No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
In vitro efficacy of aquaculture antimicrobials and genetic determinants of resistance in bacterial isolates from tropical aquaculture disease outbreaks
Abstract
Understanding the efficacy of antimicrobials against pathogens from clinical samples is critical for their responsible use. The manuscript presents in vitro efficacy and antimicrobial resistance (AMR) genes in seven species of fish pathogens from the disease outbreaks of Indian aquaculture against oxytetracycline, florfenicol, oxolinic acid, and enrofloxacin. In vitro efficacy was evaluated by minimum inhibitory concentration and minimum bactericidal concentration. The gene-specific PCR screened AMR genes against quinolones (qnrA, qnrB and qnrS) and tetracyclines (tetM, tetS, tetA, tetC, tetB, tetD, tetE, tetH, tetJ, tetG, and tetY). The results showed that Aeromonas veronii (45%) showed the maximum resistance phenotype followed by Streptococcus agalactiae (40%), Photobacterium damselae (15%), Vibrio parahaemolyticus (10%), and Vibrio vulnificus (5%). There was no resistance among Vibrio harveyi and Vibrio alginolyticus against the tested antimicrobials. The positive association between tetA, tetB, tetC, tetM, or a combination of these genes to oxytetracycline resistance and qnrS to quinolone resistance indicated their potential in surveillance studies. The prevalence of resistance phenotypes (16.43%) and evaluated AMR genes (2.65%) against aquaculture antimicrobials was low. The resistance phenotype pattern abundance was 0.143. All the isolates showed susceptibility to florfenicol. The results help with the appropriate drug selection against each species in aquaculture practices.
ResearchGate has not been able to resolve any citations for this publication.
The present study reports the comparative pharmacokinetic profiles of florfenicol and its metabolite (florfenicol amine, FFA) in Trachinotus blochii under tropical marine conditions (salinity: 35 ± 1.4‰; temperature: 28.8 ± 0.54 °C) following a single in-feed oral administration of the recommended dose (15 mg/Kg). Furthermore, the study investigated the distribution of these two compounds in nine different tissues. The maximum florfenicol concentrations (Cmax) in plasma and tissues were observed within five hours (Tmax), except for bile. The Cmax ranged from 572 to 1954 ng/g or ml and was in the intestine > bile > muscle + skin > liver > gill = heart > plasma > kidney = spleen. The elimination half-life of FFC was significantly slower in the bile (38.25 ± 4.46 h). The AUC tissue/plasma was highest for bile (3.77 ± 0.22), followed by intestine > muscle + skin > heart > liver > kidney = gill = spleen. Tmax and t1/2β were slower, and Cmax was lower for FFA than florfenicol in all tissues except Cmax of the kidney and bile. FFA t1/2β was exceptionally slower in the kidney (46.01 ± 8.2 h). Interestingly, reaching an apparent distribution rate of > 0.5 was comparatively faster in the kidney, liver, and gills than in other tissues. The highest apparent metabolic rate was in the kidney (0.95 ± 0.01) and the lowest in plasma (0.41 ± 0.01). The generated data can be applied for formulating efficient therapeutic protocols in T. blochii, a promising mariculture species.
Graphical Abstract
With the rapid growth of the aquaculture production since the 1980s, there has been a concomitant increase in disease outbreaks. The injudicious and/or incorrect use of anti-microbial agents against diseases of farmed aquatic species poses a considerable threat to the development and growth of a successful and sustainable aquaculture industry. An increase in antimicrobial resistance (AMR) is an important consequence, resulting to the difficulty in treating common bacterial diseases in populations of aquatic organisms, combined with the presence of antibiotic residues in food fish and their products, leading to import refusals and negative impacts on international trade. To reduce the frequency of AMR, good aquaculture and effective biosecurity practices should include the prudent and responsible use of antibiotics and also consider the use of alternatives to antibiotics, in addition to disease prevention management. This article reviews the literature discussing the scope of the problem pertaining to antibiotic use, the emergence of AMR in aquaculture and to consider and discuss viable alternatives (e.g., vaccination, bacteriophages, quorum quenching, probiotics and prebiotics, chicken egg yolk antibody and medicinal plant derivative). We also discuss lessons learnt, from specific case studies such as the vaccination of farmed salmon in Norway and the use of 'specific pathogen-free' seed-as primary and essential part of a biosecurity strategy.
The use of chemicals, biologicals and veterinary medicinal products (VMPs) helps in healthy and sustainable fish production. Information on the use of these products is essential for assessing farming practices, potential human health and environmental risks. A questionnaire‐based nationwide survey covering aquaculture farms (n = 2936) producing carps, tilapia, pangasius and rainbow trout in freshwater and shrimp in brackishwater estimated the use of 52 different types of inputs which included disinfectants (597 g t−1), probiotics (2.28 kg t−1), environmental modifiers (22.82 kg t−1), nutritional supplements (1.96 kg t−1), natural anti‐infective agents (293 g t−1), herbicide and piscicides (844 g t−1), antibiotics (2 mg PCU−1), antifungal (4 mg PCU−1), and antiparasitic (14 mg PCU−1) agents. The bulk of these inputs was used for soil and water quality improvement and had low environmental and human safety concerns. The multivariate analysis revealed significant variation in the frequency and quantity of compounds use among farm groups. Redundancy analysis revealed a significant association between the number of products used and stocking density. The survey also showed a considerable influence of education and farming experience on the usage pattern of aquaculture inputs. Results of the study indicated greater reliance of farmers on the use of disinfectants for biosecurity, nutritional supplements for enhanced growth and environmental modifiers for maintaining soil and water quality in culture systems. Though there was no use of restricted antibiotics and antiparasitic agents, the development and implementation of standard regulatory guidelines are essential for safe and effective use of inputs for sustainable aquaculture.
FAO Conferences in 2015 and 2019 underscored the importance and
urgency of addressing the growing global threat of antimicrobial resistance
(AMR) in all countries through a coordinated, multisectoral, One Health approach
in the context of the 2030 Agenda for Sustainable Development. They
highlighted that access to effective antimicrobials and their appropriate and
prudent use has a role in productive and sustainable agriculture and aquaculture
– and that their misuse contributes to the rising rates of AMR which
negatively impacts the advances made in medicine, public health, veterinary
care, food and agriculture production systems, and food safety. The conferences
highlighted also that access to effective antimicrobial agents constitutes
a prerequisite for productive and sustainable agriculture, particularly
animal husbandry and aquaculture and safe food, on which countless livelihoods
depend throughout the world, but that hard-won gains in animal and
human health and development are at risk due to increasing resistance to
antimicrobials.
Contributing towards the goal of building resilience in the food and
agriculture sectors by limiting the emergence and spread of AMR depends
on controlling AMR effectively as a shared responsibility among farmers,
herders, growers, fishers, prescribers and policy-makers in food and agriculture
– as well as other sectors. Preventive action will provide an economic
benefit, especially when compared to the considerable percent of GDP expected
to be lost if AMR is permitted to develop into a global emergency
through the widespread failure of medicines.
The availability and use of effective antimicrobials is essential for the
health and welfare of terrestrial and aquatic animals, and in crop production.
The overuse and misuse of antimicrobials in animal and plant
production is influenced by an interplay of factors. These serve as targets
for action to address challenges ranging from: i) treatment failures driving
production losses and food insecurity; to ii) the impacts on human health.
Once individuals become carriers of antimicrobial-resistant microorganisms,
they can easily spread AMR among communities and across
borders. AMR can also reach the general population by spilling over into
agriculture products and the environment, contaminating waterways, wildlife
and soil. Given the global interconnected web of transmission, a multisectoral
and multidisciplinary approach is critical to the success of
National Action Plans (NAPs) for delivering on the Global Action Plan on AMR
(GAP; WHO, 2015).
This FAO Action Plan on AMR 2021–2025 sets out the five objectives that
guide the programming of FAO activities. The latter will be amended
as needed to reflect progress, new challenges, and available
resources. The action plan principally is intended to help guide FAO’s
support to its Members to build capacities, and is not a policy document.
The Action Plan provides for flexibility to respond to Members’ requests and
participation of Members in activities indicated is voluntary. The activities and
support provided under the Action Plan will be guided by the latest developments
in science, international guidance and standards.
Xanthomonas arboricola pv. pruni ( Xap ) causes bacterial spot, a major worldwide disease of Prunus species. Very few chemical management options are available for this disease and frequent applications of oxytetracycline (OTC) in the United States peach orchards have raised concerns about resistance development. During 2017–2020, 430 Xap strains were collected from ten peach orchards in South Carolina. Seven OTC-resistant (OTC R ) Xap strains were found in 2017 and 2020 from four orchards about 20–270 km apart. Interestingly, the seven strains were also resistant to streptomycin (STR). Six strains grew on media amended with ≤100 μg/mL OTC, while one strain, R1, grew on ≤250 μg/mL OTC. Genome sequence analysis of four representative OTC R strains revealed a 14–20 kb plasmid carrying tetC , tetR , and strAB in each strain. These three genes were transferable to Xanthomonas perforans via conjugation, and they were PCR confirmed in all seven OTC R Xap strains. When tetC and tetR were cloned and expressed together in a sensitive strain, the transconjugants showed resistance to ≤100 μg/mL OTC. When tetC was cloned and expressed alone in a sensitive strain, the transconjugants showed resistance to ≤250 μg/mL OTC. TetC and tetR expression was inducible by OTC in all six wild-type strains resistant to ≤100 μg/mL OTC. However, in the R1 strain resistant to ≤250 μg/mL OTC, tetR was not expressed, possibly due to the presence of Tn 3 in the tetR gene, and in this case tetC was constitutively expressed. These data suggest that tetC confers OTC resistance in Xap strains, and tetR regulates the level of OTC resistance conferred by tetC . To our knowledge, this is the first report of OTC resistance in plant pathogenic xanthomonads.
This study presents the occurrence and abundance of Aeromonas antibiotic-resistant bacteria (ARB) and genes (ARGs) isolated from water, biofilm and fish in two commercial trout farms before and one week after flumequine treatment. Wild (WT) and non-wild (NWT) strains were determined for quinolones (flumequine, oxolinic acid and enrofloxacin), oxytetracycline (OXY), florfenicol (FFN), trimethoprim-sulfamethoxazole (TMP) and colistin (COL), and pMAR (presumptive multi-resistant) strains were classified. Forty-four ARGs for the mentioned antibiotics, β-lactams and multi-resistance were quantified for 211 isolates. BlaSHV-01, mexF and tetE were the dominant ARGs. A greater occurrence and abundance of tetA2, sul3, floR1, blaSHV-01 and mexF were observed for NWT compared to WT. The occurrence of pMAR and NWT Aeromonas for quinolones, OXY, FFN, TMP, COL and ARGs depended on the Aeromonas origin, antibiotic use and the presence of upstream activities. Our results revealed the impact of a flumequine treatment on Aeromonas present on a fish farm through an increase in NWT and pMAR strains. The link between fish and their environment was shown by the detection of identical ARB and ARGs in the two types of samples. There appears to be a high risk of resistance genes developing and spreading in aquatic environments.
The sustainability of aquaculture has been debated intensely since 2000, when a review on the net contribution of aquaculture to world fish supplies was published in Nature. This paper reviews the developments in global aquaculture from 1997 to 2017, incorporating all industry sub-sectors and highlighting the integration of aquaculture in the global food system. Inland aquaculture—especially in Asia—has contributed the most to global production volumes and food security. Major gains have also occurred in aquaculture feed efficiency and fish nutrition, lowering the fish-in–fish-out ratio for all fed species, although the dependence on marine ingredients persists and reliance on terrestrial ingredients has increased. The culture of both molluscs and seaweed is increasingly recognized for its ecosystem services; however, the quantification, valuation, and market development of these services remain rare. The potential for molluscs and seaweed to support global nutritional security is underexploited. Management of pathogens, parasites, and pests remains a sustainability challenge industry-wide, and the effects of climate change on aquaculture remain uncertain and difficult to validate. Pressure on the aquaculture industry to embrace comprehensive sustainability measures during this 20-year period have improved the governance, technology, siting, and management in many cases.
Streptococcosis cause severe losses for global tilapia farming, especially in developing countries. The aim of this study was to identify and characterize streptococci recovered from Nile tilapia farmed in the Philippines. Moribund and apparently healthy fish were sampled from grow-out cages, ponds and hatcheries. Clinical signs observed included exophthalmia, eye opacity, ascites, lethargy, erratic swimming and haemorrhages. Results showed that both Streptococcus iniae and Streptococcus agalactiae were associated with disease in these sites. Consistent with global reports, including those from South-East Asia, S. agalactiae was more widespread than S. iniae. Molecular serotyping of the S. agalactiae isolates identified the serotype Ia and serotype Ib. Histopathological findings were meningitis, meningoencephalitis and septicaemia. Identical virulence profiles were found for all strains of S. iniae, while S. agalactiae strains were separated into virulence profile I and profile II. All strains were susceptible to the tested antibiotics and resistant to oxolinic acid. Only S. agalactiae serotype Ib showed resistance to sulphamethoxazole-trimethoprim. This is the first study from the Philippines to characterize the streptococci involved in disease outbreaks in tilapia aquaculture. Outputs from this study will promote the development of efficacious disease control strategies in tilapia farming for the Philippines and South-East Asia.
Here, 70 potential Vibrio pathogens belonging to nine species, dominated by Vibrio harveyi, were isolated and identified from diseased aquacultured marine fish in South China. Subsequently, the prevalence of 11 virulence genes and the resistance to 15 antibiotics in these strains were determined. Most strains possessed atypical virulence genes in addition to typical virulence genes. Notably, hflk and chiA originating from V. harveyi, and flaC associated with V. anguillarum were detected in more than 40% of atypical host strains. Multidrug resistance was widespread: 64.29% strains were resistant to more than three antibiotics, and the multi-antibiotic resistance index ranged from 0.00 to 0.60. The proportions of strains resistant to the antibiotics vancomycin, amoxicillin, midecamycin, and furazolidone all exceeded 50%; nevertheless, all strains were sensitive to florfenicol, norfloxacin, and ciprofloxacin. Furthermore, both virulence genes and antibiotic resistance were more prevalent in Hainan than in Guangdong, owing to the warmer climate and longer annual farming time in Hainan. These results therefore suggest that warming temperatures and overuse of antibiotics are probably enhancing antibiotic resistance and bacterial infection. This study reveals that pathogenic Vibrio spp. with multi-antibiotic resistance are highly prevalent among marine fish in South China and thus warrant further attention. The results will provide helpful guidance for ecological regulation and local antibiotic use in the control of marine fish farming’ Vibrio diseases in South China, facilitating the implementation of national green and healthful aquaculture.
Improving the reproducibility of biomedical research is a major challenge. Transparent and accurate reporting is vital to this process; it allows readers to assess the reliability of the findings and repeat or build upon the work of other researchers. The ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) were developed in 2010 to help authors and journals identify the minimum information necessary to report in publications describing in vivo experiments. Despite widespread endorsement by the scientific community, the impact of ARRIVE on the transparency of reporting in animal research publications has been limited. We have revised the ARRIVE guidelines to update them and facilitate their use in practice. The revised guidelines are published alongside this paper. This explanation and elaboration document was developed as part of the revision. It provides further information about each of the 21 items in ARRIVE 2.0, including the rationale and supporting evidence for their inclusion in the guidelines, elaboration of details to report, and examples of good reporting from the published literature. This document also covers advice and best practice in the design and conduct of animal studies to support researchers in improving standards from the start of the experimental design process through to publication.
Aim
The main objective of this study was to emphasize on histopathological examinations and molecular identification of Streptococcus agalactiae isolated from natural infections in hybrid tilapia (Oreochromis niloticus) in Temerloh Pahang, Malaysia, as well as to determine the susceptibility of the pathogen strains to various currently available antimicrobial agents.
Materials and Methods
The diseased fishes were observed for variable clinical signs including fin hemorrhages, alterations in behavior associated with erratic swimming, exophthalmia, and mortality. Tissue samples from the eyes, brain, kidney, liver, and spleen were taken for bacterial isolation. Identification of S. agalactiae was screened by biochemical methods and confirmed by VITEK 2 and 16S rRNA gene sequencing. The antibiogram profiling of the isolate was tested against 18 standard antibiotics included nitrofurantoin, flumequine, florfenicol, amoxylin, doxycycline, oleandomycin, tetracycline, ampicillin, lincomycin, colistin sulfate, oxolinic acid, novobiocin, spiramycin, erythromycin, fosfomycin, neomycin, gentamycin, and polymyxin B. The histopathological analysis of eyes, brain, liver, kidney, and spleen was observed for abnormalities related to S. agalactiae infection.
Results
The suspected colonies of S. agalactiae identified by biochemical methods was observed as Gram-positive chained cocci, β-hemolytic, and non-motile. The isolate was confirmed as S. agalactiae by VITEK 2 (99% similarity), reconfirmed by 16S rRNA gene sequencing (99% similarity) and deposited in GenBank with accession no. KT869025. The isolate was observed to be resistance to neomycin and gentamicin. The most consistent gross findings were marked hemorrhages, erosions of caudal fin, and exophthalmos. Microscopic examination confirmed the presence of marked congestion and infiltration of inflammatory cell in the eye, brain, kidney, liver, and spleen. Eye samples showed damage of the lens capsule, hyperemic and hemorrhagic choroid tissue, and retina hyperplasia accompanied with edema. Brain samples showed perivascular and pericellular edema and hemorrhages of the meninges. Kidney samples showed hemorrhage and thrombosis in the glomeruli and tubules along with atrophy in hematopoietic tissue. Liver samples showed congestion of the sinusoids and blood vessel, thrombosis of portal blood vessel, and vacuolar (fatty) degeneration of hepatocytes. Spleen samples showed large thrombus in the splenic blood vessel, multifocal hemosiderin deposition, congestion of blood vessels, and multifocal infiltration of macrophages.
Conclusion
Therefore, it can be concluded that pathological changes in tissues and organs of fish occur proportionally to the pathogen invasion, and because of their high resistance, neomycin and gentamicin utilization in the prophylaxis or treatment of S. agalactiae infection should be avoided.
The Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca) is a manually curated resource containing high quality reference data on the molecular basis of antimicrobial resistance (AMR), with an emphasis on the genes, proteins and mutations involved in AMR. CARD is ontologically structured, model centric, and spans the breadth of AMR drug classes and resistance mechanisms, including intrinsic, mutation-driven and acquired resistance. It is built upon the Antibiotic Resistance Ontology (ARO), a custom built, interconnected and hierarchical controlled vocabulary allowing advanced data sharing and organization. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for resistome prediction from raw genome sequence. Recent improvements include extensive curation of additional reference sequences and mutations, development of a unique Model Ontology and accompanying AMR detection models to power sequence analysis, new visualization tools, and expansion of the RGI for detection of emergent AMR threats. CARD curation is updated monthly based on an interplay of manual literature curation, computational text mining, and genome analysis.
The aim of this study was to develop a multiplex PCR amplification method that simultaneously detects the presence of four bacterial pathogens (Photobacterium damselae, V. alginolyticus, V. harveyi and V. parahaemolyticus), which are often synergistically caused disease to culture fish throughout the tropical waters, and occasionally cause food poisoning and
wound infection to human. Specific multiplex PCR primers targeting conserve regions of virulence genes of the pathogens were designed and tested against different concentrations of MgCl2 and annealing temperatures. In addition to specificity against different bacterial species, the multiplex PCR was also tested against tissue and environmental samples known to harbor the pathogens. The result showed that the multiplex PCR was highly specific to the target pathogens. The optimum MgCl2 concentration and annealing temperature for successful multiplex PCR amplification of the pathogens were at 5.0 mM and 56 °C, respectively. The detection limit of the multiplex PCR was at 10 pg of DNA template. Although the concentration of the pathogens in the environment is often lower, enrichment with tryptic soy broth supplemented with 2% NaCl (w/v) has shown to enhance the growth of the bacterial pathogens and hence improved detection. The rapidity, simplicity and cost-effectiveness of the multiplex PCR amplification method described in this paper provide a useful bio-security tool for the determination of the pathogens in aquaculture farms and seafood processing industries throughout the tropical countries.
Epidemiological cut-off values were developed for application to antibiotic susceptibility data for Flavobacterium psychrophilum generated by standard CLSI test protocols. The MIC values for ten antibiotic agents against Flavobacterium psychrophilum were determined in two laboratories. For five antibiotics, the data sets were of sufficient quality and quantity to allow the setting of valid epidemiological cut-off values. For these agents, the cut-off values, calculated by the application of the statistically based normalized resistance interpretation method, were ≤16 mg L−1 for erythromycin, ≤2 mg L−1 for florfenicol, ≤0.025 mg L−1 for oxolinic acid (OXO), ≤0.125 mg L−1 for oxytetracycline and ≤20 (1/19) mg L−1 for trimethoprim/sulphamethoxazole. For ampicillin and amoxicillin, the majority of putative wild-type observations were ‘off scale’, and therefore, statistically valid cut-off values could not be calculated. For ormetoprim/sulphadimethoxine, the data were excessively diverse and a valid cut-off could not be determined. For flumequine, the putative wild-type data were extremely skewed, and for enrofloxacin, there was inadequate separation in the MIC values for putative wild-type and non-wild-type strains. It is argued that the adoption of OXO as a class representative for the quinolone group would be a valid method of determining susceptibilities to these agents.
Conventional Aeromonas species identification based on biochemical methods is challenged by the heterogeneous nature of the species. Here we present a new multiplex PCR method directed towards gyrB and rpoB genes that identifies the four species A. hydrophila, A. media, A. veronii and A. caviae and application of the method on a Danish strain collection.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
The global rise in antimicrobial resistance (AMR) among bacteria causing infectious diseases is well documented and the associated risks for human health are well known. There is much less research on AMR with regard to environmental strains, both opportunistic and pathogenic ones. The genus Aeromonas is widely distributed in the environment and causes many variable diseases in fish and humans. Infections in humans are predominantly caused by A. veronii, A. hydrophila and A. caviae (A. punctata) in a form of bacteremia, gastroenteritis or even septicaemia in immunocompetent and immunocompromised individuals. Different groups of antibiotics are used in the treatment but studies indicate that fluoroquinolones and cefotaxime are the most efficient. A disturbing consequence of antibiotic overuse is an increasing number of detections of various antibiotic resistance genes (ARG) within this genus. The water environment is one of the major modes of transmission of resistant bacteria from animals to humans, and thus the dissemination of antibiotic resistance genes, particularly those located in mobile genetic elements (MGE) such as plasmids and transposons. This review summarizes recently published information on the type, distribution, and transmission of ARG by MGE, widespread in Aeromonas strains living in various aquatic environments, including wastewaters, natural waters, aquacultures and urban drinking water. The data available indicate that the opportunistic pathogens like Aeromonas spp. might serve as important vectors of ARG for clinically relevant pathogens present in the water bodies.
Abstract The ribosome and protein synthesis are major targets within the cell for inhibition by antibiotics, such as the tetracyclines. The tetracycline family of antibiotics represent a large and diverse group of compounds, ranging from the naturally produced chlortetracycline, introduced into medical usage in the 1940's, to second and third generation semi-synthetic derivatives of tetracycline, such as doxycycline, minocycline and more recently the glycylcycline tigecycline. Here we describe the mode of interaction of tetracyclines with the ribosome and mechanism of action of this class of antibiotics to inhibit translation. Additionally, we provide an overview of the diverse mechanisms by which bacteria obtain resistance to tetracyclines, ranging from efflux, drug modification, target mutation and the employment of specialized ribosome protection proteins.
The aim of this study was to evaluate antimicrobial susceptibility of Aeromonas spp. isolates from common carp and koi carp coming from randomly chosen farms. The isolates were tested for susceptibility to 8 antimicrobial agents using the standard agar dilution susceptibility test. In all isolates, PCR was used to detect the presence of tet(A-E) genes, integrase genes, and gene cassettes. From the total 72 isolates of motile aeromonads sampled from koi carp, 36 isolates (50%) were resistant to oxytetracycline, 18 (25%) to ciprofloxacin, 5 (7%) to chloramphenicol, 5 (7%) to florfenicol, and 11 (15%) to trimethoprim. Among 49 isolates of motile aeromonads collected from common carp, 20 (41%) were resistant to oxytetracycline, 3 (6%) to chloramphenicol, and 3 (6%) to florfenicol. The resistance of aeromonads isolated from koi carp was significantly higher to ciprofloxacin (P = 0.00024). The presence of class 1 integrons was detected in these isolates only (P = 0.00024). Tet genes were detected in 40% (48/121) of isolates, with tet(E) being the most dominant. Our results demonstrated a significant difference in the incidence of resistant isolates collected from koi carp and common carp (P = 0.00042). This difference can be ascribed to a distinct antibiotic policy established on consumer fish farms versus ornamental fish farms. The potential risk for resistant bacteria to spread and transmit infection to humans should be considered in cases of technological crossover between the two types of fish farms.
Resistance to quinolones and fluoroquinolones is being increasingly reported among human but also veterinary isolates during the last two to three decades, very likely as a consequence of the large clinical usage of those antibiotics. Even if the principle mechanisms of resistance to quinolones are chromosome-encoded, due to modifications of molecular targets (DNA gyrase and topoisomerase IV), decreased outer-membrane permeability (porin defect), and overexpression of naturally occurring efflux, the emergence of plasmid-mediated quinolone resistance (PMQR) has been reported since 1998. Although these PMQR determinants confer low-level resistance to quinolones and/or fluoroquinolones, they are a favorable background for selection of additional chromosome-encoded quinolone resistance mechanisms. Different transferable mechanisms have been identified, corresponding to the production of Qnr proteins, of the aminoglycoside acetyltransferase AAC(6′)-Ib-cr, or of the QepA-type or OqxAB-type efflux pumps. Qnr proteins protect target enzymes (DNA gyrase and type IV topoisomerase) from quinolone inhibition. The AAC(6′)-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. Finally, the QepA and OqxAB efflux pumps extrude fluoroquinolones from the bacterial cell. A series of studies have identified the environment to be a reservoir of PMQR genes, with farm animals and aquatic habitats being significantly involved. In addition, the origin of the qnr genes has been identified, corresponding to the waterborne species Shewanella sp. Altogether, the recent observations suggest that the aquatic environment might constitute the original source of PMQR genes, that would secondly spread among animal or human isolates.
To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human.
Species-specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co-existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube.
Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples.
This simple, rapid and cost-effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.
Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, β-lactamase-encoding genes, bla
TEM-1 and bla
CTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another.
The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk.
Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.
In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial
species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87°C for 84 clinical
and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection
by the real-time PCR method was 1 pg of purified genomic DNA or 102 V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection
to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed
that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched
samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines.
The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within
8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses
or outbreaks caused by this pathogen.
The frequent use of antibiotics in developed countries has led to the emergence of widespread bacterial resistance. In this study, the interindividual variability of the antibiotic susceptibility of 50 putative microorganisms in aggressive periodontitis patients has been evaluated by means of VC (variation coefficient).
A total of 60 microbial samples were collected from 20 adult patients diagnosed with aggressive periodontitis (2-4 samples by patient). Bacterial strains of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, and Peptostreptococcus micros were isolated according to Slots' rapid identification method. The susceptibilities to 10 antibiotics were studied: penicillin G (PEN), ampicillin (AMP), amoxicillin (AMX), amoxicillin/clavulanate (AMC), tetracycline (TET), doxycycline (DOX), ciprofloxacin (CIP), erythromycin (ERY), spiramycin (SPI) and clindamycin (CLIN), using the Disk Diffusion Susceptibility test (DDS test: Kirby-Bauer's modified method for anaerobic bacteria). The broth microdilution Minimum Inhibitory Concentration test was carried out as a control test.
Among the 50 identified bacteria, 15 were P. gingivalis, 12 P. intermedia, 8 T. forsythia, 9 F. nucleatum, and 6 P. micros. The results of the DDS test show that penicillins (especially AMC, AMP, and AMX), cyclines (especially DOX) and CLIN are highly effective against the 50 anaerobic studied bacteria. CIP and ERY have the lowest efficacy against those bacteria. CIP shows a very variable activity according to anaerobic bacteria species, being particularly inactive against P. gingivalis and very efficient against T. forsythia and P. micros. SPI is also highly efficient but not against P. micros.
The interindividual susceptibility of principal periodontal pathogens to antibiotics is not homogeneous and seems to vary according to bacterial species and antimicrobial molecules. This variability seems to be greater with older molecules (PEN, TET, ERY) than with more recent ones, which indicates more stable results (AMC, AMX, AMP, and DOX). P. intermedia appeared to be the bacteria most resistant to penicillins and showed the highest coefficient variation. Together with scaling and root planing, the combination of two antibiotics would therefore seem to be recommended in the treatment of aggressive periodontitis, particularly in the presence of P. intermedia.
An increasing incidence of multidrug resistance amongst Aeromonas spp. isolates, which are both fish pathogens and emerging opportunistic human pathogens, has been observed worldwide. This can be attributed to the horizontal transfer of mobile genetic elements, viz.: plasmids and class 1 integrons. The antimicrobial susceptibilities of 37 Aeromonas spp. isolates, from tilapia, trout and koi aquaculture systems, were determined by disc-diffusion testing. The plasmid content of each isolate was examined using the alkaline lysis protocol. Tet determinant type was determined by amplification using two degenerate primer sets and subsequent HaeIII restriction. The presence of integrons was determined by PCR amplification of three integrase genes, as well as gene cassettes, and the qacEDelta1-sulI region. Thirty-seven Aeromonas spp. isolates were differentiated into six species by aroA PCR-RFLP, i.e., A. veronii biovar sobria, A. hydrophila, A. encheleia, A. ichtiosoma, A. salmonicida, and A. media. High levels of resistance to tetracycline (78.3%), amoxicillin (89.2%), and augmentin (86.5%) were observed. Decreased susceptibility to erythromycin was observed for 67.6% of isolates. Although 45.9% of isolates displayed nalidixic acid resistance, majority of isolates were susceptible to the fluoroquinolones. The MAR index ranged from 0.12 to 0.59, with majority of isolates indicating high-risk contamination originating from humans or animals where antibiotics are often used. Plasmids were detected in 21 isolates, with 14 of the isolates displaying multiple plasmid profiles. Single and multiple class A family Tet determinants were observed in 27% and 48.7% of isolates, respectively, with Tet A being the most prevalent Tet determinant type. Class 1 integron and related structures were amplified and carried different combinations of the antibiotic resistance gene cassettes ant(3'')Ia, aac(6')Ia, dhfr1, oxa2a and/or pse1. Class 2 integrons were also amplified, but the associated resistance cassettes could not be identified. Integrons and Tet determinants were carried by 68.4% of isolates bearing plasmids, although it was not a strict association. These plasmids could potentially mobilize the integrons and Tet determinants, thus transferring antimicrobial resistance to other water-borne bacteria or possible human pathogens. The identification of a diversity of resistance genes in the absence of antibiotic selective pressure in Aeromonas spp. from aquaculture systems highlights the risk of these bacteria serving as a reservoir of resistance genes, which may be transferred to other bacteria in the aquaculture environment.
A total of 232 isolates of gram-negative bacteria were recovered from mammals, reptiles, and birds housed at Asa Zoological
Park, Hiroshima prefecture, Japan. Forty-nine isolates (21.1%) showed multidrug resistance phenotypes and harbored at least
one antimicrobial resistance gene. PCR and DNA sequencing identified class 1 and class 2 integrons and many β-lactamase-encoding
genes, in addition to a novel AmpC β-lactamase gene, blaCMY-26. Furthermore, the plasmid-mediated quinolone resistance genes qnr and aac(6′)-Ib-cr were also identified.
The main objective of this study was to determine the prevalence of the Qnr determinants in clinical and environmental Aeromonas spp. ...
Tetracycline-resistant (Tetr) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September
2000. Of the 66 Tetr gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tetr genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tetr strains transferred Tetr genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included
strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tetr strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.
The antibiotic oxytetracycline (OTC) has been widely used for therapeutic and preventive management of bacterial diseases in finfish and shellfish. In the present study the bioavailability, pharmacokinetics, and withdrawal period of the OTC have been determined following in-feed administration in intensively cultured catfish Pangasianodon hypophthalmus. Furthermore, the pharmacokinetic parameters of oral route were also compared with parenteral route. Drug concentrations were measured in various tissues at different time intervals by LC-MS/MS. The study revealed the drug kinetics best followed the enterohepatic circulation model with very poor bioavailability and low blood concentration after oral administration. In the withdrawal study, after 10-days of in-feed administration at the therapeutic dose the drug reached very high concentrations in the liver and kidneys but did not attain minimum inhibitory concentrations (MICs) in blood or flesh. OTC concentration also did not exceed the recommended MRL value in flesh; however, considering high amounts of the chemical in the liver and kidneys a withdrawal period of 4 days (at 28 ± 1.5 oC) is recommended for consumer safety. Poor bioavailability and non-attainment of minimum therapeutic concentration in blood and flesh do not warrant in-feed administration of OTC for control of bacterial diseases in P. hypophthalmus.
Availability of data and materials
All data generated and analyzed during this study are included in this article. Raw data may be shared upon reasonable request.
Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio harveyi are the causative agents of the most severe diseases of marine and brackish aquaculture systems. These are also associated with serious ailments in humans. The present paper unravels the virulence features/genes and antimicrobial resistance (AMR) phenotypes/genes of these Vibrio spp. isolated from natural disease outbreaks of marine/estuarine fishes of India for the first time. Results on virulence features showed that V. vulnificus infected fish can pose public health risk. While, it has been found clinically that V. parahaemolyticus without tdh and trh genes are pathogenic to fish, even though they are reported to be, not associated with human diseases. It was significant to note that virulence features of V. harveyi were induced at increased salinity. Analysis based on the percentage prevalence of susceptible isolates and variation coefficient of zone diameters categorized 17 antibiotics in terms of their efficiency against each fish pathogenic species. Multiple antibiotic resistance index (MARi) of the isolates ranged between 0.058 and 0.47. Results on MARi and percentage of multidrug resistance strains indicated that >50% of the isolates were from low antibiotic usage area. The study generated tetH sequence from V. parahaemolyticus for the first time, and the sequence revealed high identity to that of clinical strains. Presence of tetB/ tetH gene was identified as the predictor for the resistance against the first generation tetracycline, the most commonly used antibiotic against Vibrio spp. in aquaculture practices. The data on associations between AMR features predicted certain cross-resistance between antimicrobials within Vibrio spp. Altogether, the paper serves as the baseline for epizootic tracking of public health significant vibrios from diseased fishes, to devise practical guidelines for antibiotic use and to formulate efficient control measures against three Vibrio spp. in aquaculture, targeting final applications in the implementation of national green and healthy aquaculture practices.
The antimicrobial susceptibilities of Turkish F. psychrophilum isolates were investigated using a Clinical and Laboratory Standards Institute (CLSI) standard microdilution testing protocol. The significance of the minimum inhibitory concentrations was determined by applying empirically and statistically determined epidemiological cut-off values calculated by normalized resistance analysis. Twenty-five F. psychrophilum isolates made over four years from three different trout species in five different regions were examined. All isolates were fully susceptible to amoxicillin and florfenicol, but 88% of our isolates were categorized as having reduced susceptibility to the two quinolones, oxolinic acid, and enrofloxacin. Eighty percent of the isolates were categorized as showing reduced susceptibility to oxytetracycline.
Sequence analysis demonstrated that the reduced susceptibility to the quinolones was associated with chromosomal mutations in the gyrA and parE genes. PCR analysis demonstrated that the genes tetA, tetB, tetC, tetE, tetH, tetL, and tetM were not associated with the reduced susceptibility to oxytetracycline and the significance of this observation is discussed. PCR analysis also showed the presence of sul2 in six isolates and floR in one isolate. This study is the first report of these genes in F. psychrophilum. It was noted, however, that the floR was detected in an isolate categorized as fully susceptible to florfenicol, but the reasons for its lack of expression in the phenotype was not investigated.
It is argued that the Turkish trout industry, in common with those of many countries world-wide, is critically dependent on the continued susceptibility of F. psychrophilum to florfenicol.
Antimicrobial resistance is one of the major threats faced in aquaculture systems. Hence the present study is mainly focused on to determine the antimicrobial susceptibility associated with pathogens derived from diseased freshwater ornamental guppy fishes. Around fifteen isolates were resolved from the infected fishes and subjected to antimicrobial susceptibility testing, phenotypic and genotypic characterization. Disc diffusion method was adopted for checking the antibiotic susceptibility using 17 antibiotic discs belonging to different classes. Dendrogram generated 5 clusters based on the biochemical tests and representative isolates were sequenced and identified as Aeromonas hydrophila, Aeromonas sobria, Pseudomonas putida, Acinetobacter soli and Kurthia gibsonii. The Shannon wiener diversity index of the resolved isolates was found to be 1.395 as determined by Primer-E software. Among the isolates, the majority of them was found to be Pseudomonas putida and exhibited higher antibiotic resistance towards antibiotics of 10 classes including third generation Cephalosporin and others showed the same against antibiotics of at least 5 classes tested. All of the recovered isolates possessed the MAR index of greater than 0.2, indicating the heavier dose of antibiotics in the farm. The detection of plasmid-mediated class I integron in Aeromonas hydrophila, Aeromonas sobria and Acinetobacter soli indicated the possibility of heavier dissemination of antimicrobial resistant genes among the ornamental fishes. Gentamycin and Ciprofloxacin were significantly effective against all the isolates and can be successfully applied in aquaculture. The occurrence of antibiotic resistance reminds the proper surveillance and continuous monitoring programmes in the fish farms and usage of other effective alternatives.
This book puts emphasis on the isolation, taxonomy, diagnosis (phenotypic, serology and molecular biology), epizootiology, pathogenicity mechanisms, and methods of disease control (by vaccination, immunostimulation, probiotics, prebiotics, plant products, and antimicrobial compounds. Co-infections, which are attributed to more than one microbial species have been discussed. Shortcomings in knowledge have been highlighted. This sixth edition is the successor to the original version, first published in 1987, and which fills the need for an up-to-date comprehensive text on the biological aspects of the bacterial taxa which cause disease in finfish.
The book is primarily targeted at researcher workers, including postgraduate students, and diagnosticians. It is anticipated that the readership will include veterinary microbiologists, public health scientists and microbial ecologists.
Photobacterium damselae is a Gram negative bacterium causes photobacteriosis, a worldwide septicemic disease in aquaculture including sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). The pathogenicity of bacterial subspecies and the disease pathological changes in natural and experimental infections have thus far yielded inconsistency of effective preventive measures. This study aimed to represent a comprehensive analysis of the potential pathogenic capacities of the two subspecies of P. damselae in cultured sea bass and sea bream in the Northwestern region of Egypt. Diseased 321 sea bass and 257 sea bream, in addition to 99 healthy sea bass fingerlings were sampled from three farms located along the Mediterranean Sea. P. damselae subspecies were isolated from diseased fish and characterized using bacteriological, molecular, and antimicrobial susceptibility methods. Healthy fish were challenged by a virulent P. damselae subsp. piscicida, monitored for disease signs and mortality, and the histopathological abnormalities and hematological disorders were carried out. Clinical signs and gross lesions in naturally infected sea bass and sea bream showed great similarities with absence of a subspecies-specific characteristic sign or lesion. The two subspecies were recovered through the entire year from individual fish sample, suggests a coexistence of two subspecies endemic infection. In diseased sea bass, 38.32% and 16.20% were positive for P. damselae subsp. piscicida and subsp. damselae, respectively. However in diseased sea bream, 44.47% and 26.46% were positive for P. damselae subsp. piscicida and subsp. damselae, respectively. High mortalities and devastating clinicopathologic abnormalities represented by sever clinical signs, hematological disorders and histological abnormalities strengthen the pathogenicity of P. damselae subspecies in the two fish species and therefore, a vaccination strategy against both subspecies should be taken into account.
Quinolone antimicrobials are synthetic and widely used in clinical medicine. Resistance emerged with clinical use and became common in some bacterial pathogens. Mechanisms of resistance include two categories of mutation and acquisition of resistance-conferring genes. Resistance mutations in one or both of the two drug target enzymes, DNA gyrase and DNA topoisomerase IV, are commonly in a localized domain of the GyrA and ParE subunits of the respective enzymes and reduce drug binding to the enzyme-DNA complex. Other resistance mutations occur in regulatory genes that control the expression of native efflux pumps localized in the bacterial membrane(s). These pumps have broad substrate profiles that include quinolones as well as other antimicrobials, disinfectants, and dyes. Mutations of both types can accumulate with selection pressure and produce highly resistant strains. Resistance genes acquired on plasmids can confer low-level resistance that promotes the selection of mutational high-level resistance. Plasmid-encoded resistance is due to Qnr proteins that protect the target enzymes from quinolone action, one mutant aminoglycoside-modifying enzyme that also modifies certain quinolones, and mobile efflux pumps. Plasmids with these mechanisms often encode additional antimicrobial resistances and can transfer multidrug resistance that includes quinolones. Thus, the bacterial quinolone resistance armamentarium is large.
© 2015 New York Academy of Sciences.
We aimed to document the risk of Aeromonas spp. in marine shrimp species cultured in inland low salinity ponds in Thailand. In 14 of 18 shrimp samples retrieved from inland grow-up ponds, Aeromonas spp. were detected at ranges from 4667 to 1,500,000 CFU/g body weight. The phylogenetic tree constructed with the gyrB and cpn60 concatenated sequences indicated that the 87 isolates consisted of Aeromonas veronii (70%), Aeromonas aquariorum (18%), Aeromonas caviae (7%), Aeromonas jandaei (2%), and Aeromonas schubertii (2%). The potential virulence of the isolates was examined by phenotypic and PCR assays. Hemolytic activity and the extracellular activity of lipase, DNase, and gelatinase were observed in most isolates (94-99%). PCR revealed the presence of 9 genes related to virulence in the 87 isolates: act (75%), aer (74%), alt (30%), ast (1%), ascV (34%), aexT (24%), fla (92%), ela (34%), and lip (24%). The susceptibility profiles to 14 antimicrobial agents of isolates were typical for the genus, but resistance to cefotaxime, a third-generation cephalosporin, and imipenem were found in two A. aquariorum and in three A. veronii isolates, respectively. These resistances were confirmed by determining minimum inhibitory concentrations. Our results indicate that the microbiological risk posed by Aeromonas should be considered for marine shrimp species that are cultured in low-salinity ponds. These shrimps may also be a vehicle for the transfer of different genotypes of Aeromonas and antibiotic-resistant determinants to regions worldwide through trade.
Copyright © 2014 Elsevier Ltd. All rights reserved.
A comparison was made of the in vitro activities of five quinolone antibacterials (nalidixic acid, piromidic acid, oxolinic acid, flumiquine and miloxacin) currently used in aquaculture in Japan and five newer quinolones (ofloxacin, norfloxacin, enoxacin, ciprofloxacin and tosufloxacin), against selected fish bacterial pathogens. Of the earlier quinolones, flumiquine and oxolinic acid showed the highest bacteriostatic and bactericidal activities against Gram-negatives. The newer quinolones, however, showed considerably higher activities, with ciprofloxacin and tosufloxacin most active. The newer quinolones were also active against strains resistant to the earlier quinolones. Grampositive bacteria were resistant to all the earlier quinolones, but some strains were sensitive to the newer quinolones, particularly to ofloxacin, ciprofloxacin and tosufloxacin. The activities of the newer quinolones were found to be less affected by serum, pH or the addition of magnesium, than the earlier quinolones. The newer quinolones were considered to have potential for the treatment of Gram-negative, and possibly Gram-positive, bacterial fish pathogens.
We found increased numbers of oxytetracycline (OTC)-resistant bacteria in sediment and seawater around a marine aquaculture site after OTC therapy. Samples were collected at an aquaculture site along the coast of the Seto Inland Sea, Japan in 2004. In April, the percentage of bacteria resistant to 60 mu g mL(-1) OTC in the surface sediment was 6.8%-20.0%. The percentages increased during OTC therapy in the summer reaching 53.3%-60.7% in September. Ninety-two days after drug cessation, the percentages decreased to below 22.9%. Tet(M)-positive bacteria were detected in the sediment and seawater samples. Tet(M) was evident in both Gram-positive and Gram-negative bacteria from various genera, and was newly identified in Paenibacillus, Sporosarcina, Shewanella, and Pseudoalteromonas. The dominant tet(M)-positive isolates were strains of Vibrio suggesting that this genus is an important reservoir for tet(M) in the marine environment. Two different alleles were found, tet(M)-A and tet(M)-B, each in isolates from five genera. The data suggests drug therapy used in the aquaculture acted as a selective pressure promoting increased numbers of resistant bacteria.
Vibrio anguillarum, also known as Listonella anguillarum, is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. In both aquaculture and larviculture, this disease is responsible for severe economic losses worldwide. Because of its high morbidity and mortality rates, substantial research has been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. This review summarizes the current state of knowledge pertaining to V. anguillarum, focusing on pathogenesis, known virulence factors, diagnosis, prevention and treatment.
The goal of this study was to estimate the distribution of pathogens, as well as their antimicrobial resistance pattern, in cows affected by clinical or subclinical mastitis in the Rhône-Alpes region of France. A total of 1770 samples were taken between January 2007 and March 2008, leading to the identification of 1631 bacterial isolates. Streptococcus uberis (22.1%), Escherichia coli (16%), and coagulase-positive staphylococci (15.8%) were identified as the major causative agents of clinical mastitis, whereas coagulase-positive staphylococci (30.2%), coagulase-negative staphylococci (13.7%), and Streptococcus dysgalactiae (9.3%) were predominantly implicated in subclinical mastitis. Yet, in both types of mastitis, about 20% of all cases were due to a large number of different bacterial species that were isolated at a low frequency (<5%), which cannot be considered as minor (e.g., Klebsiella spp.) or noncontagious (e.g., Corynebacterium spp.). The overall proportion of antibiotic resistance was low, except for penicillin G in staphylococci, as well as for macrolides and tetracycline in streptococci. Yet, these resistance proportions were much lower than those reported in human medicine. Besides providing up-to-date information on mastitis in France, this survey also indicates the prudent use of antibiotics by veterinarians. As a result, this study suggests that the risk of transmission of resistant bacteria from milk or milk products to human is very limited, even in case of consumption of raw milk. However, it also confirms the fact that attention must be maintained to avoid any emergence of such resistant bacteria.
The aim of this study was to evaluate antimicrobial susceptibility of Aeromonas spp. isolates from common carp and koi carp coming from randomly chosen farms. The isolates were tested for susceptibility to 8 antimicrobial agents using the standard agar dilution susceptibility test. In all isolates, PCR was used to detect the presence of tet(A-E) genes, integrase genes, and gene cassettes. From the total 72 isolates of motile aeromonads sampled from koi carp, 36 isolates (50%) were resistant to oxytetracycline, 18 (25%) to ciprofloxacin, 5 (7%) to chloramphenicol, 5 (7%) to florfenicol, and 11 (15%) to trimethoprim. Among 49 isolates of motile aeromonads collected from common carp, 20 (41%) were resistant to oxytetracycline, 3 (6%) to chloramphenicol, and 3 (6%) to florfenicol. The resistance of aeromonads isolated from koi carp was significantly higher to ciprofloxacin (P=0.00024). The presence of class 1 integrons was detected in these isolates only (P=0.00024). Tet genes were detected in 40% (48/121) of isolates, with tet(E) being the most dominant. Our results demonstrated a significant difference in the incidence of resistant isolates collected from koi carp and common carp (P=0.00042). This difference can be ascribed to a distinct antibiotic policy established on consumer fish farms versus ornamental fish farms. The potential risk for resistant bacteria to spread and transmit infection to humans should be considered in cases of technological crossover between the two types of fish farms.
Resistance to quinolones and fluoroquinolones has been increasingly reported among human and veterinary isolates during the last three decades related to their wide clinical use. Until recently, the mechanisms of resistance to quinolones in Enterobacteriaceae were believed to be only chromosome-encoded, i.e. related to modifications of the molecular targets (DNA gyrase and topoisomerase IV), decreased outer-membrane permeability (porin defect) and overexpression of naturally-occurring efflux. However, emergence of plasmid-mediated quinolone resistance (PMQR) has been reported since 1998. Three mechanisms are known to date: Qnr proteins, aminoglycoside acetyltransferase AAC(6')-Ib-cr, and efflux pump QepA. The Qnr proteins protect DNA gyrase and type IV topoisomerase from quinolone inhibition. Four types of Qnr protiens have been reported: QnrA (six variants), QnrB (19 variants), QnrC (one variant), and QnrS (three variants). The AAC(6')-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. The protein AAC(6')-Ib-cr contains two amino acid substitutions as compared to the wild-type enzyme AAC(6')-Ib. Both Qnr and AAC(6')-Ib proteins have been reported worldwide. Lately reported, the plasmid-encoded QepA efflux pump may extrude hydrophilic fluoroquinolones (eg. norfloxacin, ciprofloxacin, and enrofloxacin).
A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
The chloramphenicol resistance gene (pp-cat) was cloned from a transferable R plasmid of Pasteurella piscicida, pSP9351, and the sequence of the gene was determined. Subcloning and deletion analysis localized the resistance gene, pp-cat, to within a 2.3 kb HincII-BamHI fragment. The fragment as a probe hybridized with the type I chloramphenicol acetyltransferase (CAT) gene and did not hybridize to CAT types II, III, and CAT-VA. The fragment hybridized to transferable R plasmids encoded with resistance to chloramphenicol, which were detected from P. piscicida isolated in different years. Nucleotide sequences of the coding and flanking regions of pp-cat (2031 bp) identified an open reading frame coding type I CAT of a molecular mass of about 25,000 Da. Comparison analysis of the sequences outside the cat open reading frame showed also that pp-cat has homology, in part, with the gene that coding for the endonuclease EcoRII and those that flank the cat gene derived from the Acinetobacter baumannii chromosome.
Enterococcus faecalis and E. faecium isolated from humans in the community (98 and 65 isolates), broilers (126 and 122), and pigs (102 and 88) during 1998 were tested for susceptibility to 12 different antimicrobial agents and for the presence of selected genes encoding resistance using PCR. Furthermore, the presence of vancomycin resistant enterococci was examined in 38 human stool samples using selective enrichment. Widespread resistance to chloramphenicol, macrolides, kanamycin, streptomycin, and tetracycline was found among isolates from all three sources. All E. faecium isolates from humans and pigs were susceptible to avilamycin, whereas 35% of isolates from broilers were resistant. All E. faecium isolates from humans were susceptible to vancomycin, whereas 10% and 17% of isolates from broilers and pigs, respectively, were resistant. A vancomycin resistant E. faecium isolate was found in one of the 38 human fecal samples examined using selective enrichment. All vancomycin resistant isolates contained the vanA gene, all chloramphenicol resistant isolates the cat(pIP501) gene, and all five gentamicin resistant isolates the aac6-aph2 gene. Sixty-one (85%) of 72 erythromycin resistant E. faecalis examined and 57 (90%) of 63 erythromycin resistant E. faecium isolates examined contained ermB. Forty (91%) of the kanamycin resistant E. faecalis and 18 (72%) of the kanamycin resistant E. faecium isolates contained aphA3. The tet(M) gene was found in 95% of the tetracycline resistant E. faecalis and E. faecium isolates of human and animal origin, examined. tet(K) was not observed, whereas tet(L) was detected in 17% of tetracycline resistant E. faecalis isolates and in 16% of the E. faecium isolates. tet(O) was not detected in any of the isolates from pigs, but was observed in 38% of E. faecalis isolates from broilers, in two E. faecalis isolates from humans and in three E. faecium isolates from broilers. tet(S) was not detected among isolates from animals, but was observed in 31% of E. faecalis and one E. faecium isolate from humans. This study showed a frequent occurrence of antimicrobial resistance and the presence of selected resistance genes in E. faecalis and E. faecium isolated from humans, broilers and pigs. Differences in the occurrence of resistance and tetracycline resistance genes were observed among isolates from the different sources. However, similar resistance patterns and resistance genes were detected frequently indicating that transmission of resistant enterococci or resistance genes takes place between humans, broilers, and pigs.
Specific primer pairs were selected for the PCR amplification of 14 tetracycline resistant genes commonly found in Gram positive and Gram negative organisms. Combinations of primer pairs were used in multiplex PCR reactions to detect specific groups of tet genes as follows; Group I tet (B), tet (C), tet (D); Group II tet (A), tet (E), tet (G); Group III tet (K), tet (L), tet (M), tet (O), tet (S); Group IV tetA (P), tet (Q), tet (X). To test the multiplex PCR, Groups I and II were used on 25 clinical isolates of Salmonella enterica serovar Typhimurium DT104. Group III primers were used to investigate 19 clinical isolates of methicillin-resistant Staphylococcus aureus. Multiplex PCR should result in significant savings in terms of labour and cost in analysis of a large number of strains when compared with using an individual PCR for targeting each gene. It may also be a useful method to differentiate the types of tetracycline resistance when used as an additional marker for the purpose of outbreak investigation and surveillance.
A growing inefficacy of antimicrobial agents to treat infectious diseases has stimulated research on the development of antibiotic resistance in bacteria in the environment. Sustained exposure of soil microorganisms to tetracycline (TC) in flow-through columns (50mg/L influent) significantly decreased the effluent concentration of total heterotrophs and selected for TC-resistant (Tet(r)) soil bacteria. This suggests that TC released to the environment from animal farms may contribute to the development and amplification of TC resistance, with soil bacteria serving as reservoirs for antibiotic resistance continuance. Burkholderia cepacia, with genetic determinants for efflux pumps that facilitate TC excretion, was the only bacterium that grew on TC-amended R2A plates. Following 300 days of exposure, TC was removed from the influent to study the recovery pattern of the microbial community. The percentage of Tet(r) hererotrophs decreased from 25% to close to the control level of 1% within 1 month of discontinuing TC exposure. This was due both to a significant rebound in the total heterotrophic population and to a significant decrease in the concentration of Tet(r) bacteria. Thus, discontinuing TC exposure or curtailing its use should enhance natural attenuation mechanisms that mitigate the spread of resistance vectors.
Antimicrobial resistance
- WHO