Multisystemic abscesses in African green monkeys (Chlorocebus aethiops) with invasive Klebsiella pneumoniae--identification of the hypermucoviscosity phenotype.

Invasive Klebsiella pneumoniae is an emerging disease of humans characterized by abscesses in the liver or other sites involving bacteria with the unique hypermucoviscosity phenotype. Over several months, 7 African green monkeys in our research colony developed abscess formation in multiple locations and succumbed to disease. K. pneumoniae was identified by bacterial culture in 6 monkeys and immunohistochemistry in 1 additional monkey. All monkeys had been housed in, or had contact with monkeys housed in, 1 animal room in our facility. All affected monkeys had 1 or more abscesses, most notably in the abdomen, but also affecting the lungs, cerebellum, and skin. Abdominal abscesses and associated adhesions entrapped loops of bowel, forming palpable masses. Abdominal masses were located at the root of the mesentery, the ileocecocolic junction, or the pelvic inlet. In 1 case, culture, serotyping, and polymerase chain reaction (PCR) analysis of the bacterial isolate identified K. pneumoniae expressing the hypermucoviscosity phenotype and capsular serotype K2 and determined that the K. pneumonia was genetically rmpA(+)/magA(-).

Pink-pigmented non-fermentative gram-negative rods associated with human infections: a clinical and diagnostic challenge.

Over the past several decades, the appearance of pink-pigmented bacteria in clinical specimens has gone from being a microbiologic curiosity in the clinical laboratory to the recognition of these aerobic microorganisms as etiologic agents of human disease, most notably bloodstream infections. Advances in the fields of molecular taxonomy and phylogenetics indicate that at least four distinct genera and eight different species are associated with clinical infections in susceptible patient populations. However, these bacteria are slow growing and present multiple diagnostic challenges to the microbiology laboratory including culture, isolation, and identification to species rank. This article provides a current review of these unusual non-fermentative chromogenic bacteria including their disease spectrum, taxonomy, and laboratory identification. The review also highlights the pitfalls or shortcomings we currently have in our knowledge of these microbes and their disease-producing capabilities.

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.

Occurrence, characterisation and detection of potential virulence determinants of emerging aquatic bacterial pathogens from the Philippines and Thailand.

Strains of Aeromonas spp., 'non-cholera vibrios' (NCVs) and Plesiomonas shigelloides isolated from aquatic environments, fish and human diarrhoeal cases in the Philippines and Thailand were characterised for potential virulence markers. Thus, the production of cytotoxin, cell-associated and cell-free haemolysin and their capacity to adhere to human intestinal (Henle 407) cells in vitro was investigated. In addition, the occurrence of tlh and tdh haemolysin genes and urease activity among V. parahaemolyticus strains was investigated. The results showed that strains recovered from clinical sources (human and fish) produced these virulence factors, whereas these are absent in environmental strains.

Rat-bite fever (Streptobacillus moniliformis): a potential emerging disease.

OBJECTIVES: To determine the relative prevalence of human infections attributable to Streptobacillus moniliformis in California over the past 3 decades. METHODS: A retrospective analysis of all the data collected was conducted on S. moniliformis cultures identified by the Microbial Diseases Laboratory (MDL) from January 1970 to December 1998. RESULTS: Information on a total of 45 S. moniliformis isolates was analyzed. Overall, 91% of the isolates were from human sources; 58% were received since 1990. These strains were divided almost equally between males and females, with 50% of the isolates from patients 9 years old or younger. In 75% of the cases of human infections where a diagnosis was given, rat-bite fever (RBF) was suspected; 83% of these suspected cases involved either a known rat bite or exposure to rodents. CONCLUSIONS: As crowding becomes an increasing environmental reality, humans are more frequently being exposed to zoonotic diseases as a result of encounters with "wild" animals. Domesticated animals also are exposed more frequently to wild animals; thus, increasing human exposure to once rare zoonotic illnesses. Rat-bite fever is a disease that seems to be easily recognizable by clinicians, easily identified in the clinical laboratory (if suspected), and successfully treated when the appropriate therapy is administered. Physicians should consider RBF as a possible diagnosis when fever, rash, and exposure to rats are part of the patient's history.

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.

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).

Susceptibilities of Yersinia pestis strains to 12 antimicrobial agents.

Ninety-two strains of Yersinia pestis recovered over a 21-year period were evaluated for susceptibility to traditional and newer antimicrobial agents. In vitro resistance was noted only against rifampin and imipenem (approximately 20% of strains). The most active compounds (MIC at which 90% of the isolates tested are inhibited) against Y. pestis were cefixime, ceftriaxone, trimethoprim-sulfamethoxazole, and trovafloxacin.

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.

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).

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.

Antimicrobial susceptibility patterns of Aeromonas jandaei, A. schubertii, A. trota, and A. veronii biotype veronii.

Fifty-six isolates of four Aeromonas species, which have been documented as causative agents of human infections or isolated from human clinical specimens, were subjected to antimicrobial susceptibility testing using a MicroScan WalkAway conventional (overnight incubation) gram-negative panel. The four species tested and the number of isolates of each were as follows: Aeromonas jandaei, 17; A. schubertii, 12; A. trota, 15; and A. veronii biotype veronii, 12. All isolates of A. trota were susceptible to all antimicrobial agents tested, except cefazolin (20% of isolates were resistant) and cefoxitin (13% of isolates were resistant). All isolates of A. schubertii and A. veronii biotype veronii, as well as 88% of A. jandaei isolates, were resistant to ampicillin. Resistance to ampicillin-sulbactam ranged from 25% of A. schubertii strains to 100% of A. veronii biotype veronii strains. Cefazolin resistance ranged from 17% of A. veronii biotype veronii isolates to 59% of A. jandaei isolates. Imipenem resistance was detected in 65% of A. jandaei strains and 67% of A. veronii biotype veronii strains. A. jandaei displayed resistance to piperacillin and ticarcillin in 53 and 71% of the isolates, respectively. A. veronii biotype veronii strains were 100% susceptible to piperacillin and 100% resistant to ticarcillin. These antibiogram data may be useful in establishing the identification of these four species when members of the genus Aeromonas are isolated from human clinical sources.

Association of Neisseria cinerea with ocular infections in paediatric patients.

Twenty-two strains of Neisseria cinerea were recovered from paediatric patients over a 7-year period and forwarded to the Microbial Diseases Laboratory for biochemical identification and/or confirmation. Eighteen of these 22 strains (82%) were recovered from the eyes of very young children (< or = 1 year), > 50% occurring during the neonatal period. The majority of eye isolates were involved in a variety of ocular infections including orbital cellulitis, conjunctivitis, and eye discharge (most common); in four of the 13 instances (31%) where laboratory data was available, Neisseria cinerea was recovered in pure culture. Neisseria cinerea isolates were often submitted to the Microbial Diseases Laboratory as possible 'N. gonorrhoeae' or 'Neisseria species' due to problems resulting from the use of commercial assays or unfamiliarity with the organism. These observations indicate that N. cinerea can produce eye infections in very young children, who presumably acquire this organism vertically from the mother during birth. Accurate identification of N. cinerea in such infants can preclude the social trauma and possible legal ramifications which can initially result from its misidentification as N. gonorrhoeae.

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.