First fertilizing-value typology of digestates: A decision-making tool for regulation.

Defined as the residue from anaerobic digestion (AD), digestate refers to a set of materials with varied biochemical compositions. The objective of this study was to establish a digestate typology according to its fertilizing-value with data from literature and internal unpublished databases. To establish a relatively big database allowing the application of advanced statistics, usual fertilizing-value parameters were used: dry matter, volatile solids, C/N, C/Organic-N, total N (TN), total ammoniacal nitrogen (TAN), TAN/TN, total P and total K. Statistical analysis was performed on a dataset of 91 raw digestates, 34 solid fractions and 25 liquid fractions after separation. The resulting typology outlined that fertilizing-values are linked to AD feedstock and process. As case study regulations, no digestate (without any post-treatment) fulfilled French standards and the latest European Union regulation proposal on fertilizers. Options to reach regulations' product categories were discussed according to the typology. For the first time, a digestate typology was established based on fertilizing value, which can be a useful tool enhancing digestate management and policy making.

Influence of the airflow rate on heat and mass transfers during sewage sludge and bulking agent composting.

The aim of this study was to characterize the gas flow during the composting, at a pilot scale, of a mixture of sludge and bulking agent, in order to model heat and mass transfers involved in the process. Thus, a closed 300-litre cylindrical pilot was fed with a mixture of wastewater treatment sludge and pine bark. Aeration was supplied from the bottom via an air blower and gases were collected at the top. Three experiments were led with constant gas flow rates and one with varying aeration rate. Temperatures within the pilot reactor were monitored all along the trials and their evolutions were discussed in term of heat transfers and parameters influencing the heat balance. Concurrently, Retention Time Distribution curves were obtained by injecting a pulse of methane in the entering airflow and by analysing the methane concentration in the exhaust gas, every two or three days during composting. The gas flow, within the composting medium, was characterized by a dispersion model, which is a deviation of the plug flow model. The dispersive effect of the flow was correlated to the evolution of the experimental temperature, and a convective dispersion model was used to describe the heat and mass transfers through the gas flow. These equations will be, in future work, coupled with heat production and mass degradation terms in order to model the global mass and heat balances of this composting process. Finally, axial dispersion coefficients of gases were determined and correlated with the airflow rate.

A respirometric method for characterising the organic composition and biodegradation kinetics and the temperature influence on the biodegradation kinetics, for a mixture of sludge and bulking agent to be co-composted.

A respirometric method was set up to study kinetics of biological reactions involved in the treatment of organic wastes-sludge mixed with pine barks--by composting. Oxygen consumption rates of this type of mixture were monitored during 10-20 days, using a 10 l respirometric cell kept at constant temperature and moisture. Oxygen consumption kinetics were modelled and organic matter composition was characterised as biomass, easily-biodegradable, slowly-biodegradable and non-biodegradable organic matter. The influence of temperature on kinetics was tested. Results show that this respirometric method is a useful tool for the characterisation of solid organic matter biodegradability and for the modelling of the biological kinetics of the composting process.

A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets.

This study, Vetoquinol S.A. epidemiosurveillance, was conducted from 1994 to 2001 in order to determine the susceptibility (by MIC determination) to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains from infected pets originated from six European countries. Isolates were collected from urinary infections (Escherichia coli), respiratory infections (Pasteurella multocida), dermatological infections (Staphylococcus intermedius, Pseudomonas aeruginosa) and otitis (S. intermedius, P. aeruginosa). The MIC distribution for each species was the same both before and after the launch of marbofloxacin in 1995. In E. coli, a resistant population was present before the use of marbofloxacin; this resistance was induced by co- or cross-resistance to other antibiotics used previously. Over this period, there was no significant evolution of MIC(90) for any bacterial species studied and no development of resistance was observed. Marbofloxacin was the most active antibiotic against P. multocida isolates and had the lowest MIC. No difference in MIC distribution was seen between the S. intermedius (unimodal distribution) isolated from dermatological infections and those from otitis. This was also true for P. aeruginosa. The use of marbofloxacin was not found to have induced a significant increase or spread of resistant bacteria.

Seven years survey of susceptibility to marbofloxacin of bovine pathogenic strains from eight European countries.

This study was conducted from 1994 to 2001 to determine the susceptibility of bovine pathogenic bacteria to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains originated in bovine diseases from eight European countries. They were isolated from gut infections (Escherichia coli, salmonellae), mastitis (E. coli, Staphylococcus aureus, Streptococcus uberis, S. agalactiae, S. dysgalactiae) and respiratory diseases (Mannheimia haemolytica, Pasteurella multocida, Haemophilus somnus). There was no change in the MIC distributions for each species after the launch of marbofloxacin in 1997. In E. coli, a resistant population was present before the use of marbofloxacin having been induced by co- or cross-resistance to other antibiotics used previously. Over this period the only a significant change seen was an increase in MIC(90) of E. coli from the gut (1.275 microg/ml in 1994/1995 to 5.098 microg/ml in 2001). All the salmonellae were susceptible to marbofloxacin with a MIC(90) = 0.073 microg/ml in 2001 without development of high level resistance. The use of marbofloxacin seems not to have favoured a significant increase and spreading of resistant bacteria.

Antimicrobial resistance in staphylococci from animals with particular reference to bovine Staphylococcus aureus, porcine Staphylococcus hyicus, and canine Staphylococcus intermedius.

Besides their role as commensals on the skin and mucosal surfaces, staphylococci may be involved in a wide variety of diseases in animals. Staphylococcal infections in animals are mainly treated with antimicrobial agents and as a consequence, staphylococci from animal sources have developed and/or acquired resistance to the respective antimicrobial agents. Resistance statistics obtained from national monitoring programmes on staphylococci from cattle and pigs, but also from surveillance studies on staphylococci involved in diseases in dogs are reported and reviewed with regard to their comparability. This review mainly focusses on the genetic basis of antimicrobial resistance in staphylococci of animal origin. Particular attention is paid to resistance to those antimicrobial agents which are most frequently used in veterinary medicine, but also to antimicrobial agents, such as chloramphenicol and mupirocin, which are used in specific cases for the control of staphylococcal infections in pets and companion animals. In addition, plasmids and transposons associated with the respective resistance properties and their ways of spreading between members of the same or different staphylococcal species, but also between staphylococci and other gram-positive bacteria, are described.

Antimicrobial resistance in pasteurella and mannheimia: epidemiology and genetic basis.

Isolates of the genera Pasteurella and Mannheimia cause a wide variety of diseases of great economic importance in poultry, pigs, cattle and rabbits. Antimicrobial agents represent the most powerful tools to control such infections. However, increasing rates of antimicrobial resistance may dramatically reduce the efficacy of the antimicrobial agents used to control Pasteurella and Mannheimia infections. This review presents a short summary of the infections caused by Pasteurella and Mannheimia isolates in food-producing animals and the possibilities of preventing and controlling primary and secondary pasteurellosis. Particular reference is given to antimicrobial chemotherapy and the resistance properties of Pasterurella and Mannheimia isolates. The genetic basis of the most predominant resistance properties such as resistance to beta-lactam antibiotics, tetracyclines, aminoglycosides, sulfonamides, and chloramphenicol is discussed. This is depicted with reference to the role of plasmids and transposons in the spread of the resistance genes among Pasteurellaceae and members of other bacterial families and genera.

New trends in regulatory rules and surveillance of antimicrobial resistance in bacteria of animal origin.

Since the introduction in the 1940s of antibiotics as drugs against bacterial infections in human and then veterinary medicine, two major events have caused a shift in the antibiotherapy era: (1) the emergence of resistant bacteria and (2) the awareness of the limits of new drug development. It rapidly became urgent to set up measures in order to evaluate the importance of resistant bacteria and their origin as well as to limit the dissemination of resistant vectors (bacteria and bacterial genes). This led to the establishment of guidelines and regulatory rules necessary for risk assessment and clearly dependent upon monitoring and research organisations. At a veterinary level, the possible dissemination of multiresistant bacteria from animals to humans, through feeding, urged various national European and international institutions to give general recommendations to monitor and contain the emergence and diffusion of resistant strains. This paper gives an overview of the evolution of regulatory rules and monitoring systems dealing with multiresistant bacteria.

Plasmid-mediated florfenicol resistance encoded by the floR gene in Escherichia coli isolated from cattle.

A florfenicol resistance gene almost identical to floR of Salmonella enterica serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in Escherichia coli isolates of animal origin. Analysis of the floR gene flanking regions of one of the plasmids showed that they were different from those encountered in S. enterica serovar Typhimurium DT104.

The French antibiotic resistance monitoring programs.

Surveillance of antimicrobial resistance in bacteria from animal origin in France is organised by the French Agency for Food Safety (Agence Française de Sécurité Sanitaire des Aliments, AFSSA) through two types of networks. The first collects non-human zoonotic Salmonella strains in one centre (AFSSA, Paris) where they are tested for their antimicrobial susceptibility. The others, managed by AFSSA Lyon, deal with bovine pathogenic strains and are multicentric, that is they collecting antibiotic sensitivity and other data from the local public veterinary diagnostic laboratories. This requires standardisation of the methods used in each partner laboratory. Statistical analysis of any change in French resistance patterns can be monitored by these three networks either as a function of strain pathogenicity and/or of the ecological origin of the isolate. The system also encourages efficient collaboration between veterinarians and the laboratory. Such collaboration improves both the quality of routine antibiotic testing and understanding of the molecular mechanisms underlying resistance.

Monitoring of antibiotic resistance in bacteria of animal origin: epidemiological and microbiological methodologies.

The occurrence of antibiotic-resistant bacteria in food animals is a major public health threat. Information on the prevalence of resistance to specific drugs in both bacterial and animal species together with changes occurring over time, are necessary to understand the magnitude of the problem and to establish baselines for taking action. The aim of this paper is to define the minimum epidemiological and microbiological requirements for establishing a surveillance of antimicrobial resistance in bacteria of animal origin. Surveillance should involve different bacterial species, veterinary pathogens, zoonotic bacteria and commensal bacteria used as indicators. The collected data should be periodically updated and the reports distributed among practising veterinarians and regulatory authorities. These reports would be a useful tool for developing guidelines for the prudent use of antimicrobial agents in veterinary medicine and for action strategies.

Comparative studies of mutations in animal isolates and experimental in vitro- and in vivo-selected mutants of Salmonella spp. suggest a counterselection of highly fluoroquinolone-resistant strains in the field.

The occurrence of mutations in the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) of Salmonella typhimurium experimental mutants selected in vitro and in vivo and of 138 nalidixic acid-resistant Salmonella field isolates was investigated. The sequencing of the quinolone resistance-determining region of these genes in highly fluoroquinolone-resistant mutants (MICs of 4 to 16 microg/ml) revealed the presence of gyrA mutations at codons corresponding to Gly-81 or Ser-83, some of which were associated with a mutation at Asp-87. No mutations were found in the gyrB, parC, and parE genes. An assay combining allele-specific PCR and restriction fragment length polymorphism was developed to rapidly screen mutations at codons 81, 83, and 87 of gyrA. The MICs of ciprofloxacin for the field isolates reached only 2 microg/ml, versus 16 microg/ml for some in vitro-selected mutants. The field isolates, like the mutants selected in vivo, had only a single gyrA mutation at codon 83 or 87. Single gyrA mutations were also found in highly resistant in vitro-selected mutants (MIC of ciprofloxacin, 8 microg/ml), which indicates that mechanisms other than the unique modification of the intracellular targets could participate in fluoroquinolone resistance in Salmonella spp. A comparison of experimental mutants selected in vitro, field strains, and mutants selected in vivo suggests that highly fluoroquinolone-resistant strains are counterselected in field conditions in the absence of selective pressure.

A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in Salmonella typhimurium DT104.

A new chloramphenicol resistance gene from Salmonella typhimurium DT104, designated floR, also conferring resistance to florfenicol, was characterized. Sequence analysis of the deduced FloR protein suggested that it belongs to the 12-TMS (transmembrane segments) multidrug efflux pumps family. The floR gene, and the downstream sequenced tetR and tetA tetracycline resistance genes, were surrounded by two class 1 integrons. The first one contained the resistance gene aadA2 and a deleted sulI resistance gene. The second one contained the beta-lactamase gene pse1 and a complete sulI gene. Thus, the floR gene is included in a multiresistance locus of at least 12.5 kb. Its particular organization and chromosomal location could be involved in the antibioresistance pattern stability of the DT104 Salmonella typhimurium strains.

Pulsed-field gel electrophoresis is more efficient than ribotyping and random amplified polymorphic DNA analysis in discrimination of Pasteurella haemolytica strains.

One hundred thirty-three strains of Pasteurella haemolytica of both biotypes (90 and 43 strains of biotypes A and T, respectively) and almost all the serotypes were subjected to ribotyping, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE) analysis for epidemiological purposes. A total of 15 patterns recorded as ribotypes HA to HO were found for the P. haemolytica biotype A strains, with ribotypes HA, HC, and HD being encountered most often (66 strains [74%]); and 20 ribotypes, designated HA' to HT', that were clearly distinct from those observed for biotype A strains were observed for strains of biotype T. RAPD analysis generated a total of 44 (designated Rp1 to Rp44) and 15 (designated Rp1' to Rp 15') unique RAPD patterns for biogroup A and biogroup T, respectively. Analysis of the data indicated that a given combined ribotype-RAPD pattern could be observed for biotype A strains of different serotypes, whatever the zoological or geographic origin, whereas this was not the case for biotype T strains. PFGE appeared to be more efficient in strain discrimination since selected strains from various zoological or geographical origins harboring the same ribotype-RAPD group were further separated into unique entities.

Manifestation and epidemiology of contagious bovine pleuropneumonia in Europe.

The authors describe the clinical profile and epidemiology of contagious bovine pleuropneumonia in Europe. This disease, once considered to have been eradicated several years ago, has now become endemic in southern countries of Europe. The status of contagious bovine pleuropneumonia in Portugal and Italy, and the evolution of the disease during the last ten years, are analysed in detail, in addition to the measures undertaken for control and eradication. The authors also refer to hosts and possible reservoirs of the infection.

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals.

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.

CAT III chloramphenicol resistance in Pasteurella haemolytica and Pasteurella multocida isolated from calves.

Chloramphenicol, which had been used extensively for antimicrobial veterinary therapy, was prohibited in Europe in 1994. Soon after it became available, resistance to this drug was detected, generally conferred by plasmids encoding inactivating enzymes, the chloramphenicol acetyltransferases (CAT), in Gram-negative as well as in Gram-positive bacteria. In the last few years, resistance to antibiotics emerged in Pasteurella strains from breeding herds and this evolution was followed by a national surveillance network. Chloramphenicol-resistance was more recently detected in multiresistant strains. We studied 25 strains of Pasteurella, selected for their resistance to chloramphenicol. Production of a CAT was demonstrated in all these strains. PCR amplification indicated that the CAT produced was of type III for 23 of them. In these strains, chloramphenicol-resistance was mediated by plasmids of about 5.1 kb. Southern blots on restriction fragments suggested a high degree of homology between these 5.1 kb plasmids. In the two other strains, production of a CAT type I was demonstrated, and the corresponding genes were either shown on a plasmid of 17 or 5.5 kb.

[Evolution of antimicrobial susceptibility in salmonellas from bovine origin in France.]. / Evolution de la sensibilité aux antibiotiques des salmonelles d'origine bovine en France.

Animals are a wide source of salmonellas for humans and their environment. Domestic ruminants play an important role because of their high susceptibility to salmonellas infection giving an acute disease with massive salmonella excretion and mortality if lack of treatment. As in human medicine use of antibiotics at therapeutic doses in animals can lead to the selection of resistant strains may be pathogenic for humans by direct contact or through environment and food. Taking into account the importance of salmonellosis on calves in rearing units, CNEVA Lyon has set up since 1982 a national network for antimicrobial resistance monitoring of main bacterial pathogens in bovine. This network allowed to detect a recent evolution of antimicrobial susceptibility in salmonella from dairy cows related with new problem of salmonellosis in cattle.