Luc Devriese

Antimicrobial Resistance of Old and Recent Staphylococcus aureus Isolates from Poultry: First Detection of Livestock-Associated Methicillin-Resistant Strain ST398

ABSTRACT The susceptibilities of 12 antimicrobial agents for two collections of Staphylococcus aureus, isolated in the 1970s and in 2006 from poultry, were determined. For eight antibiotics, the percentage of resistance was significantly higher in the recent isolates. Ten recent isolates were methicillin resistant and had spa types t011 and t567, belonging to multilocus sequence type 398. This is the first report of “livestock-associated” methicillin resistant S. aureus from healthy poultry.

Application and Evaluation of the Interlaboratory Reproducibility of tRNA Intergenic Length Polymorphism Analysis (tDNA-PCR) for Identification of Streptococcus Species

ABSTRACT The discriminatory power, speed, and interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) combined with capillary electrophoresis was evaluated for the identification of streptococci. This method was carried out in three different laboratories under highly standardized conditions for 54 strains belonging to 18 different species. It was concluded that interlaboratory reproducibility of tDNA fingerprints produced by means of capillary electrophoresis was sufficiently high to permit the exchange between different laboratories and the construction of common libraries which can be consulted for comparison with fingerprints obtained independently in separate laboratories. In a second step, 17 other species were included in the study and examined in one of the participating laboratories. All Streptococcus species studied, except S. mitis, S. oralis, S. parasanguinis, S. pneumoniae, S. thermophilus, and S. vestibularis, showed distinguishable tDNA fingerprints. A database of well-characterized strains was constructed to enable computer-aided identification of unknown streptococcal isolates.

The Gram-positive tonsillar and nasal flora of piglets before and after weaning

Aims:u2002To investigate Gram‐positive nasal and tonsillar microbial flora of piglets before and after weaning.

Rabbit staphylococcosis: difficult solutions for serious problems

Staphylococcus aureus infections are a major problem in rabbitries. The main manifestations are subcutaneous abscesses, mastitis, pododermatitis and septicaemia. Two patterns of infection can be distinguished. In the first type, clinical signs remain limited to a small number of rabbits in a flock. This type has little economic importance and is caused by low-virulence S. aureus strains. In the second type, the disease shows an epidemic spread. Consequences are poor production results, infertility and death. This leads to chronic problems and a subsequent decline in production. The latter type is caused by high-virulence strains. Biotyping, phage typing and RAPD typing contribute to the characterisation of high-virulence S. aureus strains. Administration of antibiotics, disinfection of the environment and vaccination are not able to solve the problems. Therefore, the only effective measure is to cull the entire flock and to restart with a new rabbit population after thorough disinfection. Limiting the introduction of new rabbits in existing rabbitries and reducing contacts between rabbitries to an absolute minimum are currently the only way to face this most difficult problem.

Identification of Lactobacillus species using tDNA-PCR.

tDNA intergenic spacer PCR (tDNA-PCR) using consensus primers complementary to the conserved edges of the tRNA genes can amplify the intergenic spacers. Separation of the PCR products with capillary electrophoresis enables discrimination between fragments differing only one basepair in length. This method was applied to a collection of 82 Lactobacillus strains belonging to 37 species in order to evaluate the discriminatory power of this technique within this genus. Twenty-one species could be distinguished to species level on the basis of a unique tDNA fingerprint pattern. The other species grouped by two (e.g. L. fermentum and L. cellobiosus) or three (L. acidophilus, L. gallinarum and L. helveticus). Inclusion of the resulting fingerprints in a numerical database containing fingerprints of numerous other Gram-positive and Gram-negative species makes the identification of unknown strains possible.

Identification and antimicrobial susceptibility of Staphylococcus chromogenes isolates from intramammary infections of dairy cows.

Staphylococcus chromogenes is a highly prevalent species in subclinical mastitis with a well-established impact on somatic cell count. Few data are available on its antimicrobial susceptibility. The objective of this study was three-fold: (1) to evaluate simple identification tests by comparing them with a genomic method; (2) to determine minimal inhibitory concentrations (MICs) of different antibiotics; (3) to search for the presence of important resistance mechanisms and resistance-determining genes.Seventy-three staphylococcal strains, all collected on different dairy farms, were tentatively identified as S. chromogenes based on their lack of hemolysis and their characteristic intermediate DNase activity. The identification of 70 strains was confirmed as S. chromogenes by tRNA intergenic spacer PCR (tRNA PCR). Three strains were identified as S. sciuri, a species that is naturally cloxacillin- and lincomycin-resistant. All 70 S. chromogenes strains were found to be normally susceptible to neomycin, gentamicin, erythromycin, enrofloxacin, and to penicillinase-stable penicillins and cephalosporins, represented in this study by cloxacillin. The latter result was confirmed by the absence of the mecA gene in each of 13 strains in which this gene was searched for. Twenty-seven (38%) strains were penicillinase producers. Three lincomycin-resistant S. chromogenes strains were found to carry the linA gene. It was concluded that S. chromogenes can be identified reliably in routine mastitis bacteriology, and that the only resistance of importance is against penicillinase-susceptible penicillins.

Screening of genes encoding adhesion factors and biofilm formation in Staphylococcus aureus isolates from poultry

Staphylococcus aureus isolates recovered from poultry between 1970 and 1972 (90 old isolates) and in 2006 (81 recent isolates) were screened for the presence of bap, icaA and icaD genes associated with biofilm formation, and for bbp, cna, ebpS, eno, fib, fnbA, fnbB, clfA and clfB genes that encode microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Most of the old isolates were collected from broiler breeders affected by staphylococcal tenosynovitis and arthritis, whilst nearly all recent isolates were derived from the nose and cloaca of healthy broilers. Ten recent isolates belonged to the animal-associated methicillin-resistant S. aureus (MRSA) ST398. All isolates were positive for clfA, clfB, eno and fnbA, and were negative for bap and bbp. All recent isolates were also positive for ebpS and cna, and all old isolates were positive for fib. The average number of genes encoding adhesins and biofilm-associated proteins in both groups was eight per isolate. All MRSA ST398 isolates in this study were positive for icaD, cna, ebpS, eno, fnbA, fnbB, clfA and clfB. No relevant differences were found between the presence of adhesin and biofilm formation genes in old and recent S. aureus isolates or in isolates from healthy compared with diseased chickens. From this study, there is no indication that the presence of these genes has changed over time and no specific association could be found between the presence of certain MSCRAMM or biofilm genes in poultry S. aureus isolates and the isolates capacity to cause disease.

Comparison of the efficacy of four antimicrobial treatment schemes against experimental Ornithobacterium rhinotracheale infection in turkey poults pre-infected with avian pneumovirus

The clinical efficacy of drinking-water administration of enrofloxacin for 3 and 5 days, amoxicillin for 5 days and florfenicol for 5 days for the treatment of respiratory disease induced by an experimental Ornithobacterium rhinotracheale infection in turkeys pre-infected with avian pneumovirus (APV) was assessed based on clinical, bacteriological and histopathological examinations. Experimental groups of 15 susceptible 3-week-old turkeys were each inoculated oculonasally with APV subtype A and 3 days later with susceptible O. rhinotracheale bacteria. Antimicrobial treatment started 1 day after O. rhinotracheale inoculation. After infection, the birds were examined and scored for clinical signs, swabbed daily and weighed at different times. Five birds were euthanized and examined for macroscopic lesions at necropsy at 5 days post bacterial inoculation, and the remainder at 15 days post bacterial inoculation. Samples of the turbinates, trachea, lungs, air sacs, heart and pericardium were collected for bacteriological and/or histological examination. Recovery from respiratory disease caused by an APV/O. rhinotracheale dual infection was most successful after enrofloxacin treatment, irrespective of treatment duration, followed by florfenicol. Amoxicillin treatment was not efficacious. Clinical signs and the number of O. rhinotracheale organisms re-isolated from the trachea and the different respiratory organs were significantly reduced by enrofloxacin treatment for 3 and 5 days. O. rhinotracheale bacteria were not re-isolated from the tracheas of the birds treated with enrofloxacin except for one bird in the 5-day group, as early as 1 day after medication onset. In the group treated with enrofloxacin for 5 days, O. rhinotracheale organisms with a higher minimal inhibitory concentration value (×8) were isolated starting 2 days following treatment onset, initially from a single turkey and subsequently from the other animals. Comparaison de lefficacité de quatre traitements antibiotiques vis-à-vis dune infection expérimentale à Ornithobacterium rhinotracheale chez des dindonneaux pré infectés par un pneumovirus aviaire Lefficacité clinique de ladministration dans leau de boisson, de lenrofloxacine pendant 3 et 5 jours, de lamoxicilline pendant 5 jours et du florfénicol pendant 5 jours pour le traitement dune maladie respiratoire induite par une infection expérimentale à Ornithobacterium rhinotracheale (ORT) chez des dindes pré infectées avec un pneumovirus aviaire (AVP) a été évalué à laide dexamens cliniques bactériologiques et histopathologiques. Des groupes de 15 dindes sensibles, âgées de 3 semaines, ont été inoculées par voie oculonasale avec un AVP de type A et 3 jours plus tard avec un ORT sensible. Le traitement antibiotique a démarré le 1er jour après linoculation de l ORT. Après linfection, les symptômes ont été enregistrés et des écouvillons ont été réalisées tous les jours, de plus les animaux ont été pesés à différents moments. Cinq jours après linoculation de la bactérie (dpbi) 5 animaux ont été euthanasiés puis autopsiés et les lésons macroscopiques ont été recherchées. Il en a été de même sur les survivants 15 dpbi. Les échantillons de cornets nasaux, trachée, poumons, sacs aériens, cæur, et de péricarde ont été prélevés pour des examens bactériologiques et/ou histologiques. Suite à la maladie respiratoire causée par une double infection à APV/ORT, le traitement ayant donné les meilleurs résultats de guérison a été lenrofloxacine, quelle que soit la durée du traitement, suivi par le florefénicol. Le traitement à lamoxicilline na pas été efficace. Les symptômes et le nombre de germes dORT ré isolés de la trachée et des différents organes respiratoires ont été significativement réduits après les traitements à lenrofloxacine pendant 3 et 5 jours. ORT na pas été réisolé des trachées des oiseaux traités à lenrofloxacine à lexception dun sujet dans le groupe traité durant 5 jours, juste le jour après le début du traitement. Dans le groupe traité à lenrofloxacine durant 5 jours, des germes dORT présentant des valeurs de concentration minimale inhibitrice plus élevées (x 8) ont été isolés à partir du deuxième jour après le début du traitement, à partir dun seul sujet puis ensuite à partir des autres sujets. Vergleich der Wirksamkeit von vier antimikrobiellen Behandlungsschemata gegen eine experimentelle Infektion mit Ornithobacterium rhinotracheale von Putenküken nach vorheriger Infektion mit aviärem Pneumovirus Die klinische Wirksamkeit einer Medikation mit Enrofloxacin für 3 oder 5 Tage, Amoxycillin für 5 Tage und Florfenicol für 5 Tage über das Trinkwasser als Behandlung einer durch eine experimentelle Infektion mit Ornithobacterium rhinotracheale (ORT) verursachte Respirationserkrankung in Puten, die vorher mit aviärem Pneumovirus (APV) infiziert worden waren, wurde durch klinische, bakteriologische und histopathologische Untersuchungen ermittelt. Versuchsgruppen mit jeweils 15 empfänglichen dreiwöchigen Putenküken wurden okulonasal mit APV-Subtyp A und drei Tage später mit empfindlichen ORT-Bakterien infiziert. Die antimikrobielle Behandlung begann einen Tag nach der ORT-Inokulation. Nach der Infektion wurden die Küken auf klinische Veränderungen untersucht, täglich mittels Tupferabstrichen beprobt und in bestimmten Abständen gewogen. Fünf Tiere pro Gruppe wurden 5 Tage nach der bakteriellen Infektion (dpbi) und die restlichen Tiere 15 dpbi euthanasiert und seziert. Für die bakteriologische und/oder histologische Untersuchung wurden Proben von Nasenmuscheln, Trachea, Lunge, Luftsäcke, Herz und Perikard entnommen. Eine Genesung von der durch eine Doppelinfektion mit APV/ORT verursachten Respirationserkrankung trat am schnellsten nach der Enrofloxacin-Behandlung unabhängig von der Behandlungsdauer ein, gefolgt von Florfenicol. Die Amoxicillin-Behandlung war nicht erfolgreich. Die klinischen Symptome und die Zahl der ORT-Organismen, die aus der Trachea und den verschiedenen Respirationgsorganen isoliert wurden, konnten durch die drei- oder fünftägige Enrofloxacin-Behandlung signifikant reduziert werden. ORT-Bakterien wurden mit einer Ausnahme in der 5-Tage-Gruppe am ersten Behandlungstag nicht aus den Tracheen der Enrofloxacin-behandelten Putenküken reisoliert. In der Gruppe, die 5 Tage lang mit Enrofloxacin behandelt wurde, konnten ab dem zweiten. Behandlungstag anfangs aus einer und nachfolgend auch aus den anderen Küken ORT-Organismen mit einem 8-fach höheren minimalen Hemmkonzentration-Wert isoliert werden. Comparación de la eficacia de cuatro tratamientos antibióticos frente a la infección experimental con Ornithobacterium rhinotracheale en pavos previamente infectados con pneumovirus aviar Se evaluó la eficacia clínica de la administración de enrofloxacina durante 3 y 5 días, amoxicilina durante 5 días y florfenicol durante 5 días en agua de bebida como tratamientos de la enfermedad respiratoria causada por la infección experimental de Ornithobacterium rhinotracheale (ORT) en pavos previamente infectados con pneumovirus aviar (APV) en base a observaciones clínicas, bacteriológicas e histopatológicas. Los grupos experimentales formados por 15 pavos susceptibles de 3 semanas de edad se inocularon vía oculonasal con APV del subtipo A y tres días más tarde con bacteria ORT susceptible. Los tratamientos antibióticos se iniciaron un día después de la inoculación de ORT. Tras la infección, las aves se examinaron, se valoraron sus signos clínicos y se tomaron hisopos diariamente y se pesaron en distintas ocasiones. Se sacrificaron y se examinaron las lesiones macroscópicas en la necropsia de 5 aves a los 5 días post inoculación bacteriana (dpbi) y de las restantes a los 15 dpbi. Se tomaron muestras de cornetes nasales, tráquea, pulmones, sacos aéreos, corazón y pericardio para estudios bacteriológico y/o histológico. La recuperación de la enfermedad respiratoria causada por la infección mixta de APV/ORT fue más satisfactoria tras el tratamiento con enrofloxacina, independientemente de la duración de este tratamiento, seguido por el tratamiento con cloranfenicol. El tratamiento con amoxicilina no fue eficaz. Los signos clínicos y el número de organismos ORT reaislados de la tráquea y de distintos órganos respiratorios se redujo de manera significativa con el tratamiento con enrofloxacina durante 3 y 5 días. No se reaislaron bacterias ORT de tráqueas de aves tratadas con enrofloxacina incluso en períodos tan breves de medicación como un día, a excepción de un ave en el grupo de 5 días. A partir de los dos días de tratamiento, en el grupo tratado con enrofloxacina, solamente se aislaron organismos ORT con mayores (x8) concentraciones mínimas inhibitorias (MIC) en un pavo, y posteriormente en el resto de los animales.

Identification of a new biotype of Actinomyces hyovaginalis in tissues of pigs during diagnostic bacteriological examination

Bacterial strains isolated from a large variety of necropsy samples of pigs and previously described as a phenotypical homogeneous group were shown to belong to the species Actinomyces hyovaginalis. This was unexpected because their colonial characteristics, as well as their origins, were very different from those originally reported for the vaginal strains on which the species description of A. hyovaginalis was based. Colonial morphology, as well as fermentation of cellobiose, reactions in hippurate and nitrate and production of beta-glucuronidase, allowed separation of the strains studied here from the vaginal strains. Analysis of tRNA intergenic length polymorphisms (tDNA-PCR), 16S rRNA-gene sequencing and DNA-DNA hybridizations were carried out and led to the proposal of a separate biotype within the species A. hyovaginalis. Since, the strains were isolated from different body sites, this biotype has been designated as the general biotype of A. hyovaginalis, while the strains on which the original species description was based are designated as the vaginal biotype.