Predicting virulence of Aeromonas isolates based on changes in transcription of c-jun and c-fos in human tissue culture cells

Aims:  To screen for the virulence potential of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2.
Methods and Results:  Aeromonas cells were added to Caco-2 cells at a ratio of approx. 1 : 1. After 1-, 2- and 3-h incubation at 37°C, mRNA was extracted from the cells and gene expression of two host genes, c-jun and c-fos, quantified. Aeromonas isolates which were pathogenic in the neonatal mouse model demonstrated up-regulation of c-jun and c-fos compared to avirulent isolates.
Conclusions:  Human cell culture results showed that c-jun and c-fos were predictive of Aeromonas virulence.
Significance and Impact of the Study:  An Aeromonas relative virulence scale is proposed for use in the testing of Aeromonas drinking water isolates.     

Alternative host model to evaluate Aeromonas virulence

Bacterial virulence can only be assessed by confronting bacteria with a host. Here, we present a new simple assay to evaluate Aeromonas virulence, making use of Dictyostelium amoebae as an alternative host model. This assay can be modulated to assess virulence of very different Aeromonas species.     

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.     

Distribution of virulence genes in Aeromonas spp. isolated from Sardinian waters and from patients with diarrhoea

AIMS: To characterize 46 isolates of different Aeromonas spp. strains (26 Aeromonas hydrophila, 13 Aeromonas sobria and 7 Aeromonas salmonicida) isolated from coastal water and clinical sources in Sardinia, Italy. METHODS AND RESULTS: The isolates were analysed for the production of the following virulence properties: slime, haemolysin, gelatinase and protease production, and adhesion to eucaryotic epithelial cells. The presence of known virulence genes: A. hydrophila cytolytic enterotoxin gene AHCYTOEN; type IV pilus gene Tap; Bundle forming pilus genes BfpA and BfpG were investigated using polymerase chain reaction (PCR). In addition the enterobacterial repetitive intergenic consensus sequences (ERIC)-PCR fingerprinting was used to further differentiate the strains. CONCLUSIONS: This study confirms the presence of virulent Aeromonas strains in the Mediterranean sea. The study also found a greater prevalence of haemolysin, protease and gelatinase production, as well as a higher adhesion capacity, among strains isolated from patients with diarrhoea. SIGNIFICANCE AND IMAPCT OF THE STUDY: This is the first time that Aeromonads have been isolated and characterized from Sardinian waters and from patients with diarrhoea in Sardinia. This study adds to our knowledge of the ecology of this micro-organism and may in the future help prevent infections both in fish and in humans.     

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.     

Measurement of virulence of aeromonads using a suckling mouse model of infection

Abstract Virulent and avirulent strains of Aeromonas spp. were identified and virulence quantified using an animal model. Virulence was measured by determining a 50% lethal dose (LD₅₀) 43 h after oral administration of live bacteria. The LD₅₀ of virulent Aeromonas isolates ranged from log₁₀ 7.53 (mean) organisms to log₁₀ 8.88 (mean). Some isolates were avirulent in this model. Detection of cytotoxic activity in culture supernatants correlated with virulence (Fisher exact test, P = 0.0029). There was no correlation between LD₅₀ and the source of the isolate, β-haemolysis or lipopolysaccharide (LPS) banding profile on SDS-PAGE. In this animal model, virulence was multifactorial in that: (i) bacterial multiplication in the gut was associated with fatal infection; (ii) the increase in bacterial numbers in the gut of mice administered a lethal dose of bacteria was accompanied by accumulation of fluid; and (iii) there was evidence of extraintestinal spread of infection. Protection of suckling mice by rabbit antiserum to Aeromonas cell envelopes was observed.     

Impact of continuous axenic cultivation in Leishmania infantum virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species.     

Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification

AIMS: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes. METHODS AND RESULTS: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates. New primer sets were designed for all the target genes, except for act. The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% (ast) of the strains, respectively. Of the 205 isolates tested only one isolate had all the virulence genes. There was a variety of combinations of virulence factors within different strains of the same species. However, a dominant strain having the same set of virulence factors, was usually isolated from any given tap in different rounds of sampling from a single tap. CONCLUSIONS: These results show that Aeromonas bacteria found in drinking water possess a wide variety of virulence-related genes and suggest the importance of examining as many isolates as possible in order to better understand the health risk these bacteria may present. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a rapid method for characterizing the virulence factors of Aeromonas bacteria and suggests that municipally treated drinking water is a source of potentially pathogenic Aeromonas bacteria.     

Coevolution of parasite virulence and host life history

Most models about the evolutionary interactions between a parasite's virulence and its host's life history neglect two potentially important aspects: epidemiological and coevolutionary feedback. We emphasize their importance by presenting models that describe the coevolution of a semelparous host's age at reproduction and a parasite's virulence in different environmental conditions. In particular, we first show that an epidemiological feedback will lead to a nonmonotonic response of the host's age at reproduction as virulence increases. We then show that the coevolutionary pressure on virulence can lead to complex associations between the host's life history and the parasite's virulence, which would not be expected with more traditional models of host or parasite evolution. Thus, for example, a high mortality rate of the host favours avirulent parasites and late reproduction of the host when the environmental conditions allow the host to grow rapidly, but early reproduction and high virulence when growth is slow.     

Yersinia virulence depends on mimicry of host Rho-family nucleotide dissociation inhibitors

Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. A critical virulence determinant is the Yersinia protein kinase A, or YpkA, a multidomain protein that disrupts the eukaryotic actin cytoskeleton. Here we solve the crystal structure of a YpkA-Rac1 complex and find that YpkA possesses a Rac1 binding domain that mimics host guanidine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits nucleotide exchange in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and impair in vivo YpkA-induced cytoskeletal disruption. In cell culture experiments, the kinase and the GDI domains of YpkA act synergistically to promote cytoskeletal disruption, and a Y. pseudotuberculosis mutant lacking YpkA GDI activity shows attenuated virulence in a mouse infection assay. We conclude that virulence in Yersinia depends strongly upon mimicry of host GDI proteins by YpkA.     

Trade-offs in the evolution of virulence in an indirectly transmitted macroparasite

The adaptive trade-off theory for the evolution and maintenance of parasite virulence requires that virulence be genetically correlated with other fitness characteristics of the parasite. Many theoretical models rely on a positive correlation between virulence and transmissibility. They assume that high parasite replication rates are associated with a high probability of transmission (and, hence, increased parasite fitness), but also with high levels of damage to the host (high virulence). Schistosomes are macroparasites with an indirect life cycle involving a mammalian and a molluscan host. Here we demonstrate, through the development of five substrains, a genetic basis for schistosome virulence. We used these substrains further in order to investigate the presence of parasite fitness traits that were genetically correlated with virulence. High virulence in the (mouse) definitive host was, as predicted, positively correlated with parasite replication. In contrast, in the (snail) intermediate host high virulence was associated with low parasite replication rates. Variation in infectivity to and parasite replication in the definitive host was suggested as a compensating mechanism for the maintenance of virulence in the snail host. This is the first report of a trade-off in parasite reproductive success across hosts in an indirectly transmitted macroparasite.     

Intracellular vacuolation induced by culture filtrates of Plesiomonas shigelloides isolated from environmental sources

AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. METHODS AND RESULTS: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila. All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. CONCLUSIONS: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.     

Biochemical characteristics and virulence of environmental group F bacteria isolated in the United States.

Bacteria phenotypically resembling Aeromonas hydrophila, but requiring NaCl for growth, have been isolated form the New York Bight. The bacteria proved to be identical to group F organisms isolated from cases of human diarrhea in Indonesia and Bangladesh. Anaerogenic strains initiated responses in Y-1 tissue culture and rabbit ileal loop, consistent with those associated with cytotoxin- and enterotoxin-producing Aeromonas spp. strains. Separation on the basis of production of gas from glucose by group F strains was correlated with differences in mean guanine-plus-cytosine deoxyribonucleic acid base composition and in deoxyribonucleic acid relative reassociation. Both aerogenic and anaerogenic strains reassociated to a significantly greater extent with Vibrio spp. than with Aeromonas spp. and indeed should be considered a new species of the genus Vibrio.     

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.     

Molecular characterization and distribution of virulence-associated genes amongst Aeromonas isolates from Libya

AIMS: The aim of the study was to type 52 Aeromonas spp. isolates from chicken carcasses, children with diarrhoea and a hospital environment in Libya, and to determine the distribution of putative virulence genes amongst them. METHODS AND RESULTS: Macrorestriction analysis using pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S rRNA and aroA genes were used to type the isolates. Whereas 30 of 32 chicken isolates were identified as Aeromonas veronii, eight of 12 environmental isolates were Aer. caviae. Three species were identified amongst the eight isolates from children. Aeromonas veronii and Aer. caviae isolates could be divided into eight and five PFGE types, respectively. All species could be further subtyped into one of 21 aroA PCR-RFLP groups. Aerolysin-like haemolysin or enterotoxin gene sequences were detected in all the isolates. Overall carriage rates for hlyA and alt were 77 and 75%, respectively. CONCLUSIONS: Seven of eight isolates from children were of different subtypes, indicating a lack of any common source of acquisition. Isolates of common molecular type did not share identical distributions of putative virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the effectiveness of using molecular typing to identify and study genetic variation amongst Aeromonas isolates.     

Oxygen and contact with human intestinal epithelium independently stimulate virulence gene expression in enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) are important intestinal pathogens causing acute and persistent diarrhoeal illness worldwide. Although many putative EAEC virulence factors have been identified, their association with pathogenesis remains unclear. As environmental cues can modulate bacterial virulence, we investigated the effect of oxygen and human intestinal epithelium on EAEC virulence gene expression to determine the involvement of respective gene products in intestinal colonisation and pathogenesis. Using in vitro organ culture of human intestinal biopsies, we established the colonic epithelium as the major colonisation site of EAEC strains 042 and 17‐2. We subsequently optimised a vertical diffusion chamber system with polarised T84 colon carcinoma cells for EAEC infection and showed that oxygen induced expression of the global regulator AggR, aggregative adherence fimbriae, E. coli common pilus, EAST‐1 toxin, and dispersin in EAEC strain 042 but not in 17‐2. Furthermore, the presence of T84 epithelia stimulated additional expression of the mucinase Pic and the toxins HlyE and Pet. This induction was dependent on physical host cell contact and did not require AggR. Overall, these findings suggest that EAEC virulence in the human gut is modulated by environmental signals including oxygen and the intestinal epithelium.     

Potential virulence and antimicrobial susceptibility of Aeromonas popoffii recovered from freshwater and seawater

Aeromonas popoffii is the most recent species within the genus Aeromonas described from freshwater. In our study this species was also recovered from this habitat and for the first time from seawater. Most of the virulence factors known in Aeromonas spp. (aerolysin/hemolysin, serine protease, lipases and DNases) were highly prevalent in this species. Third-generation cephalosporins and quinolones were the most active antimicrobial agents against A. popoffii.     

Differences in production of several extracellular virulence factors in clinical and food Aeromonas spp. strains

C. PIN, M.L. MARÍN, D. SELGAS, M.L. GARCÍA, J. TORMO AND C. CASAS. 1995. Production of several extracellular virulence factors (lipase, protease and haemolysin) was compared in 15 Aeromonas spp. isolated from faeces of patients with Aeromonas -associated gastroenteritis and 81 strains isolated from food. Strains from food did not show differences in production of these factors when compared with strains isolated from faeces. However, if strains were considered in relation to autoagglutination (AA) character, the AA⁺ differed from AA^- strains in lipase and protease production. Supernatant fluids of AA⁺ food and human strains showed 2·5-fold more protease production than that observed in AA^- strains. These two characteristics of certain Aeromonas strains could be related with the more virulent capacity.     

Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain

AIMS: To study the phenotypic characteristics of Aeromonas spp. from environmental and clinical samples in Spain and to cluster these strains by numerical taxonomy. METHODS AND RESULTS: A collection of 202 Aeromonas strains isolated from bivalve molluscs, water and clinical samples was tested for 64 phenotypic properties; 91% of these isolates were identified at species level. Aeromonas caviae was predominant in bivalve molluscs and Aerom. bestiarum in freshwater samples. Cluster analyses revealed eight different phena: three containing more than one DNA-DNA hybridization group but including strains that belong to the same phenospecies complex (Aerom. hydrophila, Aerom. sobria and Aerom. caviae), Aerom. encheleia, Aerom. trota and three containing unidentified Aeromonas strains isolated from bivalve molluscs. CONCLUSIONS:Aeromonas spp. are widely distributed in environmental and clinical sources. A selection of 16 of the phenotypical tests chosen allowed the identification of most isolates (91%), although some strains remain unidentified, mainly isolates from bivalve molluscs, suggesting the presence of new Aeromonas species. Numerical taxonomy was not in total concordance with the identification of the studied strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerical taxonomy of Aeromonas strains isolated from different sources revealed the presence of potentially pathogenic Aeromonas spp., especially in bivalve molluscs, and phena with unidentified strains that suggest new Aeromonas species.