Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments.

Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.     

Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.     

斑点叉尾鲖源杀鲑气单胞菌无色亚种分离鉴定及药敏特性

研究从患溃疡症斑点叉尾鲙中分离到一株致病性菌株ry01, 生理生化鉴定和16S rDNA基因序列分析表明ry01株为鲑气单胞菌无色亚种(Aeromonas salmonicida subsp. achromogenes)。人工感染该菌后发病鱼表现为与自然发病类似症状, 且从组织中再分离的细菌特性与原感染菌相同。腹腔注射后该菌株对斑点叉尾鲙的半数致死量为4.17×106 CFU/mL。菌株ry01对强力霉素及左氧氟沙星等高度敏感, 为斑点叉尾鲙该病防控提供了理论依据。     

Plant transformation by coinoculation with a disarmed Agrobacterium tumefaciens strain and an Escherichia coli strain carrying mobilizable transgenes

Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.     

Characterization of an F18+ enterotoxigenic Escherichia coli strain from post weaning diarrhoea of swine, and of its conjugative virulence plasmid pTC

The enterotoxigenic Escherichia coli (ETEC) strain Ec2173, causing post weaning diarrhoea in swine, harbours six plasmids ranging from 13 to 200 kb in size. The heat stable toxin genes sta, stb and a tetracycline resistance gene were located on a self conjugative 120-kb plasmid, called pTC. In the cloned ColE1 type origin of replication of pTC a deletion was detected compared to other ColE1 replicons affecting the replication modulator gene rom. Epidemiological studies on ETEC isolates showed that pTC-like plasmids are widely distributed among porcine ETEC strains; thus representing an example of co-evolution of antibacterial resistance and virulence in pathogenic E. coli.     

Antibiotic resistance and survival of E coli in the alimentary tract

Some antibiotics tend to select for R-factor-carrying Escherichia coli in the human gut, with complex long-term consequences. Some resistant strains disappear rapidly when treatment ends, while others persist for months in the absence of obvious antibiotic selection pressure, and the performance of individual resistant strains seems to depend more on the nature of the strain than on the plasmid carried. R plasmids are relatively rare in those E coli that colonize well in the gut and resistant bacteria therefore tend to disappear when treatment ends, but this situation could change dramatically if R plasmids became prevalent among those strains of E coli that colonize effectively.     

Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments

In this study we assessed the occurrence, diversity and conjugative potential of plasmids in integron-carrying Aeromonas and Enterobacteriaceae from wastewaters. Sixty-six strains were included as donors in mating assays using rifampicin-resistant Escherichia coli and Pseudomonas putida recipient strains. The diversity of plasmids from donors and transconjugants (resistant to tetracycline or streptomycin) was evaluated by restriction analysis and replicon typing targeting 19 incompatibility groups. Restriction patterns revealed a diverse plasmid pool present in these strains. Plasmids were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E.?coli, Enterobacter sp.), FIC (A.?salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A.?veronii, Aeromonas sp., E.?coli), HI1 (E.?coli) and U (Aeromonas media) replicons. Nevertheless, 50% of the plasmids could not be assigned to any replicon type. Among integron-positive transconjugants, FrepB, I1 and HI1 replicons were detected. Results showed that wastewaters enclose a rich plasmid pool associated with integron-carrying bacteria, capable of conjugating to different bacterial hosts. Moreover, replicons detected in this study in Aeromonas strains expand our current knowledge of plasmid diversity in this genus.     

Studies on Inc-P plasmids in Erwinia carotovora subsp. carotovora

Abstract Inc-P plasmids, RP4, R751, pMO850, and pRK2013 were transferred to Erwinia carotovora . These plasmids were stably maintained in E. carotovora and the transconjugants were efficient donors of respective plasmids to other strains of E. carotovora and Escherichia coli . These plasmids were not able to mobilize chromosomal markers from one strain of E. carotovora to another strain of E. carotovora even in the presence of homologous DNA sequences on the plasmid and the bacterial chromosome. The presence of Inc-P plasmid does not affect the pathogenic phenotype of E. carotovora . A broad host range Inc-P cosmid, pLAFR1, was transferred to E. carotovora with the help of pRK2013, suggesting the potential use of a binary plasmid system for genetic complementation in E. carotovora .     

PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification

In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.     

Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates

Despite the critical role of plasmids in horizontal gene transfer, few studies have characterized plasmid relatedness among different bacterial populations. Recently, a multiplex PCR replicon typing protocol was developed for classification of plasmids occurring in members of the Enterobacteriaceae. Here, a simplified version of this replicon typing procedure which requires only three multiplex panels to identify 18 plasmid replicons is described. This method was used to screen 1,015 Escherichia coli isolates of avian, human, and poultry meat origin for plasmid replicon types. Additionally, the isolates were assessed for their content of several colicin-associated genes. Overall, a high degree of plasmid variability was observed, with 221 different profiles occurring among the 1,015 isolates examined. IncFIB plasmids were the most common type identified, regardless of the source type of E. coli. IncFIB plasmids occurred significantly more often in avian pathogenic E. coli (APEC) and retail poultry E. coli (RPEC) than in uropathogenic E. coli (UPEC) and avian and human fecal commensal E. coli isolates (AFEC and HFEC, respectively). APEC and RPEC were also significantly more likely than UPEC, HFEC, and AFEC to possess the colicin-associated genes cvaC, cbi, and/or cma in conjunction with one or more plasmid replicons. The results suggest that E. coli isolates contaminating retail poultry are notably similar to APEC with regard to plasmid profiles, with both generally containing multiple plasmid replicon types in conjunction with colicin-related genes. In contrast, UPEC and human and avian commensal E. coli isolates generally lack the plasmid replicons and colicin-related genes seen in APEC and RPEC, suggesting limited dissemination of such plasmids among these bacterial populations.     

Insights into the environmental resistance gene pool from the genome sequence of the multidrug-resistant environmental isolate Escherichia coli SMS-3-5

The increasing occurrence of multidrug-resistant pathogens of clinical and agricultural importance is a global public health concern. While antimicrobial use in human and veterinary medicine is known to contribute to the dissemination of antimicrobial resistance, the impact of microbial communities and mobile resistance genes from the environment in this process is not well understood. Isolated from an industrially polluted aquatic environment, Escherichia coli SMS-3-5 is resistant to a record number of antimicrobial compounds from all major classes, including two front-line fluoroquinolones (ciprofloxacin and moxifloxacin), and in many cases at record-high concentrations. To gain insights into antimicrobial resistance in environmental bacterial populations, the genome of E. coli SMS-3-5 was sequenced and compared to the genome sequences of other E. coli strains. In addition, selected genetic loci from E. coli SMS-3-5 predicted to be involved in antimicrobial resistance were phenotypically characterized. Using recombinant vector clones from shotgun sequencing libraries, resistance to tetracycline, streptomycin, and sulfonamide/trimethoprim was assigned to a single mosaic region on a 130-kb plasmid (pSMS35_130). The remaining plasmid backbone showed similarity to virulence plasmids from avian-pathogenic E. coli (APEC) strains. Individual resistance gene cassettes from pSMS35_130 are conserved among resistant bacterial isolates from multiple phylogenetic and geographic sources. Resistance to quinolones was assigned to several chromosomal loci, mostly encoding transport systems that are also present in susceptible E. coli isolates. Antimicrobial resistance in E. coli SMS-3-5 is therefore dependent both on determinants acquired from a mobile gene pool that is likely available to clinical and agricultural pathogens, as well, and on specifically adapted multidrug efflux systems. The association of antimicrobial resistance with APEC virulence genes on pSMS35_130 highlights the risk of promoting the spread of virulence through the extensive use of antibiotics.     

Association of Type III secretion genes with virulence of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida possesses a number of potential virulence factors, including a recently identified plasmid-encoded Type III secretion system. A number of field isolates of A. salmonicida subsp. salmonicida were examined for the presence of Type III secretion genes. Using in vitro experiments, it was found that field isolates containing such genes are cytotoxic to fish cell lines, whereas those that lack these genes are not. Using a rainbow trout in vivo model, the virulence of a wild type A. salmonicida subsp. salmonicida strain (Strain JF2267), which possesses Type III secretion genes, was compared to that of a laboratory derivative of the same strain that has lost these genes. While Strain JF2267 was virulent towards rainbow trout, its derivative was not. The A. salmonicida subsp. salmonicida Type Strain ATCC 33658T, which also lacks Type III secretion genes, was also found to be avirulent by this challenge model. The findings from both the in vitro and in vivo experiments suggest that the presence of Type III secretion genes is associated with the virulence of this important fish pathogen.     

Chromate-resistance genes in plasmids from antibiotic-resistant nosocomial enterobacterial isolates

The presence of chromate-resistance genes in enterobacteria was evaluated in a collection of 109 antibiotic-resistant nosocomial isolates from nine major cities in México. Results were compared with the presence of mercury-resistance genes. Susceptibility tests showed that 21% of the isolates were resistant to chromate (Cr(R)), whereas 36% were resistant to mercury (Hg(R)). Cr(R) levels were high in Klebsiella pneumoniae (61%), low in Enterobacter cloacae (12%) and Escherichia coli (4%), and null in Salmonella sp. isolates. Colony hybridization demonstrated that the majority of metal-resistant isolates hybridized with chrA gene (87% of Cr(R) isolates), encoding a CHR transporter homologue, and merA gene (74% of Hg(R) isolates), encoding MerA mercuric reductase, suggesting that most isolates expressed these widespread metal-resistance systems. Southern blot hybridization of Cr(R) isolates showed that plasmids of 80, 85, and 95 kb from K. pneumoniae isolates, and of 100 kb from an E. cloacae isolate, contained chrA-related sequences. These plasmids belonged to IncN or IncP incompatibility groups, and conferred Cr(R), as well as multiple antibiotic resistance, when transferred by conjugation to an E. coli standard strain. These data indicated that Cr(R) genes may be distributed among clinical enterobacteria via conjugative plasmids, probably by coselection with antibiotic-resistant genes.     

Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.     

长江上游网箱养殖圆口铜鱼杀鲑气单胞菌杀鲑亚种的分离与鉴定

研究首次报道了圆口铜鱼(Coreius guichenoti)疥疮病, 从患病圆口铜鱼的肝脏中分离到优势菌株YTL1, 并运用形态学观察、生理生化检测、16S rRNA和6个管家基因的系统发育分析等对分离菌株进行鉴定。基于以上实验结果, YTL1被最终鉴定为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp. salmonicida)。通过标准Kirby-Bauer纸片扩散法进行抗菌药物敏感性试验, 筛选治疗该暴发病的有效药物, 结果显示YTL1对氟苯尼考, 诺氟沙星和氨苄青霉素等13种抗生素敏感, 对6种抗生素如杆菌肽, 链霉素和卡那霉素有耐药性, 对红霉素具有中等敏感性。因此, 氟苯尼考被建议用来伴饵投喂, 并取得了较好的疾病控制效果。草鱼幼鱼(Ctenopharyngodon idella)和斑马鱼(Danio rerio)的人工感染试验结果显示, 经腹腔注射7.6×106—7.6×108 CFU/mL的YTL1菌液后, 感染鱼的症状与患病圆口铜鱼症状相似。研究证明基于6个管家基因的多序列位点分型是鉴定杀鲑气单胞菌至亚种水平的一种有效方法, 杀鲑气单胞菌是圆口铜鱼人工养殖的最大威胁之一, 并发现鲤科鱼类, 如草鱼和斑马鱼均是杀鲑气单胞菌杀鲑亚种的易感宿主。     

Trimethoprim resistance plasmids in Escherichia coli isolated from cases of diarrhoea in cattle, pigs and sheep

A total of 1572 isolates of Escherichia coli obtained from the faeces of young farm animals with diarrhoea over the period 1980–1983 were screened for resistance to trimethoprim (Tp). Resistance to Tp was detected in263/954 (28%) of bovine isolates,59/441 (13%) of porcine isolates and15/177 (9%) of ovine isolates. Seventy-five resistant isolates from separate outbreaks of infection on farms within a 25 mile radius of Nottingham were examined in detail. Sixty-eight (91%) of the 75 isolates were resistant to > 1024 mg Tp/1 and 34 (50%) of these 'highly resistant' isolates (45% of total resistant isolates) transferred their Tp resistance to E. coli K12. A further 13 (17%) isolates were demonstrated to carry non-self-transferable plasmids which were capable of being mobilized to E. coli K12 by the broad host range plasmid RP4. Thirty-one self-transferable Tp R plasmids were divided between the following incompatibility groups: IncB (14 plasmids), IncF_***H (4 plasmids), IncH₂ (1 plasmid), IncIaP (10 plasmids), IncIdT (1 plasmid) and IncP (1 plasmid). In terms of antibiotic resistance patterns and incompatibility properties, many of these plasmids closely resembled those isolated from human patients in the same area, suggesting that there may be a common pool of Tp R plasmids.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Genetic diversity of the fish pathogen Aeromonas salmonicida demonstrated by random amplified polymorphic DNA and pulsed-field gel electrophoresis analyses

The current taxonomy of Aeromonas salmonicida includes 4 subspecies. A. salmonicida subsp. salmonicida is associated with salmonid furunculosis, and A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and A. salmonicida subsp. smithia are strains that show variation in some biochemical properties. This classification does not readily encompass isolates from a wide range of fish hosts currently described as atypical A. salmonicida. This study examined 17 typical strains, 39 atypical strains and 3 type A. salmonicida subspecies strains for genetic similarity using the random amplified polymophic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) techniques. On the basis of RAPD- and PFGE-derived profiles, similarity matrices and dendrograms were constructed. The results showed that species A. salmonicida constituted a genetically heterogeneous group of strains, encompassing within an homogeneous or clonal lineage comprised solely of typical strains and the A. salmonicida subsp. salmonicida type strain.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

The interaction of trimethoprim and quinolones against Gram-negative fish pathogens

The effect of combination of trimethoprim with other non-sulphonamide antibacterial agents, in particular oxolinic acid and nalidixic acid, was evaluated against Gram-negative fish pathogens. The species included Aeromonas salmonicida, Yersinia ruckeri , some Vibrio spp. and Escherichia coli as a reference. The extent of synergy found by other workers with these substances against human Gram-negative bacteria was not apparent here. Some positive interaction between trimethoprim and oxolinic acid was found with Aer. salmonicida, Y. ruckeri and E. coli and between trimethoprim and nalidixic acid with Y. ruckeri in double disc diffusion tests but was not supported by fractional inhibitory concentration indices. The combinations were not effective in preventing emergence of resistance in passage on a drug gradient. Trimethoprim-resistant isolates of Aer. salmonicida were inhibited by low levels of oxolinic acid but the converse did not apply.