Lateral Flagella and Swarming Motility in Aeromonas Species

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.     

Unusual Fermentative, Gram-Negative Bacilli Isolated from Clinical Specimens: II. Characterization of Aeromonas Species

Morphological and physiological characterization of 12 Aeromonas strains isolated from clinical specimens demonstrated several important diagnostic features. These included polar arrangement of the flagella; production of oxidase, deoxyribonuclease, amylase, gelatinase, and lipase; lack of ornithine decarboxylase; and sensitivity to polymyxin. Awareness of these features should result in more frequent differentiation of aeromonads from the physiologically similar members of the genus Plesiomonas and the late- or non-lactose-fermenting species of Enterobacteriaceae.     

Distribution of Aeromonas Species in the Intestinal Tracts of River Fish

Aeromonas isolates were obtained from fish intestines, water, and sediments from an urban river and identified by the DNA-DNA microplate hybridization method. The isolates were Aeromonas veronii (22%), Aeromonas caviae (18%), Aeromonas hydrophila (13%), Aeromonas sobria (8%), Aeromonas jandaei (7%), and other Aeromonas spp. (33%). Aeromonas species occurred at high densities with high incidences, regardless of season. The results strongly suggest that aeromonads are indigenous in fish intestines, water, and sediments of rivers and have the potential to be predominant in aquatic environments.     

Temporal Occurrence of Vibrio Species and Aeromonas hydrophila in Estuarine Sediments

Marine sediments were assayed for their concentration of Vibrio spp. and Aeromonas hydrophila over 1 year. A temporal variation was observed in which A. hydrophila, and to a lesser degree V. fluvialis, were found in the winter months, V. parahaemolyticus and V. vulnificus predominated in the spring and summer, with non-O-1 V. cholerae and V. alginolyticus detected in the late summer and fall. These organisms were found in greatest numbers in the top 5 cm of sediment, but were detectable down to 15 cm. Epidemiological data revealed a predominance of non-O-1 V. cholerae infections at the time the organisms were observed to flourish in the sediments.     

Clinical involvement of Aeromonas hydrophila.

Aeromonas hydrophila has for some time been regarded as an opportunistic pathogen in hosts with impaired local or general defence mechanisms. Infections in such individuals are generally severe. The organism is also being isolated with increasing frequency throughout the world from a variety of focal and systemic infections of varying severity in persons that are apparently immunologically normal. Most commonly it causes acute diarrheal disease by producing an enterotoxin. Thus the organism appears to have greater clinical significance that was hitherto suspected. The organism has been infrequently reported from humans in Canada, but its correct laboratory identification, together with increased awareness that it can contribute to illness, will undoubtedly lead to more reports of its isolation in Canada.     

Identification of Aeromonas schubertii and Aeromonas jandaei by using a polymerase chain reaction-probe test

Abstract Two oligonucleotide primers were used in a polymerase chain reaction-protocol to amplify a region (approx. 850 bp) of the 16S rRNA gene of Aeromonas schubertii and Aeromonas jandaei . Hybridization of the polymerase chain reaction products to specific internal probes provided a highly specific method for the identification of these two species.     

Aeromonas and Pseudomonas Species Carriers of ampC FOX Genes in Aquatic Environments

Resistance to beta-lactam antibiotics is evolutionarily ancient, and resistance to new drugs develops and quickly spreads. Most studies on beta-lactam resistance concentrate on pathogens in medical and veterinary settings. We show that ampC FOX genes can be found in natural environmental isolates.     

Hemolytic Activity and Siderophore Production in Different Aeromonas Species Isolated from Fish

The hemolytic activity and siderophore production of several strains of motile aeromonads were determined. The hemolytic activity of Aeromonas caviae and Aeromonas eucrenophila was enhanced after trypsinization of the samples. The enhancement of hemolysis was observed in strains that carried an aerolysin-like gene, detected by a PCR procedure. Siderophore production was demonstrated in all but one strain of Aeromonas jandaei. No apparent relationship was observed between the presence of plasmid DNA and hemolysis or siderophore production.     

Identification of Flavones in Thirteen Liliaceae Species

Changes in the levels of free and protein amino acids in the callus of Hiproly barley were studied during differentiation. Adventitious roots were formed in the callus after 90 days of cultivation on modified White’s medium containing 1 μm indoleacetic acid (IAA) and 200 μm adenine sulfate, and callus placed on Murashige–Skoog’s medium without plant hormones formed adventitious roots after 30 days of cultivation. During differentiation, protein amino acids in the callus decreased, then increased, without an appreciable compositional change in the protein amino acids. The amount of free amino acids in the callus increased with root formation. The major free amino acids during differentiation were glutamine, asparagine, alanine, and proline. Glutamine increased until roots were found in the callus after cultivation. Asparagine gradually increased during differentiation.     

Identification of the major outer membrane proteins of Aeromonas salmonicida

Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the etiological agent of furunculosis, a serious infectious disease of salmonids. Aeromonas spp. are ubiquitous waterborne bacteria responsible for a wide spectrum of diseases among aquatic organisms and humans. Bacterial outer membrane proteins (OMPs) play a significant role in virulence as they comprise the outermost surface in contact with host cells and immune defense factors. To identify the major OMPs of A. salmonicida a proteomic analysis was undertaken using a carbonate OMP-enrichment protocol. The enriched OMP-extracts were separated by 2-dimensional electrophoresis (2-DE) and the spots identified using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) via an electrospray ionization source. In total, 76 unique proteins were identified from the 125 spots observed on the 2-D gel. The surface layer (S-layer) VapA protein dominated the A. salmonicida OMP 2-D profile, accounting for 60% of the protein on the 2-D gels. Among the other outer membrane proteins identified were at least 10 porins and various receptors involved in nutrient acquisition. Also identified in the carbonate insoluble fraction were phosphoglycerate kinase, enolase and others that lacked classical export sorting signals. The putative association of these proteins with the cell surface might provide new insights concerning the biological and pathogenic roles of these molecules in A. salmonicida infection. This work represents the first systematic attempt to characterize the cell surface of A. salmonicida.     

Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia

Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions—exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.     

鳗鲡病原性维氏气单胞菌的分离与鉴定

从养殖患病的日本鳗鲡(Anguilla japonica)肾脏分离得到1株优势菌株,形态特征和生理生化测试结果显示,分离菌在4%羊血平板培养基上呈α型溶血,革兰氏阴性,有运动性,氧化酶阳性,在pH6.0和1%NaCl中生长等。结合Biolog自动微生物鉴定系统和16S rRNA、gyrB基因序列分析结果,确定该分离菌为维氏气单胞菌(Aeromonas veronii)。人工腹腔注射感染鳗鲡的试验结果表明,分离菌对鳗鲡的半数致死量LD50为2.15×107CFU/mL。药敏试验结果显示,在13种抗菌类药物中,分离菌对左氧氟沙星、氟哌酸、阿奇霉素和萘啶酸等8种药物敏感,而对利福平和新生霉素等5种药物敏感度低。     

Problems in the Identification of Species of Eimeria

ABSTRACT. The paper is concerned with the principles upon which coccidia of the genus Eimeria may be characterized. Reference strains for comparative purposes usually are not available and the limitations of morphological data for speciation are discussed. The value of other parameters are considered such as host and site specificity, pathogenicity, immunological specificity, pre-patent period, sporulation time, enzyme variation, and DNA buoyant density. The weight afforded to each of these parameters for specific identification may vary according to the parasite and host studied. Determinations of physiological and behavioral characteristics that are now becoming available should be included in species definitions wherever possible.     

Rapid Identification of Mutans Streptococcal Species

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS-blue dextran-PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranase gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species-specific PCR products in the reference species (15 strains of 5 species) of mutans streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS-blue dextran-PAGE also showed species-specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.     

Identification of Flavone Compounds in Eighteen Gramineae Species

Conditions for tryptophan synthesis from pyruvic acid, indole and NH₄Cl by Enterobacter aerogenes AHU 1540 having a high tryptophanase activity, were investigated using a reaction mixture containing 1.7% of pyruvic acid. Under optimum conditions, 16.4g/liter of tryptophan was accumulated after 24 hr of incubation.

Agaricus campestris AHU 9382 produced pyruvic acid in amounts of 22 ~ 26.5 g/liter from 5% of glucose after 3-days shaking culture. When E. aerogenes was added to this fermentation broth together with indole and NH₄Cl, pyruvic acid produced was rapidly converted to tryptophan and yields of tryptophan as high as 15 g/liter were obtained after 12 hr of incubation. Furthermore, pyruvic acid fermentation by Saccharomyces exiguus AHU 3110 or Corynebacterium sp. 37-3A could also be used as a pyruvic acid source for subsequent tryptophan production.     

Distribution of Virulence Factors and Molecular Fingerprinting of Aeromonas Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-Human Transmission

A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.Aeromonas species cause both intestinal and extraintestinal infections (25, 33, 78), and the latter include septicemia, cellulitis, wound infections, urinary tract infections, hepatobiliary tract infections, soft tissue infections, and, occasionally, meningitis and peritonitis (25, 30, 78). In immunocompromised children, these pathogens can cause even more severe forms of infections, such as hemolytic-uremic syndrome (HUS) and necrotizing fasciitis (3, 23), although detailed studies are needed to establish such associations. Worldwide, the rate of isolation of Aeromonas from diarrheic stools has been reported to be as high as 10.8%, compared to 2.1% for healthy controls (25, 37, 78). An increased rate of isolation of Aeromonas species was reported in flood water samples during Hurricane Katrina in New Orleans (58), and skin and soft tissue infections caused by Aeromonas species were among the most common infections in the survivors of the 2004 tsunami in southern Thailand (28). In particular, Aeromonas salmonicida causes fish infections that result in huge economical losses in the fishing industry (6, 22). The ability of aeromonads, as well as other bacteria, to survive in chlorinated water when they are in biofilms and their resistance to multiple antibiotics are major public health concerns (46).Aeromonas-related gastroenteritis remains somewhat controversial (24, 36). There have been a number of well-described cases and a few documented outbreaks, but whether all aeromonad fecal isolates from symptomatic persons are the actual causes of diarrheal disease is still questionable. One theory for this conundrum was posed in 2000 by two of us, who suggested that only specific subsets of Aeromonas strains within and between species are actually pathogenic for humans (38). This highlights the importance of developing accurate biotyping, molecular fingerprinting, and virulence factor analysis methods for differentiating environmental and clinical aeromonads from one another and for comparing them (38).Of the 19 currently recognized Aeromonas species, A. hydrophila, A. caviae, and A. veronii biovar sobria are the most common species known to cause the majority of human infections, and they account for more than 85% of all clinical isolates (34). The pathogenesis of Aeromonas infections is multifactorial, as aeromonads produce a wide variety of virulence factors, including hemolysins, cytotonic and cytotoxic enterotoxins, proteases, lipases, leucocidins, endotoxin, adhesions, and an S layer, that act in concert to cause disease in the host (12-14, 50, 51). The cytotoxic enterotoxin Act, which has some similarities to aerolysin (31), is one of the most significant virulence factors in diarrheal isolate SSU of A. hydrophila and was first characterized in our laboratory (12). Act is secreted by the type II secretion system (T2SS) and has hemolytic, cytotoxic, and enterotoxic activities (12). In addition, our laboratory recently sequenced and characterized two other secretion systems, T3SS and T6SS, that were found to contribute to the virulence of A. hydrophila SSU (66, 67, 72). We recently characterized an effector of the T3SS, which was designated AexU, and found that it was associated with ADP ribosylation of host cell proteins, a rounded phenotype in HeLa cells, inhibition of phagocytosis, induction of apoptosis, and mouse mortality (66, 67). In recent studies, we also investigated the role of two T6SS-associated effectors, the valine-glycine repeat G (VgrG) family of proteins and hemolysin-coregulated protein (Hcp), in the virulence of A. hydrophila (71, 72). We demonstrated that VgrG1 of A. hydrophila had actin-ADP ribosylation activity that induced host cell cytotoxicity (71). Based on the model for T6SS, the VgrG1 protein must assemble with the highly homologous VgrG2 and VgrG3 proteins to form a cell-puncturing device to deliver effector proteins into the host cells (59). We also obtained evidence that expression of the hcp gene in HeLa cells led to their apoptosis, and animals immunized with recombinant Hcp were protected from subsequent challenge with a lethal dose of wild-type A. hydrophila SSU (72).In addition, cytotonic enterotoxins (e.g., Alt [heat labile] and Ast [heat stable]) were identified in a diarrheal A. hydrophila SSU isolate (14, 63) that induced fluid secretion in the ligated small intestinal loops of animals (47). More recently, we identified some additional virulence factor-encoding and regulatory genes, such as the enolase, hlyA (hemolysin), gidA (glucose-inhibited division A), vacB (virulence-associated protein B), dam (DNA adenine methyltransferase), and tagA (ToxR-regulated lipoprotein) genes, which modulated the virulence of A. hydrophila SSU (19-21, 57, 62, 64). The production of such a wide array of virulence factors by Aeromonas species is indicative of their potential to cause severe diseases in humans. These virulence factor-encoding genes might be differentially expressed in Aeromonas species depending on the environmental conditions, such as water or the human host.A cell-to-cell signaling system, known as quorum sensing (QS), might play an important role in sensing physiological conditions and helping bacteria express the virulence genes at an appropriate time under the appropriate conditions. Thus far, at least three QS circuits have been identified in Gram-negative bacteria, and they were designated LuxRI (autoinducer 1 [AI-1]), LuxS (AI-2), and AI-3 (epinephrine/norepinephrine). All of these QS systems were detected in our SSU clinical strain of A. hydrophila, and we recently demonstrated that N-acyl homoserine lactone (AHL) (AI-1) and AI-2-mediated QS controlled the virulence of A. hydrophila SSU (40, 43). Further, we observed decreased production of N-acyl homoserine lactones when we deleted two major virulence factor-encoding genes, the act gene and the gene encoding an outer membrane protein (aopB), an important component of the T3SS (65), from A. hydrophila SSU. Likewise, we observed that N-acyl homoserine lactone production was also modulated by regulatory genes, such as dam and gidA, in A. hydrophila SSU (18). Thus, differential expression of genes might also be an important factor in the pathogenesis of Aeromonas species.The presence of any virulence gene in strains of Aeromonas isolated from water should be carefully scrutinized, as such genes could be expressed better in a human host, which could lead to devastating outcomes. In addition, it is possible that in the environment certain Aeromonas clones may predominate and cause human diseases more frequently than other clones. Thus, it is important to determine the clonal variation of a range of Aeromonas species isolated from various sources and identify predominant clones by a polyphasic approach that includes biochemical phenotyping, virulence marker detection, and molecular fingerprinting techniques.In the present study, we compared 199 Aeromonas isolates, 146 of which were from water sources and 53 of which were from human patients with diarrhea in the Unites States. In addition, 28 reference and classical strains that were obtained from various culture collections and/or were isolated from specimens obtained in diverse geographical areas of the world, including water and clinical specimens, were also characterized. All isolates were biochemically identified to the phenospecies group level, examined for the presence of a set of 11 virulence factors by using DNA colony blot hybridization, and fingerprinted by using pulsed-field gel electrophoresis (PFGE). Some of the virulence factors selected, including T6SS effectors, were also examined by using functional assays. Our data provide the first suggestive evidence of water-to-human transmission, i.e., of successful colonization and infection by particular strains of certain Aeromonas species.