Yves Millemann

Carbapenemase-producing Acinetobacter spp. in Cattle, France.

To the Editor: Multidrug resistance in bacteria isolated from animals is an emerging phenomenon, mirroring what is happening among humans. During the past decade, expanded-spectrum β-lactamases in Enterobacteriaceae from humans (1) and animals (2) worldwide have been reported. Among humans, as a consequence of this high rate, use of carbepenems is increasing selection pressure; carbapenem-resistant gram-negative organisms are increasingly reported, including carbapenemase-producing Enterobacteriaceae and Acinetobacter spp (3). The most commonly acquired carbapenemases identified in Acinetobacter spp. correspond to carbapenem-hydrolyzing class D β-lactamases (3). In particular, the worldwide spread of OXA-23–producing A. baumannii is considered a serious threat; those strains are frequently involved in nosocomial outbreaks for which therapeutic options are extremely limited (3,4). Our study objective was to evaluate the possible occurrence of carbapenemase-producing gram-negative bacteria in dairy cattle in France. In August 2010, at a dairy farm 30 km from Paris, France, rectal swabs were collected from 50 cows. Samples were precultured in buffered peptone water and incubated for 18 h at 37°C. Cultures were inoculated by streaking 100 μL of the suspensions onto Drigalski agar plates (bioMerieux, Balmes-les-Grottes, France) containing 1 μg/mL of imipenem to select for carbapenem-resistant gram-negative isolates. Of the 50 samples, 9 produced growth on imipenem-containing plates. All colonies tested (10 colonies/sample) by using the API 20 NE (bioMerieux) system were first identified as A. lwoffii. Molecular techniques based on sequencing of the gyrA, gyrB, and rpoB genes (5) enabled more precise identification and indicated that all isolates belonged to the Acinetobacter genomospecies (DNA group) 15TU, which is known to be phylogenetically related to A. lwoffii and which has been reportedly isolated from sewage, freshwater aquaculture habitats, trout intestines, and frozen shrimp (6). One colony per sample was retained for further investigation (isolates BY1 to BY9). Susceptibility testing and MIC determinations were performed by disk-diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, France) and Etest (AB bioMerieux, Solna, Sweden) (Table). All isolates except 1 were resistant to penicillins, combinations of penicillins and β-lactamase inhibitors, and carbapenems but susceptible to cefotaxime and of reduced susceptibility to ceftazidime. Isolate BY1 showed higher MICs for carbapenems (Table). In addition, all isolates were resistant to tetracycline, kanamycin, and fosfomycin and remained susceptible to fluoroquinolones, chloramphenicol, gentamicin, amikacin, tobramycin, and sulfonamides. Susceptibility profiles of 3 Acinetobacter genomospecies 15TU reference strains showed that they were fully susceptible to penicillins, carbapenems, tetracycline, and kanamycin. Table Antimicrobial drug MICs for Acinetobacter genomospecies 15TU isolates from cows and reference strains, France, August 2010 Clonal diversity between the isolates was assessed by pulsed-field gel electrophoresis (5), which showed 6 distinct genotypes. Isolate BY1 corresponded to a single clone (data not shown), which indicated that the occurrence of Acinetobacter genomospecies 15TU strains among these animals was not the result of dissemination of a single clone. PCR detection and sequencing of genes that encode carbapenem-hydrolyzing class D β-lactamases (5) showed that the 9 Acinetobacter genomospecies 15TU isolates harbored a blaOXA-23 gene, whereas the 3 reference strains remained negative. Sequencing confirmed that all isolates expressed β-lactamase OXA-23, which is known to be widespread in A. baumannii. Mating-out assays and plasmid electroporation assays were performed by using blaOXA-23–positive Acinetobacter spp. isolates as donors and rifampin-resistant A. baumannii BM4547 isolates as a recipient strain (5); however, these assays were unsuccessful. Plasmid DNA analysis (5) gave uninterpretable results, with DNA degradations. The genetic structures surrounding the blaOXA-23 gene were investigated by PCR mapping (7), which identified transposon Tn2008 in isolate BY2 only. Tn2008 is a major vehicle for the spread of the blaOXA-23 gene in A. baumannii in the People’s Republic of China (8) and the United States (9). In the other isolates, the ISAba1 element of Tn2008 had been truncated by a novel insertion sequence termed ISAcsp2 (www-is.biotoul.fr). The dairy farmer indicated that most animals from which OXA-23 producers had been identified had received antimicrobial drugs in the previous weeks. Although 1 animal had received amoxicillin-clavulanate, most of the others had been given oxytetracycline and neomycin to treat mastitis. β-lactamase OXA-23 is a common source of carbapenem resistance in A. baumannii (5). Infections with multidrug-resistant OXA-23–producing A. baumannii or A. junii have been reported from hospitals but not from the community. Our study showed that OXA-23–producers in particular, and carbapenemase producers in general, may be isolated from animals. Among the hypotheses that could explain the selection of this carbapenemase, use of penicillins or penicillin–β-lactamase inhibitor combinations could create selective pressure for β-lactamases because OXA-23 does confer, in addition to decreased susceptibility to carbapenems, a high level of resistance to those compounds. We have previously shown that A. radioresistens, an environmental species, was the progenitor of the blaOXA-23 gene (10). Studies are needed to determine to what extent and at which locations Acinetobacter genomospecies 15TU and A. radioresistens might co-reside and therefore where the blaOXA-23 gene exchange might have occurred.

A comparison of antimicrobial usage in human and veterinary medicine in France from 1999 to 2005

OBJECTIVES The antimicrobials allowed and amounts sold in veterinary and human medicine in France were compared to see if the same antimicrobial drugs are used in veterinary and human medicine, and to the same extent. METHODS Registers of all approved antimicrobial commercial products, kept by the French Agency for Veterinary Medicinal Products (AFSSA ANMV) for animals and the French Health Products Safety Agency (AFSSAPS) for humans, were compared to determine whether the same antimicrobials were approved in 2007 for use in both human and animal populations. Sales data were collected from pharmaceutical companies between 1999 and 2005 by the AFSSA ANMV and AFSSAPS. Usage of the different antimicrobial anatomical therapeutic chemical (ATC) classes in human and veterinary medicines was recorded. Data were expressed in tonnes of active ingredients and were then related to the animal and human biomasses to compare usages expressed in mg/kg. RESULTS All antimicrobial ATC classes were used in both human and veterinary medicine. Tetracyclines accounted for the most sales in veterinary medicine. beta-Lactams predominated in human medicine. A decrease in the amounts consumed by both human and animal populations was observed during the study. In 2005, 760 tonnes were used in human medicine and 1320 tonnes in veterinary medicine, corresponding to 199 and 84 mg/kg of live weight in human and animal populations, respectively. CONCLUSIONS The same antimicrobial drugs were used in human and veterinary medicines but the quantitative patterns of use were different. Expression of antimicrobial usage is a key point to address when comparing usage trends.

Evaluation of humoral response and protective efficacy of two inactivated vaccines against bluetongue virus after vaccination of goats.

Bluetongue serotype 8 has become a major animal health issue in the European Union and the European member States have agreed on a vaccination strategy, which involves only inactivated vaccines. In this study, the efficacy of two inactivated vaccines against bluetongue virus serotype 8 (BTV-8) used in Europe since 2008, BTVPUR ALSAP(®) 8 (MERIAL) and BOVILIS(®) BTV8 (Intervet/SP-AH), was evaluated in goats immunized and challenged with BTV-8 field isolates under experimental conditions. Serological, virological and clinical examinations were conducted before and after challenge. Three groups of 10 goats each (groups A, B and C) were randomly constituted and 2 groups (A and C) were subcutaneously vaccinated twice with one dose of the two commercial vaccines BTVPUR ALSAP 8 (group A) or BOVILIS BTV8 (group C) respectively. Animals of the groups A, C and B (B: controls) were challenged with a virulent inoculum containing BTV-8. During the experiment, it was found out that the BTV-8 challenge inoculum was contaminated with another BTV serotype. However, results demonstrated that vaccination of goats with two injections of BTVPUR ALSAP 8 or BOVILIS BTV8 provided a significant clinical protection against a BTV-8 challenge and completely prevented BTV-8 viraemia in all vaccinated animals. Qualitative data showed no difference in the kinetics and levels of the humoral response induced by these two inactivated vaccines.

Ceftriaxone-resistant salmonella enterica serotype Newport, France.

The multidrug-resistant (MDR) Salmonella enterica serotype Newport strain that produces CMY-2 beta-lactamase (Newport MDR-AmpC) was the source of sporadic cases and outbreaks in humans in France during 2000-2005. Because this strain was not detected in food animals, it was most likely introduced into France through imported food products.

Epidemiological analysis of Salmonella enterica from beef sampled in the slaughterhouse and retailers in Dakar (Senegal) using pulsed-field gel electrophoresis and antibiotic susceptibility testing

Seventy-eight isolates of Salmonella spp. isolated from beef sampled from the official city slaughterhouse and from retailers in Dakar, Senegal were analyzed using serotyping, antimicrobial testing and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (PFGE). These analyses were done to identify clonal relationships and potential transmission routes in beef channel. XbaI macrorestriction allowed defining 17 genotypes among the six main analyzed serotypes: Salmonella bredeney (3 genotypes), S. muenster (6), S. waycross (1), S. corvallis (3), S. kentucky (1) and S. brandenburg (3). The cross analysis of PFGE profiles and origin of the beef samples reveals a wide range of contamination sources in the beef channel in Dakar. Comparison of PFGE and antimicrobial resistance types shows that the Salmonella contamination sources are equally shared by the slaughterhouse (56% of the isolates) and by the distribution channel (44% of the isolates) by handlings and houseflies.

Characteristics of Salmonella contamination of broilers and slaughterhouses in the region of Constantine (Algeria).

The present study provides the first data about the prevalence of Salmonella contamination of broilers and slaughterhouses in the region of Constantine, Algeria. The serotypes and anti‐microbial resistance phenotypes of the isolates were determined, and risk factors contributing to the contamination were evaluated. A total of 2490 samples, 1800 originating from 30 broiler farms and 690 from 15 slaughterhouses, were taken during two periods: March 2005–June 2006 and September 2006–March 2007. Salmonella contamination concerned 37% of the broiler farms and 53% of the slaughterhouses. Among the 55 isolates recovered, 10 different serotypes were identified. The most frequently recovered serotypes in both slaughterhouses and breeder farms were S. Hadar (36%, n = 20), S. Virchow (16%, n = 9), S. Infantis (10.9%, n = 6), S. Albany (11%, n = 6) and S. Carnac (7%, n = 4). Isolates belonging to S. Heidelberg (2%, n = 1) and S. Rissen (2%, n = 1) were found only in farms, while those belonging to S. Typhimurium (9%, n = 5), S. Enteritidis (4%, n = 2) and S. Montevideo (2%, n = 1) were recovered only from slaughterhouses. Thirty‐nine isolates (80%) were resistant to at least one anti‐microbial and 51% were multi‐resistant, i.e. resistant to two or more anti‐microbial molecules. About 58% (n = 32) were resistant to streptomycin, 36% (n = 20) to tetracyclines, 27% (n = 15) to nalidixic acid, 13% (n = 7) to ofloxacin and one isolate to enrofloxacin. Finally, seven distinct anti‐microbial resistance profiles were identified. In parallel, four risk factors were found to be significantly associated with Salmonella contamination. Together with the huge spread of Salmonella in the broiler production chain in Constantine, Algeria, these risk factors highlight the hazards of the broiler channels, particularly linked to poor technical and hygiene practices.

Extended-Spectrum β-Lactamase CTX-M-15-Producing Klebsiella pneumoniae of Sequence Type ST274 in Companion Animals

ABSTRACT Screening of extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacteria in companion animals living in the Paris area in France identified a high rate of CTX-M-15-producing Klebsiella pneumoniae. Those isolates were recovered during the 2010-2011 period from both infections and asymptomatic colonizations. Sequence typing revealed that most of these isolates belonged to sequence type ST274. Interestingly, the blaCTX-M-15 gene was located on a specific and novel plasmid scaffold. These findings highlight that companion animals may be reservoirs for CTX-M-15-producing K. pneumoniae evolving separately from the human reservoir of CTX-M-15 producers.

Ocular and cardiac malformations associated with maternal hypovitaminosis A in cattle

VITAMIN A deficiency in cattle usually occurs in France as outbreaks in beef cattle fed a ration with little green forage, especially when no mineral and vitamin supplement is provided. Clinical signs are mainly seen in male cattle (Paulsen and others 1989) and may vary: blindness, exophthalmia or dilatation of the pupil can occur, with good body condition and normal temperature (Meyus and others 2003). Convulsions, ataxia, stillbirths, poor growth and diarrhoea have also been associated with vitamin A deficiency (Divers and others 1986, Booth and others 1987, Paulsen and others 1989). This condition has also recently been associated with the birth to heifers of calves exhibiting ocular and cardiac abnormalities (Mason and others 2003). This short communication describes an ‘outbreak’ of marked ocular deformations in suckling calves, born to heifers or cows that were deficient in vitamin A. During November 2003, a small Charolais herd experienced the consecutive births of four animals presenting with ocular malformations and/or apparent blindness. The farm, in Burgundy, comprised 16 Charolais dams and approximately 200 ha of land on which cereals were grown. The calving period lasted from October 15 until the end of February each year and artificial insemination was never used, and heifers were presented to the bull at the end of February. The farmer rarely bought in animals, although he had replaced the bull one year previously. On average, a new bull was bought every three years. Animals at pasture also received hay; when indoors, they were fed maize silage, beet pulp pellets, and hay at night. Calves were fed alfalfa. Each year, new calves were sold in October. Deworming was based on the use of fenbendazole (Panacur; Intervet) once a year, at housing, although it had not yet taken place at the time of the investigation. Newborn calves remained with their dams. There was possible contact with two neighbouring farms: one dairy farm and one farm with suckling cows. The farm with the problems had a meadow without a neighbouring meadow, and one meadow with a double enclosure. All the animals had been housed, since November 1, in an old building that had been transformed into a free-housing stable. Cows and heifers were kept in separate houses but received the same feed. The bull was in an individual box. Any sick animals had been separated from others (since November 1) and placed in another building 200 m away. Two abortions had occurred in 2002. The initial hypotheses to explain the cases of blindness were bovine viral diarrhoea (BVD) virus in utero infection, hypovitaminosis A or an inherited condition, as has been described in Hereford cattle (Barkyoumb and Leipold 1984). As the offspring of two successive bulls were affected, the genetic hypothesis was dismissed. The farm was visited on November 22. The farmer reported that two or three sporadic cases of blindness had occurred several years previously. No clinical signs other than ocular signs were noticed, except for the abortions. A serological investigation for Brucella species, BVD virus, Chlamydia species and bovine respiratory syncytial virus had been carried out in 2002, without any positive result. There were no problems in the animals with regard to growth (good body condition), dermatology (coat quality) or balance (no ataxia). The four affected animals were a two-year-old heifer, which was blind without any visible ocular lesion, a one-yearold heifer with ocular malformations, an apparently blind four-month-old male calf and a three-week-old calf (calf 4) (Table 1). Another calf exhibiting ocular lesions had died the previous week. Their appetite and body conditions were not affected. The range of clinical signs can be seen in Fig 1. Other clinical signs included the absence of a menace reflex, but there was no observation of any dilated pupils or papilloedema. Complementary to the examination, investigation for antibodies to BVD virus and for BVD virus antigens was undertaken in the four affected animals as well as in their dams. All results were negative. Calf 4 was hospitalised in the Alfort National Veterinary School on December 1. Blood was sampled in order to verify its BVD virus status and to establish a biochemical ‘check-up’. A complete clinical examination was performed and the animal was monitored during the whole hospitalisation period. The same clinical signs of microphthalmos, blindness and gingival ulcerations were observed; the ulcers progressively recovered. In addition, tachycardia with a leftand rightsided heart murmur was present. The calf again tested negative for BVD antibodies and antigens in the blood. Also, viral cultures failed to isolate BVD virus. No major biochemical abnormalities were recorded. In parallel, a serological investigation for BVD and infectious bovine rhinotracheitis was undertaken on all 28 animals in the herd; all the animals were seronegative for both viruses. Calf 4 was euthanased on December 20. At postmortem examination, the following lesions were noted: microphthalmos, which was more severe in the left eye (Fig 2), hypogenesia of the optical nerves (especially the left one), abnormal liquid in ocular globes when cut (red in the left eye, brown in the right eye), bilateral retina atrophy, aphakia and the absence of vitreous body and anterior uvea. Examination of the heart showed a ventricular septal defect, right ventricular FIG 1: Ocular deformations seen in animals born to heifers or cows that were deficient in vitamin A. (a) Fully haired corneal dermoid on the right eye (heifer 2), (b) cutaneous proliferation on the left eyelids (heifer 2), and (c) right-sided microphthalmos (calf 4) (a) (b)

Evidence of transplacental transmission of bluetongue virus serotype 8 in goats

During the incursion of bluetongue virus (BTV) serotype 8 in Europe, an increase in the number of abortions in ruminants was observed. Transplacental transmission of BTV-8 in cattle and sheep, with subsequent foetal infection, is a feature of this specific bluetongue serotype. In this study, BTV-8 ability to cross the placental barrier at the beginning of the second third of pregnancy and at the end of pregnancy was investigated in goats in two separate experiments. In the first experiment, nine goats were experimentally infected with BTV-8 at 61 days of pregnancy. Foetuses were collected 21 dpi. BTV-8 was evidenced by real time RT-PCR and by viral isolation using blood from the umbilical cord and the spleens of 3 out of the 13 foetuses. All dams were viraemic (viral isolation) at the moment of sampling of the foetuses. Significant macroscopic or histological lesions could not be observed in foetuses or in their infected dams (notably at the placenta level). In the second experiment, 10 goats were infected with BTV-8 at 135 days of pregnancy. Kids were born by caesarean section at the programmed day of birth (15 dpi). BTV-8 could not be detected by rt-RT-PCR in blood or spleen samples from the kids. This study showed for the first time that BTV-8 transplacental transmission can occur in goats that have been infected at 61 days of pregnancy, with infectious virus recovered from the caprine foetuses. The observed transmission rate was quite high (33%) at this stage of pregnancy. However, it was not possible to demonstrate the existence of BTV-8 transplacental transmission when infection occurred at the end of the goat pregnancy.

Co-occurrence of aminoglycoside resistance gene armA in non-Typhi Salmonella isolates producing CTX-M-15 in Algeria

Centre Universitaire Eltarf, Eltarf, Algerie; Universite Paris-Est, Ecole Nationale Veterinaire d’Alfort, Unite Microbiologie Alimentaire, Securite et Qualite des Aliments (MASQ), 94704 Maisons-Alfort Cedex, France; Service de Microbiologie, Centre Hospitalier Universitaire Dorban, Annaba, Algerie; Unite Caracterisation et Epidemiologie Bacterienne (CEB), Laboratoire de Securite des Aliments de Maisons-Alfort, ANSES, 94706 MaisonsAlfort Cedex, France; INSERM U914 ‘Emerging Resistance to Antibiotics’, Service de Bacteriologie-Virologie, Hopital de Bicetre, Faculte de Medecine Paris-Sud, 94275 Le Kremlin-Bicetre, Paris, France