Toxinogenicity and markers of pathogenicity of Pseudomonas aeruginosa strains isolated from patients with tumor diseases

Potential virulence factors (elastase, proteinase, lipase, phospholipase C, alginate) as well as surface properties (hydrophobicity, motility) were determined in 103Pseudomonas aeruginosa strains isolated from patients with cancer. Nontypable strains were the dominant group (60%), followed by serotypes O11 (17%), O12 (7%) and O4 (5%). Seventy-one strains (69%) produced high level of elastase (10–60 mg/L), 87% of the strains possessed high activity of proteinase (bacterial) (10–250 mg/L) and 69% of the strains demonstrated higher level of lipase (20–150 U/mL); these elevated levels of enzymes were associated mainly with nontypable strains. On the other hand, 79% of the strains did not produce or produced only a low level of phospholipase C and 60% of isolates did not manifest any or very low production of alginate. Hydrophobicity demonstrated by adherence of the bacteria to xylene was shown by 69% of strains; 94% of strains aggregated with ammonium sulfate. Motility in the range of 31–80 mm was found in 76 strains (74%). The considerable virulence of testedP. aeruginosa strains was confirmed. The nontypable strains manifested the most frequent group with high level of elastase, proteinase, lipase, hydrophobicity and motility.     

Some properties of<Emphasis Type="Italic">Plesiomonas shigelloides</Emphasis> treated with aminoglycosides

The effect of aminoglycoside antibiotics (amikacin, gentamicin, netilmicin and tobramycin) at sublethal concentrations (sub-MICs) on some properties of Plesiomonas shigelloides strains was evaluated. All agents decreased the bacterial surface hydrophobicity. Amikacin (1/4 of the MIC) and netilmicin (1/4 and 1/8 of the MIC) changed the hydrophobic character of P. shigelloides surface to a hydrophilic one. Treatment of the strains with aminoglycosides decreased also motility, netilmicin being the most effective. No significant changes were found in lipolytic activity of antibiotic-treated strains. In the majority of cases aminoglycosides increased sensitivity of bacteria to hydrogen peroxide. The tested antibiotics did not induce production of short-chained N-acylhomoserine lactones signal molecules. Aminoglycosides at sub-MICs affected important activities of P. shigelloides potentially associated with their virulence in dependence on strain, antibiotic and concentration.     

碳源及温度对食源假单胞菌群体感应信号分子产生的影响

许多革兰氏阴性菌通过产生N-酰基-高丝氨酸内酯（AHLs）类信号分子来调控某些性状的表达,即群体感应（quorum sensing）。假单胞菌是一种导致食品腐败的重要腐败细菌,也产生AHLs。本文研究了不同温度及碳源对食源假单胞菌AHLs产生的影响。结果表明,该假单胞菌在25℃条件下,产生两种AHL信号分子,而在4℃条件下,所产生的短链AHL分子消失,主要产生长链AHL分子。而且在不同碳源（葡萄糖,果糖,木糖,麦芽糖等）的培养基中生长,所产生的AHLs分子种类也不同。同时发现当pH>7.5时,AHLs的稳定性下降。由此得出,在不同的环境条件(碳源及温度)下假单胞菌所产生的AHLs种类不同。为进一步研究群体感应现象在食品腐败中的作用以及开发基于干扰腐败菌群体感应的新型食品防腐技术提供研究基础。     

食源假单胞菌群体感应信号分子的研究

从市售鲜鱼中分离的3株革兰氏阴性菌,经16S rDNA鉴定为假单胞菌属,该菌是一种导致食品腐败的重要腐败细菌。N-酰基-高丝氨酸内酯(AHLs)是革兰氏阴性菌群体感应(QS)系统中一类重要的信号分子,以密度依赖的方式调控某些生理性状的表达。利用AHLs检测菌株对3株假单胞菌进行检测发现,均产生AHLs类信号分子,且FML05-1和FML05-2至少产生两种AHLs,主要的信号分子是N-3-氧代-辛酰基-高丝氨酸内酯(N- 3-oxo-C_8-HSL)。同时对菌株FML05-2在生长过程中所产生的AHLs的活性变化进行研究,发现AHLs活性在菌体生长至12h时达到最大。首次对食源假单胞菌所产生的AHLs进行了研究,为以干扰腐败细菌群体感应为靶点的食品防腐保鲜策略提供研究基础。     

Acyl homoserine lactones from culture supernatants of Pseudomonas aeruginosa accelerate host immunomodulation

The virulence of Pseudomonas aeruginosa is multifactorial and under the control of quorum sensing signals, such as acyl homoserine lactones (AHLs). The importance of these molecules in the establishment of infection has been previously reported. These molecules either improve the virulence potential of P. aeruginosa or modulate the host immune response. To establish the immune modulating potential of quorum sensing signal molecules, previous studies have only used synthetic AHLs. However, there can be differences in the biological properties of synthetic and natural AHLs. The use of naturally extracted AHLs from the culture supernatant of P. aeruginosa is likely to simulate natural conditions more than the use of synthetic AHLs. Therefore, in the present study, the immune modulating potential of synthetic and naturally extracted AHLs was compared using a thymidine uptake assay, immunophenotyping and sandwich ELISA in order to assess mouse T-cell proliferation and production of Th1 and Th2 cytokines. Natural AHLs were able to suppress T-cell proliferation, even at low concentrations, compared to synthetic AHLs. The majority of cells undergoing proliferation were CD4+, as revealed by immunophenotyping. The inhibition of T-cells was stronger with natural AHLs compared to synthetic AHLs. Moreover, the natural AHLs were also able to shift immune responses away from host protective Th1 responses to pathogen protective Th2 responses.     

Synthesis of multiple N-acylhomoserine lactones is wide-spread among the members of the Burkholderia cepacia complex

Seventy strains of the Burkholderia cepacia complex, which currently comprises six genomic species, were tested for their ability to produce N-acylhomoserine lactone (AHL) signal molecules. Using thin layer chromatography in conjunction with a range of AHL biosensors, we show that most strains primarily produce two AHLs, namely N-octanoylhomoserine lactone (C8-HSL) and N-hexanoylhomoserine lactone (C6-HSL). Furthermore, some strains belonging to B. vietnamiensis (genomovar V) produce additional long chain AHL molecules with acyl chains ranging from C10 to C14. For B. vietnamiensis R-921 the structure of the most abundant long chain AHL was confirmed as N-decanoylhomoserine lactone (C10-HSL) by liquid chromatography-mass spectrometry (LC-MS) in combination with total chemical synthesis. Interestingly, a number of strains, most notably all representatives of B. multivorans (genomovar II), did not produce AHLs at least under the growth conditions used in this study. All strains were also screened for the production of extracellular lipase, chitinase, protease, and siderophores. However, no correlation between the AHL production and the synthesis of these exoproducts was apparent. Southern blot analysis showed that all the B. cepacia complex strains investigated, including the AHL-negative strains, possess genes homologous to the C8-HSL synthase cepI and to cepR, which encodes the cognate receptor protein. The nucleotide sequence of the cepI and cepR genes from one representative strain from each of the six genomovars was determined. Furthermore, the cepI genes from the different genomovars were expressed in Escherichia coli and it is demonstrated that all genes encode functional proteins that direct the synthesis of C8-HSL and C6-HSL. Given that cepI from the B. multivorans strain encodes a functional AHL synthase, yet detectable levels of AHLs were not produced by the wild-type, this suggests that additional regulatory functions may be present in members of this genomovar that negatively affect expression of cepI.     

Production of acylated homoserine lactones by psychrotrophic members of the Enterobacteriaceae isolated from foods.

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5 degrees C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10(6) CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5 degrees C under an N(2) atmosphere when mean cell densities increased to 10(6) CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10(6) CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.     

Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.     

Enzymatic characterization of Vibrionaceae strains isolated from environment and cold-blooded animals.

Enzymatic profiles were determined by the API ZYM system for 15 strains of non 01 Vibrio cholerae, 4 strains of V. metschnikovii, 9 strains of V. anguillarum, 6 strains of Plesiomonas shigelloides and 115 strains motile Aeromonas sp. All of the tested strains produced alkaline phosphatase, leucine aminopeptidase and did not possess alpha-fucosidase and alpha-mannosidase. Some differences in enzymatic activities among the tested Vibrionaceae strains were noted. The strains of non 01 V. cholerae, V. metschnikovii, V. anguillarum and P. shigelloides did not produce trypsin, whereas all of the tested Vibrio sp. strains appeared to be positive for this enzyme. Only the strains of P. shigelloides produced BI-Phospho-hydrolase. The lack of acid phosphatase activity was observed among the strains of V. anguillarum.     

Chemical identification of<Emphasis Type="Italic"> N</Emphasis>-acyl homoserine lactone quorum-sensing signals produced by<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> strains in defined medium

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.     

N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.     

Detection of different quorum-sensing signal molecules in a virulent Edwardsiella tarda strain LTB-4

Aims:  The aim of this study was to elucidate the potential quorum-sensing (QS) signal molecules of an emerging pathogen ( Edwardsiella tarda strain LTB-4) of cultured turbot ( Scophthalmus maximus ).
Methods and Results:  A sensitive and rapid double-layer plate method using biosensor strain Agrobacterium tumefaciens KYC55 was developed to detect the N-acylhomoserine lactone (AHL)-related compounds in bacteria. LTB-4 was found to have two QS systems, one was based on the AHLs and the other was based on the autoinducer-2 (AI-2). The AI-2 activity produced by LTB-4 was growth phase dependent and topped at OD₆₀₀ of 1·0. The protocol to detect cholerae autoinducer 1 (CAI-1) activity in bacteria was modified, lowering the background luminescence of biosensor strain Vibrio harveyi JAF375. CAI-1 activity could not be detected in LTB-4.
Conclusion:  Edwardsiella tarda LTB-4 produced at least four kinds of AHLs during its whole growth phase. In comparison with the AHL-inducing QS, AI-2 may be the first predominant signal, functioning at early exponential phase. LTB-4 did not produce any CAI-1 activity.
Significance and Impact of the Study:  Different QS signal molecules of Edw. tarda LTB-4 were clarified by improved bioassays. In contrast to earlier studies detecting two types of AHLs, strain LTB-4 produced at least four kinds of AHLs, which seemed to be C₄-HSL, C₆-HSL, 3-oxo-C₆-HSL and an uncharacterized AHL molecule.     

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.     

Prevalence and virulence properties of Vibrio cholerae non-O1, Aeromonas spp. and Plesiomonas shigelloides isolated from Cambé Stream (State of Paraná, Brazil)

The incidence of Vibrio cholerae, Aeromonas spp. and Plesiomonas shigelloides was determined in water samples from Cambé Stream. The samples were collected from seven different sites. The serogroups, virulence markers and drug resistance profiles were also evaluated. Twelve Aer. hydrophila, 12Aer. caviae, eight Aer. sobria, seven Ple. shigelloides and two V. cholerae non-O1 were isolated. They belonged to different serogroups and all produced haemolysis in different assays. Five of the Aeromonas strains and one of V cholerae non-O1 were positive for enterotoxin activity. Haemagglutination and its inhibition, using erythrocytes of different origins, was variable for Aeromonas spp. and V. cholerae, while none of the Ple. shigelloides haemagglutinated in association with any type of erythrocyte. All isolates exhibited multiple drug resistance. These results indicate that the occurrence of V. cholerae non-O1, Aeromonas spp. and Ple. shigelloides, in water used for vegetable irrigation, human recreation and animal consumption, among others, represents a potential risk for humans.     

青岛近海沉积物生物被膜中密度感应及密度感应淬灭细菌多样性

【背景】细菌密度感应(Quorum sensing,QS)是指细菌利用分泌的信号分子进行相互交流的现象,而密度感应淬灭(Quorumquenching,QQ)是指通过干扰信号分子的产生、释放、积累或应答从而阻抑密度感应通路。【目的】探究青岛近海沉积物生物被膜中密度感应和密度感应淬灭细菌的多样性。【方法】采用海水培养基2216E从青岛近海沉积物生物被膜中分离获取可培养细菌,采用平板交互划线和高通量筛选的方法分别筛选具有密度感应和密度感应淬灭的菌株。【结果】共分离获得83株共54种具有密度感应和密度感应淬灭的细菌,分属于四大细菌门类:变形菌门、拟杆菌门、厚壁菌门和放线菌门。其中,38株(45.8%)可以产生酰基高丝氨酸内酯(Acyl-homoserine lactone,AHL)类信号分子,它们分属于变形菌门(37株,15种)和拟杆菌门(1株,1种),优势属为弧菌属和鲁杰氏菌属;能够降解AHL类信号分子的有57株(68.7%),在变形菌门(41株,23种)、拟杆菌门(14株,10种)、厚壁菌门(5株,5种)以及放线菌门(1株,1种)中均有分布。【结论】在青岛近海沉积物生物被膜可培养细菌中,具有密度感应和密度感应淬灭现象的菌株具有很高的丰度和多样性,为后续生态学意义的研究与海洋微生物的开发提供了参考。     

Detection and characterization of bacteria from the potato rhizosphere degrading N-acyl-homoserine lactone

Quorum sensing plays a role in the regulation of soft rot diseases caused by the plant pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. The signal molecules involved in quorum sensing in P. carotovorum subsp. carotovorum belong to the group of N-acyl homoserine lactones (AHLs). In our study, we screened bacteria isolated from the potato rhizosphere for the ability to degrade AHLs produced by P. carotovorum subsp. carotovorum. Six isolates able to degrade AHLs were selected for further studies. According to 16S rDNA sequence analysis and fatty acid methyl ester profiling, the isolates belonged to the genera Ochrobactrum, Rhodococcus, Pseudomonas, Bacillus, and Delftia. For the genera Ochrobactrum and Delftia, for the first time AHL-degrading isolates were found. Data presented in this study revealed for the first time that Ochrobactrum sp. strain A44 showed the capacity to inactivate various synthetic AHL molecules; the substituted AHLs were inactivated with a lower efficiency than the unsubstituted AHLs. Compared with the other isolates, A44 was very effective in the degradation of AHLs produced by P. carotovorum subsp. carotovorum. It was verified by polymerase chain reaction, DNA-DNA hybridization, and a lactone ring reconstruction assay that Ochrobactrum sp. strain A44 did not possess AHL lactonase activity. AHL degradation in Ochrobactrum sp. strain A44 occurred intracellularly; it was not found in the culture supernatant. AHL-degrading activity of A44 was thermo sensitive. Experiments in planta revealed that Ochrobactrum sp. strain A44 significantly inhibited the maceration of potato tuber tissue. Since A44 did not produce antibiotics, the attenuation of the decay might be due to the quenching of quorum- sensing-regulated production of pectinolytic enzymes. The strain can potentially serve to control P. carotovorum subsp. carotovorum in potato.     

Reliable enzyme-linked immunosorbent assay systems for pathogenic factors of Pseudomonas aeruginosa alkaline proteinase, elastase, and exotoxin A: a comparison of methods for labeling detection antibodies with horseradish peroxidase

Sensitive sandwich enzyme-linked immunosorbent assay (ELISA) systems for the quantification of 3 pathogenic factors of Pseudomonas aeruginosa-alkaline proteinase (aeruginolysin), elastase (pseudolysin ), and exotoxin A-were developed. The maleimide-pyridyl disulfide method was applied for the labeling of rabbit anti-each antigen IgG with horseradish peroxidase (HRP) and the conjugates were used as secondary antibodies (detection antibodies) in the ELISA systems. The EDTA, a chelating agent, was added to the buffers for sample and detection antibody, which inhibited the degradation of IgG by elastase derived from P. aeruginosa for improving the assay precision. The ELISA systems using the HRP-labeled detection antibodies produced by the maleimide-pyridyl disulfide method exhibited higher sensitivity than previously reported methods. The detection limits for alkaline proteinase, elastase, and exotoxin A were 18 pg/ml, 34 pg/ml, and 22 pg/ml, respectively. The intra-assay coefficients of variation for alkaline proteinase, elastase, and exotoxin A were 3.4%-5.0%, 1.9%-3.5%, and 1.3%-5.4%, respectively. These ELISA systems exhibited good inter-assay precision, non-cross-reactivity, dilution linearity, and recovery . Employing these ELISA systems, we revealed that pathogenic factor concentrations were different among the P. aeruginosa strains tested, which may relate to the different pathogenicity of each strain.     

Production of Acylated Homoserine Lactones by Psychrotrophic Members of the Enterobacteriaceae Isolated from Foods

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10⁶ CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5°C under an N₂ atmosphere when mean cell densities increased to 10⁶ CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10⁶ CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.     

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.