Caviae Strains Research Articles

Colistin Resistance Mediated by Mcr-3-Related Phosphoethanolamine Transferase Genes in Aeromonas Species Isolated from Aquatic Environments in Avaga and Pakro Communities in the Eastern Region of Ghana.

Colistin is classified by the World Health Organization (WHO) as a critically important and last-resort antibiotic for the treatment of infections caused by carbapenem-resistant bacteria. However, colistin resistance mediated by chromosomal mutations or plasmid-linked mobilized colistin resistance (mcr) genes has emerged. Thirteen mcr-positive Aeromonas species isolated from water samples collected in Eastern Ghana were analyzed using whole-genome sequencing (WGS). Antimicrobial susceptibility was tested using the broth microdilution method. Resistome analysis was performed in silico using a web-based platform. The minimum inhibitory concentration (MIC) of colistin for all except three isolates was >4 µg/mL. Nine new sequence types were identified and whole-genome analysis revealed that the isolates harbored genes (mcr-3-related genes) that code for Lipid A phosphoethanolamine transferases on their chromosomes. BLAST analysis indicated that the amino acid sequences of the mcr-3-related genes detected varied from those previously reported and shared 79.04-99.86% nucleotide sequence identity with publicly available mcr-3 variants and mcr-3-related phosphoethanolamine transferases. Analysis of the genetic context of mcr-3-related genes revealed that the genetic environment surrounding mcr-3-related genes was diverse among the different species of Aeromonas but conserved among isolates of the same species. Mcr-3-related-gene-IS-mcr-3-related-gene segment was identified in three Aeromonas caviae strains. The presence of mcr-3-related genes close to insertion elements is important for continuous monitoring to better understand how to control the mobilization and dissemination of antibiotic resistance genes.

Analysis of global Aeromonas caviae genomes revealed that strains carrying T6SS are more common in human gastroenteritis than in environmental sources and are often phylogenetically related.

Aeromonas caviae is an emerging human enteric pathogen. However, the genomic features and virulence genes of A. caviae strains from human gastroenteritis and other sources have not been fully elucidated. Here, we conducted a genomic analysis of 565 global A. caviae strains isolated from different sources, including 261 strains isolated from faecal samples of gastroenteritis patients, of which 18 genomes were sequenced in this study. The presence of bacterial virulence genes and secretion systems in A. caviae strains from different sources was compared, and the phylogenetic relationship of A. caviae strains was assessed based on the core genome. The complete genome of A. caviae strain A20-9 isolated from a gastroenteritis patient was obtained in this study, from which 300 putative virulence factors and a T4SS-encoding plasmid, pAC, were identified. Genes encoding T4SS were also identified in a novel genomic island, ACI-1, from other T4SS-positive strains. The prevalence of T4SS was significantly lower in A. caviae strains from gastroenteritis patients than in environmental strains (3 %, P<0.0001 vs 14 %, P<0.01). Conversely, the prevalence of T6SS was significantly higher in A. caviae strains isolated from gastroenteritis patients than in environmental strains (25 %, P<0.05 vs 13 %, P<0.01). Four phylogenetic clusters were formed based on the core genome of 565 A. caviae strains, and strains carrying T6SS often showed close phylogenetic relationships. T3SS, aerolysin and thermostable cytotonic enterotoxin were absent in all 565 A. caviae strains. Our findings provide novel information on the genomic features of A. caviae and suggest that T6SS may play a role in A. caviae-induced human gastroenteritis.

Epidemiological characteristics, virulence potential, antimicrobial resistance profiles, and phylogenetic analysis of Aeromonas caviae isolated from extra-intestinal infections.

Aeromonas caviae (A. caviae) is one of the major etiological agents in human intestinal infections reported to be associated with a broad spectrum of extra-intestinal infections with increasing incidence over recent years. Although previous studies have established its significance as a causative agent of both bloodstream and gastrointestinal infections, the characteristics of A. caviae that cause extra-intestinal infections remain unilluminated.In this single-center retrospective study, we investigated epidemiological characteristics, antimicrobial resistance genes and phenotypes, virulence genes, and phyloevolution of 47 clinical A. caviae isolated from patients with extra-intestinal infections from 2017 to 2020. A. caviae strains were identified by biochemical tests and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS), ultimately confirmed to species level by whole-genome sequencing (WGS). Antimicrobial resistance and virulence genes were identified using the Comprehensive Antibiotic Resistance Database (CARD) and the virulence factor database (VFDB), respectively. Phylogenetic analysis of 47 clinical strains was performed by combining with 521 A. caviae strains from NCBI database. A. caviae was an opportunistic pathogen in immunocompromised patients, especially those with underlying hepatobiliary diseases and malignancies. 19 out of 47 isolates were identified as multidrug resistance (MDR) strains. Piperacillin-tazobactam, levofloxacin, gentamicin, amikacin with a resistance rate of less than 10% remained as options to treat extra-intestinal infections. 24 out of 47 isolates exhibited non-susceptibility to cephalosporins and cephamycins, all of which carried β-lactamase gene, including bla MOX, bla PER-3, bla OXA, bla NDM, and bla CphA. Most stains (98%, 46/47) carried at least one of the virulence genes, but extra-intestinal infections had a low mortality rate. Phylogenetic analysis indicated the risk of nosocomial transmission but revealed no outbreak. However, the emergence of MDR and β-lactamase resistance genes in extra-intestinal isolates of A. caviae is becoming an increasing risk to public health and requires attention. This study strengthen our understanding of A.caviae isolated from extra-intestinal infections. It may contribute to the management of extra-intestinal infections as well as the prevention and control of drug resistance.

Whole-Genome Sequences of Antibiotic-Resistant Aeromonas caviae Strains Isolated from Treated Wastewater.

The presented data provide new information on antibiotic resistance and virulence genes in the genomes of Aeromonas caviae strains TW-2 and TW-6, isolated from treated wastewater. The results confirm the presence of multi-antibiotic-resistant Aeromonas caviae strains with virulence properties as "high-risk isolates" in treated wastewater.

Phylogenetic characteristics, virulence and antibiotic resistance of Aeromonas caviae isolated from foodborne and environmental samples

Objective To identify 49 Aeromonas caviae strains isolated form foodborne and environmental samples and analyze their virulence and antibiotic resistance. Methods All strains were identified by VITEK and API 20NE. Two housekeeping genes, gryB and rpoD, were amplified by PCR for phylogenetic analysis. Virulence genes including act, alt, ast, lip, ahp, aerA, hlyA, ompA1 and fla were detected by PCR. In vitro antimicrobial susceptibility of these strains was tested with AST-GN16 kit. Results Fifty-four Aeromonas caviae/Aeromonas hydrophila strains were identified by biochemical tests. Phylogenetic analysis revealed that there were 49 Aeromonas caviae strains, four Aeromonas hydrophila strains and one Aeromonas taiwanensis strain. The positive rates of alt, lip, ompA1, fla, act, aerA and hlyA genes were 100.00%, 100.00%, 79.59%, 14.29%, 2.04%, 2.04% and 2.04%, respectively. None of the isolates carried ast or ahp gene. A total of four virulence gene combination patterns were detected, and the predominant pattern was alt/lip/ompA1 (32/49). Antibiotic resistance rates of the Aeromonas caviae strains to ampicillin, cefazolin, cefoxitin, amoxicillin/clavulanic acid, ceftriaxone, trimethoprim/sulfamet hoxoazole and aztreonam were 79.59%, 14.29%, 10.20%, 6.12%, 4.08%, 4.08% and 2.04%, respectively. All strains were susceptible to piparacillin/tazobactam, imipenem, amikacin, gentamicin, levofloxacin, tigacycline and nitrofurantoin. Two multidrug-resistant strains were detected. Conclusions Aeromonas caviae/Aeromonas hydrophila can be effectively identified by the housekeeping genes gyrB and rpoD, and the closely related Aeromonas caviae and Aeromonas taiwanensis strains can be identified by rpoD. All Aeromonas caviae strains carried two or more virulence genes and one strain isolated from environment was positive for seven virulence genes. Aeromonas caviae strains were generally resistant to penicillin, which was mainly relate to the production of β-lactamase. No strain was resistant to carbapenems, aminoglycosides, fluoroquinolones, tetracyclines or furans. Multidrug-resistant strains were observed in food and drinking water. Key words: Aeromonas caviae; Aeromonas hydrophila; Phylogenetic analysis; gyrB gene; rpoD gene; Virulence gene; Antibiotic resistance

High-resolution genome-wide analysis is essential for the identification of ambiguous Aeromonas strains.

Aeromonads are mainly opportunistic pathogens; however, many species are emerging as important human pathogens. Therefore, monitoring these bacteria and their accurate characterization of its species is highly important. Aeromonas Aer593 strain was recovered from a diarrhoea outbreak and did not group with any previously described Aeromonas species by housekeeping gene sequencing. To clarify the taxonomic position of Aer593, its genome was sequenced and analysed by multilocus phylogenetic analysis (MLPA), in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI) and core genome-based phylogenetic analyzes. The MLPA with the housekeeping genes gyrB, rpoD, recA, dnaJ, gyrA and dnaX ranked the Aer593 isolate into an independent branch suggesting that it could represent a new species. However, the identity percentages of Aer593 to A. caviae strains using robust genomic analysis by isDDH and ANI were at least 81.3% and 97.8%, respectively, defining Aer593 as A. caviae. Multilocus sequence typing (MLST) presented an exact match against only a single allele (groL96) and the novel ST648 was assigned for this strain. The core genome-based phylogenetic analyses with a total of 863 orthologous genes also grouped the Aer593 isolate with A. caviae reference strains. These findings warn about the possibility of misidentification of some Aeromonas strains by MLPA and show that high-resolution genome-wide analysis is essential for the correct identification of ambiguous Aeromonas strains.

Genetic characterization and potential molecular dissemination mechanism of tet(31) gene in Aeromonas caviae from an oxytetracycline wastewater treatment system

Recently, the rarely reported tet(31) tetracycline resistance determinant was commonly found in Aeromonas salmonicida, Gallibacterium anatis, and Oblitimonas alkaliphila isolated from farming animals and related environment. However, its distribution in other bacteria and potential molecular dissemination mechanism in environment are still unknown. The purpose of this study was to investigate the potential mechanism underlying dissemination of tet(31) by analysing the tet(31)-carrying fragments in A. caviae strains isolated from an aerobic biofilm reactor treating oxytetracycline bearing wastewater. Twenty-three A. caviae strains were screened for the tet(31) gene by polymerase chain reaction (PCR). Three strains (two harbouring tet(31), one not) were subjected to whole genome sequencing using the PacBio RSII platform. Seventeen A. caviae strains carried the tet(31) gene and exhibited high resistance levels to oxytetracycline with minimum inhibitory concentrations (MICs) ranging from 256 to 512 mg/L. tet(31) was comprised of the transposon Tn6432 on the chromosome of A. caviae, and Tn6432 was also found in 15 additional tet(31)-positive A. caviae isolates by PCR. More important, Tn6432 was located on an integrative conjugative element (ICE)-like element, which could mediate the dissemination of the tet(31)-carrying transposon Tn6432 between bacteria. Comparative analysis demonstrated that Tn6432 homologs with the structure ISCR2-∆phzF-tetR(31)-tet(31)-∆glmM-sul2 were also carried by A. salmonicida, G. anatis, and O. alkaliphila, suggesting that this transposon can be transferred between species and even genera. This work provides the first report on the identification of the tet(31) gene in A. caviae, and will be helpful in exploring the dissemination mechanisms of tet(31) in water environment.

Detection of some virulence genes in A. hydrophila and A. caviae isolated from fresh water fishes at Qalubia Governorate

The study was conducted on 225 diseased fish samples, 125 Nile tilapia (Oreochromis niloticus)and 100 Catfish(Claris gariepinus) , collected from different fish markets at Qalubia Governorateduring the period from January (2016) to May (2017) for detection of Aeromonas species. Thesamples were taken from apparently pathognomonic lesions in muscle, kidney, liver, intestine andspleen after clinical and postmortem examination for bacteriological examination. The resultsrevealed that, 125 Aeromonas species were isolated from the examined samples where A.hydrophila and A. caviae were identified. Accurately 114 (91.2 %) A. hydrophila strains, 63(50.4%) and 51 (40.8%) were isolated from C. gariepinus and O. niloticus fishes respectively.Meanwhile, 11(8.8 %) A. caviae strains, 7 (5.6%) and 4 (3.2%) from C. garicpinus and O. niloticusfishes respectively. Further PCR results for virulence genes in isolated Aeromonas strains indicatedthat, aero gene was detected in 9 out of 10 A .hydrophila studied strains and in 3 out of 6 A. caviae ;hly gene in 7 out of 10 A .hydrophila and in 2 out of 6 A. caviae; Ahcytoen gene in 6 out of 10 A.hydrophila and in 1 out of 6 A. caviae ; act gene in 6 out of 10 A .hydrophila and in 3 out of 6 A.caviae and ast gene in 7 out of 10 A .hydrophila and in 3 out of 6 A. caviae studied strains. Finally,the production of a wide array of virulence factors by isolated strains is indicative of their potentialto cause diseases in fishes and humans.

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi.

Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). β-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7–3.4%. The average gyrB sequence similarity between all species was 93%, demonstrating its acceptable taxonomic resolution. The presence of multiple species of potential pathogenic Aeromonas in fresh retail sushi raises new food safety issues related to the increased consumption of ready-to-eat food composed of raw ingredients.

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.

Adhesion and cytotoxicity of Aeromonas caviae to rabbit intestinal epithelium ex vivo.

Aeromonads are considered potential pathogens for humans and animals and are responsible for the etiology of intestinal and extraintestinal diseases. The presence of Aeromonas spp. in food and water shows that it is an important vehicle of infection in humans. The pathology caused by these bacteria involves several virulence factors, such as the ability to produce toxins, adhesion and invasion. The present study investigated the interaction of five Aeromonas caviae strains isolated from human diarrheic faeces with rabbit ileal and colonic mucosa ex vivo, using in vitro organ culture model. The in vitro adhesion assays using cultured tissue were performed with A. caviae strains co-incubated with intestinal fragments of ileum and colon over a period of 6h. The fragments were analyzed by light and electron microscopy. All strains adhered to rabbit ileal and colonic mucosa ex vivo, with higher degree of adherence presented on colonic mucosa. The typical aggregative adherence pattern was observed among strains studied. Through electron and light microscopy, we observed extensive colonization of ileal and colonic mucosa, large mucus production, biofilm formation and morphological alterations such as intense vacuolization, structural disorganization, cell extrusion and destruction of the villi. These results demonstrate that in vitro organ culture of intestinal mucosa from rabbit may be used to investigate Aeromonas spp. Finally, our results support the pathogenic potential of Aeromonas emphasising their importance in public health.

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.

Development of a PCR Assay Based on a Single-Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene.

Aeromonas caviae is a bacterial pathogen that causes various infectious diseases in both humans and animals. To facilitate its detection, we developed species-specific primer sets targeting polymorphisms in the gyrB gene for use in a PCR assay. The technique was able to detect 100% (29/29) of the A. caviae strains tested using either of two sets of primers (designated ACF1-ACR and ACF3-ACR), which produced 293-bp and 206-bp amplicons, respectively. Another set of primers (designated ACF2-ACR) yielded a 237-bp amplicon and exhibited 90% (26/29) positive results with respect to A. caviae. None of the primer sets exhibited cross-reactivity with 12 non-A. caviae isolates and 52 other non-Aeromonas bacteria. The detection limit using the ACF2-ACR and ACF3-ACR primer sets in pure culture was 1.6 × 10(3) CFU/mL, or 6 CFU per reaction, whereas that of the ACF1-ACR primer set was 1.6 × 10(4) CFU/mL, or 60 CFU per reaction. In the case of spiked Nile Tilapia Oreochromis niloticus, the sensitivity of all primer sets without enrichment was 1.8 × 10(4) CFU/g, or 30 CFU per reaction. Primer set ACF3-ACR was the best for a PCR assay targeting the gyrB gene, and the PCR technique developed was rapid, specific, and sensitive for the identification of A. caviae.

The Aeromonas caviae AHA0618 gene modulates cell length and influences swimming and swarming motility.

Aeromonas caviae is motile via a polar flagellum in liquid culture, with a lateral flagella system used for swarming on solid surfaces. The polar flagellum also has a role in cellular adherence and biofilm formation. The two subunits of the polar flagellum, FlaA and FlaB, are posttranslationally modified by O-linked glycosylation with pseudaminic acid on 6–8 serine and threonine residues within the central region of these proteins. This modification is essential for the formation of the flagellum. Aeromonas caviae possesses the simplest set of genes required for bacterial glycosylation currently known, with the putative glycosyltransferase, Maf1, being described recently. Here, we investigated the role of the AHA0618 gene, which shares homology (37% at the amino acid level) with the central region of a putative deglycosylation enzyme (HP0518) from the human pathogen Helicobacter pylori, which also glycosylates its flagellin and is proposed to be part of a flagellin deglycosylation pathway. Phenotypic analysis of an AHA0618 A. caviae mutant revealed increased swimming and swarming motility compared to the wild-type strain but without any detectable effects on the glycosylation status of the polar flagellins when analyzed by western blot analysis or mass spectroscopy. Bioinformatic analysis of the protein AHA0618, demonstrated homology to a family of l,d-transpeptidases involved in cell wall biology and peptidoglycan cross-linking (YkuD-like). Scanning electron microscopy (SEM) and fluorescence microscopy analysis of the wild-type and AHA0618-mutant A. caviae strains revealed the mutant to be subtly but significantly shorter than wild-type cells; a phenomenon that could be recovered when either AHA0618 or H. pylori HP0518 were introduced. We can therefore conclude that AHA0618 does not affect A. caviae behavior by altering polar flagellin glycosylation levels but is likely to have a role in peptidoglycan processing at the bacterial cell wall, consequently altering cell length and hence influencing motility.

Mesophilic strains of Aeromonas spp. can acquire the multidrug resistance plasmid pRAS1 in horizontal transfer experiments at low temperatures

Both biotic and abiotic characteristics of an ecosystem play an important role in the horizontal transfer of DNA in nature. The abiotic factor temperature has a great impact on such transfers as it controls the metabolic activity of mesophilic microorganisms. Moreover, psychrophilic bacteria, which are not affected by low temperatures, are considered to be potential donors of DNA to mesophilic bacteria under temperature stress conditions. In our study, mesophilic Aeromonas spp. strains isolated from fresh fish were genotypically identified and used as recipients in in vitro conjugal transfer experiments using plasmid pRAS1 from psychrophilic strain Aeromonas salmonicida 718 at three different temperatures (8, 15 and 20 °C). The transfer of the plasmid was confirmed by identifying the elements of the integron in pRAS1. A low temperatures did not prevent the transfer of the pRAS1 plasmid to Aeromonas veronii, A. media, A. hydrophila and A. caviae strains, which showed detectable conjugation frequencies of 10–8 at 8 °C. In other strains of the same species, transconjugants were not detected, which indicated that the genetic background of each strain directly affected the ability to be a recipient of this plasmid at the temperatures tested. Our results demonstrate that mesophilic Aeromonas spp. strains are potential reservoirs of extrachromosomal genetic material. Implications of this plasmid transfer at low temperatures and its possible consequences for human health are discussed.

Enumeration and Characterization of Aeromonas spp. Isolated from Milk and Some Dairy Products in Sharkia Governorate, Egypt

A survey was conducted to determine the prevalence of Aeromonas spp. in 75 random samples (25 each of raw cow\'s milk, local plain yoghurt and Domiati cheese) collected from different dairy shops, supermarket and street peddlers in Diarb Negm and Zagazig cities, Sharkia Governorate, Egypt. Investigations involved proteolytic and lipolytic activities of isolated Aeromonas spp. and the effect of heat- treatment, acidity, pH and sodium chloride concentration on prevalence of Aeromonas bacteria. Prevalence of Aeromonas spp. was proved in 32.0%, 44.0% and 20.0% of examined raw cow\'s milk, local plain yoghurt and Domiati cheese samples with a mean count of 9.8 x103, 1.4 x105 and 6.9 x103/ml, respectively. Classification of confirmed raw cow\'s milk isolates revealed that A. trota, A. hydrophila, A. janda and A. caviae were the predominant strains with percentages of 40.0%, 25.0%, 25.0% and 10.0%, respectively. While, local plain yoghurt isolates could be classified as A. caviae, A. sobria, A. hydrophila, A. trota and A. schubertii with percentages of 36.4%, 22.7%, 18.2%, 13.6% and 9.1%, respectively. Meanwhile, Classification of 10 confirmed Domiati cheese cultures revealed that the predominant strains were A. hydrophila, A. caviae and A. trota with percentages of 30%, 50% and 20%, respectively. All laboratory pasteurized milk samples revealed no count and there is marked decrease in the count of Aeromonas spp. as the acidity % of the examined raw cow’s milk samples increase, the pH value of the examined local plain yoghurt samples decrease and the NaCl % of the examined Domiati cheese samples increase. Characterization of isolated Aeromonas strains pointed that 50% of A. hydrophila, 60% of A. caviae, 40% of A. sobria, 53.8% of A. trota, 100% of A. janda and 50% of A. schubertii were psychrotrophic. A. hydrophila exhibited proteolytic and lipolytic activities at the percentage of 41.7% and 16.7%, respectively but in case of A. caviae strains the percentages were 46.7% and 20%, respectively and with A. trota were 30.8% and 15.4%, respectively. 60% of A. sobria and 100% of A. janda and A. schubertii strains showed proteolytic activity only. The public health importance and economic significance of existing microorganisms as well as the suggestive measures for improving the quality of raw milk and dairy products were discussed.

Aeromonas spp. simultaneously harbouring blaCTX-M-15, blaSHV-12, blaPER-1 and blaFOX-2, in wild-growing Mediterranean mussel (Mytilus galloprovincialis) from Adriatic Sea, Croatia

Aeromonas species are becoming renowned as emerging pathogens by increasingly giving rise to a wide spectrum of food and waterborne infections in humans. Another worrisome feature of aeromonads is the growing frequency of antibiotic resistance as a consequence of their prominent diversity in terms of resistance determinants. This study aimed at determining the antimicrobial resistance pattern, prevalence and characterization of acquired β-lactamases, including extended-spectrum-β-lactamases (ESBLs) and AmpC cephalosporinases, as well as the presence of class 1 and 2 integrons, in Aeromonas isolates from wild-growing Mediterranean mussel (Mytilus galloprovincialis) of the eastern coast of Adriatic Sea, Croatia. Isolates were tested for susceptibility to 16 antibiotics and β-lactam/β-lactamase inhibitor combinations. Cephalosporin-resistant isolates were further screened by PCR for genes encoding AmpC (blaFOX, blaCMY, blaMOX, blaLAT, blaBIL, blaDHA, blaACC, blaMIR, blaACT), ESBLs (blaTEM, blaSHV, blaCTX-M, blaPER, blaVEB, blaGES/IBC, blaOXA) and integrases (intI1, intI2, intI3). Location of bla genes was characterized by plasmid DNA fingerprinting and Southern blot hybridization. Plasmids carrying ESBL genes were investigated for transferability by conjugation and PCR-based replicon typed. Out of 147 Aeromonas isolates recovered, 30 (20%) demonstrated multiple resistance profile, with co-resistance most frequently detected against penicillins, piperacillin/sulbactam and tetracycline. ESBL-encoding genes were detected in 21 (13 Aeromonas caviae and 8 Aeromonas hydrophila) isolates, with blaCTX-M-15 gene identified in 19 and blaSHV-12 in 12 isolates. Among them, 10 isolates simultaneously harboured blaCTX-M-15 and blaSHV-12, while 3 isolates additionally carried an AmpC β-lactamase blaFOX-2 gene. blaPER-1 gene was identified in a single isolate also harbouring the blaCTX-M-15 gene. While blaSHV-12 was chromosomally encoded, blaCTX-M-15 was located on conjugative IncFIB-type plasmids of ~40kb in A. caviae isolates. IntI1 and intI2 genes were detected in 57.1% and 33.3% of ESBL-producing isolates.To the best our knowledge, this is the first report of environmental A. caviae isolates producing CTX-M-15, and isolation of SHV-12-producing A. hydrophila and A. caviae strains worldwide. This is also believed to be the first report of the FOX-2, CTX-M-15 and SHV-12 simultaneous production in aeromonads, highlighting both the potential risk for human health, and a role of these foodborne pathogens as reservoirs of resistance determinants in coastal marine environment.

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.

Association of Aeromonas caviae polar and lateral flagella with biofilm formation

To evaluate the association of the polar and lateral flagella with biofilm formation on plastic surfaces in 76 Aeromonas caviae strains isolated from environment (lagoon water), food (vegetables, fish and cheese) and human source (faeces). Both polar (flaA) and lateral (lafA) flagellin genes have been investigated by means of PCR and colony blot hybridization assays. The ability to form biofilm in polystyrene microtitre plates was evaluated and correlated with the presence and absence from these genes. The flaA and lafA genes had a frequency of 94% and 71%, respectively. All lafA(+) strains were also flaA(+) . Biofilm formation was observed in 72% of strains. Ninety-four per cent of flaA(+) lafA(+) strains could form biofilm and those that presented an intense biofilm production harboured both genes. All flaA(-) lafA(-) isolates, as well as 76% of flaA(+) lafA(-) strains, were incapable of forming biofilm. All the fish strains were flaA(+) lafA(+) and displayed higher biofilm formation (88%). Lagoon water samples exhibited lower positivity rate for the lafA gene (57%) and decreased ability to produce biofilm (39%). Both polar and lateral flagellar function contribute to biofilm formation in Aer. caviae strains. This study provides evidence for the association of both flagella with biofilm formation, a factor required for pathogenicity of Aer. caviae strains of varied sources, especially food and human.