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Over the past decade, the emergence of Aeromonas species as bona fide human pathogens and their probable role as etiologic agents of bacterial gastroenteritis have resulted in an explosion of scientific interest in the genus. Major accomplishments occurring in this field during that interval include a more refined taxonomy, identification of new cell-associated factors (surface layers, pili), and the molecular analysis of selected extracellular gene products that may play a critical role in pathogenesis (hemolysins, enterotoxins). This review provides an updated overview of recent systematic, clinical, and pathophysiologic advances and defines key areas of medical and scientific interest in which major questions remain unanswered.

The pathogenic characteristics of 35 Edwardsiella strains from clinical and environmental sources were investigated. Overall, most Edwardsiella tarda strains were invasive in HEp-2 cell monolayers, produced a cell-associated hemolysin and siderophores, and bound Congo red; many strains also expressed mannose-resistant hemagglutination against guinea pig erythrocytes. Edwardsiella hoshinae strains bound Congo red and were variable in their invasive and hemolytic capabilities while Edwardsiella ictaluri strains did not produce either factor; neither E. hoshinae nor E. ictaluri expressed mannose-resistant hemagglutination nor elaborated siderophores under the tested conditions. Selected strains of each species tested for mouse lethality indicated strain variability in pathogenic potential, with E. tarda strains being the most virulent; 50% lethal doses in individual strains did not correlate with plasmid content, chemotactic motility, serum resistance, or expression of selected enzyme activities. The results suggest some potential important differences in pathogenic properties that may help explain their environmental distribution and ability to cause disease in humans.

Commercial DNA hybridization assays (Syngene, Inc., San Diego, Calif.) utilizing alkaline phosphatase-labeled oligonucleotide probes for the identification of Mycobacterium tuberculosis complex and M. avium complex (MAC) were evaluated with 261 isolates of mycobacteria. On the basis of biochemical criteria, the test for MAC was 98% specific and more sensitive (95 of 99, 95%) than Gen-Probe (88 of 99, 89% sensitivity); the major difference in sensitivity noted between the two systems was related to the hybridization of seven MAC strains to the SNAP X probe. The M. tuberculosis complex probe correctly identified all 62 isolates of M. tuberculosis and all 11 isolates of M. bovis, for a sensitivity of 100%. There were two discrepant reactions with mycobacteria other than M. tuberculosis complex isolates.

Previous DNA hybridization studies established 12 Aeromonas genospecies, from which nine phenotypic species have been proposed: Aeromonas hydrophila, A. sobria, A. caviae, A. media, A. veronii, A. schubertii, A. salmonicida, A. eucrenophila, and A. jandaei. We have delineated a new Aeromonas genospecies, A. trota, on the basis of 13 strains isolated primarily from fecal specimens from southern and southeastern Asia. All strains were highly related to the proposed type strain, AH2 (ATCC 49657T): 51 to 100% (60 degrees C) and 49 to 99% (75 degrees C), with 0.2 to 2.2 divergence. AH2 was only 16 to 41% (60 degrees C) related to all other Aeromonas type strains and DNA group definition strains. The unique profile of A. trota includes negative reactions for esculin hydrolysis, arabinose fermentation, and the Voges-Proskauer test, positive reactions for cellobiose fermentation, lysine decarboxylation, and citrate utilization, and susceptibility to ampicillin, as determined by the broth microdilution MIC method and the Bauer-Kirby disk diffusion method (10 micrograms). Nine of the A. trota strains were from a single study of 165 geographically diverse aeromonads. This finding questions the efficacy of screening fecal specimens for Aeromonas spp. with ampicillin-containing media and suggests a previously unrecognized prevalence of this new species.

The genetic characteristics and biochemical and structural properties of a number of autoagglutinating (AA) strains of Aeromonas associated with invasive and noninvasive disease in humans and infections in animals and from environmental sources were investigated. Of 27 strains analyzed by multilocus enzyme typing and DNA hybridization studies, 25 (93%) were confirmed to belong to either hybridization group 1 (phenospecies and genospecies Aeromonas hydrophila) or 8 (phenospecies Aeromonas sobria; genospecies Aeromonas veronii). Further analysis of 19 of these strains indicated that four major groups could be identified on the basis of serologic and surface characteristics, protein and lipopolysaccharide composition, and virulence properties; these groupings held true regardless of the site of isolation or disease process involved. The major AA+ group identified was serogroup O:11, whose strains possessed an S layer, were resistant to the bactericidal activity of normal serum, and were pathogenic in mice. The results suggest a set of useful phenotypic and structural markers for identification of specific subsets of mesophilic Aeromonas involved in a wide range of infections in the animal kingdom.

The in vitro susceptibilities of 22 isolates of Edwardsiella tarda were studied with 22 antibiotics and antibiotic-beta-lactamase-inhibitor agents. Results indicated that all isolates were susceptible to the aminoglycosides, cephalosporins, penicillins, imipenem, aztreonam, ciprofloxacin, and antibiotic-beta-lactamase-inhibitor agents. Each strain produced a beta-lactamase even though no resistance was detected to the beta-lactams.

The pathogenic properties of 16 Plesiomonas shigelloides strains recovered from humans with extraintestinal and intestinal illnesses, infected animals, and environmental sources were investigated. Most strains possessed a high cell charge and low surface hydrophobicity analogous to those of Shigella spp.; additionally, serogroup O:17 strains reacted with Shigella group D antisera. However, unlike the shigellae, P. shigelloides strains did not universally bind Congo red, were noninvasive in HEp-2 cell assays, and did not produce a Shiga-like toxin on Vero cells. On HEp-2, Y1, and possibly Vero cells, a low-level cytolysin was consistently produced by all 16 P. shigelloides strains when grown in either Evan Casamino Acids-yeast extract or Penassay broth. The median 50% lethal dose for all 16 P. shigelloides strains in outbred Swiss Webster mice was 3.5 x 10(8) CFU (range, 3.2 x 10(7) to greater than 1 x 10(9) CFU). Animal pathogenicity did not correlate with cytolysin expression, possession of a greater than or equal to 120-MDa plasmid, protein profile, or resistance to complement-mediated lysis. No strain analyzed produced siderophores or a heat-stable enterotoxin. The results suggest that members of the genus Plesiomonas have an overall low pathogenic potential, irrespective of the site of isolation or phenotypic, serologic, or surface properties shared with other traditional enteropathogens.

A number of different techniques were used to analyse classic and atypical serogroup O:11 Aeromonas isolates. Five of seven atypical O:11, S layer-negative strains lacking a homogeneous LPS side-chain pattern exhibited varying degrees of mouse pathogenecity. One virulent atypical strain (AH-77) synthesized a surface array protein (SAP) but was unable to anchor it to the cell surface in an intact form, presumably due to a defect in its LPS architecture. Proteinase K digestion to remove the SAP, or growth at elevated temperatures (42 degrees C) to reduce the proportion of SAP synthesized from classic O:11 S layer-positive strains, did not alter their LD50 values in outbred mice. In addition, a spontaneous mutant, AS-180-1, that was S layer-negative was as virulent as the parental S layer-positive strain in the mouse model. These results suggest neither the SAP nor the characteristic serogroup O:11 homogeneous LPS side-chain pattern are directly involved in mouse pathogenicity.

The ability of 22 Edwardsiella strains to penetrate and replicate in cultured epithelial cells was initially evaluated by light microscopy methods and by the recovery of gentamicin-resistant (Gmr) bacteria from the Triton X-100 cell lysates of HEp-2-infected monolayers. Giemsa-stained HEp-2 cells revealed the presence of numerous internalized bacteria 3 h postinfection, often appearing as parallel rows of replicated bacteria within the cytosol and sometimes obliterating the cytoplasm because of the large numbers of bacilli present. Invasive bacteria were also sometimes found within cytoplasmic vacuoles in infected cells; thin-section electron micrographs of HEp-2-infected cells supported these conclusions. Results of light microscopy studies and cell lysate assays indicated that most Edwardsiella tarda (92%) and some Edwardsiella hoshinae strains were invasion positive on one or more occasions, while Edwardsiella ictaluri isolates were uniformly negative. HEp-2 invasion by E. tarda was a microfilament-dependent (cytochalasin B- and D-sensitive) process, with maximum numbers of Gmr CFU recorded between 3 and 6 h postinfection. The small percentage (0.01 to 1.0%) of the challenge inoculum recoverable as Gmr progeny 3 to 6 h postinfection was attributed to a strong cell-associated (not filterable) hemolysin that was produced by a majority (85%) of the E. tarda strains but not by E. ictaluri and only minimally by E. hoshinae. This cytolysin/hemolysin was responsible for the toxic effects observed in HEp-2 cells during the infection-replication process of edwardsiellae and appears to play a role in the release of internalized and replicated bacteria from infected cells. The results suggest an invasion strategy with some similarities to and differences from those of other recognized enteroinvasive pathogens.

The protein and lipopolysaccharide (LPS) compositions of 10 autoagglutinating Aeromonas hydrophila and Aeromonas sobria strains were studied; one group consisted of five serogroup O:11 strains that contained an S layer, while a second group was composed of diverse serogroups that were S layer negative by transmission electron microscopy. All serogroup O:11 strains were found to contain a predominant 52,000- to 54,000-molecular-weight protein that was present on both whole-cell and outer membrane protein profiles; this protein was found to be glycine extractable under low-pH (pH 4) conditions and was identified as the surface array protein. LPS analysis revealed that all O:11 strains exhibited homogeneous-length O-polysaccharide side chains characterized primarily by two or three major bands. In contrast, S-layer-negative autoagglutinating strains of other serogroups lacked this predominant surface array protein, and silver stain analysis of LPS indicated that such profiles mainly consisted of core antigens and were deficient in or devoid of O-polysaccharide side chains. These collective results offer potential explanations for observed differences between these two groups in virulence, disease spectrum, and pathogenic properties.

A comparative study to determine beta-D-galactosidase activity among 171 strains of aerobic actinomycetes (including mycobacteria and rhodococci) was performed by using two growth media and four O-nitrophenyl-beta-D-galactopyranoside (ONPG) substrates. The ONPG test was found to be a valuable screening test to differentiate between the ONPG-positive Nocardia spp. and the rapidly growing ONPG-negative mycobacteria and rhodococci. However, ONPG results varied significantly depending on the growth medium and test substrate used.

We evaluated the extent to which serogroup O:11 Aeromonas strains could be recovered from both clinical and environmental specimens and the cultural parameters that affected the phenotypic marker (autoagglutination) associated with this group. Of over 200 Aeromonas strains screened, serogroup O:11 was identified only among the phenospecies A. hydrophila and A. sobria and was associated with clinical isolates more frequently than with environmental strains. Blood and wound isolates accounted for almost 50% of all O:11 strains identified. The autoagglutination phenotype associated with O:11 strains could be detected in most commercial liquid media, under a wide range of growth temperatures, and within 15 min of incubation at 100 degrees C. The results suggest that clinical laboratories can recognize this important group of Aeromonas strains by two simple tests.

Five commercial kit systems for the rapid identification of Neisseria gonorrhoeae were evaluated. The systems were tested with various pathogenic and saprophytic Neisseria and Branhamella strains having reactions typical for their individual species. Three systems (RIM-N, quadFERM, and RapID NH) were found to be 100% sensitive and specific.

During an 11-year period (1978 to 1989), over 300 strains of Yersinia spp. (excluding Y. pestis and Y. pseudotuberculosis) were recovered from a variety of gastrointestinal and extraintestinal sites in patients in California. Over the 11-year period, Y. enterocolitica serogroup O:3 predominated, although a shift in the relative frequency of this serogroup was observed during this interval, increasing dramatically during the years 1984 to 1989. Of the remaining Y. enterocolitica isolates, over 40% were identified as belonging to serogroups generally considered to be nonpathogenic, although many of these isolates were recovered in association with milder cases of gastroenteritis. The results suggest a changing and expanding spectrum of Y. enterocolitica serogroups associated with various gastrointestinal and systemic infections.

The antibiotic susceptibilities of 29 isolates of Plesiomonas shigelloides were studied with 24 antibiotics and antibiotic-inhibitor combinations. Results indicated that all isolates were susceptible to the cephalosporins, penicillins combined with a beta-lactamase inhibitor, aztreonam, and ciprofloxacin. Most isolates were resistant to the penicillins, possibly via production of a penicillinase.

Pseudomonas aeruginosa, a free-living bacterial species, is a major nosocomial pathogen, especially of compromised patients within medical facilities. Numerous factors contribute to the ecological selection of this bacterial species within the hospital environment, among which the expression of newly acquired or quiescent enzymatic capability seems par-amount. The emergence of pathogenic strains ofP. aeruginosa appears to be gradual, embodying a transition of strains from their natural aquatic environment, to establishing inanimate (hospital) and animate (human) reservoirs. In this stepwise transition, subsets ofP. aeruginosa may evolve which express a survival trait, for example, gentamicin resistance, but concomitantly suffer a loss of invasive potential. In this study,P. aeruginosa strains from natural [22], hospital [11], and stool [17] sources were evaluated for their physiological and exoenzymatic activity and compared with gentamicin-resistantP. aeruginosa (GRPA) strains [49] of clinical origin. As a whole, environmental and hospital isolates showed reduced enzymatic potential, for example, frequency of production of elastase, lipase, deoxyribonuclease, and pyocyanin production. Human fecal isolates most closely resembled the prototype of human invasiveP. aeruginosa in their gentamicin susceptibility (95%) and increased frequencies of exoenzymes, including elastase production. On the other hand, GRPA were frequently apyocyanogenic (9/49), lacked extracellular enzymes correlated with pathogenicity, and were rarely isolated from systemic sites. When encountered, these strains appeared to represent colonization of a body site rather than incitants of overt infection. As a "subset" ofP. aeruginosa, gentamicin resistance was seen predominantly among serotype 11 strains, and encountered most frequently from patients with localized urinary tract infections.

Vibrio vulnificus was isolated from a bacteremic patient. This strain, together with other isolates of V. vulnificus, was compared with V. alginolyticus, V. fluvialis, and V. parahaemolyticus with regard to growth characteristics on enteric agar media (enabling isolation and identification) and production of exoenzymes which could correlate with invasive potential. V. vulnificus grew well on MacConkey. Endo, xylose-lysine deoxycholate, and Hektoen enteric agar plates. Because V. vulnificus colonies resembled those of lactose-fermenting strains of the family Enterobacteriaceae, however, isolation of this vibrio from mixed specimens or stools may require the use of thiosulfate-citrate-bile salts-sucrose agar. V. vulnificus produced numerous exoenzymes (protease, DNase, lipase, and esterase) but not elastase or lecithinase. Although differences in exoenzyme production were observed among the four vibrio species, no single exoenzyme could be linked to the invasive potential of V. vulnificus.

Colonial variants of Pseudomonas aeruginosa have received renewed interest because of their occurrence in sputum cultures of patients with cystic fibrosis. We encountered 11 strains of P. aeruginosa from various body sites of non-cystic fibrosis patients. The strains showed two to three colonial variants, including smooth, rough, and iridescent morphotypes that arose from subculture of a single colony of P. aeruginosa originating from a primary source. The colonial segregants differed in antibiotic susceptibility (resistance to gentamicin, carbenicillin, chloramphenicol, and tetracycline), presence or absence of exoenzymes (gelatinase and elastase), degree of proteolytic activity (caseinase), pigmentation, and antigenicity. These observations suggest that in vivo dissociation with concomitant changes in enzymatic and surface properties might greatly enhance invasiveness. Concurrent differences in antimicrobial susceptibility among the colonial variants could account in some instances for the failure of antibiotic treatment in P. aeruginosa infections in which one would anticipate a positive therapeutic response.

A blood isolate of Pseudomonas aeruginosa was encountered which produced, on subculture to Mueller-Hinton agar, markedly adherent, tenacious colonies which were characterized microscopically by the presence of serpentine rows of interlocking bacilli. Factors accounting for the observed morphologic aberration, which was lost upon subculture, remain unknown.

A genetic probe (Gen-Probe) was used to evaluate potential epidemiologic and susceptibility differences of Mycobacterium avium complex (MAC) strains isolated from 154 patients with and without the acquired immunodeficiency syndrome (AIDS). Genetic analysis revealed that 98% of the 45 patients with AIDS harbored only M. avium regardless of the anatomic or geographic source of the isolate; in contrast, approximately 40% of MAC isolates recovered from 109 patients without AIDS were M. intracellulare. Most M. intracellulare of respiratory origin recovered from patients without AIDS were involved in infectious processes. When 95 MAC isolates (M. avium, n = 53; M. intracellulare, n = 42) were evaluated for in vitro susceptibility to primary or secondary antimycobacterial drugs, significant differences were noted. M. intracellulare was more susceptible to streptomycin, rifampin, and ethambutol than M. avium; the converse was true for ethionamide. The results of this study suggest potentially important differences in disease spectrum and in vitro susceptibility profile for M. avium and M. intracellulare.

Enterotoxin production correlated with biotype in a study of 686 strains of Aeromonas spp. from Indonesia, Thailand, the United States, and Western Australia. Most strains were isolated from feces but nonfecal human isolates and environmental strains were also included. More than 80% of Voges-Proskauer (VP)-positive strains, classified as A. hydrophila, were enterotoxigenic in the suckling mouse assay as were 90% of VP-positive, arabinose-negative strains. An association between positive VP, arabinose fermentation, and failure to produce enterotoxins was found only with environmental strains. VP-negative strains which did not oxidize gluconate or produce gas from glucose were classified as A. punctata subsp. caviae. Only 2 of the 286 strains produced enterotoxins, and both were from Indonesian fecal samples. There were few remaining VP-negative strains, classified as A. punctata subsp. punctata and, of these, about half were enterotoxigenic. Regardless of source and species, 97% of Aeromonas spp. were correctly classified in relation to enterotoxin production with a hemolysin assay. A combination of biochemical testing and hemolysin assay should be suitable for diagnostic laboratories to identify enterotoxigenic Aeromonas spp. which, in children, are associated with diarrhea, unlike non-enterotoxigenic strains.

Over a one-year period, 32 strains (31 clinical, 1 environmental) of Aeromonas sp. were recovered. Chief sources of isolation were the gastrointestinal tract (48%), wounds (19%), and blood (13%). Gastrointestinal isolates were most often recovered from young (less than 5 yrs) children with diarrhea; wound or blood isolates were recovered more often from an older (avg. 56 yrs) population with one of several underlying disorders. Regardless of body site of isolation, most strains of Aeromonas appeared to be community acquired and not nosocomially transmitted. Over 70% of all isolates recovered during this year period were isolated during summer or fall months, suggesting a seasonal distribution of this microorganism. Speciation of Aeromonas isolates revealed A. hydrophila to be the predominant species isolated from clinical specimens, although significant percentages of other Aeromonas sp. were also recovered from clinical material.

The saprophytic bacterium Serratia plymuthica was recovered from a facial wound (burn) site of a pediatric patient. The clinical significance of the organism was undetermined due to its apparent eradication from this location by therapy with topical 1% silver sulfadiazine. Seeding of the burn with S. plymuthica may have occurred from contaminated moisture sometimes found on and around steam radiators.

The importance of the genus Aeromonas in human disease has recently become better appreciated through the use of improved methodologies for the recovery and identification of aeromonads from biologic specimens and as a result of medical studies aimed at determining the clinical significance of these bacteria when isolated from localized and invasive infections of humans. Taxonomic schemes currently allow for the identification of six Aeromonas species (five mesophilic and one psychrophilic), four of the five mesophilic species having been recovered from infectious processes or isolated from clinical material of human origin. Two major categories of human illness attributed to Aeromonas species have been observed: acute gastroenteritis in both pediatric and adult populations and disseminated disease (e.g., bacteremia) in persons with underlying hematologic malignancies or hepatic dysfunctions. Although the role of mesophilic aeromonads as important agents of bacterial gastroenteritis remains unconfirmed because of the inability to fulfill Koch's postulates (no animal model), several lines of compelling clinical and laboratory evidence indicate that these microorganisms are significant enteric pathogens.

Nineteen Plesiomonas shigelloides strains were evaluated for their stability at acidic and slightly alkaline pHs and for their susceptibility to antimicrobial agent-like compounds produced by enteric flora. Most P. shigelloides isolates were rapidly inactivated under high-acid (pH 4 or less) conditions. Screening of enteric bacteria for elaboration of factors active against P. shigelloides revealed two organisms (Pseudomonas aeruginosa and Streptococcus [Enterococcus] faecium) capable of secreting such inhibitory substances. The results of this study suggest some factors potentially important in regulating gastrointestinal colonization by P. shigelloides from environmental sources.

Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.

The acceptability of different lots of commercial components which constitute our basal medium for susceptibility testing of mycobacteria was evaluated. The basal medium consisted of Middlebrook 7H10 agar supplemented with 10% oleic acid-albumin-dextrose-catalase and 0.5% glycerol. Studies were performed by using three separate microbiologic assays, and results were compared with parallel tests on previously standardized and acceptable lots of media. Components were rejected if comparison with standardized medium showed a major change in growth support or susceptibility status of any reference strain to any antimicrobial agent tested. Of the components tested in such a manner, 7 of 23 (30%) lots of 10% oleic acid-albumin-dextrose-catalase, 2 of 13 (15%) lots of Middlebrook 7H10 agar, and 0 of 5 lots of glycerol were found to be unacceptable. This study demonstrates that individual lots of components of this basal medium may vary significantly in their suitability for susceptibility testing, and failure to detect such variation may dramatically affect susceptibility profiles.

Autoagglutination (AA phenotype) of mesophilic aeromonads in broth was found to be a virulence-associated marker. There were two kinds of AA+ strains: those that spontaneously pelleted (SP+), and those that pelleted only after boiling (PAB+). Of 79 strains tested, 24 (30%) were AA+, and 18 of these were recovered from clinical specimens. Most of the AA+ strains (n = 21) were identified as either Aeromonas sobria or Aeromonas hydrophila. Of the well-documented clinical isolates of A. sobria and A. hydrophila available, 5 (46%) of 11 from invasive disease and 4 (14%) of 29 from noninvasive disease were SP- PAB+. The SP- PAB+ phenotype was significantly associated with invasive infections (e.g., bacteremia and peritonitis [chi 2, P less than 0.05]). All seven of the SP- PAB+ A. sobria and A. hydrophila strains tested killed mice within 48 h after intraperitoneal infection with 1 x 10(7) to 3 x 10(7) CFU, whereas only two of four SP+ PAB+ strains tested were lethal. All of the SP- PAB+ A. sobria and A. hydrophila isolates examined shared common O somatic antigens and possessed an external layer peripheral to the cell wall as determined by thin-section electron micrography. The LL1 strain of A. hydrophila used by Dooley et al. (J. S. G. Dooley, R. Lallier, and T. J. Trust, Vet. Immunol. Immunopathol. 12:339-344, 1986) to demonstrate an S membrane protein component in aeromonads virulent for fish also was SP- PAB+ and possessed the peripheral membrane, suggesting an association between these two components. Seven AA- and three SP+ strains tested lacked this layer; furthermore, 22 (71%) of 31 such isolates did not kill mice. The AA phenotype was a stable characteristic upon long-term passage of isolates in vitro. Study of SP+ and PAB+ aeromonads by surface charge and hydrophobicity analyses indicated that neither property correlated with either virulence or the presence of an external layer.

We used DNA restriction fragments, derived from the exotoxin A gene and surrounding sequences, as an epidemiological marker for Pseudomonas aeruginosa. Using these DNA fragments as probes in Southern blot hybridizations and/or total genomic digestions, we were able to distinguish greater than 100 different strains of P. aeruginosa. The stability of the marker in vitro was established by using well-characterized strains, which were stored under different conditions and subjected to chemical mutagenesis. The stability of the marker within a given strain in vivo was established during experimental infection in the chronic rat lung model of pseudomonas pneumonia. P. aeruginosa serially cultured from individual patients with cystic fibrosis were examined by using this marker. Isolates that varied in colonial morphology, serotype, and biotype were identical when analyzed by Southern blot hybridization using the fragment as a probe. Indistinguishable isolates (by serotyping, biotyping, and antibiograms) cultured from two unrelated patients were easily distinguished by using Southern blot analysis.

Aeromonas hydrophila gastroenteritis was detected in 12 pediatric patients during a 5-month period. Chief complaints included bloody diarrhea, fever, vomiting, and abdominal pain. Severe symptoms in two patients necessitated hospitalization and supportive care. Phenotypic characteristics associated with enterotoxigenicity of A. hydrophila strains demonstrated that all 12 isolates were cytotoxic to HeLa cells and most were lysine decarboxylase positive (75%). A correlation existed between the presence of the five virulence-associated markers of two isolates of A. hydrophila and the severity of disease. Although the length and symptoms of gastroenteritis varied among all 12 patients, most had self-limiting diarrhea. The frequent occurrence of A. hydrophila gastroenteritis in pediatric patients warrants a greater appreciation of this agent as a significant cause of diarrhea, especially in summer.

Group G streptococci were isolated from throat and extrapharyngeal cultures from 75 patients during an 18-month period. Of 29 throat isolates, 18 were recovered from patients with pharyngitis, 8 were of unknown significance, and 3 were of questionable etiology. Clinical significance could be ascribed to 13 of 46 extrapharyngeal isolates recovered from wound, urinary tract, blood, and conjunctival cultures. Extrapharyngeal isolates recovered from stool, sputum, and vaginal cultures were considered nonsignificant. A total of 96 group G streptococcal strains (including 21 human and 14 bovine strains from outside sources) were tested for exoenzyme production and subjected to a large battery of biochemical tests. Bovine and human isolates could be distinguished on the basis of trehalose fermentation, litmus milk reduction, and production of beta-D-glucuronidase, hyaluronidase, and fibrinolysin. Eight distinct biotypes could be discerned on the basis of fermentation of trehalose, raffinose, and lactose and esculin hydrolysis. All isolates that fermented raffinose were associated with infection. These results support the concept of two distinctly different epidemiological reservoirs of group G streptococci in humans and bovines.

The enzymatic potential of 54 clinical and 22 environmental isolates of Pseudomonas aeruginosa from soil and water were evaluated by substrate plate assays. Clinical isolates produced substantial levels of 9 of the 11 enzymes assayed, whereas strains recovered from soil or water were relatively inert enzymatically. Elastase, deoxyribonuclease, and elevated protease activities were associated preferentially with clinical isolates of systemic origin; these activities were found twice as frequently in clinical isolates as in strains derived from sputum or the urogenital tract. Our data suggest that these factors may play an important role in the dissemination of P. aeruginosa from local or superficial sites. A comparison of the enzyme profiles of the environmental and clinical isolates indicated that colonization or infection by environmental strains of P. aeruginosa is a rare event and that environmental and clinical strains comprise separate biovars. Epidemiologically, enzyme profiles permitted the fingerprinting and differentiation of clinical strains from various sources.

The simultaneous recovery of Aeromonas hydrophila and Aeromonas caviae from the stool of a 49-year-old woman with watery diarrhea was facilitated through the use of a blood agar medium which detected the hemolytic capability of A. hydrophila. In vitro phenotypic tests support the conclusion that only the A. hydrophila isolate was clinically significant.

We examined a group of 23 recent clinical isolates of Yersinia enterocolitica recovered from symptomatic patients residing in the New York, N.Y. area. These isolates were tested for the presence of plasmids, exoenzyme activity, mouse lethality, and phenotypic properties postulated to correlate with virulence. Of the 23 isolates, 17 harbored a 60- to 65-kilobase (kb) plasmid. Six isolates were lethal for white mice, showed the phenotypic markers of autoagglutination and calcium dependence for growth at 37 degrees C, and contained a 60- to 65-kb plasmid. Restriction endonuclease analysis with several different enzymes revealed the presence of three distinct plasmid profiles in these isolates. Isolates with a single plasmid of 60 to 70 kb, typical for this species, were detected, but these were of three distinct types as judged from restriction enzyme digestion. One strain was unusual among clinical isolates of Y. enterocolitica in that it contained at least four distinct plasmids. In addition, this nontypable strain showed exoenzymatic activity similar to that of serogroup O8 isolates, was not lethal to mice, and did not require calcium for growth at 37 degrees C.

Examination of 45 human fecal isolates of Vibrio parahaemolyticus revealed the emergence of an unusual bioserovar (O4:K12, urease positive) associated with cases of gastroenteritis which appear to be domestically acquired on the West Coast of the United States and Mexico.

Dialyzed brain heart infusion-skim milk agar medium facilitated the recovery of colonial dissociants of Pseudomonas aeruginosa. Differences in colonial morphology as well as in proteolytic activity were readily visualized, thus permitting facile isolation of segregating colony types for further biochemical, serological, and susceptibility studies.

Vibrio alginolyticus, an extremely halophilic member of this genus, was isolated from multiple sets of blood cultures drawn during a septic crisis of a patient with osteogenic sarcoma. No identifiable source of this infection could be determined.

Plesiomonas shigelloides is a gram-negative, facultatively anaerobic rod whose appropriate taxonomic position is presently under investigation. The isolation and identification of this microorganism in contaminated specimens (e.g., feces) by a clinical laboratory depend on the screening of gram-negative colonies for oxidase and indole positivity and the appropriate use of selective and differential agars. Plesiomonads have been associated with extraintestinal diseases (bacteremia, meningitis) on rare occasions; they have been recovered sporadically from patients presenting with acute gastroenteritis. Although case reports and epidemiologic data support a role for P. shigelloides in diarrheal disease, laboratory investigations have failed to identify an enteropathogenic mechanism in these bacteria consistently or to reveal an animal model that faithfully reproduces the disease. Moreover, studies with volunteers have failed to establish an etiologic relation between Plesiomonas and bacterial gastroenteritis. An accurate picture of the role of this bacterium in human disease must await future studies.

Previous reports have suggested that clinical strains of gentamicin-resistant Pseudomonas aeruginosa (GRPA) generally produce only superficial infections (wounds, urinary tract infections) in contrast to their more invasive gentamicin-susceptible counterparts (GSPA). In view of this finding, a comparative study of a number of phenotypic properties of 20 GRPA and GSPA strains (10 isolates) was assessed to determine how closely related these two populations are and how their phenotypic properties might reflect virulence. GRPA isolates were found to be more adherent to buccal cells than their susceptible counterparts (P = 0.0001). Motility, however, was generally restricted in the GRPA population when compared with GSPA isolates (P = 0.02), although on the basis of zone diameters, some strains overlapped into the other group. Enzymatically, GSPA isolates produced significantly more lipase activity against C-14 lipids than GRPA strains (P = 0.04). No differences were recorded between the two populations in dye sensitivity or in their ability to grow on minimal media at 37 and 42 degrees C. Only 35% of the GRPA isolates agglutinated in 1 of the 17 monospecific antisera reactive for P. aeruginosa. The results of this study suggest that in vivo-generated GRPA strains possess phenotypic properties intermediate between those described for in vitro-derived GRPA isolates and their progenitor GSPA strains. The increased adherence of clinical GRPA isolates to buccal cells may explain their predilection to produce wound and urinary tract infections, whereas their lack of systemic dissemination may be partially due to decreased motility and to reduced lipase production.

Fifty-four motile Aeromonas strains, composing the three currently recognizable species, were tested for susceptibility to cephalothin by broth dilution and disk agar diffusion assays. Cephalothin susceptibility was significantly associated with Aeromonas sobria (P less than 0.001) and may be an additional phenotypic marker useful in the identification of this species.

A new agent, 9-chloro-9-(diethylaminophenyl)-10-phenylacridan (C-390), was compared to currently available selective agars for the recovery of Pseudomonas aeruginosa. Media containing C-390 were highly selective for P. aeruginosa and enhanced recovery of the organism from clinical specimens because of the low background level of contaminating microorganisms.

Evaluation of Giemsa-stained smears of scrapings from the base of vesicles or ulcers ( Tzanck preparation) for the presence of multinucleated giant cells and/or intracellular inclusions were diagnostic for herpesvirus in 18 of 21 cases (86%) of culturally proved herpesvirus infections. Smears from four patients with varicella-zoster infection also revealed cytologic alterations characteristic of herpesvirus.

The efficacy of eight routine enteric agars for supporting the growth of 32 strains of Aeromonas spp. (17 A. hydrophila strains, 8 A. sobria strains, and 7 A. caviae strains) was investigated. The plating efficiency of Aeromonas spp. on these media varied greatly (range, 0 to 100%), as did their colony size when compared with that on noninhibitory medium (5% sheep blood agar). Plating efficiency on seven of these eight media appeared to be strain- and not species dependent. Overall, eosin-methylene blue and Hektoen enteric agars showed low plating efficiencies for A. hydrophila, whereas both A. sobria and A. caviae were severely inhibited on brilliant green agar. When all these species are considered collectively, deoxycholate, MacConkey, and xylose lysine deoxycholate appeared to be the most satisfactory routine agars for Aeromonas spp. recovery when used in conjunction with blood agar.

Ecthyma gangrenosum has not been described during the course of blood stream invasion with Pseudomonas maltophilia, although it occurs with a 30% frequency in Pseudomonas aeruginosa septicemia. We isolated P. maltophilia from the blood and an ecthyma lesion in a leukemic patient. The organism was an avid protease and elastase producer and hence mimicked the exoenzyme profile of invasive P. aeruginosa. The patient responded to moxalactam to which the isolate was susceptible in vitro. On the basis of this report, P. maltophilia may be included among an emerging number of gram-negative bacillary species capable of producing severe cutaneous manifestations of bacteremia.

A group of 147 Aeromonas isolates from diverse clinical and environmental sources was subjected to the biotyping scheme of Popoff and Veron. Of the 147 isolates biotyped, 137 (93%) could be identified, with Aeromonas hydrophila predominating (48%) and equal percentages (25 to 27%) of the other two species (Aeromonas sobria and Aeromonas caviae). A number of additional biochemical properties were found to be significantly associated with one or more of these three species. These included lysine decarboxylase activity, hemolysis of sheep erythrocytes, lecithinase production, staphylolytic activity, arbutin hydrolysis, and acid production from utilization of various carbohydrates. By incorporating these phenotypic properties into an extended biotyping system, 98% of the isolates were identified. Selective distribution of individual species with respect to certain body sites was noted.

Aeromonas spp. show patterns of hemagglutination with human group O cells in the presence of fucose, galactose, and mannose. These patterns are related to biotype as well as to the source of isolates. There was good correlation between hemagglutination pattern and the presence of diarrhea among strains isolated in Western Australia, which was the only source with adequate data for classification of children with an without diarrhea. Most of the environmental and other nonfecal isolates produced patterns different from those in strains associated with diarrhea. These results suggest that hemagglutinins should be considered with enterotoxins as virulence factors in Aeromonas spp.

The differential and selective sodium dodecyl sulfate-polymyxin B-sucrose medium (SPS) of Kitaura et al. (T. Kitaura, S. Doke, I. Azuma, M. Imaida, K. Miyano, K. Harada, and E. Yabuuchii, FEMS Microbiol. Lett. 17:205-209, 1983), which highlights alkylsulfatase activity, was evaluated for its potential use in the direct isolation and enumeration of Vibrio vulnificus from shellfish. V. vulnificus was detected by this method in six of nine shellfish samples collected from diverse geographic locales during the summer of 1986. Direct enumeration of V. vulnificus at 7.0 X 10(2) to 2.2 X 10(4) CFU/g of shellfish was achieved on SPS agar. All sample results were confirmed in parallel examinations by using conventional glucose-salt-Teepol (Shell Oil Co.) broth and alkaline peptone water enrichment with plating onto thiosulfate-citrate-bile salts-sucrose agar. Additionally, alkylsulfatase activity was evaluated in vitro for 97 strains representing 14 Vibrio spp. V. vulnificus and Vibrio cholerae-01 were the only species consistently found to possess this activity. The range of plating efficiencies for random V. vulnificus strains analyzed on SPS was 11 to 74% (mean, 39%). The use of SPS shows great promise for the study of shellfish and other environmental sources for V. vulnificus.