Antibiotic Resistance of Aeromonas spp. Isolated from Tropical Fish Imported from Singapore

Abstract

To determine the scope of antibacterial resistance among Aeromonas spp., bacterial cultures were taken from a variety of tropical fish species imported from Singapore. The samples were plated on Rimler-Shotts medium for bacterial isolation and identified with both the API20E and Nonfermenter Test Strip systems. Aeromonas hydrophila, A. sobria, and A. caviae were identified in monomicrobic and concomitant infections. Following identification of bacterial isolates, 11 antimicrobials routinely used in the tropical fish industry and 1 new fluoroquinolone were tested for their effectiveness against Aeromonas spp. with the Kirby-Bauer disk-diffusion technique. Aeromonas sobria proved to be the most resistant, often showing susceptibility to only 3 of 12 test drugs. Aeromonas hydrophila was consistently the least resistant. Based on these results, antimicrobial resistance appears to be a rapidly emerging problem in the pet fish industry.     

Communications: Viability of Bacterial Pathogens in Frozen Fish

Abstract

Channel catfish Ictalurus punctatus injected with Aeromonas hydrophila, Pseudomonas fluorescens, Edwardsiella tarda, or E. ictraluri were frozen at ?20°C after death. Bacterial isolation at 2-d intervals after freezing indicated that A. hydrophila could be recovered for 20 d, P. fuorescens for 60 d, E. tarda for 50 d, and E. ictaluri for 30 d in frozen fish.     

Early Changes in Pigmented Macrophages in Head Kidney of Channel Catfish Infected with Aeromonas hydrophila

Abstract

Channel catfish Ictalurus punctatus with scarified skin were exposed to the bacterial pathogen Aeromonas hydrophila. Systemic infections developed in 80% of the exposed fish, and the remaining exposed fish had infections limited to the cutaneous lesion. Skin of control fish was injured in the same manner as for the exposed fish, but control fish were not exposed to A. hydrophila nor was A. hydrophila recovered from them. None of the fish died during the 3-d experiment, and gross lesions in head kidneys, including changes in the size or relative weight, were not found in infected fish. Macrophage aggregates in head kidney of systemically infected fish increased 68% in mean area, 28% in number, and 111% in total relative volume. Volume of lipofuscin, the predominate pigment found in channel catfish head kidney, in systemically infected fish was about twice that of control fish. Volume of hemosiderin was about four times as great and macrophages containing hemosiderin were about three times as numerous in systemically infected fish as in control fish. The amount of melanin in macrophages did not change in infected fish. Significant differences in macrophage aggregates and pigments were not found between controls and fish with only superficial infections. Microscopic lesions, other than changes in macrophage aggregates and pigments, were not found in the head kidney. Increases in macrophage aggregates, lipofuscin, and hemosiderin in head kidney of channel catfish were useful for quantification of injury caused by a systemic bacterial infection.     

Isolation of Bdellovibrio as Biological Therapeutic Agents Used For the Treatment of Aeromonas hydrophila Infection in Fish

Fourteen strains of Bdellovibrio‐like organisms were isolated from cultured fish ponds using Aeromonas hydrophila J‐1 as host, one of them formed large plaques after 48 h of incubation at 28°C on a double layer plate, designated as Bdellovibrio C‐1. The Bdellovibrio was confirmed by electron microscopy and PCR amplification of Bdellovibrio‐specific 16S rDNA. The optimum temperature for the growth of BdC‐1 was between 15–37°C and with optimal activity at temperatures of 25–30°C. The ability of BdC‐1 to lyse A. hydrophila was similar in the pH range of 6.5 to 8.5. It can lyse 23 Gram‐negative bacterial strains comprising three genera of fish pathogens and one strain of Escherichia coli but cannot lyse Gram‐positive bacteria such as Bacillus subtillis and Staphylococcus aureus. Immersion of fish in water containing different concentrations of BdC‐1 was used in protection against an experimental infection of A. hydrophila J‐1. Results show that the mortality of groups immersed with BdC‐1 was lower than the group without BdC‐1. These results suggest that it may be possible to use Bdellovibrio to control the disease caused by A. hydrophila.     

The occurrence and antibiotic resistance of motile Aeromonas in livestock

The present study was carried out to assess the prevalence of motile Aeromonas spp. in the faeces of clinically healthy sheep, cattle and horses and evaluate their susceptibility to some anti-microbial agents. Rectal swabs from 120 sheep, 85 cattle and 20 horses were examined for Aeromonas species using alkaline peptone water (pH 8.4) as the enrichment medium and Aeromonas Selective Agar containing 5 mg/l ampicillin as the isolation medium. Identification and antibiotic resistance of motile Aeromonas strains was performed using Gram Negative Enteric ID panel. Motile aeromonads were isolated from 12 (10%) sheep, 7 (8.2%) cattle and 1 (5%) horse. Of these 20 aeromonad isolates, 13 were A. caviae, 6 were A.sobria and 1 was A. hydrophila. Aeromonas species in the faeces of livestock might pose a public health problem for humans who are in direct contact with contaminated animals. However, further studies should be performed on aeromonads relating to their transmission between animals and humans.     

Experiments on Virulence Dose and Portals of Entry for Aeromonas hydrophila in Walking Catfish

Abstract

Aeromonas hydrophila, isolated from chevron snakehead Ophicephalus (=Channa) striatus affected with epizootic ulcerative syndrome (EUS), was injected intramuscularly into healthy walking catfish Clarias batrachus at varying 10-fold serial dilutions from 10⁸ to 0 colony-forming units (cfu) per fish. Only 10⁶ or more cfu/mL induced dermomuscular lesions. Initial healing of lesions was observed by day 7 but complete healing was not apparent until day 16. Experiments were also conducted on possible portals of entry of A. hydrophila into walking catfish: Intramuscular (IM) injection, gastric gavage, fish food, and immersion of injured fish in rearing water inoculated with the test bacteria. Injuries were caused by skin or muscle cut, dermal scraping or incision, fish bite, and cohabitation of fish with golden snails Ampullarius sp. Only IM injection treatment induced dermomuscular pathology in the test catfish. This suggests that a localization of A. hydrophila to a level of 10⁶ cfu/mL in the musculature must be established for dermal lesions to develop.     

Phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. in Malaysia

Mycobacteriosis due to mycobacteria is one of the most common bacterial diseases in ornamental fish. We describe here the phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. using ATCC Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium chelonae as references. A total of four isolates (M1, M2, M3, M4) were obtained from four out of 106 fish samples using selective agar, and identified to Mycobacterium genus using acid-fast staining and 16s rRNA gene-based genus specific polymerase chain reaction. DNA sequencing and NCBI-BLAST analysis further identified isolate M1 as M. marinum and isolates M2, M3, M4 as M. fortuitum. Morphological, physiological and biochemical tests were carried out for phenotypic characterizations. Universal M13 and wild-type phage M13 RAPD dendogram was generated to illustrate the genetic relationship of the isolates and reference strains.     

Growth Inhibition of Bacterial Fish Pathogens and Quorum-Sensing Blocking by Bacteria Recovered from Chilean Salmonid Farms

The main goal of this study was to find bacterial isolates with the ability to inhibit the growth of the fish pathogens Aeromonas hydrophila, Vibrio anguillarum, and Flavobacterium psychrophilum and to inhibit the blockage of the quorum-sensing (QS) system. A total of 80 gram-negative strains isolated from various freshwater Chilean salmonid farms were studied. We determined that 10 strains belonging to the genus Pseudomonas inhibited at least one of the assayed fish pathogens. Of these, nine strains were able to produce siderophores and two strains were able to inhibit the growth of all assayed pathogenic species. When the 80 strains were examined for QS-blocking activity, only the strains Pseudomonas sp. FF16 and Raoultella planticola R5B1 were identified as QS blockers. When the QS-blocker strains were analyzed for their ability to produce homoserine lactone (HSL) molecules, thin-layer chromatography analysis showed that both strains were able to produce C6-HSL– and C8-HSL–type molecules. Strain R5B1 did not show growth inhibition properties, but strain FF16 also led to inhibition of growth in A. hydrophila and F. psychrophilum as well as to siderophore production. Pseudomonas sp. FF16 exhibited potentially useful antagonistic properties and could be a probiotic candidate for the salmon farming industry.

Received July 31, 2014; accepted December 17, 2014     

Phenotypic and Genotypic Heterogeneity among Streptococcus iniae Isolates Recovered from Cultured and Wild Fish in North America,Central America and the Caribbean Islands

Abstract

Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.

Received April 20, 2014; accepted July 7, 2014     

Predominant Bacteria Associated with Red Snapper from the Northern Gulf of Mexico

Abstract

Since the Deepwater Horizon oil spill in 2010, anecdotal observations of Red Snapper Lutjanus campechanus from the northern Gulf of Mexico exhibiting unusual external lesions have been reported. Two opportunistic bacterial fish pathogens, Vibrio vulnificus and Photobacterium damselae, were recovered from the fish and were deemed responsible for the abnormalities. However, the culturable microbiota of healthy Red Snapper has not yet been characterized. We analyzed the heterotrophic bacteria associated with healthy Red Snapper caught off the Louisiana coast. In total, 179 isolates from 60 fish were recovered from skin and mucus, and 43 isolates were obtained from anterior kidney. All isolates were identified by 16S ribosomal RNA gene sequencing. The Proteobacteria was the predominant phylum in both external and internal samples, followed by the Firmicutes and the Actinobacteria. Within the Proteobacteria, most isolates were members of the genera Vibrio and Photobacterium, and V. natriegens and P. damselae were the predominant species. The results of this study suggest that both Vibrio spp. and Photobacterium spp. are associated with the normal microbiota of healthy Red Snapper. Thus, the opportunistic fish pathogens recovered in previous studies cannot be deemed lesion-forming until Koch's postulates are fulfilled.

Received March 1, 2013; accepted September 4, 2013     

Screening for the Fish Disease Agent Aeromonas salmonicida with an Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract

Serological detection of bacterial pathogens in fish tissue is an important tool for surveying epidemiological situations. Whenever antibacterial treatment of fish is recommended, it becomes necessary, however, to culture the pathogen for sensitivity testing. An enzyme-linked immunosorbent assay (ELISA) was developed to identify Aeromonas salmonicida in culture. This serological identification can partly substitute for biochemical characterization of the organism and thus decrease the time between isolation and sensitivity testing by at least 3 d. The ELISA works with only one bacterial colony and yields results within 4 h. During this time, a bacterial suspension can be prepared for the resistance test. The specificity of an antiserum, raised in rabbits, against whole cells of A. salmonicida can be increased by adsorption with strains of cross-reacting species. However, difficulties arise when serologically heterogeneous species (e.g., A. hydrophila) are used as the cross-reacting bacterium. In the present study, severalfold adsorption with four isolates did not totally rule out cross-reactivity against additional strains. Therefore, the strain in question should also be checked for colony morphology, production of pigment, or presence of cytochrome oxidase to validate the serologically obtained result.     

Improved Broth Microdilution Method for Antimicrobial Susceptibility Testing of Francisella Noatunensis Orientalis

In this project we optimized a minimal inhibitory concentration testing protocol for Francisella noatunensis orientalis. Thirty-three F. noatunensis orientalis isolates recovered from different fish species and locations were tested, and Escherichia coli ATCC 25922 was used as a quality control reference strain. A modified cation-adjusted Mueller Hinton broth supplemented with 2% IsoVitalex and 0.1% glucose (MMH) was tested at a pH of 6.4 ± 0.1, 7.1 ± 0.1, and 7.3 ± 0.1. Growth curves generated for F. noatunensis orientalis indicated that MMH at a pH of 6.4 ± 0.1 provided optimal growth. There were no significant differences in the growth curves obtained from isolates recovered from different fish species or from fresh or marine water. The pH of 6.4 ± 0.1 in the MMH media interfered with the inhibitory properties of the potentiated sulfonamides (ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole) when using the E. coli ATCC reference strain. Minimal inhibitory concentrations of eight antimicrobials (gentamicin, enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, and oxolinic acid) were similar for all F. noatunensis orientalis isolates. The in vitro susceptibility data provided here can provide a baseline for monitoring the development of antimicrobial resistance among F. noatunensis orientalis isolates, as well as provide valuable data in the development of potential therapeutics.

Received October 27, 2015; accepted April 13, 2016 Published online August 2, 2016     

Impact of Thiamine Deficiency on T-cell Dependent and T-cell Independent Antibody Production in Lake Trout

Abstract

Lake trout Salvelinus namaycush on thiamine-replete and thiamine-depleted diets were evaluated for the effects of thiamine status on in vivo responses to the T-dependent antigen trinitophenol (TNP)-keyhole limpet hemocyanin (TNP-KLH), the T-independent antigen trinitrophenol-lipolysaccaharide (TNP-LPS), or Dulbecco's phosphate-buffered saline (DPBS; negative control fish). Plasma antibody concentrations were evaluated for possible differences in total anti-TNP activity as well as differences in response kinetics. Associations between anti-TNP activity and muscle and liver thiamine concentrations as well as ratios of muscle-to-liver thiamine to anti-TNP activity were also examined. Thiamine-depleted lake trout that were injected with TNP-LPS exhibited significantly more anti-TNP activity than thiamine-replete fish. The depleted fish injected with TNP-LPS also exhibited significantly different response kinetics relative to thiamine-replete lake trout. No differences in activity or kinetics were observed between the thiamine-replete and -depleted fish injected with TNP-KLH or in the DPBS negative controls. Anti-TNP activity in thiamine-depleted lake trout injected with TNP-KLH was positively associated with muscle thiamine pyrophosphate (thiamine diphosphate; TPP) concentration. A negative association was observed between the ratio of muscle-to-liver TPP and T-independent responses. No significant associations between anti-TNP activity and tissue thiamine concentration were observed in the thiamine-replete fish. We demonstrated that thiamine deficiency leads to alterations in both T-dependent and T-independent immune responses in lake trout.

Received July 25, 2011; accepted July 12, 2012     

Host factors associated with the detection of Aeromonas salmonicida and Yersinia ruckeri in Ontario, Canada government fish hatcheries

This paper presents an epidemiological investigation of Ontario Ministry of Natural Resources Fish Health Laboratory data from 1981 to 1997, to determine whether fish species and age were associated with lot-level detection of Aeromonas salmonicida and Yersinia ruckeri in hatchery fish. In stepwise logistic regression, the species brook trout and back-cross (lake trout crossed with the hybrid “splake”) were more likely to test A. salmonicida-positive compared to all other species reared in the hatcheries. Similarly, the species brook trout was significantly more likely to test Y. ruckeri-positive compared to all other species. For both pathogens, the 1–5-month age group was associated significantly with detection. These findings suggest that purposive sampling of higher-risk fish lots could increase the likelihood of detecting both study pathogens.     

Evaluation of different API systems for identification of porcine Pasteurella multocida isolates

An exhaustive biochemical characterisation of 60 porcine Pasteurella multocida clinical isolates recovered from lesions indicative of pneumonia, previously confirmed by PCR and all belonging to the capsular serogroup A, was performed by means of four commercial systems. The API 20NE correctly identified almost all isolates (95%), but only 60% could be ascribed to this species by the API 20E method. The high diversity exhibited by the API 50CHB/E system, with six different patterns, does not advise its use as additional system for a definitive identification at the species level, but this method could be a potential tool for characterising P. multocida isolates below this level. The more uniform reactions yielded by the API ZYM test make this system helpful in the confirmatory identification of this organism. The high variability (20 profiles) obtained when the four systems are taken together also suggests their usefulness for epidemiological purposes in order to sub-type P. multocida isolates.     

Detection and genotypic differentiation of Campylobacter jejuni and Campylobacter coli strains from laying hens by multiplex PCR and fla-typing

In total, 26 Campylobacter (C.) strains, isolated from liver, spleen, caecal or jejunal content of laying hens from different flocks were examined. In these flocks a drop in egg production, an increasing mortality and livers with whitish-grey lesions as post-mortem finding were observed. Suspected Campylobacter colonies were differentiated using a modified m-PCR in 13 Campylobacter jejuni and 13 Campylobacter coli strains. All isolates were characterised by typing of the flaA and flaB gene each with two restriction enzymes. To compare the four different profiles for all strains an artificial “fla-type” was generated. Different and identical fla-types of C. jejuni and C. coli were recovered from both intestinal and extra-intestinal organs of the laying hens and even from individual birds. One significant observation is that some fla-types of C. jejuni or C. coli were detected in intestinal and systemic sites but not all fla-types of both species appeared to be equally able to invade internal organs.     

Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study

Antimicrobial therapy is a useful tool to control bovine mastitis caused by Staphylococcus aureus, as consequence an increase in staphylococci resistant cases has been registered. Alternative strategies are desirable and bacteriocins represent attractive control agents to prevent bovine mastitis. The aim of this work was to evaluate the activity of five bacteriocins synthesized by Bacillus thuringiensis against S. aureus isolates associated to bovine mastitis. Fifty S. aureus isolates were recovered from milk composite samples of 26 Holstein lactating cows from one herd during September 2007 to February 2008 in México and susceptibility of those isolates to 12 antibiotics and 5 bacteriocins from B. thuringiensis was evaluated. S. aureus isolates were mainly resistant to penicillin (92%), dicloxacillin (86%), ampicillin (74%) and erythromycin (74%); whereas susceptibility to gentamicin, trimethoprim and tetracycline was detected at, respectively, 92%, 88%, and 72%. All S. aureus isolates showed susceptibility to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287 followed by kenyacin 404, entomocin 420 and tolworthcin 524. Our results showed that S. aureus isolates had differences in the antimicrobial resistance patterns and were susceptible to bacteriocins produced by B. thuringiensis, which could be useful as an alternative method to control bovine mastitis.     

Enterotoxigenicity,hemagglutination and cell-surface hydrophobicity in Aeromonas hydrophila,A. Sobria and A. Salmonicida

Thirty-one Aeromonas hydrophila, 13 A. sobria and two A. salmonicida strains of diverse sources were tested for enterotoxigenicity, hemagglutination and cell surface hydrophobicity. Although 93% of the culture supernatant fluids of the Aeromonas strains exhibited cytotoxic effects on Y1 adrenal and Chinese hamster ovary (CHO) cells, typical rounding of Y1 adrenal cells was reproducibly observed before cytotoxicity for 80% of the isolates within 1 h of exposure.Twenty-eight strains were positive for delayed permeability factor (DPF) activity in rabbit skin. Culture filtrates of 16 of 20 strains that were positive both in the Y1 adrenal cell test and for DPF activity elicited fluid accumulation in rabbit ileal loops. The DPF and ileal loop activities were neutralizable by cholera antitoxin. All, except two strains each of A. sobria and A. hydrophila, produced a heat-stable, rapid permeability factor (RPF) detected in rabbit skin. Heat-treated culture supernatant fluids of two A. hydrophila and one A. sobria isolate gave positive responses in the infant mouse assay. Nine other strains gave borderline reactions.When A. hydrophila and A. sobria isolates were grown in broth, approximately 90% agglutinated bovine, chicken, human group A and guinea-pig erythrocytes in the presence of mannose at 4°C and/or 20°C. The two A. salmonicida isolates produced mannose resistant hemagglutination (MRHA) of these four blood types.Hydrophobic interaction chromatography indicated adhesive potential in 61% A. hydrophila and 100% A. sobria strains expressing weak to strong hydrophobic cell surface properties. The results of these investigations strongly imply that the Aeromonas strains produce a cytotonic enterotoxin immunologically related to cholera toxin. Adhesive characteristics were commonly found in both clinical and routine isolates.     

Genetic organization and preferential distribution of putative pilus gene clusters in Streptococcus suis

Recent analyses of Streptococcus suis isolates using multilocus sequence typing (MLST) suggested the importance of sequence type (ST) 1 and ST27 complexes for animal hygiene and public health. In this study, to investigate whether pilus-associated genes in S. suis can be used as novel genetic markers for important clonal groups, we examined the correlation between STs and putative pilus-associated gene profiles in S. suis. Genomic searches using sequenced genomes and sequence data determined in several isolates revealed the presence of at least four distinct putative pilus gene clusters in S. suis (srtBCD, srtE, srtF, and srtG clusters). On the basis of the presence or absence of genes in the four clusters, 108 S. suis isolates from various origins were classified into 12 genotypes (genotypes A–L). Genotypes A and B, which possessed srtBCD plus srtF clusters and srtF plus srtG clusters, respectively, were the most common in isolates from diseased pigs and humans, and 29.9% and 59.8% of the isolates belonged to genotypes A and B, respectively. In contrast, only 4.8% and 28.6% of isolates from healthy carriers were genotypes A and B, respectively. MLST analysis showed the associations of genotypes A and B with ST1 and ST27 complexes, respectively. In addition, srtBCD and srtG clusters were preferentially distributed to ST1 and ST27 complex members, respectively. These results suggest that profiling of selected pilus-associated genes could be used as an easy screening method to monitor isolates important for S. suis infection.     

Molecular and virulence characteristics of multi-drug resistant Salmonella Enteritidis strains isolated from poultry

Forty-six Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strains were isolated from chicken meat, faeces, and eggshells collected from hatcheries throughout Korea. The strains were examined for the presence of antimicrobial resistance and virulence genes. All 46 isolates were resistant to at least one of 21 antibiotics used in this study, 30 (65.2%) were resistant to three or more antimicrobials, and a single remarkable isolate was resistant to 15 antimicrobials. The isolates were primarily resistant to penicillins, sulfisoxazole, streptomycin, tetracycline and quinolones.The high rate of resistance in S. Enteritidis strains, sometimes to multiple drugs, may complicate future options for treating human infections. Nineteen of the 21 penicillin resistant isolates carried the bla_TEM gene, while one strain, resistant both to penicillins and ceftriaxone, carried the bla_CTX-M gene. Thirty-seven of the 45 sulfisoxazole resistant isolates carried sul2, and 23/24 streptomycin resistant isolates carried both strA and strB. All 10 tetracycline resistant isolates carried the tet(A) gene. Most isolates harboured both SPI-1 and SPI-2-associated genes, and the spv operon, which are known to be associated with human infections. The presence of these genes suggests that these strains could give rise to public health problems if dispersed in the general human population.