Hydrophila Isolates Research Articles

AEROMONAS HYDROPHILA SENSITIVITY TO DISINFECTANTS

The aim of the present study was to determine the sensitivity of Aeromonas hydrophila to dis-infectants and to propose an efficient method for control of fish diseases caused by this patho-gen The tested A. hydrophila strains were inactivated by the combinations of glutaraldehyde, formaldehyde and quaternary ammonium salts (HMI® Glicofin) and glutaraldehyde, (ethylene-dioxy) dimethanol and benzalkonium chloride (Aldesept®). Iodine (Desinfekt B®), n-alkyl-dimethyl-benzyl-ammonium chloride (HMI® Roda Super), ethanol (40%, 50% and 70%) and potassium permanganate (1000 mg/l) were also found effective against A. hydrophila. At a concentration of 5 mg/L, potassium permanganate reduced A. hydrophila (?TCC 7965) counts by 3% (log10 0.25), while the strain was completely inactivated by 100 mg/L. Under the influ-ence of 5 mg/L and 100 mg/L potassium permanganate, the clinical A. hydrophila isolate counts were reduced by 0.7% (log10 0.06), and 2.3% (log10 0.19), respectively. Most tested disinfectants were effective against A. hydrophila, with the exception of potassium permanga-nate at 5 mg/L.

Isolation of <i>Aeromonas hydrophila</i> from EUS Affected Shing <i>Heteropneustes fossilis</i> of a Fish Farm in Mymensingh

Aeromonas hydrophila bacteria was isolated from the suspected EUS-affected shing fish, Heteropneustes fossilis (Bloch). The disease investigations were primarily based on clinical signs and subsequently confirmed by the isolation of bacterial pathogen Aeromonas hydrophila from lesion of liver and kidney. The A. hydrophila isolates were identified by a series of morphological, physiological and biochemical tests. The total bacterial load in liver, intestine and kidney were 1.67 × 104 to 6.46 × 108 CFU/g, 1.71 × 103 to 1.18 × 109 CFU/g and 1.47 × 104 to 3.70 × 108 CFU/g respectively.DOI: http://dx.doi.org/10.3329/pa.v19i1.17362 Progress. Agric. 19(1): 117 - 124, 2008

Implication of Lateral Genetic Transfer in the Emergence of Aeromonas hydrophila Isolates of Epidemic Outbreaks in Channel Catfish

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.

Protective effects of the Aeromonas phages pAh1-C and pAh6-C against mass mortality of the cyprinid loach (Misgurnus anguillicaudatus) caused by Aeromonas hydrophila

To investigate methods to control the mass mortality of cyprinid loaches (Misgurnus anguillicaudatus) caused by multiple-antibiotic-resistant Aeromonas hydrophila on a private fish farm in Korea, we isolated bacteriophages (phages), designated pAh1-C and pAh6-C, from the Han River in Seoul. The two isolated phages were morphologically classified as Myoviridae and showed similar infection patterns for A. hydrophila isolates. The two phages possessed approximately 55kb (pAh1-C) and 58kb (pAh6-C) of double-stranded genomic DNA, and their gDNAs showed different restriction endonuclease digestion patterns. Both phages showed efficient bacteriolytic activity against fish-pathogenic A. hydrophila from loaches. The latent periods of the phages were estimated to be approximately 30min (pAh1-C) and 20min (pAh6-C), while the burst sizes were 60PFU/cell (pAh1-C) and 10PFU/cell (pAh6-C). The phages proved to be efficient in the inhibition of bacterial growth, as demonstrated by their in vitro bactericidal effects. Additionally, a single administration of either phage to cyprinid loaches resulted in noticeable protective effects, with increased survival rates against A. hydrophila infection. These results suggest that the phages pAh1-C and pAh6-C constitute potential therapeutic agents for the treatment of A. hydrophila infection in fish.

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.

Virulence Factors and Molecular cloning of Outer Membrane Protein (OMP) gene from virulent Aeromonas hyrophila isolated from infected gold fish Carassius auratus

Aeromonas hyrophila strains AHV1, AHV2 and AH3 were isolated and identified from Muscle tissue, intestine, body fluid and gills of infected gold fish Carassius auratus. In order to study their virulence, LD50 tests against normal gold fish, proteolytic, haemolytic and challenge studies were performed. The virulence studies revealed that, both AHV1 and AHV2 strains are highly positive for proteolytic and haemolytic properties. The LD50 data showed that, the fish C. auratus are highly susceptible to A. hyrophila strains AHV1 and AHV2 at cent percent lethal rate. The survival of C. auratus significantly (P<0.05) decreased when challenged with virulent strains of A. hydrophila AHV1 and AHV2. The outer membrane protein (OMP-TS) gene was successfully amplified generating an the amplicon size of 1008 bp. The amplified product from the genomic DNA of AHV1 strain was cloned in to pTZ57R/T vector, transformed into DH5? cells and sequenced. The sequenced clone is resembling to various A. hydrophila isolates and submitted to NCBI GenBank database (accession no:HQ331525). DOI: http://dx.doi.org/10.3329/bjm.v28i2.11819 Bangladesh J Microbiol, Volume 28, Number 2, December 2011, pp 70-75

Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish

Multiple factors can be involved in the virulence processes of Aeromonas hydrophila. The objective of the present paper was to verify the presence of aerolysin, hidrolipase, elastase and lipase virulence genes through the polymerase chain reaction (PCR) in A. hydrophila isolates obtained from fish of the São Francisco River Valley, and to evaluate virulence according to the presence of these genes in Nile tilapia fingerlings. One hundred and fourteen isolates from the bacteria were used. DNA was heat extracted and PCR undertaken using specific primers described in the literature. For in vivo tests Nile tilapia fingerlings were used. From the PCR tests, negative isolates for all genes tested were selected, positive isolates for two genes (aerolysin and elastase) and positive for the four genes tested. These were inoculated at a concentration of 10(8) UFC/ml into the tilapias, considered as treatments; another group of animals was used as control (with inoculation of saline solution). In all, 12 distinct standards regarding the presence of virulence factors in isolates from A. hydrophila, were observed. Of the 114 isolates analyzed, 100 (87.72%) presented at least one of the virulence factors under study. The virulence factors were widely distributed among the A. hydrophila isolates. Aerolysin was the most frequent virulence factor present in the isolates analyzed. A. hydrophila led to the mortality of the Nile tilapia fingerlings, regardless of the absence or quantity of virulence genes tested.

Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China

To investigate the species distribution in Aeromonas isolates from diseased fish, healthy controls and water environment in China; to evaluate the frequency of the aerolysin (aer), cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), temperature-sensitive protease (eprCAI) and serine protease (ahp) genes in Aeromonas isolates; and to determine the potential pathogenicity of these isolates. Two hundred and two Aeromonas isolates from diseased fish (n = 42), healthy fish (n = 120) and water environment (n = 40) in China were identified to species levels based on sequencing of the housekeeping gene gyrB, while the distribution of five virulence factors, including aer, alt, act, eprCAI and ahp, was investigated by PCR. Aeromonas veronii (25/42; 60%) and Aeromonas hydrophila (14/42; 33%) were the species most commonly isolated from diseased fish, while Aer. veronii was the most common species in healthy fish (90/120; 75%) and water samples (25/40; 62·5%). All the five virulence genes were present in 9% (19/202), among which 10 strains were from diseased fish and nine were identified as Aer. hydrophila. For the strains carrying five virulence genes, the average 50% lethal doses (LD(50s) ) of strains from diseased fish were lower when compared with the strains from healthy fish and water environment. Aeromonas veronii is the most common species, but no significant difference exists in the isolates obtained from diseased fish and from healthy fish. However, Aer. hydrophila isolates were significantly more frequent from diseased fish than from healthy fish. aer+alt+act+eprCAI+ ahp+ was more frequent virulence genotype in Aeromonas isolates from diseased fish than from healthy fish and water environment, and the aer+alt+act+eprCAI+ahp+ isolates were more virulent to zebrafish comparing to the other genetic profiles. Aeromonas species in aquatic environments are various and have considerable virulence potential, and therefore, there is a need for more careful and intensive epidemiology studies.

Grass carp (Ctenopharyngodon idellus) infected with multiple strains of Aeromonas hydrophila

Ctenopharyngodon idellus is an important commercial fish species in China and many other countries to meet human food habits. However, there is no definitive data to indicate the multi-infection of pathogenic Aeromonas hydrophila strains in cultured C. idellus. In this study, four strong virulent strains with five virulence genes were simultaneously isolated from the cultured C. idellus suffering from septicemia, and identified as different A. hydrophila isolates using the ATB 32GN system, phylogenetic analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). They were susceptible to chloramphenicol, tobramycin, kanamycin, norfloxacin, spectinomycin, furantoin, azithromycin, and resistant to carbenicillin, penbritin, clindamycin. In addition, to further control the multi-infection of A. hydrophila, norfloxacin, the well-known fishery drug widespread used in aquaculture, was employed to treat this disease. Its minimum inhibitory concentrations (MICs) were 2~4 mg/L and good protection effects were also exhibited on the naturally infected fish at a final concentration of 1 and 2 g/kg in the feed (cal. 20 and 40 mg/kg of fish). As an important pathogen of a zoonotic and foodborne disease, the multi-infection of A. hydrophila strains may be an emerging threat in grass carp farming and food safety, and more importance should be attached to their multi-infection in fish farming. Key words: Ctenopharyngodon idellus, Aeromonas hydrophila, multi-infection, control.

Distribution and phenotypic and genotypic detection of a metallo-β-lactamase, CphA, among bacteraemic Aeromonas isolates

The objectives of the study were to investigate the distribution of cphA-related genes (cphA) encoding a CphA metallo-β-lactamase (MBL) among 51 consecutive Aeromonas blood isolates and to compare different phenotypic methods for detecting CphA. The presence of cphA was detected by PCR. Four phenotypic methods, the imipenem-EDTA combined disc test, imipenem-EDTA MBL Etest, agar dilution test and modified Hodge test (MHT), were used to detect imipenem susceptibility and MBL production. The results showed that 35 (69%) blood isolates had cphA. All (100%) of 16 Aeromonas aquariorum isolates and 12 Aeromonas veronii isolates, and 4 (80%) of 5 Aeromonas hydrophila isolates, carried cphA, but none of 15 Aeromonas caviae isolates did. With the standard inocula, irrespective of the presence or absence of cphA, all but one (50, 98%) isolates were susceptible to imipenem tested by disc diffusion, Etest and agar dilution (10(4) c.f.u. spot inocula), and did not exhibit MBL production by the imipenem-EDTA combined disc test and MBL Etest. By the agar dilution test using large inocula (10(7) c.f.u.), 34 (97%) of 35 cphA(+) isolates had imipenem MICs of ≥16 µg ml(-1), higher than the susceptible breakpoint (4 µg ml(-1)), and demonstrated positive results for the MHT, while one cphA(+) and all 17 cphA(-) isolates had imipenem MICs of ≤4 µg ml(-1). In conclusion, the distribution of cphA among aeromonads is species-specific, found in A. aquariorum, A. veronii and A. hydrophila, and the MHT may be a phenotypic screening test for CphA production.

Class 1 Integrons in <i>Aeromonas hydrophila</i> Isolates from Farmed Nile Tilapia (<i>Oreochromis nilotica</i>)

The aim of this study was to determine antimicrobial resistance of Aeromonas hydrophila isolated from farmed Nile Tilapia. A total of 50 A. hydrophila isolates from clinical cases were screened for the presence of class 1, 2 and 3 integrons and all the strains resistant to enrofloxacin and/or ciprofloxacin (n=19) examined for mutation in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The intI1 gene was detected in 23 A. hydrophila strains (46%) but no intl2 and intl3 were detected. Among these, 14 isolates (60.8%) carried gene cassettes inserted in variable regions i.e., partial aadA2, aadA2, dfrA1-orfC and dfrA12-aadA2, of which the most common gene cassette array was dfrA12-aadA2 (26.09%). Conjugal transfer of class 1 integrons with resistance gene array was detected. All the A. hydrophila strains resistant to enrofloxacin and/or ciprofloxacin possessed mutations in the QRDRs of gyrA and parC. Only a Ser-83-Ile substitution was identified in GyrA and only a Ser-80-Ile amino change was found in ParC. The data confirms that A. hydrophila from farm-raised Nile Telapia serve as a reservoir for antimicrobial resistance determinants.

Detection of Aeromonas hydrophila in water using PCR

Aeromonas genus is considered an emerging pathogen in drinking water supplies as is evident from surveillance studies carried out in Punjab, India. This study proposes the detection of Aeromonas by a molecular method. A total of 544 isolates of Aeromonas hydrophila were isolated from 690 drinking water samples and were further characterized phenotypically and confirmed by polymerase chain reaction (PCR) amplification using specific primers targeting a 685 base‐pair fragment of the 16S rRNA gene. Validation of the assay was done for all 544 biochemically characterized A. hydrophila isolates. At the assay time of 6 h, the PCR protocol showed reproducibility, specificity, and sensitivity, and proved to be an inexpensive alternative to conventional phenotypic assay, which has limitations (e.g., duration of incubation, antagonistic organism interference, lack of specificity, poor detection of viable but nonculturable microorganisms). The isolates showed multiple antibiotic resistances with a score > 0.5 on the multiple antibiotic resistance indexes. This may be ascribed as a potential human health hazard.

Virulence of Aeromonas hydrophila to channel catfish Ictaluras punctatus fingerlings in the presence and absence of bacterial extracellular products

We investigated the virulence of three 2009 west Alabama isolates of Aeromonas hydrophila (AL09-71, AL09-72 and AL09-73) to channel catfish Ictalurus punctatus fingerlings (4.6 +/- 1.3 g) in the presence and absence of extracellular products (ECPs) from overnight bacterial culture using both bath immersion and intraperitoneal injection routes. At a concentration of 1.65 x 10(8) colony-forming units (CFU) ml(-1), AL09-73 without its ECPs killed 100% of the catfish fingerlings within 2 h by bath immersion. However, at a similar concentration, AL09-73 in the presence of its ECPs killed only 23 +/- 6% catfish fingerlings. The absence of ECPs in the bath immersion experiment also significantly (p < 0.05) increased the virulence of AL09-71, AL09-72, and AL98-C1B, a 1998 Alabama strain of A. hydrophila, suggesting that the virulence of the 4 A. hydrophila isolates was mainly due to bacterial cells, not to their overnight ECPs. Filter-sterilized ECPs failed to kill any catfish by bath immersion or injection. The virulence order of the 4 A. hydrophila isolates, by both bath immersion and intraperitoneal injection, was: AL09-73 > or = AL09-71 > AL09-72 > or = AL98-C1B. At 2 h post bath immersion, all 4 isolates of A. hydrophila were found in all tissues studied (skin, intestine, liver, spleen, kidney, gill and brain), with the highest bacteria count being in the gill and kidney.

Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in west Alabama (USA) in 2009

Three isolates (AL09-71, AL09-72, and AL09-73) of Aeromonas hydrophila were cultured from infected channel catfish Ictalurus punctatus during a disease outbreak in west Alabama, USA, in August 2009. Sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR), cpn60, gyrB, and rpoD genes of the 3 strains revealed that the 3 strains were closely related to each other, sharing 97 to 99% nucleotide sequence similarities. However, ISR sequences of the 3 isolates from 2009 shared only 64% nucleotide sequences with AL98-C1B, a 1998 isolate of A. hydrophila cultured from diseased fish in Alabama. Sequences of cpn60, gyrB, and rpoD from the 3 isolates from 2009 shared 91 to 95% homologies with AL98-C1B. Based on both LD50 and LD95 values of intraperitoneal injection assays, the virulences of the 3 isolates from 2009 were not significantly different from each other, but were at least 200-fold more virulent than AL98-C1B, indicating that the 3 west Alabama isolates of A. hydrophila from 2009 were highly virulent to channel catfish.

Molecular cloning, sequence analysis and structure modeling of OmpR, the response regulator of Aeromonas hydrophila

The ability of bacteria to survive and proliferate in changing environmental conditions, and during host cell invasion is the key to their pathogenicity. In order to achieve this, the bacteria use a signal transduction system, the two component regulatory system, which consists of a sensor kinase and a response regulator. The EnvZ/OmpR system regulates the porin genes ompF/ompC in response to changes in osmolarity. In the present study, the ompR gene of Aeromonas hydrophila (isolate Ah17) was cloned, sequenced and characterized. Further an attempt was made to analyze the structural characteristics of the OmpR protein from Aeromonas hydrophila. The three dimensional structure of the protein was predicted by homology modeling and the modeled structure was compared to other members of two component response regulators. This study would be helpful for structure based drug design approaches to generate drugs against this harmful pathogen to control its proliferation in both human and fish hosts.

Identification of unique DNA sequences present in highly virulent 2009 Alabama isolates of Aeromonas hydrophila

In 2009, a disease outbreak caused by Aeromonas hydrophila occurred in 48 catfish farms in West Alabama, causing an estimated loss of more than 3 million pounds of food size channel catfish. Virulence studies have revealed that the 2009 isolates of A. hydrophila are at least 200-fold more virulent than a 1998 Alabama isolate AL98-C1B. However, up to now, no molecular markers have been identified to differentiate the highly virulent 2009 isolates from other isolates of A. hydrophila. To understand the genetic differences between the highly virulent 2009 isolates and the less virulent AL98-C1B at molecular level, PCR-select bacterial genome subtractive hybridization was used in this study. A total of 96 clones were selected from the subtractive genomic DNA library. Sequencing results revealed that the 96 clones represented 64 unique A. hydrophila sequences. Of the 64 sequences, three (hypothetical protein XAUC_13870, structural toxin protein RtxA, and putative methyltransferase) were confirmed to be present in the three virulent 2009 Alabama isolates but absent in the less virulent AL98-C1B. Using genomic DNAs from nine field isolates of A. hydrophila with different virulence as templates, two sequences (hypothetical protein XAUC_13870 and putative methyltransferase) were found to be only present in highly virulent A. hydrophila isolates, but absent in avirulent isolates.

Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebrafish

To evaluate the frequency of the aerolysin (aerA), cytotoxic enterotoxin (alt) and serine protease (ahp) genes in Aeromonas hydrophila isolates from different sources, and to determine the relationship between the presence of these genes and virulence of A. hydrophila in zebrafish. Aeromonas hydrophila isolates from clinical cases (n=40), from healthy fish (n=22) and from water environment (n=21) were analysed with respect to the prevalence of aerA, alt and ahp genes by PCR assay. These virulence factors occur among clinical isolates as well as among isolates from healthy fish and water environment. The majority (97·6%) of the strains examined carried one or more virulence genes. The isolates were divided into seven genetic profiles on the basis of PCR result: aerA(+) alt(+) ahp(+) (62·7%), aerA(+) alt(+) ahp(-) (13·3%), aerA(+) alt(-) ahp(+) (10·8%), aerA(-) alt(+) ahp(+) (4·8%), aerA(-) alt(-) ahp(+) (3·6%), aerA(+) alt(-) ahp(-) (2·4%) and aerA(-) alt(-) ahp(-) (2·4%). A higher frequency of genetic group aerA(+) alt(+) ahp(+) was determined in the isolates from diseased animals compared to those from healthy fish or water environments. Virulence properties of 26 representative strains belonging to the seven genetic profiles were further characterized. Results demonstrated that as the present of virulence genes increased, the proteolytic, haemolytic and cytotoxic activities of extracellular products also increased. And the 50% lethal doses (LD(50)s) of aerA(+) alt(+) ahp(+) isolates (<10(5)) in zebrafish were lower when compared with the strains expressing one or combinations of two virulence genes (>10(6)). Virulence properties of A. hydrophila correlated well with the presence of virulence genes tested. aerA(+) alt(+) ahp(+) was more frequent virulence genotype in A. hydrophila isolates from clinical diseases than from healthy fish and water environment, and the aerA(+) alt(+) ahp(+) isolates were more virulent to zebrafish compared to the other six genetic profiles. The detection for aerA, alt and ahp can be used for virulence typing of A. hydrophila isolates.

Simple and direct detection of Aeromonas hydrophila infection in the goldfish, Carassius auratus (L.), by dot blotting using specific monoclonal antibodies

A combination of eight isolates of Aeromonas hydrophila was used to produce monoclonal antibodies (MAbs). Ten different groups of MAbs specific to Aeromonas were selected. The first five groups of MAbs demonstrated high specificity and bound to only one or two isolates of A. hydrophila. The sixth and the seventh groups of MAbs were A. hydrophila specific. They recognized seven of eight A. hydrophila isolates (AH1, 2, 3, 4, 5, 6, 8); however, the MAb in the seventh group also showed cross-reactivity to one isolate of Aeromonas caviae (AC3). The eighth MAb group recognized two isolates of A. hydrophila (AH2 and AH5) and demonstrated cross-reactivity to one isolate of Aeromonas sobria (AS1) and one isolate of A. caviae (AC3). The tenth group of MAbs bound to all isolates of Aeromonas spp. tested (AH1-8, AS1-6, AC1-5, Aeromonas veronii and Aeromonas jandaei) without cross-reactivity to any of the other bacteria tested. MAbs in the ninth group showed similar specificity to those in the tenth group but did not recognize two isolates of A. sobria (AS4 and AS6) or A. jandaei. All the MAbs could be used to identify Aeromonas by dot blotting with a sensitivity ranging from 10⁵ to 10⁷ CFU mL⁻¹. However, the sensitivity of detection was increased to 10²-10³ CFU mL⁻¹ after inoculation of the sample in tryptic soy broth for 3-6 h before performing the dot blotting. The dot blot method can be used for the direct detection of A. hydrophila infection in symptomatic and asymptomatic goldfish. This study demonstrated a convenient immunological tool that can be used for the direct detection of A. hydrophila and Aeromonas infections in a complex sample without the requirement for separation of the bacteria or isolation and biochemical tests.

Identity, virulence genes, and clonal relatedness of Aeromonas isolates from patients with diarrhea and drinking water

Among 800 stool specimens from patients with diarrhea submitted by Primary Care Centers for routine analysis to the Hospital of León (NW Spain) Microbiology and Parasitology Service, 32 (4%) were tested positive for Aeromonas spp. Mixed infections with other enteric pathogens occurred in 12 patients. A. caviae was isolated from 23 clinical specimens. There were also patients infected with A. media, A. hydrophila, A. bestiarum, and A. veronii biovar veronii. All but three isolates carried one or more of the virulence genes. The incidence of the alt, hlyA, aerA, ast, and laf genes was 71.9, 28.1, 25.0, 18.8, and 9.4%, respectively. The alt(+)/ast(+) combination was detected in four isolates and the aerA(+)/hlyA(+) combination was detected in the two A. hydrophila isolates. None of the strains harbored the TTSS, stx1, or stx2 genes and nine bore plasmids. Thirty clinical isolates and a collection of 12 A. caviae and A. media strains obtained from León municipal drinking water over the study period were typed by pulsed-field gel electrophoresis (PFGE). PFGE patterns revealed genetic relatedness and persistence over time among water isolates and some clinical isolates. Interestingly, one A. caviae (aerA(-)/hlyA(-)/alt(+)/ast(-)/laf(+)) human isolate and two A. caviae (aerA(-)/hlyA(-)/alt(+)/ast(-)/laf(+)) drinking water isolates had indistinguishable PFGE patterns, suggesting waterborne infection.

Immunoproteomics of extracellular proteins of theAeromonas hydrophilaChina vaccine strain J-1 reveal a highly immunoreactive outer membrane protein

Aeromonas hydrophila is a gram-negative bacterium that can infect a variety of aquatic and terrestrial animals. It is essential to develop a vaccine to reduce the economic losses caused by this bacterium in aquaculture worldwide. Here, an immunoproteomic assay was used to identify the immunogenic extracellular proteins of the Chinese vaccine strain J-1. Ten unique immunogenic proteins were identified from the two-dimensional electrophoresis immunoblot profiles by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS. One protein of interest, Omp38, was detected by antisera on two-dimensional immunoblots, suggesting that it might be located both extracellularly and in the membrane. In exploring the potential of Omp38 as a vaccine candidate in fish, we found the omp38 gene to be prevalent by PCR among different (36/48) A. hydrophila isolates. The recombinant Omp38 induced a strong antibody response in rabbits, and the polyclonal antibody could recognize a band of approximately 38 kDa in the immunoblots of outer membrane protein extracts from most (24/40) of the A. hydrophila strains, including different predominant serotypes in China. These results indicated that the outer membrane antigen identified in this study could be developed as a vaccine candidate to induce protective immunity against A. hydrophila infection.