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.     

Effect of Incubation Temperature and Salinity on Expression of the Outer Membrane Protein Profile of Edwardsiella tarda

Abstract

Outer membrane proteins (OMP) of 10 isolates of Edwardsiella tarda were compared by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The OMP profile of the type strain E. tarda ATCC 15947 cultured at 25°C had five major protein bands of 40, 36.5, 34, 28.5, and 25 kDa and a large number of minor proteins ranging in size from approximately 10 to 120 kDa. Differences between the OMP profiles of the isolates of E. tarda included the inconsistent presence of the 34- or 36.5-kDa proteins in five isolates of E. tarda and two major bands of 47 and 44 kDa that were present in only two isolates of E. tarda. There were no differences in the outer membrane protein profiles of 9 out of 10 isolates of E. tarda incubated at a temperature of 25°C compared with those at 35°C. To evaluate the effect of salinity, 10 isolates of E. tarda were cultured in brain heart infusion broth containing 0.5, 1.5, and 3.0% sodium chloride. Reactions of isolates of E. tarda to the different salinity levels were placed into three groups. The first group expressed more or fewer protein bands at 1.5% sodium chloride. The second group lost major bands at 3% salinity, whereas the third group had no change in the OMP profile with salinity. The OMP profile differences and the different reactions to salinity levels suggest that the isolates are heterogeneous.     

Role of Low-Molecular-Weight Ciliostatic Toxins in Vibriosis of Bivalve Mollusks

Abstract

Toxins play a major role in bacillary necrosis of hatchery-reared bivalve larvae. Two principal types of toxin of Vibrio spp. have been identified: A proteinase (molecular weight = 40,000) that degrades connective tissue and at least one ciliostatic toxin (molecular weight = 500–1,000). The ciliostatic toxin is stable at 100°C for 10 min and probably accounts for the cessation of feeding and swimming in the early stages of bacillary necrosis. Ciliostatic toxins were produced by 17 of 20 Vibrio spp. that are pathogenic to fish or that were isolated from diseased mollusks and included Vibrio tubiashii, V. anguillarum, and V. alginolyticus. Of 53 Vibrio and Aeromonas isolates not associated with bivalve infections, only 15 produced a ciliostatic toxin.     

Biochemical,Biophysical, and Serological Homogeneity of Edwardsiella ictaluri

Abstract

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish, which, during the past 5 years, has become the most serious infectious disease problem of cultured channel catfish Ictalurus punctatus. We compared 40 isolates of E. ictaluri from different geographical regions and host fish species. From the biophysical tests, a pH of 7.0–7.5 and a temperature of 25–30°C were optimum growth conditions for all E. ictaluri isolates. All isolates grew well in media with an NaCl concentration of 0.5% or less, but none of the E. ictaluri isolates grew in media with a concentration of 2.0 or 5.0% NaCl. Biochemically, 42 out of 46 tests gave the same reaction for all 40 isolates. The only observed differences were in gas production at 25°C, the o-nitrophenylbeta-D-galactopyranoside test, ornithine decarboxylation, and D-mannose utilization. Serologically, identical agglutinin titers (1:80) to E. ictaluri-specific rabbit antisera were observed, and all isolates cross-agglutinated with four different antisera. Based on the biophysical, biochemical, and serological reactions of 40 isolates of E. ictaluri, identification of distinct strains was not possible, although some were slightly different biotypically.     

Characterization of ammonia-assimilating bacteria in a lagoon for wastewater from a paddock of dairy cattle

We investigated microorganisms that assimilated ammonia in lagoon treatment processes. Ammonia‐assimilating microorganisms were detected by nitrogen‐limited medium that contained ammonia as the sole nitrogen source. Numbers of ammonia‐assimilating aerobes (log CFU/g) were 3.4, 4.8, 5.0, 4.8 and 5.0 (log CFU/mL) on the culture plate incubated at 4°C, 10°C, 15°C, 20°C and 25°C, respectively. Many isolates used ammonia in high rates when they were purely cultivated in nitrogen‐limited medium added to sterilized lagoon extract. Many of them used ammonia even when they were cultivated in media containing viable microbial flora of the lagoon. Among them, enterobacteriaceae and Pseudomonas sp. were identified by analysis of 16S ribosomal DNA.     

Isolation and Characterization of Potentially Human Pathogenic,Cytotoxin‐producing Aeromonas Strains from Retailed Seafood in Berlin,Germany

The presence of potentially human pathogenic strains of Aeromonas was investigated in 84 samples of seafood which were purchased from retail traders in Berlin, Germany in spring 2000. A total of 134 Aeromonas strains were isolated on selective [GSP agar and Aeromonas (Ryan) agar] and unselective (standard count agar and enterohaemolysin agar) media from 27 (32.1%) of the samples and were classified as Aeromonas hydrophila (67.9%), A. caviae (26.1%) and A. sobria (6.0%) by biotyping. Thirteen (48.1%) of the 27 positive samples contained more than one species of Aeromonas. Production of haemolysins on enterohaemolysin agar was found with 132 (98.5%) of the strains at 28°C and with 130 strains (97.0%) at 37°C growth temperature. Vero cytotoxins were produced by 99 (73.9%) of the strains when grown at 28°C but only by 24 of the strains (17.9%) at 37°C. The latter strains were identified as A. hydrophila (n = 22) and A. sobria (n = 2) which came from 17 (20.2%) samples of raw seafood and from ready‐to‐eat salted herring ‘Matjes’ products. Cytotoxin‐encoding genes for aerolysin (aer) and haemolysin A (hlyA) were investigated by PCR. Aer and hlyA genes were detected in both, strains which produced toxins only at 28°C and strains which produced toxins at 37°C. Our data indicate that raw seafood and ready‐to‐eat fish products can harbour potential human pathogenic, cytotoxin producing Aeromonas strains.     

Cell-Surface-Associated Properties of Fish Pathogenic Bacteria

Abstract

Pathogenicity assays showed that 33 of 42 potentially pathogenic strains of bacteria tested were virulent to rainbow trout Oncorhynchus mykiss. Regardless of their degree of virulence to fish, strains of motile Aeromonas, A. salmonicida, and Vibrio anguillarum were moderately hydrophobic. Only 46 and 25°10 of the strains were able to hemagglutinate human and trout erythrocytes, respectively. Hydrophobicity and hemagglutination were practically absent in isolates of Yersinia ruckeri. A notable number of the strains positively adhered to salmonid (51%) and nonsalmonid (55%) fish cells. Whereas the treatment of the bacteria with proteinase K or trypsin did not decrease the hydrophobicity of the isolates, within motile Aeromonas and A. salmonicida species, strains with both protease-sensitive and -resistant hemagglutinating and adhesive abilities occurred. The effects of heat and sugars on hemagglutinating and hydrophobic properties varied within all bacterial groups. Although treatment of strains with D-mannose or L-fucose had distinct effects on adhesiveness according to the bacterial species and the cell system used, none of the heat-treated (80°C for 15 min) bacteria lost their capacity to adhere to cultured fish cells. The results showed that there was no direct relationship between any of the cell surface properties analyzed and the degree of virulence of the strains.     

Comparison of the API 20E and BBL Crystal E/NF Identification Systems for Differentiating Bacterial Isolates from Apparently Healthy Reared Sea Bass (Dicentrarchus labrax)

The ability of two commercial rapid identification systems, API 20E and BBL Crystal E/NF, to reliably identify bacterial isolates from the internal organs of reared sea bass were compared. The tests gave different results: API 20E identified bacteria as Pseudomonas spp. with 37% accuracy, while BBL Crystal E/NF identified them as Flavobacterium odoratum with 99% accuracy. Although F. odoratum is not a marine fish pathogen, conventional tests conducted with the same isolates were more indicative of them being Flavobacterium spp. than Pseudomonas spp., suggesting that BBL Crystal E/NF was more reliable in this identification. Both systems were found to be applicable for diagnostics of marine fish pathogens, but should be used with caution because of possible misinterpretation.     

Influence of temperature on the reproduction of the earthworm Eisenia foetida (Oligochaeta)

An experimental investigation revealed that cocoon production by E. foetida (Oligochaeta) increased linearly with increase in temperature in the range 10 to 25 °C. Diurnally fluctuating temperature did not have a marked influence on total cocoon production at the temperature levels of 10, 15 and 25 °C. A highly significant difference in cocoon production occurred between a constant temperature of 20 °C and a mean temperature of 20 °C (which fluctuated diurnally between 12 and 28 °C). Maximum cocoon production was obtained at 25 °C with each worm producing a cocoon every second day. The investigation also showed that temperature does influence the number of hatchlings per cocoon. At a temperature of 25 °C fewer worms hatched per cocoon than at 20 °C.     

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.     

Isolating and evaluating lactic acid bacteria strains for effectiveness on silage quality at low temperatures on the Tibetan Plateau

Four lactic acid bacteria (LAB) strains isolated from straw silages on the Tibetan Plateau were characterized, and their effects on the fermentation quality of Italian ryegrass (Lolium multiflorum Lam.) at different temperatures (10°C, 15°C and 25°C) were studied. These LAB isolates were evaluated using the acids production ability test, morphological observation, Gram staining, physiological, biochemical and acid tolerance tests. All the isolates (M1, LM8, LO7 and LOG9) could grow at 5‐20°C, pH 3.5‐7.0 and NaCl (3.0%, 6.5%). Strains M1, LM8, LO7 and LOG9 were identified as Lactobacillus plantarum, L. coryniformis, Pediococcus pentosaceus and P. acidilactici, respectively, by sequencing 16S ribosomal DNA. The four isolates were added to Italian ryegrass for ensiling for 30 days at various temperatures. Compared with the corresponding control, inoculating with isolates M1, LM8 and LO7 could improve the silage quality of Italian ryegrass at low temperatures, indicated by significantly (P < 0.05) higher lactic acid (LA) contents and ratios of lactic acid/acetic acid (LA/AA), and significantly (P < 0.05) lower pH and ammonia nitrogen/total nitrogen (AN/TN). Compared with other isolates, LM8 performed better at 10°C and 15°C, indicated by the higher (P < 0.05) LA content and ratio of LA/AA, and the lower (P < 0.05) pH and AN/TN.     

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.     

Quantitative and Qualitative Bacterial Flora of Giant Freshwater Prawn Macrobrachium rosenbergii Cultured in Earthen Ponds in Saudi Arabia

Abstract

Quantitative and qualitative analyses of bacterial flora associated with the digestive tract of the giant freshwater prawn Macrobrachium rosenbergii cultured in earthen ponds of Saudi Arabia were carried out. Bacterial counts and flora of prawn-culture pond water, sediment, and prawn carapaces, along with important physicochemical parameters, were investigated, and the isolates were identified to the genus or species level. Total viable counts (TVC; mean ± SD) varied between (1.0 ± 2.3) × 10⁴ and (1.7 ± 0.8) × 10⁵ colony-forming units (cfu) per milliliter in pond water, (5.8 ± 1.7) × 10⁷ and (1.1 ± 2.9) × 10⁹ cfu/g in sediment, (1.5 ± 0.9) × 10⁵ and (1.6 ± 2.4) × 10⁶ cfu/cm² in the carapace, and (9.1 ± 1.5) × 10⁶ and (8.7 ± 1.8) × 10⁷ cfu/g in the digestive tract of freshwater prawns. The bacterial flora was predominantly gram-negative, accounting for 80% of total isolated strains. Altogether, 21 bacterial species of 16 genera were identified. Aeromonas hydrophila, Shewanella putrefaciens, other Aeromonas spp., Vibrio spp., and Enterococcus spp. were the most abundant bacterial species (prevalence ≥10%) in pond water; A. hydrophila, S. putrefaciens, Vibrio spp., and Pseudomonas spp. were the most abundant in sediment; S. putrefaciens, A. hydrophila, Vibrio spp., Enterococcus spp., and Aeromonas spp. were the most abundant on the prawn carapace; and A. hydrophila, S. putrefaciens, Vibrio spp., Enterococcus spp., and Pasteurella spp. were the most abundant in the digestive tracts. In every population studied, Aeromonas spp., S. putrefaciens, Enterococcus spp., Vibrio spp., Pasteurella spp., Chryseomonas spp., and Pseudomonas spp. were present.     

Bacteriological investigation on “Mauro” sold in Catania

Seaweeds known as “Mauro” are traditionally used fresh or to prepare omelettes in Sicily (Italy). Twenty samples sold in Catania between May 2005 and September 2007 were analyzed for Escherichia coli, Total Enterobacteria, Aeromonas spp., Pseudomonas spp., Vibrio spp., and Salmonella spp. Thirty Vibrio strains were examined for the presence of the virulence genes, toxR, toxRS, tl, tdh, and trh in the genomes of the isolates. Total Enterobacteria ranged between 2.23 and 6.85 log CFU/g, and in six samples, E. coli ranged between 0.70 and 2.74 log CFU/g. Aeromonas spp. was present in samples between 1.0 and 5.90 log CFU/g, while Pseudomonas spp. ranged between 2.70 and 7.27 log CFU/g. Vibrio spp. was present in 75% of samples at values between 1.30 and 4.60 log CFU/g. The most frequently isolated species was V. alginolyticus (76.66% of isolates), of which 82.60% were positive for toxR and the remaining 17.40% of strains were positive for toxRS. V. parahaemolyticus was identified in 13.33% of strains; all were positive for toxR and, in one case, for both toxR and toxRS. V. coralliitycus was isolated in 6.66% of strains (all positive for toxR), and one was identified as V. mimicus (positive for toxRS). The results of this study suggest that there is need for stringent quality control during harvesting and distribution of Mauro.

     

Bacteriology of the horizontal ear canal of dogs

Of 57 dogs included in a survey of the bacteriology of the horizontal ear canal, 31 showed clinical signs consistent with ear irritation. Twenty-six dogs were presented for other reasons and were asymptomatic for ear irritation. All dogs sampled were anaesthetized and sterile moistened swabs used to sample the horizontal ear canal. Swabs were introduced through a sterilized (121°C for 15 min) polypropylene otoscope cone and care was taken to prevent contamination of the cone with material from elsewhere in the ear canal. Swabs were processed within 20 min of sampling and cultures were incubated aerobically and anaerobically. Swabs from the ears of asymptomatic dogs were mixed in broth prior to plating to ensure optimal elution of small numbers of organisms from the swabs. Very small numbers of bacteria and yeasts were isolated from asymptomatic dogs while Pseudomonas spp. and Proteus spp. were frequent isolates from symptomatic dogs with chronic irritation. The significance of these findings is discussed in relation to the sampling and culturing techniques used in this and other surveys.     

Evaluation of a multi-test microtube system for the identification of veterinary isolates of Enterobacteriaceae

Four hundred and twenty-two isolates of Enterobacteriaceae from veterinary sources were used to evaluate the API20E system. The accuracy of the individual tests, compared to the conventional equivalent was established at a minimum of 88%. The identification level of the system was determined to be 86%, a level considerably lower than that recorded for human clinical and food based studies. If the API20E system is adopted for use in veterinary laboratories, then the limitations of the system must be clearly understood.     

Development of Similar Broth Microdilution Methods to Determine the Antimicrobial Susceptibility of Flavobacterium columnare and F. psychrophilum

Flavobacterium columnare and F. psychrophilum are major fish pathogens that cause diseases that may require antimicrobial therapy. Choice of appropriate treatment is dependent upon determining the antimicrobial susceptibility of isolates. Therefore we optimized methods for broth microdilution testing of F. columnare and F. psychrophilum to facilitate standardizing an antimicrobial susceptibility test. We developed adaptations to make reproducible broth inoculums and confirmed the proper incubation time and media composition. We tested the stability of potential quality-control bacteria and compared test results between different operators. Log phase occurred at 48 h for F. columnare and 72–96 h for F. psychrophilum, confirming the test should be incubated at 28°C for approximately 48 h and at 18°C for approximately 96 h, respectively. The most consistent susceptibility results were achieved with plain, 4-g/L, dilute Mueller–Hinton broth supplemented with dilute calcium and magnesium. Supplementing the broth with horse serum did not improve growth. The quality-control strains, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, yielded stable minimal inhibitory concentrations (MIC) against all seven antimicrobials tested after 30 passes at 28°C and 15 passes at 18°C. In comparison tests, most MICs of the isolates agreed 100% within one drug dilution for ampicillin, florfenicol, and oxytetracycline. The agreement was lower with the ormetoprim–sulfdimethoxine combination, but there was at least 75% agreement for all but one isolate. These experiments have provided methods to help standardize antimicrobial susceptibility testing of these nutritionally fastidious aquatic bacteria.

Received June 24, 2015; accepted October 2, 2015.     

Isolation of Campylobacter spp. from Client‐Owned Dogs and Cats,and Retail Raw Meat Pet Food in the Manawatu,New Zealand

Campylobacter causes acute gastroenteritis in people worldwide and is frequently isolated from food, animals and the environment. The disease is predominately food‐borne but many routes of transmission and sources of infection have been described, including contact with pets. The prevalence of Campylobacter spp. in dogs and cats varies widely, and data on New Zealand pets are limited. This study aimed to investigate the prevalence of Campylobacter spp. in dogs, cats and retail raw meat pet food products in New Zealand and to characterize Campylobacter jejuni isolates using multilocus sequence typing (MLST). Ninety dogs and 110 cats examined at the Massey University Veterinary Teaching Hospital for elective procedures, and fifty locally purchased retail raw meat pet diets were sampled. Two culture protocols combining Bolton broth enrichment and mCCDA and CAT agars in a microaerobic atmosphere at 42°C and 37°C with species identification using PCR were performed. The prevalence of Campylobacter spp., C. jejuni, Campylobacter upsaliensis and Campylobacter helveticus was 36%, 13%, 23% and 1% in dogs and 16%, 5%, 5% and 7% in cats, respectively. One dog had Campylobacter lari confirmed, and three dogs and one cat had multiple Campylobacter spp. detected. Significantly more animals tested positive using CAT than mCCDA agar (P < 0.001). Being neutered, vaccinated for Bordetella bronchiseptica, fed dry diets and brought in for neutering were protective factors for dogs, whereas attendance for dental treatment was a risk factor for cats. Campylobacter spp. were isolated from 28%, C. jejuni 22%, C. lari 6% and Campylobacter coli 6% of food samples. Six isolates positive by Campylobacter genus PCR were identified as Arcobacter butzleri. Poultry meat was more likely to be positive than non‐poultry meat (P = 0.006). Of the 13 C. jejuni pet isolates with full MLST profiles, eight were of different sequence types (ST) and all nine food isolates were of different STs.     

Biological efficacy and stability of diluted ticarcillin-clavulanic acid in the topical treatment of Pseudomonas aeruginosa infections

Topical compounded Timentin^® diluted with an inactive vehicle has been reported to be effective in the treatment of otitis externa caused by Pseudomonas aeruginosa. The aims of this study were to determine the biological efficacy of Timentin^® (ticarcillin and clavulanic acid) when diluted in the carrier vehicle Methopt^® against P. aeruginosa and to determine the efficacy and stability of Timentin^® aqueous stock concentrate solution. Timentin^® stock concentrate was tested against four P. aeruginosa isolates on days 0, 7, 14, 21 and 28; then after 2, 3, 4, 5, 6, 9 and 12 months of storage at 4 or ?20°C. The diluted Timentin^®–Methopt^® solutions were tested against all isolates after 0, 2, 4, 6, 8, 10, 12, 14, 17, 21, 24 and 28 days of storage at 24 or 4°C. Minimal inhibitory concentration (MIC) levels for all strains were determined using the broth microdilution method. The MIC of the stock solution remained relatively constant and acceptable throughout the study when stored at ?20°C and was also acceptable for shorter time periods (6–9 months) when stored at 4°C. The MIC for the diluted Timentin^®–Methopt^® solution remained relatively constant and acceptable throughout the study for all four bacterial strains, with no difference between the solutions stored at 4 or 24°C. The results of this study indicate that storage of the Timentin^® stock solution at ?20°C does not compromise efficacy for at least 12 months and that Timentin^® diluted in Methopt^® was stable for 28 days when stored at either 4 or 24°C.