Abstract

Vibrio spp have been identified in Hawaiian waters from a variety of sources including: 1) recreational/coastal areas 2) bays and estuaries, 3) fish, prawns and shrimp in aquaculture systems, and 4) marine mammals in open ocean pens. Vibrios are important pathogens in marine and brackish water aquaculture systems, in marine mammal and human infections. Certain antibiotics are used in marine mammals and humans for treatment of these infections and can be used in aquaculture situations as a preventative measure to decrease the likelihood of infection among stressed animals. Selected Vibrio isolates from all sources were evaluated for resistance to 16 antibiotics used in treatment of bacterial infections, with emphasis on those used in treatment against gram-negative bacteria. Marine mammals (Tursiops truncatus) were undergoing antibiotic treatment during Vibrio-associated infections creating a possible selective pressure for antibiotics used in therapy while those isolated from the untreated sources maintained a high degree of sensitivity to the same antibiotics. A high degree of resistance to four antibiotics was noted in Vibrios isolated from all sources.

Introduction

Bacteria of the genus Vibrio are indigenous to marine, estuarine, and brackish water environments. Vibrios are known to cause epidemics and death in hatcheries used to raise aquatic animals(1,2), as well as infections in marine mammals(3,4) and in humans(5). Marine mammals constitute a large group of mammals indigenous to the aquatic environment. The work here addresses bottlenose dolphins, which are common representatives of this mammal group. We have previously isolated V. alginolyticus in monoculture from slow-healing wounds in male bottlenose dolphins (Tursiops truncatus) housed in Kaneohe Bay, Hawaii(6,7). A slow-healing wound id defined as one which has not healed completely within a two week period. In past studies we have also isolated Vibrio spp. from cold-blooded animals is aquaculture systems on the island of Oahu (unpublished data). Vibrio isolates were primarily recovered from wounds/lesions associated with shrimp (Penaeus spp), prawns (Macrobrachium spp.), and fish (Chaetodon and Forcipiger spp.) and from environmental waters where the animals are housed. We will collectively identify these as cold-blooded animals in future references. Vibrio isolates were previously isolated from recreational- waters, bays and estuaries (unpublished data).

The objective of this study was: 1. to identify vibrios isolated from marine mammal, cold-blooded animal and environmental sources; 2. to determine antibiotic susceptibility patterns in the isolates from these sources; 3. to compare antibiotic susceptibility patterns of Vibrio spp. from different isolate sources.

Materials and Methods

Sample Sites and Sampling/Recovery Techniques

Sample sites selected were: 1. bottlenose dolphin slow-healing wounds; 2. bottlenose dolphin uninfected, unbroken skin; 3. surface seawater from pens housing the dolphins; 4. wounds/lesions from cold-blooded animals (shrimp, prawns and fish); 5. environmental waters associated with the cold-blooded animals; 6. recreational, bay, and estuarine surface waters. Water samples were recovered using sterile plastic sampling bottles. Samples were maintained at eight degrees C and processed within two hours of collection.

Recovery of vibrios from seawater was accomplished using the membrane filtration technique(8). All recovery and other media types used were commercially available from Difco, Detroit, Michigan. The media used for recovery of vibrios was thiosulfate-citrate-bile salts-sucrose (TCBS) agar.

Unbroken skin and slow-healing wound sites in bottlenose dolphins as well as s hrim prawn, and fish wound sites were pretreated with 70% alcohol and betadine, swabbed, with the swabs placed in Amies transport media (Precision Dynamics, Burbank California) and transported to the laboratory at eight degrees C within two hours for processing. Selected swabs were also placed in enrichment cultures (alkaline peptone water) to enhance Vibrio recovery. Dolphin unbroken skin and environmental seawater isolates were taken simultaneously with the dolphin slow-healing wound isolates and from dolphins and seawater in the same sea pen.

Isolate identification and Determination of Antibiotic Resistance

Presumptive Vibrios and TCBS isolated from all sources were initially identified using the API-20E system (Analytab Products, Plainview New York) with diluent containing 2% marine salts (Instant Ocean, Mentor, Ohio). Isolates picked for identification from TCBS plates were selected to insure representation of all differing colony morphologies evident after 48 hours of incubation at 37 and 15 degrees C. More extensive identification was accomplished on all isolares using a method based on the one described by West and Colwell in 1984 9). A modification of the Kirby-Bauer antimicrobial disk diffusion method was employed with the addition of 2% marine salts to the Mueller-Hinton agar(10,11). 16 antibiotics that can be used in treatment of infections caused by gram-negative rods were selected for evaluation. In addition, two antibiotics were also evaluated because of their usefulness taxonomic differentiation (penicillin G and vibriostatic agent 0/129).

Results

Vibrio spp. were identified from all sample sources. Four unspeciated vibrios that were very similar in biochemical characteristics were isolated from bottlenose dolphin slow-healing wounds and not from any other site,.These dolphin wound sites also yielded the only isolates of V. gazogenes and V. metchnikovii. Five of seven V. harveyi isolates or iginated from wounds in dolphins or cold-blooded animals. V. alginolyticus was the most frequently isolated Vibrio spp. V. vulnificus, was recovered from environmental sources exclusively. V. marinus was strongly-associated with dolphin healthy skin (seven of eight isolates).

Antibiotic resistance in Vibrio spp. from marine mammal sources was evaluated. Slow healing wounds from four bottlenose dolphins were sampled while the animals were undergoing treatment with orally administered trimethoprim/sulfadiazine. Resistance patterns to trimethoprim and sulfadiazine indicate a marked difference between the wound isolates and those from unbroken skin and environmental sources. 0/8 wound isolates were sensitive to either trimethoprim or sulfadiazine. 8 healthy skin isolates were sensitive to sulfadiazine while 2/8 were sensitive to trimethoprim. 8/8 environmental isolates were sensitive to sulfadiazine while 4/8 were sensitive to trimethoprim. The majority of isolates from all sources were resistant and intermediately sensitive to penicillin G, cephalothin, kanamycin, erythromycin, and streptomycin and sensitive to chloramphenicol and the vibriostatic agent 0/129.

Antibiotic resistance in Vibrio spp. isolated from cold-blooded animal was also evaluated. None of the environmental waters or animals were treated with antibiotics at any time. There was no marked difference in resistance between the environmental waters and the cold-blooded animal wound isolates. The majority of isolates in both groups were resistant or intermediately sensitive to cephalothin, penicillin G, carbenicillin, ampicillin, kanamycin and streptomycin and sensitive to chloramphenicol, sulfadiazine, trimethoprim/sulfamethoxazole and vibriostatic agent 0/129.

The final evaluation addressed antibiotic resistance to Vibrio spp. isolated from environmental marine waters. These sources had no known exposure to antibiotics of any kind. Isolates from bays/estuaries and recreational waters yielded similar patterns of resistance to one another as well as to environmental waters from marine mammal and cold-blooded animal sources. The majority of isolates were resistant and intermediately sensitive to cephalothin penicillin G, ampicillin, carbenicillin, kanamycin, and streptomycin and sensitive to chloramphenicol, sulfadiazine, trimethoprim/sulfamethoxazole, and vibriostatic agent 0/129. With the exception of the dolphin slow-healing wound isolates and their relative resistance to sulfadiazine and trimethoprim, isolates from all sources yielded very similar and characteristic patterns of resistance.

Discussion

We compared isolated Vibrio spp and their antibiotic resistance from animal and environmental sources and correlated the ongoing antibiotic therapy for marine mammals. V. marinus, V. harveyi, and an unspeciated vibrio were predominantly associated with the bottlenose dolphins or cold-blooded animals and these strains could have a specific relationship with these hosts as normal flora or etiological agents of infection. V. alginolyticus, V. campbellii, V. nigripulchritudo, and V. pelagius I and II were predominately associated with environmental samples and could have a specific association with environmental waters only. Bottlenose dolphins with slow-healing wounds and undergoing orally administered trimethoprim/sulfadiazine yielded isolates with a high degree of resistance to these antibiotics when compared to healthy skin and environmental isolates from all sources. This could be as a result of selective pressure from these antibiotics in the wound area thus leaving surviving resistant strains as the predominant population. This observation could prove useful as one parameter to use in identifying etiological agents of bacterial infection in aquatic animals since it can of ten be difficult to distinguish between many contaminating isolates. Isolated environmental vibrios were consistently resistant or intermediately sensitive to penicillin ampicillin, carbenicillin streptomycin and kanamycin and sensitive to chloramphenicol, sulfadiazine and trimethoprim/sulfamethoxazole. This distinctive pattern of resistance and sensitivity should be useful in determining selection of antibiotics for treatment of Vibrio infections in Hawaii and nearby locations. Further work is focusing on the association between plasmid profiles specific patterns of antibiotic resistance and virulence for diagnostic purposes in aquatic animal bacterial infections.

References

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