Sobria Strains Research Articles

Investigation of the Antibacterial Effect of Astaxanthin and the Prevalence of Virulence and Antimicrobial Resistance Genes of Aeromonas spp.

In the study, in addition to the antibacterial effect of astaxanthin on Aeromonas hydrophila and A. sobria strains, the presence of virulence genes (Aero, act, ast, and hylA) and antibiotic resistance genes (tetC and sulI) in the strains was investigated. Antibiotic profiles of the strains were also investigated as part of the study. Strains were identified by conventional biochemical tests and PCR assay using a 16S rDNA primer pair specific for A. hydrophila. According to the results of bacteriological and molecular studies, two of the six Aeromonas strains were identified as A. hydrophila and four of them as A. sobria. The Aero virulence gene and the act virulence gene were found in all strains, while the ast and hylA virulence genes were detected only in A. hydrophila strains. All strains were resistant to chloramphenicol, tetracycline, nalidixic acid, and ampicillin in the standard disk diffusion test. Although all strains showed resistance to tetracycline and moderate resistance to oxytetracycline in the antibiogram tests, tetC antibiotic resistance gene was not detected in the strains and sulI antibiotic resistance gene was not detected in the strains. In the study, acetone solutions containing 0.1 g and 0.5 g of astaxanthin were found to have an antibacterial effect on A. hydrophila strains. Acetone solutions containing 0.1 g, 0.5 g, and 1.0 g of astaxanthin showed antibacterial effects on A. sobria strains. It was found that 0.1 g, 0.5 g, and 1.0 g astaxanthin solutions prepared with methanol and distilled water had no antibacterial effects on the strains.

Motile aeromonads recovered from tilapia (Oreochromis niloticus) cultured in earthen ponds in the Philippines: assessment of antibiotic susceptibility and multidrug resistance to selected antibiotics

A total of 177 motile Aeromonas spp., comprised of A. hydrophila (n=162) and A. sobria (n=15), isolated from the water, sediment, gills and intestines of tilapia collected from the grow-out culture earthen ponds in the Philippines were investigated for their antimicrobial susceptibility to 10 antibiotics commonly used in fish aquaculture. All motile Aeromonas spp. tested generally exhibited a very high percentage of resistance to amoxicillin (A. hydrophila: 95%; A. sobria: 100%), erythromycin (99%;100%), neomycin (83%;100%), and oxytetracycline (92%;100%), respectively. On the contrary, they showed substantial susceptibility to chloramphenicol, nitrofurantoin, doxycycline, enrofloxacin and norfloxacin. Results of the antimicrobial susceptibility testing demonstrated that 94.4% and 100% of A. hydrophila and A. sobria isolates tested were resistant to antibiotics belonging to at least 3 or more categories and therefore considered as multiple drug resistant strains. The multiple antibiotic resistance (MAR) indexing of A. hydrophila and A. sobria strains showed that they originated from high-risk sources.

Antibacterial Activity of Commercial Phytochemicals against Aeromonas Species Isolated from Fish.

Antimicrobial activities of phytochemicals—trans-cinnamaldehyde (TC), ferulic acid (FA), p-coumaric acid (p-CA), caffeic acid (CA), chlorogenic acid (CHA), Thymus vulgaris essential oil (TO), Eugenia caryophyllus essential oil (ECO), and Melaleuca alternifolia oil (TTO) against Aeromonas species—were assessed. Growth of all Aeromonas salmonicida subsp. salmonicida and almost all Aeromonas sobria strains was inhibited by TC at concentration 0.01 mg/mL, and for most Aeromonas hydrophila strains minimal inhibitory concentrations (MIC) ranged from 0.01 to 0.19 mg/mL. The inhibitory effect of TC against A. salmonicida subsp. salmonicida was comparable to the effect of oxytetracycline, and in the case of A. salmonicida subsp. salmonicida and A. sobria was higher compared to gentamicin. MIC of FA, p-CA, and CA for most strains ranged from 1.56 to 3.12 mg/mL, and MIC values of TO for most strains ranged from 0.39 to 0.78 mg/mL. TO and TC at the concentrations below ½ MIC values used in mixtures exhibited strong synergism. ECO and TC showed synergy in mixture of ⅛ MIC of ECO and ¼ MIC of TC. TC and TO exhibited the strongest inhibitory and bactericidal effect against investigated Aeromonas species, and they are a promising alternative to the use of antibiotics in controlling the growth of these fish pathogens.

Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

Aeromonas sobria is a pathogen causing food-borne illness. In immunocompromised patients and the elderly, A. sobria can leave the intestinal tract, and this opportunistically leads to severe extraintestinal diseases including sepsis, peritonitis, and meningitis. To cause such extraintestinal diseases, A. sobria must pass through the intestinal epithelial barrier. The mechanism of such bacterial translocation has not been established. Herein we used intestinal (T84) cultured cells to investigate the effect of A. sobria serine protease (ASP) on junctional complexes that maintain the intercellular adhesion of the intestinal epithelium. When several A. sobria strains were inoculated into T84 monolayer grown on Transwell inserts, the strain with higher ASP production largely decreased the value of transepithelial electrical resistance exhibited by the T84 monolayer and markedly caused bacterial translocation from the apical surface into the basolateral side of T84 monolayer. Further experiments revealed that ASP acts on adherens junctions (AJs) and causes the destruction of both nectin-2 and afadin, which are protein components constituting AJs. Other studies have not revealed the bacterial pathogenic factors that cause the destruction of both nectin-2 and afadin, and our present results thus provide the first report that the bacterial extracellular protease ASP affects these molecules. We speculate that the destruction of nectin-2 and afadin by the action of ASP increases the ability of A. sobria to pass through intestinal epithelial tissue and contributes to the severity of pathological conditions.

Strong Genomic and Phenotypic Heterogeneity in the Aeromonas sobria Species Complex.

Aeromonas sobria is a mesophilic motile aeromonad currently depicted as an opportunistic pathogen, despite increasing evidence of mutualistic interactions in salmonid fish. However, the determinants of its host-microbe associations, either mutualistic or pathogenic, remain less understood than for other aeromonad species. On one side, there is an over-representation of pathogenic interactions in the A. sobria literature, of which only three articles to date report mutualistic interactions; on the other side, genomic characterization of this species is still fairly incomplete as only two draft genomes were published prior to the present work. Consequently, no study specifically investigated the biodiversity of A. sobria. In fact, the investigation of A. sobria as a species complex may have been clouded by: (i) confusion with A. veronii biovar sobria because of their similar biochemical profiles, and (ii) the intrinsic low resolution of previous studies based on 16S rRNA gene sequences and multilocus sequence typing. So far, the only high-resolution, phylogenomic studies of the genus Aeromonas included one A. sobria strain (CECT 4245 / Popoff 208), making it impossible to robustly conclude on the phylogenetic intra-species diversity and the positioning among other Aeromonas species. To further understand the biodiversity and the spectrum of host-microbe interactions in A. sobria as well as its potential genomic diversity, we assessed the genomic and phenotypic heterogeneity among five A. sobria strains: two clinical isolates recovered from infected fish (JF2635 and CECT 4245), one from an infected amphibian (08005) and two recently isolated brook charr probionts (TM12 and TM18) which inhibit in vitro growth of A. salmonicida subsp. salmonicida (a salmonid fish pathogen). A phylogenomic assessment including 2,154 softcore genes corresponding to 946,687 variable sites from 33 Aeromonas genomes confirms the status of A. sobria as a distinct species divided in two subclades, with 100% bootstrap support. The phylogenomic split of A. sobria in two subclades is corroborated by a deep dichotomy between all five A. sobria strains in terms of inhibitory effect against A. salmonicida subsp. salmonicida, gene contents and codon usage. Finally, the antagonistic effect of A. sobria strains TM12 and TM18 suggests novel control methods against A. salmonicida subsp. salmonicida.

DETECTION OF AEROMONAS HYDROPHILA IN RAW MILK AND SOME MILK PRODUCTS WITH REFERENCE TO ITS PUBLIC HEALTH HAZARD

This study aimed to determine Aeromonas spp . in raw milk and some milk products. A total of 100 raw milk, kareish cheese, ice cream and baladi yoghurt (25 samples, each) were collected from different dairy shops and street peddlers in Assiut city, Egypt and were bacteriologically examined for presence and count of Aeromonas spp . The incidences of counted Aeromonas spp . in raw milk, kareish cheese, ice cream and baladi yoghurt were 36, 32, 24 and 0.0%, respectively, with average counts of 1.0×105, 3.2×104, 5.0×102 and < 100 cfu/ml, respectively. The incidences of counted Aeromonas hydrophila, in raw milk, kareish cheese and ice cream were 16, 12 and 8%, while for Aeromonas caviae, the incidences were 12, 16 and 12%, respectively. Moreover, the incidences of counted Aeromonas sobria in raw milk, kareish cheese and ice cream were 8, 4 and 4%, respectively. Baladi yoghurt samples were negative for Aeromonas spp. in this study. All the recovered Aeromonas hydrophila organisms were confirmed by PCR assay for the presence of 16S rRNA gene and 100% of the tested strains harboured this gene. The aerA and ahh1 virulence genes were present in Aeromonas hydrophila in percentages of 66.67 and 77.78%, respectively. All the recovered Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria strains, in this study, exhibited 100% virulence properties on bases of proteolytic, lioplytic, psychrotrophic and β-haemolytic activities. The recovered Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria exhibited 100% resistance towards Ampicillin, Amoxicillin and Erythromycin antibiotics, while, they exhibited 100% sensitivity towards Ciprofloxacin. The public health hazards of occurrence of Aeromonas spp. in milk and its products as well as the suggestive control measures were discussed.

Biofilm formation on polystyrene and glass surface byAeromonasspecies isolated from different sources

Biofilm forming bacteria can be the major sources of food contamination which can lead to potential foodborne disease. This study, a total of 58 Aeromonas strains isolated from food and sediment samples were investigated for their biofilm-forming ability in different mediums (TSA/TSB and BHIA/BHIB) and on different surfaces (polystyrene and glass). The biofilm development was determined by using microtiter plate and tube test method. While A. veroni biovar sobria strains produced strong biofilm (87.1–59.3%) by microtiter plate and tube test, respectively, and it is interesting to note moderate to high degree of adherence of A. hydrophila by microtiter plate and tube test (56.5–65.2%), respectively. In addition to this A. caviae strains showed only weak biofilm forming on glasses surface while there was no biofilm forming on polystyrene surface. The agar medium was more effective than broth medium in promoting biofilm production by the tested Aeromonas strains. Practical applications In food industry, biofilms are a potential source of product contamination and may lead to food spoilage and serious fouling problems in equipment and biofilm forming bacteria can lead to potential foodborne disease. Aeromonas-associated cases of gastroenteritis are generally considered food and waterborne. Many Aeromonas strains are capable of adhering and forming biofilm on certain surfaces. Therefore, it might be useful information to understand the biofilm forming capacity of Aeromonas spp. and to compare the biofilm formation of Aeromonas strains with different surfaces and mediums.

Infection status and virulent genes of Aeromonas in diarrhea patients in Pudong New Area, Shanghai

To investigate the infection status and virulent genes of Aeromonas in patients with acute diarrhea in Pudong New Area, Shanghai. In 2012, stool samples were collected from diarrhea patients in 12 sentinel hospitals in Pudong for the detections of 13 pathogens causing diarrhea, and the detections of 5 diarrhea related virulent genes were conducted for Aeromonas isolates. A total of 101 patients were infected with Aeromonas in 2533 patients (4.0%). A total of 101 Aeromonas strains were isolated, including 17 Aeromonas hydrophila strains (18.8%), 44 Aeromonas veronii biovar sobria strains (52.5%) and 12 Aeromonas caviae strains (29.7%). And 44 coinfections with other pathogens were detected. Aeromonas infection mainly occurred in summer and in people aged ≥20 years. Among the patients infected with Aeromonas, 71 (70.3%) had watery diarrhea, 20 (19.8%) had vomiting and 11 (10.9%) had fever. Virulent genes detection showed that 95.0% of the Aeromonas. strains carried virulent genes, and the detection rates of hlyA, aerA, act, alt, and ast genes were 5.9%, 6.9%, 67.3%, 42.6% and 13.9%, respectively. High incidence of Aeromonas infection was found in the patients with acute diarrhea in Pudong, and a high proportion of coinfections with other pathogens was detected too. Most Aeromonas strains carried virulent genes, and the distribution varied.

Characterization of Aeromonas sobria Isolated from Fish Rohu (Labeo rohita) Collected from Polluted Pond

One of the major threats to fish aquaculture sector is the infection by Aeromonas Spp. The current study assesses the phenotypic characteristics and biochemical characterization of the A. sobria strains from the fish cases with septicaemia in order to understand the frequency and occurrence of this infection in the state of Jammu and Kashmir. Clinically the infected fish Rohu (Labeo rohita), one of the Indian Major carps (IMC), was observed for symptoms like loss of escape reflex and skin darkness associated with skin haemorrhages. We isolated 30 colonies of A. sobria strain from 10 cultured Labeo rohita collected from a controlled fish pond in District Poonch of the state. The pond was affected by mismanagement practices, elevated pollution levels and anthropogenic activities. Microscopic examination revealed that the strain was rod-shaped and gram negative. The revealed percent probability identification of A. sobria from the biochemical characterization in Vitek system was 93% with GN card. This study could give us clues for understanding A. sobria harbouring in fish species and shall help in better understanding of the threat prevalent to the fish species of the region by this infection.

Molecular and chemical analysis of the lipopolysaccharide from Aeromonas hydrophila strain AH-1 (Serotype O11).

A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described; these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wbO11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waaO11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.

The Comparative Analysis of Phenotypic and Genotypic Properties of Aeromonas sobria Strains Isolated from Rainbow Trout (Oncorhynchus mykiss, Walbaum, 1972)

The Comparative Analysis of Phenotypic and Genotypic Properties of Aeromonas sobria Strains Isolated from Rainbow Trout (Oncorhynchus mykiss, Walbaum, 1972)

First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene

Isolation and full sequence analysis of ColE-type plasmid, which carries the qnrS2 gene. Quinolone resistance (qnrS2) gene-carrying plasmids were isolated from Aeromonas sobria and Aeromonas hydrophila strains, and plasmid sequencing was achieved by a primer-walking approach. The total sizes of these plasmids (pAQ2-1 and pAQ2-2) were 6900 bp and 6903 bp, respectively, and they were 99·1% identical to each other. The genes (oriV and repA) for plasmid replication were organized similar to the corresponding genes in the ColE2-type plasmids, pAsa3 and pAsa1, isolated from Aeromonas salmonicida subsp. salmonicida, but the gene (mobA) for mobilization was homologue to ColE1-type plasmid (pAsa2) from Aer. salmonicida subsp. salmonicida. Additionally, the qnrS2 gene was part of a mobile insertion cassette element in the plasmid. Two plasmids were assumed to be the same plasmid, and this identification of a plasmid-mediated qnrS2 gene from the two different strains underlines a possible diffusion of these resistance determinants in an aquaculture system. This is the first finding of the ColE-type plasmid carrying the qnrS2 gene.

Molecular characterization of fluoroquinolone-resistant Aeromonas spp. isolated from imported shrimp.

Sixty-three nalidixic acid-resistant Aeromonas sp. isolates were obtained from imported shrimp. Phylogenetic analysis of gyrB sequences indicated that 18 were A. enteropelogenes, 26 were A. caviae, and 19 were A. sobria. Double missense mutations in the quinolone resistance-determining region (QRDR) of gyrA at codon 83 (Ser→Val/Ile) and codon 92 (Leu→Met) coupled with a point mutation of parC at codon 80 (Ser→Ile/Phe) conferred high levels of quinolone resistance in the isolates. A majority of A. enteropelogenes and A. caviae strains harbored toxin genes, whereas only a few A. sobria strains harbored these genes. The fluoroquinolone-resistant Aeromonas spp. exhibited higher cytotoxicity than fluoroquinolone-sensitive, virulent Aeromonas spp. to rat epithelial cells.

Heterogeneity of Aeromonas populations in wild and farmed perch, Perca fluviatilis L.

The aeromonads comprise a diverse group of waterborne bacteria found within the gamma-proteobacteria. The last two decades have seen marked advances in Aeromonas taxonomy, and over 20 validated species are now recognized (for a recent review see Janda & Abbott 2010). Aeromonas species are well-known agents of fish disease, and this is reflected in the number of Aeromonas species originally described in diseased fish including Aeromonas allosaccharophila (Martinez-Murcia et al. 1992), Aeromonas aquariorum (Martinez-Murcia et al. 2008), Aeromonas bestiarum (Ali et al. 1996), Aeromonas piscicola (Beaz-Hidalgo et al. 2009), Aeromonas sobria (Popoff & Veron 1976) and four subspecies of Aeromonas salmonicida: A. salmonicida subsp. salmonicida (Griffin, Snieszko & Friddle 1953), A. salmonicida subsp. achromogenes (Smith 1963), A. salmonicida subsp. masoucida (Kimura 1969a,b) and A. salmonicida subsp. smithia (Austin, McIntosh & Austin 1989). Probably the best characterized of these is A. salmonicida subsp. salmonicida, the aetiological agent of furunculosis, which affects salmonid fish. In cases of acute disease, onset of mortality is rapid and mortality rates are high. Other species including Aeromonas hydrophila and A. veronii biovar sobria have been associated with ulcerative disease in farmed fish such as carp, catfish, koi and trout (Rahman et al. 2002; Kozinska 2007; Orozova et al. 2009; BeazHidalgo et al. 2010). Most recently, A. sobria has been identified as a causative agent of tail rot in tilapia (Li & Cai 2011), and A. veronii has been associated with infectious dropsy in oscar (Sreedharan, Philip & Singh 2011). In Switzerland, A. sobria was isolated from the skin lesions and internal organs of perch, Perca fluviatilis L., farmed in a freshwater lake. Diseased animals presented focal ulcers and fin rot (Wahli et al. 2005). Isolates taken from these diseased, farmed perch were able to induce similar clinical signs under experimental conditions indicating A. sobria was the causative agent of the disease (Wahli et al. 2005). A subsequent study, however, found A. sobria and A. veronii were among the dominant members of the intestinal microbiota of wild perch caught in the same lake and presenting no signs of disease (Goldschmidt-Clermont et al. 2008). The study described here aimed to determine whether these wild perch served as a reservoir of infection of farmed animals, or whether the A. sobria strains causing disease in farmed perch were distinct from those found to colonize the wild perch population. Fifty-two Aeromonas strains were isolated, from 2001 to 2005, from two distinct perch populations; a population of farmed fish reared in open pens on a freshwater lake and a population of wild perch caught in the same lake. Strains were initially isolated from material taken from perch organs (skin, intestine, kidney, spleen and liver; Table 1) with sterile loops and inoculated onto Columbia Journal of Fish Diseases 2012, 35, 607–613 doi:10.1111/j.1365-2761.2012.01388.x

Aeromonas spp. induce apoptosis of epithelial cells through an oxidant-dependent activation of the mitochondrial pathway

We investigated interactions of Aeromonas caviae, Aeromonas veronii biotype sobria and Aeromonas hydrophila strains, isolated from faecal specimens of humans with gastroenteritis, with HT29 intestinal epithelial cells. All strains were found to be cytotoxic to the cells. Bacterial infection caused generation of reactive oxygen species (ROS) and nitric oxide radical (NO(·)). The maximal levels of ROS and NO(·) were 14 and 35 times, respectively, greater in cells infected with Aeromonas spp. than in those incubated with non-pathogenic Escherichia coli. The cells incubated with cytolytic enterotoxin isolated from A. veronii biotype sobria induced the highest level of ROS and caused the highest cytotoxicity. We observed that increased accumulation of intracellular ROS leads to a loss of mitochondrial membrane potential (ΔΨ(m)). Analyses of cellular morphology and DNA fragmentation revealed characteristic features of cells undergoing apoptosis. The process was dependent on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. Treatment of infected HT29 cells with three distinct antioxidants prevented intracellular ROS production, mitochondrial damage and apoptosis. The Pearson linear test revealed positive correlations between apoptotic index at 24 h and percentage cytotoxicity, ROS production, NO(·) production and loss of ΔΨ(m). This study has provided new insights into the mechanisms contributing to the development of Aeromonas-associated gastroenteritis. The results indicate that bacteria-induced apoptosis of epithelial cells results from mitochondrial depolarization due to oxidative stress.

Molecular mechanisms of quinolone resistance in clinical isolates of Aeromonas caviae and Aeromonas veronii bv. sobria.

Mutations in quinolone targets were studied together with quinolone efflux pump activation and plasmid-mediated quinolone resistance determinants in nalidixic-acid-resistant isolates of Aeromonas caviae and Aeromonas veronii. Among 135 clinical Aeromonas spp. isolated from stools of patients with gastrointestinal symptoms, 40 nalidixic acid-resistant strains belonging to A. caviae and A. veronii were selected and their susceptibility to different quinolones (ciprofloxacin, norfloxacin, ofloxacin) further evaluated. Susceptibility to nalidixic acid and ciprofloxacin in the presence/absence of Phe- Arg-β-naphthylamide was also determined. The 16 nalidixic-acid-resistant strains identified as A. caviae were more resistant than the 24 A. veronii bv. sobria strains to ciprofloxacin, norfloxacin, and ofloxacin. All strains showed a mutation (single or double) at position 83 of the QRDR sequence of gyrA, with Ser-83 → Ile as the most frequent substitution. By contrast, no mutations were found at position 87 of gyrA. Double substitutions (GyrA-ParC) were detected in 50% of A. veronii bv. sobria isolates and in 43.75% of A. caviae strains. Both species showed decreases in the MICs of ciprofloxacin. A qnrS gene was found in an A. caviae strain. Thus, in the two species of nalidixic-acid-resistant Aeromonas isolates examined, resistance mediated by efflux pumps contributed only slightly to ciprofloxacin resistance. While two isolates were positive for the aac(6')-Ib gene, no -cr variants were detected.

Siderophore, hemolytic, protease, and pyrazinamidase activities and antibiotic resistance in motile Aeromonas isolated from fish

A total of 120 fresh water fish samples were evaluated for the presence of Aeromonas spp. A. hydrophila, A. caviae, and A. veronii bv. sobria were isolated from the gills, intestines, livers, and skins of fish, and 78 isolated Aeromonas spp. strains were further examined for siderophore, hemolytic, protease, and pyrazinamidase activities and antibiotic resistance. Siderophore production was demonstrated in almost all of the A. hydrophila and A. caviae strains except for 2 strains of A. veronii bv. sobria. The strains identified as A. hydrophila and A. veronii bv. sobria were the stronger producers of hemolysin, whereas the A. caviae strains were nonhemolytic. It was found that 92% of A. hydrophila and 91% of A. caviae strains hydrolyzed pyrazinamide in under 48 h, whereas 5% of A. veronii bv. sobria strains hydrolyzed pyrazinamide. Protease activity was found in 100% of isolates of A. hydrophila and A. veronii bv. sobria, but in 81% of A. caviae isolates. In addition, all Aeromonas species were examined for antibiotic resistance patterns and were found to be resistant to ampicillin and tetracycline.

Rosmarinus officinalis L.: Chemical Modifications of the Essential oil and Evaluation of Antioxidant and Antimicrobial Activity

Rosmarinus officinalis essential oil was separated into its hydrocarbon and oxygenated fractions. The major compounds in the hydrocarbon fraction were alpha-pinene (44.2%), camphene (24.5%), and limonene (11.7%), while in the oxygenated fraction they were 1,8-cineole (37.6%), camphor (16.5%), and bornyl acetate (21.4%). The hydrocarbon fraction was submitted to a hydroformylation process and the antioxidant activity of the product was screened by the DPPH and beta-carotene/linoleic acid tests. The hydroformylated fraction maintained the antioxidant activity of the whole oil. The MIC (minimal inhibitory concentration) and the MBC (minimal bactericidal concentration) of the essential oil, hydrocarbon, oxygenated and hydroformylated fractions were also tested on several microorganisms. Aeromonas sobria and Candida strains were the most susceptible micro-organisms. The hydroformylated fraction exhibited a MBC against Candida strains resistant to the other fractions.

High frequency of hemolytic and cytotoxic activity in Aeromonas spp. isolated from clinical, food and environmental in Rio de Janeiro, Brazil

Molecular study of aerolysin and cytotonic enterotoxin genes by PCR and colony blot hybridization was performed in 117 strains of Aeromonas spp. isolated from different sources. Homogeneous distribution of these genes in A. hydrophila complex strains was observed. For A. caviae and A. sobria complex strains, aerolysin genes were more frequent than cytotonic enterotoxins genes. Of 64 A. caviae complex strains, only one (1.5%) amplified the 451 bp product for the aer gene, however, the same primers detected a 400 bp product in 50 (78%) strains. This product was sequenced and had two short regions with homology to several hemolysin genes. The genotype aer (+)/aerA(+)/hly (+)/ast (+)/alt (+) was detected in six A. hydrophila strains from food and environmental source. The most common genotype found in A. hydrophila strains was hly (+) (85%) and aerA(+) (78.7%), while in A. caviae complex strains was aerA(+) (32.8%). All A. veronii complex sobria strains were aer (+)/aerA(+). All A. caviae and A. hydrophila were positive when tested with aer probe using the colony blot test. Thirty-seven percent of A. hydrophila and 53% of A. caviae tested were positive for ast probe. Eighty-nine percent of samples were cytotoxic in Vero cells. Our data demonstrated that Aeromonas spp. can harbor and express virulence genes and reinforce the potential of Aeromonas as a human pathogen.

Characterization of Vibrio cholerae isolated from the aquatic basins of the State of Pernambuco, Brazil

Through a continuous bacteriological monitoring programme carried out by the Health Secretariat of the State of Pernambuco, Brazil, two isolates of Vibrio cholerae O1 El Tor Ogawa were discovered in an endemic area in 2001, during a cholera inactive period, along with six V. cholerae non-O1/non-O139 strains and two Aeromonas veronii biovar sobria strains showing an unusual characteristic of agglutination with O1 antiserum. Between that time and 2005, eight other O1 isolates were found. The virulence genes present in the V. cholerae differed among strains, with only three O1 strains harboring the ctxA gene. The O1 and some non-O1/non-O139 strains displayed identical patterns of amplification of the 16S-23S intergenic spacer region. RAPD of the 10 V. cholerae O1 strains, with the two primers used, revealed heterogeneity. The presence of V. cholerae carrying virulence genes in the aquatic basins examined confirms that they constitute a vibrio reservoir during a cholera inactive period, thus strengthening the argument for a continuous monitoring programme and preventative measures for cholera, mainly in the areas where the supply of drinking water is deficient.