Enhanced growth and decreased mortality in Atlantic salmon (Salmo salar) after probiotic bath.

AIMS: Disease in farmed Atlantic salmon occurs in all its life stages. Salmon are particularly vulnerable to infectious diseases at transition from the freshwater stage to the saltwater stage. Our aim in these studies reported was to investigate the possibility that waterborne delivery of a probiotic comprised of naturally occurring marine bacterial species would reduce the mortality and improve the health and growth of farmed Atlantic salmon. METHODS AND RESULTS: In three trials at two aquaculture production sites in Norway, isolates of Aliivibrio bacteria were added to the rearing water of Atlantic salmon. The fish were followed in 4-6 months after one single bath with observations and samplings. Growth, ulcers and survival were recorded. At the end of the studies growth was up to 31% larger in the probiotic enhanced groups and in trial 1 both mortality and prevalence of ulcer were significantly lower in the probiotic enhanced group compared to the control. Feed conversion rates were recorded in trial 1 and 2 and were from 9 to 28 % better for the probiotic enhanced groups compared to the control groups. CONCLUSION: Bathing of Atlantic salmon with probiotic Aliivibrio strains increased growth, reduced mortality and improved FCR in the postsmolt period. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the potential to enhance growth, prevent ulcers and decrease mortality in Atlantic salmon after adding probiotic strains of Aliivibrio spp. into the rearing water. The study can have impact on animal welfare, economy and sustainability in the aquaculture industry.

The future of therapeutic agents in aquaculture.

The implementation and monitoring of fish health regulations vary extensively in aquaculture throughout the world. In the main salmon-producing countries, there is strict regulatory oversight of the use of pharmaceutical drugs. Such controls have supported the sustainable growth of the aquaculture industry and, in Norway, aquaculture has been able to reduce its total consumption of antibiotics by more than 99% between 1995 and today, yet there has been a 20-fold rise in production volume. Other countries on other continents may have less control, with no mandatory prescription regulations and variable quality of the pharmaceutical products available. A good regulatory framework, with control and monitoring systems, should be established in all countries where aquaculture is practised and veterinary medicinal products should only be available under veterinary prescription and supervision. Many drug resistance genes have been identified, and molecular methods should be applied to control drug resistance in the microbial and parasitic populations of all major aquaculture-producing regions.

Les niveaux de mise en place et de suivi des réglementations applicables à la santé des poissons en aquaculture sont extrêmement variables d'une région à l'autre. Les pays où la salmoniculture prédomine pratiquent un contrôle réglementaire strict de l'utilisation de produits médicamenteux. Ces contrôles ont permis une croissance durable du secteur ; ainsi, en Norvège l'aquaculture a pu réduire sa consommation totale d'antibiotiques de plus de 99 % depuis 1995 tout en multipliant par vingt le volume de production. Le niveau des contrôles est parfois moindre dans d'autres pays d'autres continents, où aucune réglementation n'impose l'obligation d'une prescription vétérinaire et où les produits pharmaceutiques disponibles sont de qualité variable. Tous les pays ayant une production aquacole devraient mettre en place un cadre réglementaire solide doté de systèmes de contrôle et de suivi ; par ailleurs, les produits pharmaceutiques vétérinaires ne devraient pouvoir être obtenus que sous prescription et supervision vétérinaires. Plusieurs gènes responsables de résistances aux agents médicamenteux ont été identifiés ; il conviendrait que toutes les régions dédiées à l'aquaculture recourent à des méthodes moléculaires pour contrôler le phénomène de la résistance aux agents médicamenteux dans les populations de microbes et de parasites.

La aplicación de reglamentos de salud piscícola y las actividades de control reglamentario en la acuicultura varían sobremanera según el lugar del mundo de que se trate. En los principales países productores de salmón, el uso de productos farmacéuticos está sujeto a una estricta supervisión reglamentaria. Estos controles han favorecido el crecimiento sostenible del sector de la acuicultura, sector que en Noruega ha logrado reducir su consumo total de antibióticos en más de un 99% entre 1995 y la actualidad, pese a haber multiplicado por veinte su producción. En otros países de otros continentes hay a veces un menor control, debido a la ausencia de reglamentos de carácter prescriptivo y a la irregular calidad de los productos farmacéuticos disponibles. Sería menester que todos los países donde se practica la acuicultura contaran con un buen ordenamiento reglamentario, dotado de sistemas de control y seguimiento, y que en ellos solo se pudieran obtener productos de uso veterinario con receta y bajo supervisión veterinarias. Sabiendo que se han encontrado muchos genes que determinan resistencia a los medicamentos, convendría aplicar métodos moleculares para controlar la aparición de farmacorresistencias en las poblaciones microbianas y parasitarias de todas las regiones que albergan un importante sector acuícola.

Cold-water vibriosis. The current status of knowledge.

The current review for the first time summarizes the findings of the 30 years of research on cold-water vibriosis (CWV). The diseased caused by Aliivibrio salmonicida (earlier known as Vibrio salmonicida) was for the first time described in 1986 and became one of the most important bacterial diseases in salmon aquaculture. The lack of appropriate vaccine hampered development of Atlantic salmon aquaculture until the late 1980s when a novel vaccine allowed dramatic increase in the Atlantic salmon farming. In December 2007, the genus Vibrio was split into two genera and several bacterial species including V. salmonicida were transferred to genus Aliivibrio. The change of the names create significant difficulties with the designation of the CWV disease agent since its abbreviation A. salmonicida became similar to another well-known salmon pathogen Aeromonas salmonicida (A. salmonicida). The disease was considered as controlled by vaccination, but reappeared at Atlantic salmon farms in 2011, this time affecting vaccinated Atlantic salmon. The current review summarizes the knowledge on pathogenesis, vaccination and treatment of CWV and proposes further directions for studying the disease.

The environmental and host-associated bacterial microbiota of Arctic seawater-farmed Atlantic salmon with ulcerative disorders.

The Norwegian aquaculture of Atlantic salmon (Salmo salar L.) is hampered by ulcerative disorders associated with bacterial infections. Chronic ulceration may provide microenvironments that disturb the normal microbial biodiversity of external surfaces. Studying the composition of microbial communities in skin ulcers will enhance our understanding of ulcer aetiology. To achieve this, we tested marine farmed Atlantic salmon and sampled the base and edge of ulcers at the end of winter (April) and end of summer (September), in addition to skin mucus of healthy individuals. In order to assess microbiota associated with the host and obtain insight into the environmental ecology, we also sampled sea water, the sediment layer underneath the farm facility and the distal intestine of Atlantic salmon. The skin microbiota of Atlantic salmon was different from that of the surrounding water. Residential Tenacibaculum and Arcobacter species persistently dominated the cutaneous skin and ulcer mucus surfaces of Atlantic salmon during both winter and summer periods. The intestinal microbiota was dominated by Mycoplasma with an increase in Aliivibrio and Alcaligenes abundance in the intestine of fish with ulcerative disorder at the end of winter. These findings suggest the presence of resilient microbes in the mucus surfaces of Atlantic salmon.

vapA (A-layer) typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes.

Sequence variation in a region of the virulence array protein gene (vapA; A-layer) was assessed in 333 ('typical' and 'atypical') isolates of the fish pathogenic bacterium Aeromonas salmonicida. Resulting similarity dendrograms revealed extensive heterogeneity, with nearly all isolates belonging to either of 14 distinct clusters or A-layer types. All acknowledged A. salmonicida subspecies (except ssp. pectinolytica, from which no vapA sequence could be obtained) were clearly separated, and notably, all isolates phenotypically identified as ssp. salmonicida formed a distinct and exclusive A-layer type. Additionally, an array of un-subspeciated atypical strains formed several equally prominent clusters, demonstrating that the concept of typical/atypical A. salmonicida is inappropriate for describing the high degree of diversity evidently occurring outside ssp. salmonicida. Most representatives assessed in this study were clinical isolates of spatiotemporally diverse origins, and were derived from a variety of hosts. We observed that from several fish species or families, isolates predominantly belonged to certain A-layer types, possibly indicating a need for host-/A-layer type-specific A. salmonicida vaccines. All in all, A-layer typing shows promise as an inexpensive and rapid means of unambiguously distinguishing clinically relevant A. salmonicida subspecies, as well as presently un-subspeciated atypical strains.

Multilocus variable number tandem repeat analysis of Edwardsiella piscicida isolates pathogenic to fish.

This study describes a novel multilocus variable number tandem repeat analysis (MLVA) based on six variable number of tandem repeat (VNTR) loci for genotyping of 37 Edwardsiella piscicida (previously Edwardsiella tarda) isolates from multiple sources. The number of alleles identified for each of the six VNTR loci ranged from 3 to 5 with VNTR loci 1 (DI = 0.632) and 3 (DI = 0.644), displaying the highest degrees of polymorphism. MLVA typing of the 37 E. piscicida isolates resulted in the identification of five major clusters consistent with their geographical origins, and were designated as MLVA types I, II, III, IV and V. Types III and V were resolved further into subtypes largely consistent with outbreak source. An MLVA profile comprising a string of integers representing the number of tandem repeats for each allele provided a unique identification for each MLVA type and/or strain. The MLVA protocol described in the current study is robust, relatively simple, has a higher power of resolution than multilocus sequence analysis (MLSA) and is capable of discriminating closely related isolates.

Edwardsiella piscicida sp. nov., a novel species pathogenic to fish.

AIMS: This study describes a novel species within the genus Edwardsiella based on phenotypic and genetic characterization of fish pathogenic Edwardsiella isolates previously identified as E. tarda. METHODS AND RESULTS: Phenotypic characterization, DNA-DNA hybridization and phylogenetic analysis of representative Edwardsiella isolates from fish previously identified as E. tarda were conducted and compared with E. tarda type strain (ATCC 15947(T)). Phenotypically, strains from fish grow with pin-point colonies producing slight ß-haemolysis under the colony. In contrast to the E. tarda type strain, fish strains did not [corrected] degrade ß-methyl-D-glucoside [corrected] (with the exception of NCIMB 2034), citric acid and L-proline. [corrected]. With the exception of strain ETK01, all fish strains were highly pathogenic to zebra fish, while ATCC 15947(T) and NCIMB 2034 were nonpathogenic. DNA-DNA hybridization (DDH) levels between representative fish isolates and the E. tarda type strain ranged from 15 to 43·6%, while NCIMB 2034 hybridised with the type strain at the level of 63·2%. DDH values between the various fish isolates ranged from 68·2 to 93·9% defining a new and separate DNA hybridization group differing from the E. tarda type strain consistent with the findings of phylogenetic analysis, in which the fish isolates comprised a separate clade. CONCLUSIONS: Phenotypical and genetic characterizations demonstrated that Edwardsiella isolates from fish described in this study do not belong to the species E. tarda or any of the previously established taxa within the genus Edwardsiella. The fish related strains studied here (excluding NCIMB 2034) represent, therefore, a novel species within the genus Edwardsiella for which we propose the name Edwardsiella piscicida sp. nov, with strain ET883(T) (NCIMB 14824(T) = CCUG 62929) as the type strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The current finding will improve the diagnosis, understanding of the epidemiology and in establishment of effective control measures against this serious fish pathogen.

Tenacibaculum sp. associated with winter ulcers in sea-reared Atlantic salmon Salmo salar.

Coldwater-associated ulcers, i.e. winter ulcers, in seawater-reared Atlantic salmon Salmo salar L. have been reported in Norway since the late 1980s, and Moritella viscosa has been established as an important factor in the pathogenesis of this condition. As routine histopathological examination of winter ulcer cases in our laboratory revealed frequent presence in ulcers of long, slender rods clearly different from M. viscosa, a closer study focusing on these bacteria was conducted. Field cases of winter ulcers during 2 sampling periods, 1996 and 2004-2005, were investigated and long, slender rods were observed by histopathological examination in 70 and 62.5% of the ulcers examined, respectively, whereas cultivation on marine agar resulted in the isolation of yellow-pigmented colonies with long rods from 3 and 13% of the ulcers only. The isolates could be separated into 2 groups, both identified as belonging to the genus Tenacibaculum based on phenotypic characterization and 16S rRNA sequencing. Bath challenge for 7 h confirmed the ability of Group 1 bacterium to produce skin and cornea ulcers. In fish already suffering from M. viscosa-induced ulcers, co-infection with the Group 1 bacterium was established within 1 h. Ulcers from field cases of winter ulcers and from the transmission experiments tested positive by immunohistochemistry with polyclonal antiserum against the Group 1 bacterium but not the Group 2 bacterium. Our results strongly indicate the importance of the Group 1 bacterium in the pathogenesis of winter ulcers in Norway. The bacterium is difficult to isolate and is therefore likely to be underdiagnosed based on cultivation only.

Atlantic salmon bath challenged with Moritella viscosa--pathogen invasion and host response.

The Gram-negative bacterium Moritella viscosa is considered to be the main causative agent of winter ulcer, a disease that primarily affects salmonid fish in sea water during cold periods. The disease is initially characterised by localised swelling of the skin followed by development of lesions. To gain more knowledge of the role of M. viscosa in the pathogenesis of winter ulcer, 159 Atlantic salmon (80-110 g) were exposed to a bath challenge dose of 7 x 10(5) cfu ml(-1) for 1 h at 8.9 degrees C. The first mortalities were registered two days post-challenge and the mortality rate increased rapidly. Multi-organ samples were taken throughout the challenge for culture, immunohistochemistry and PCR analysis. Using real-time PCR, M. viscosa DNA was first detected in the gills of all fish examined 2, 6 and 12 h after challenge. From day 2, the bacterium was detected in the muscle/skin, head kidney, spleen and liver. This was in correlation with positive cultured samples and confirmed systemic infection. The early and consistent detection of M. viscosa DNA in gill samples, and less or not in muscle/skin or intestine, could suggest gills as a port of entry for the bacterium. Immunohistochemical analysis using a polyclonal antiserum against M. viscosa demonstrated generalised staining in the lumen of blood vessels and some positive mononuclear cells. The antigens recognised by the antiserum may have originated from extracellular bacterial products and be part of a bacterial invasion strategy. To better understand the immune response in salmon to M. viscosa infection, the expression profiles of the immune genes IL1 beta, C3, ISG15 and CD83 were studied. Increased expression of IL1 beta and C3 was not induced until day 7, which may suggest that M. viscosa might utilize escape mechanisms to evade the host's immune system by suppressing relevant immune responses.

Zinc oxide enriched peat influence Escherichia coli infection related diarrhea, growth rates, serum and tissue zinc levels in Norwegian piglets around weaning: five case herd trials.

BACKGROUND: Zinc oxide (ZnO), commonly used to control post-weaning diarrhea in piglets, has been highlighted as of potential concern from an environmental perspective. The aim of this field trial was to examine effects of different sources and levels of ZnO added to peat on average daily weight gain (ADG), fecal score in pens and serum and tissue zinc (Zn) levels around time of weaning in order to reduce the environmental impact without loss of the beneficial effect of ZnO on intestinal health and growth. Five case herds with enterotoxic colibacillosis challenges were included. The piglets entered the study aged three or five weeks. All piglets received a commercial diet containing <150 mg Zn/ per kg of complete feed. Four treatment groups received commercial peat added A: uncoated ZnO, B: lipid microencapsulated ZnO, C: solely commercial peat or D: no peat (Farms 2 and 3). RESULTS: At Farms 1, 2 and 3, a significant effect of treatment was identified for fecal score (P < 0.05). Treatment A led to lower fecal scores compared to treatments C (P < 0.05) and D (P < 0.01). At Farms 2 and 3, there was a significant difference in individual average daily weight gain (iADG) between treatment A and D (P < 0.05). The iADG of piglets receiving treatment B did not differ significantly from treatment A. CONCLUSIONS: In 2016, The European Medicines Agency's Committee on Veterinary Medicinal Products concluded that the benefits of ZnO for the prevention of diarrhea in pigs do not outweigh the risks to the environment. Effective alternative measures to reduce the accumulation of Zn in the environment have not been identified. Our results imply that peat added low concentration of both coated and uncoated ZnO influences the gut health of weaned piglets reflected by enhanced weight gain and reduced occurrence of diarrhea. This preventive approach certainly represents a favourable alternative in the "One Health" perspective. It will also contribute to reduced antibiotic use in pig farming while diminishing the environmental consequences caused by ZnO.

Cloning, characterisation and phylogenetic analysis of the fur gene in Vibrio salmonicida and Vibrio logei.

The gene encoding the ferric uptake regulator protein (fur gene) of Vibrio salmonicida 87/09/1193 was located following hybridisation of an EcoRI digest of chromosomal V. salmonicida DNA with a 316 base pairs (bp) probe internal to the fur gene of Vibrio anguillarum. A 2088 bp fragment including an open reading frame of 441 bp, encoding a protein of 147 amino acids, and homologous with fur, was identified, cloned and sequenced. A plasmid bound V. salmonicida fur gene was found capable of complementing the fur mutation of Escherichia coli H1681. Although no 'iron-box' was identified upstream of the start-codon, beta-galactosidase activity in E. coli H1681 was regulated by iron availability in the media, indicating that in V. salmonicida fur, as in other fur genes, iron functions as a co-repressor. Southern blot hybridizations demonstrated that fur is conserved amongst V. salmonicida strains and several other closely related Vibrio strains in which fur remains as yet, uncharacterized. The fur gene of Vibrio logei NCIMB 2252 was subsequently amplified using polymerase chain reaction primers external to the V. salmonicida fur gene. Comparison of phylogenetic analyses using fur and 16S DNA coding for rRNA sequences, confirmed the usefulness of fur as an evolutionary marker within the genus Vibrio.

A unique plasmid profile characterizing Salmonella enteritidis isolates from patients and employees in a hospital.

Plasmid profiling was used as an epidemiological tool during a period of frequent Salmonella enteritidis infection in a hospital. S. enteritidis was isolated from 22 patients and employees. Isolates from 18 persons harbored one 29 and one 36 megadalton (MDa) plasmid. The 29 MDa plasmid has not been previously described in this species and was not found in 54 control strains of S. enteritidis from other sources. The respective restriction endonuclease digest fragments of the 36 and the 29 MDa plasmids were always identical. This plasmid pattern thus served as a marker for the isolates from the outbreak.

Self-transmissible multidrug resistance plasmids in Escherichia coli of the normal intestinal flora of healthy swine.

The resistance genes and their surroundings on three self-transmissible plasmids found in Escherichia coli of the enteric normal flora of healthy pigs have been characterized. The resistance elements found are similar to those commonly found in clinical isolates, like the transposon Tn1721 including the Tet A tetracycline resistance determinant, Tn10 with the Tet B determinant, Tn21 including a class 1 integron with the aadA1a cassette inserted, sulII encoding sulfonamide resistance, and the strA-strB genes responsible for streptomycin resistance. The plasmids were able to mobilize into various recipients, including swine pathogens, zoonotic bacteria, and commensals when conjugation experiments were carried out. Transfer of plasmids did not require optimal conditions concerning nutrition and temperature as plasmids were transferred in 0.9% saline at room temperature, suggesting that in vivo transfer might be possible. This study shows that transferable resistance elements appearing in normal flora bacteria from animals are similar to those commonly found in clinical isolates of human origin. The results indicate a probable communication between pathogens and the normal flora with respect to exchange of resistance factors.

Characterization of integrons in Escherichia coli of the normal intestinal flora of swine.

Multiresistant Escherichia coli isolates of the normal intestinal flora of healthy fattening pigs were examined for the presence of integron class 1 by XL (extra long) PCR. The class 1 integron was detected in 17 isolates originating from 14 healthy animals on seven different farms. One isolate contained two class 1 integrons. The inserted gene cassettes were characterized by DNA sequencing and PCR. The ant(3")-Ia gene responsible for resistance to streptomycin/spectinomycin was inserted in all integrons detected. Fifteen isolates contained this gene cassette as the only inserted cassette. Three isolates contained integrons with two gene cassettes. Two isolates contained integrons with the trimethoprim resistance gene dfr1 and one isolate contained the oxa1 beta-lactamase gene upstream to the ant(3")-Ia gene. Detection of these three different resistance gene cassettes in bacteria from swine shows that cassettes occurring in integrons in human clinical isolates also appear in bacteria of the normal intestinal flora of healthy swine. Two integron-harboring strains were obtained from each of three different animals. These strains were probably not clonal derivatives of each other, suggesting the existence of different multiresistance clones within the intestinal normal flora of one specific animal. The oxa1 nucleotide sequence found in E. coli from swine differ by seven nucleotides from the oxa1 nucleotide sequence of the gene from the R-plasmid RGN238. The fact that these two sequences are not identical might indicate that the two genes have evolved separately in different surroundings from the common ancestor. Transmissible plasmids of approximately 200 kb containing integron class I were detected in eight of the isolates when conjugation experiments were performed with E. coli DH5 as recipient strain. The transfer frequency ranged from 4x10(-4) to 6x10(-2) transconjugants per recipient cell. This study shows that the enteric commensals of domestic animals may be considered as a reservoir of integron-containing transmissible plasmids and gene cassettes that might be transferable to the pathogens of swine and to important zoonotic bacteria associated with the enteric flora of swine such as Salmonella typhimurium DT104.

Antibiotic resistance in Escherichia coli of the normal intestinal flora of swine.

Twelve hundred enterobacterial Escherichia coli isolates of porcine origin were screened phenotypically for antibiotic resistance. The bacteria were isolated from 10 herds of swine with different histories of exposure to antimicrobial agents for therapeutic purposes. The bacterial isolates were part of the normal bacterial flora of the intestines of the animals because they were isolated from healthy individuals. The strains were tested for phenotypic antibiotic resistance against sulfonamides, trimethoprim, streptomycin, ampicillin, neomycin, chloramphenicol, and tetracycline. Resistance against streptomycin was found to be most common, followed by resistance against sulfonamides and tetracycline. The highest number of resistant bacteria was found in herds where the use of antimicrobial agents was considered to be high. A selection of multiresistant bacterial isolates were further genetically characterized by hybridization with probes specific for the antibiotic resistance genes; sulI, sulII, dfrI, dfrIIb, dfrIX, and the class A, B, C, and D tetracycline resistance determinants. A PCR was developed and used for detection of the strA-strB gene pair encoding streptomycin resistance in gram-negative bacteria. The strA-strB gene pair was the most frequent resistance determinant in the isolates examined. This study indicates that nonpathogenic E. coli from swine may represent a considerable reservoir of antibiotic resistance genes that might be transferable to pathogens.

Comparison of genes involved in penicillin resistance in staphylococci of bovine origin.

Ten penicillin-resistant and -susceptible staphylococci, isolated from bovine mastitis milk, were studied for the presence of genes that are, or may be, involved in resistance against penicillin. The repressor (blaI), antirepressor (blaR1), and structural (blaZ) genes of the beta-lactamase-operon were found to be closely linked in all penicillin-resistant strains. The beta-lactamase gene cluster was more commonly located on chromosomal rather than plasmid DNA in the strains studied. The transposase (p480) gene, which has been identified in the Staphylococcus aureus beta-lactamase transposon Tn552, was found in only one single penicillin-resistant S. aureus strain. The other penicillin-resistant S. aureus isolates contained IS1181 in close location with the beta-lactamase gene cluster. In only one S. haemolyticus isolate was the beta-lactamase gene cluster found in close association with IS257. Penicillin-resistant S. aureus strains, which were additionally resistant to tetracycline, contained IS257 in close association with the tetracycline resistance gene (tetK). Sequence analysis of blaI, blaR1, and blaZ in two penicillin-resistant S. aureus strains revealed 94-96% sequence homology with bla in staphylococci of human origin. The results indicate a predominance of class I bla transposons rather than Tn3 family class II transposons in the isolates used in this study.

Class 1 integrons mediate antibiotic resistance in the fish pathogen Aeromonas salmonicida worldwide.

The presence of class 1 integrons was investigated in 38 sulfonamide-resistant strains of Aeromonas salmonicida subsp. salmonicida, atypical A. salmonicida and Escherichia coli conjugants with R plasmids originating from A. salmonicida. The strains originated from Finland, France, Japan, Norway, Scotland, Switzerland, and the United States. Additional resistance determinants in strains with class 1 integrons were also determined. Of 21 strains containing a class 1 integron, 19 had a single gene cassette, 1 strain had two cassettes, and 1 strain was found to lack an integrated gene cassette. In the integrons with single cassettes, aadA2 was present in eight strains, dfr16 in five strains, and aadA1 and dfrIIc in three strains each. In the integron with two cassettes, qacG and orfD were present. Tetracycline resistance was observed in 20 of the integron-positive strains, caused by the determinants Tet A and Tet E, in which Tet A frequently was associated with Tn1721. Class 1 integrons seem to be important in mediating antibiotic resistance also in the marine environment. The gene cassettes reported in this study are all described in bacteria associated with humans, and this demonstrates once more how the common gene pool is shared between organisms belonging to different environments.

Genetic linkage between resistance to quaternary ammonium compounds and beta-lactam antibiotics in food-related Staphylococcus spp.

Little is known about the occurrence of antimicrobial resistance determinants in staphylococci isolated from food and food processing industries. Quaternary ammonium compound (QAC)-resistant coagulase-negative staphylococci (CNS) isolated from food and food-processing industries were investigated for the presence of genetic determinants (qacA/B and qacC/smr) encoding resistance to the QAC benzalkonium chloride (BC), several antibiotic resistance genes, and staphylococcal insertion sequences IS257 and IS256. Six qacA/B-harboring strains were resistant to penicillin and hybridized to a blaZ probe. The qacA/B and blaZ probes hybridized to plasmids of similar size in three isolates. Molecular and genetic characterization of the 23-kb plasmid (pST6) of Staphylococcus epidermidis St.6 revealed the presence of qacB adjacent to an incomplete beta-lactamase transposon Tn552 encoding the gene cluster blaZ, blaR, and blaI. Sequence analysis of flanking regions and the intergenic region between blaZ and qacB revealed the presence of IS257 downstream of blaZ as well as sin and binR between blaZ and qacB. In the three other BC and penicillin-resistant strains, the qacA/B and blaZ genes were located on separate plasmids. A qacC harboring S. epidermidis strain (St.17) also hybridized to tetK (tetracycline resistance) and ermB (erythromycin resistance) genes. The individual genes were located on separate plasmids, suggesting no linkage between QAC and antibiotic resistance determinants. Plasmid-free Staphylococcus aureus RN4220 allowed uptake of the pST6 plasmid DNA, indicating that the resistance genes could potentially be transferred to pathogens under selective stress. In conclusion, presence of both resistance determinants could lead to co-selection during antimicrobial therapy or disinfection in hospitals or in food industries.

A transferable multiple drug resistance plasmid from Vibrio cholerae O1.

Ten multiple antimicrobial-resistant isolates of Vibrio cholerae O1 isolated from patients in Uganda were characterized, and the transferability of resistance to bacteria of diverse origins was investigated. The isolates were toxigenic and belonged to biotype E1 Tor, serotype Ogawa, and ribotype 8, and possessed a 130-MDa plasmid of incompatibility group 6-C. This plasmid, designated pRVC1, was shown to confer resistance to trimethoprim (mediated by a dhfrI gene), sulfonamides (a suII gene), tetracycline [a tet(C) gene], chloramphenicol (a catI gene), ampicillin (a beta-lactamase gene other than blaTEM or blaSHV), and streptomycin. pRVC1 proved to be transmissible at frequencies between 1 x 10(-1) and 5 x 10(-6) transconjugants per recipient to a variety of bacterial pathogens, including those of humans, animals, and fish. Most efficient transfer was observed from V. cholerae to strains of Shigella flexneri, Escherichia coli, Vibrio parahaemolyticus, and three Aeromonas species. The present in vitro study suggests that pRVC1 may spread from V. cholerae to other bacteria pathogenic to man, animals, and fish in natural environments.

Use of magnetic beads for Gram staining of bacteria in aqueous suspension.

A Gram staining technique was developed using monodisperse magnetic beads in concentrating bacteria in suspension for downstream application. The technique does not require heat fixation of organisms, electrical power, or a microscope. Gram-negative and Gram-positive bacteria were identified macroscopically based on the colour of the suspension. The bacteria concentrated on magnetic beads may also be identified microscopically.