia species were evaluated for their ability to invade HEp-2 monolayers based upon microscopic and semi-quantitative assays. Of 14 P. alcalifaciens strains tested, 3 (17%) were found to be highly invasive, 4 (22%) moderately invasive, and the remaining 61% weakly or noninvasive. HEp-2 invasion results were confirmed by thin-section electron microscopy. Invasive capabilities of P. alcalifaciens were greater at higher MOIs (100 to 1000) than at lower inocula (<10 MOI). No strain of P. stuartii or P. rettgeri tested invaded HEp-2 cells. Quantitative assays of Triton X-100-lysed, HEp-2-invaded cells indicated that between 0.001% and 0. 013% of the initial bacterial inoculum was gentamicin resistant. Further testing of select strains on various cell lines indicated the efficiency of invasion was Vero > Y1 > INT-407 > HEp-2. Two isolates recovered from a father and son with prolonged diarrhea after returning from Mexico were found to be identical on the basis of biotype, serotype, and genotype. These results provide additional evidence that some P. alcalifaciens strains cause gastroenteritis.

Evolving concepts regarding the genus Aeromonas: an expanding Panorama of species, disease presentations, and unanswered questions.

It has been almost 10 years since a major review on the association of Aeromonas with human disease has been published. During that period the number of valid species in the genus has grown to 14, with a new family (Aeromonadaceae) established to house this genus. Despite this explosion in the number of new genomospecies, only five (Aeromonas hydrophila, A. caviae, A. veronii, A. jandaei, and A. schubertii) are currently recognized as human pathogens. New syndromes attributed to this genus include hemolytic uremic syndrome, burn-associated sepsis, and a variety of respiratory tract infections, including epiglottitis. Convincing evidence suggests that some aeromonads do cause gastroenteritis, but it is presently unclear whether many of the strains isolated from feces are involved in diarrheal disease. Many questions regarding this genus remain unanswered.

Misidentification of unusual Aeromonas species as members of the genus Vibrio: a continuing problem.

Two unusual cases of Aeromonas infection are described, one associated with bacteremia (Aeromonas schubertii) and another in which the organism was recovered from an infected gall bladder (Aeromonas veronii biotype veronii). These strains were initially identified as Vibrio damsela and Vibrio cholerae by the Vitek and API 20E systems, respectively. Use of appropriate screening tests and familiarity with the newer Aeromonas species could prevent initial misidentifications and potential public health consequences.

Citrobacter farmeri bacteremia in a child with short-bowel syndrome.

A case of sepsis in a pediatric patient due to the newly described Citrobacter species C. farmeri is described. Factors predisposing this child to infection included short-bowel syndrome requiring total parenteral nutrition.

Multicenter evaluation of the MicroScan Rapid Gram-Negative Identification Type 3 Panel.

The accuracy and performance of the revised MicroScan Rapid Gram-Negative Identification Type 3 Panel (Dade MicroScan Inc., West Sacramento, Calif.) were examined in a multicenter evaluation. The revised panel database includes data for 119 taxa covering a total of 150 species, with data for 12 new species added. Testing was performed in three phases: the efficacy, challenge, and reproducibility testing phases. A total of 405 fresh and stock gram-negative isolates comprising 54 species were tested in the efficacy phase; 96.8% of these species were identified correctly in comparison to the identification obtained either with the API 20E system (bioMérieux Vitek, Hazelwood, Mo.) or by the conventional tube method. The number of correctly identified isolates in the challenge phase, including new species added to the database, was 221 of 247, or 89.5%, in comparison to the number correctly identified by the conventional tube method. A total of 465 isolates were examined for intra- and interlaboratory identification reproducibility and gave an agreement of 464 of 465, or 99.8%. The overall reproducibility of each individual identification test or substrate was 14,373 of 14,384, or 99.9%. The new Rapid Gram-Negative Identification Type 3 Panel gave accurate and highly reproducible results in this multiple-laboratory evaluation.

Analysis of the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes in urease-positive strains of Vibrio parahaemolyticus isolated on the West Coast of the United States.

Urease-positive (Ure+) and urease-negative (Ure-) strains of Vibrio parahaemolyticus isolated from patients on the West Coast of the United States between 1979 and 1995 were analyzed for the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes (trh1 and trh2). The DNA colony hybridization method with the polynucleotide probes was used to determine the distribution of the genes. Of 60 Ure+ strains, 59 strains (98%) had the trh (either trh1 or trh2) gene and 54 strains (90%) carried the tdh gene. The absence of the trh gene or a related sequence in an exceptional Ure+ strain was confirmed by Southern blot analyses. The stronger correlation with the trh gene than with the tdh gene was mostly attributable to strains possessing only the trh2 gene. Of 25 Ure- strains, 20 strains (80%) had the tdh gene but none had the trh gene. These results indicate a very strong correlation between the Ure+ phenotype and the trh gene and are consistent with those reported for strains isolated in Asia. The Ure+ strains carrying the trh genes were not restricted to a unique group of the strains. The O4:K12 strains carrying the trh1 gene have predominantly been isolated since 1979. However, strains of various non-O4:K12 serovars carrying either the trh1 or the trh2 gene became predominant after 1992. In addition, analysis by the arbitrarily primed PCR method revealed two subgroups within the selected Ure+ O4:K12 strains. Hybridization tests with oligonucleotide probes demonstrated that the trh1 sequences of the West Coast strains differ to some extent from those of Asian strains. Nevertheless, a PCR method previously established to detect both the trh1 and the trh2 genes in Asian strains could detect 98% of those genes in the West Coast strains.

Enterobacter cancerogenus ("Enterobacter taylorae") infections associated with severe trauma or crush injuries.

Five cases of Enterobacter cancerogenus infections (wound, n = 4; bacteremia, n = 1) in adults are described. All infections seemed to be community acquired and occurred after precipitating events such as multiple trauma to the head or severe crush injuries. All five strains of E cancerogenus were recovered in pure culture, and three of these were isolated on multiple occasions. The results indicate that E cancerogenus can cause wound infections and septicemia in persons environmentally exposed to these organisms during traumatic events.