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Amycolatopsis coloradensis ATCC 53629 is the producer of the glycopeptide antibiotic avoparcin. While setting up the production of the avoparcin complex, in view of its use as analytical standard, we uncovered the production of a to-date not described ristosamynil-avoparcin. Ristosamynil-avoparcin is produced together with α- and β-avoparcin (overall indicated as the avoparcin complex). Selection of one high producer morphological variant within the A. coloradensis population, together with the use of a new fermentation medium, allowed to increase productivity of the avoparcin complex up to 9 g/L in flask fermentations. The selected high producer displayed a non-spore forming phenotype. All the selected phenotypes, as well as the original unselected population, displayed invariably the ability to produce a complex rich in ristosamynil-avoparcin. This suggested that the original strain deposited was not conforming to the description or that long term storage of the lyovials has selected mutants from the original population.

WasielewskaMagdalena. Influence of feed additives for chickens on the quality of eggs laid and related risks.Journal of Education, Healthand Sport. 2021;11(9):246-258. eISSN 2391-8306. DOIhttp://dx.doi.org/10.12775/JEHS.2021.11.09.031 https://apcz.umk.pl/czasopisma/index.php/JEHS/article/view/JEHS.2021.11.09.031 https://zenodo.org/record/5513613 The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. § 8. 2) and § 12. 1. 2) 22.02.2019. © The Authors 2021; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received:05.09.2021. Revised: 12.09.2021. Accepted: 16.09.2021. Influence of feed additives for chickens on the quality of eggs laid and related risks Magdalena Wasielewska; ORCID: 0000-0001-8638-3732; E-mail: w.magdalena1@gazeta.pl Institution:Nicolaus Copernicus University in Toruń Collegium Medicum in Bydgoszcz, ul. Jagiellońska 13-15, 85-067 Bydgoszcz, Major Pharmacy Keywords: antibiotics, phytobiotics, antibiotic growth stimulants, chicken eggs, poultry Abstract A chicken egg is a valuable animal product that has served people for centuries. Research carried out over the last dozen or so years shows that society's knowledge about them is still relatively small. The research cited in the text shows that only 23% of respondents know that the color of a hen's egg shell depends on the breed of the hen, and not on the way it is fed. As a result, some consumers choose eggs in the store by choosing their color instead of the official markings on each individual egg. Chickens' nutrition has a direct impact on the quality of the eggs they lay. Chickens fed with fodder with antibiotics lay eggs in which we can find the same antibiotics that the animal ate with the food. It has many side effects, however, the greatest of which seems to be bacterial drug resistance, caused by the use of the same strong antibiotics in the nutritional industry and in human hospital medicine. Bacteria have developed various resistance mechanisms. For example, Enterococcus spp. Has developed resistance to vancomycin, Salmonella Typhimurum to tetracyclines, sulfonamides and fluoroquinolones. However, it is possible to use antibiotics in poultry treatment, but it is very important in this case to strictly observe the grace periods necessary to eliminate the antibiotic from the animal's body. Some of the world's governments, aware of the dangers of such a state of affairs, are trying to combat the phenomenon of feeding poultry with antibiotic growth stimulants by introducing new norms, orders and bans in their countries. Antibiotics such as avoparcin, zinc-bacitracin and spiromycin have been discontinued. Chicken egg producers are finding newer and newer ways to circumvent these recipes. Non-antibiotic feed additives such as probiotics, phytobiotics, and specific herbs are used. An effective way to combat this phenomenon may be to educate the public on this topic.

Background There is ongoing debate regarding potential associations between restrictions of antimicrobial use and prevalence of antimicrobial resistance (AMR) in bacteria. Objectives To summarize the effects of interventions reducing antimicrobial use in food-producing animals on the prevalence of AMR genes (ARGs) in bacteria from animals and humans. Methods We published a full systematic review of restrictions of antimicrobials in food-producing animals and their associations with AMR in bacteria. Herein, we focus on studies reporting on the association between restricted antimicrobial use and prevalence of ARGs. We used multilevel mixed-effects models and a semi-quantitative approach based on forest plots to summarize findings from studies. Results A positive effect of intervention [reduction in prevalence or number of ARGs in group(s) with restricted antimicrobial use] was reported from 29 studies for at least one ARG. We detected significant associations between a ban on avoparcin and diminished presence of the vanA gene in samples from animals and humans, whereas for the mecA gene, studies agreed on a positive effect of intervention in samples only from animals. Comparisons involving mcr-1, blaCTX-M, aadA2, vat(E), sul2, dfrA5, dfrA13, tet(E) and tet(P) indicated a reduced prevalence of genes in intervention groups. Conversely, no effects were detected for β-lactamases other than blaCTX-M and the remaining tet genes. Conclusions The available body of scientific evidence supported that restricted use of antimicrobials in food animals was associated with an either lower or equal presence of ARGs in bacteria, with effects dependent on ARG, host species and restricted drug.

Introduction Worsening antimicrobial resistance (AMR) threatens the practice of modern human and veterinary medicine. For decades, medicine and agriculture have blamed each other for the rise of resistant microbes. Widespread use and misuse of antibiotics in both medicine and agriculture have increased the prevalence and expression of resistance genes. Vre The rise of vancomycin-resistant Enterococcus faecium (VRE) in the European Union (EU) led to the ban of avoparcin, an antibiotic that is chemically related to vancomycin. In the years post-ban, VRE surveillance data of EU hospitals showed no obvious reduction in VRE rates. The USA never approved avoparcin, yet VRE has been an enormous problem in its hospitals. AMR surveillance data showed zero rates of VRE in US livestock. Whole-genome sequencing data suggest that VRE might have evolved from ampicillin-resistant E. faecium from dogs. Companion animals have been completely ignored in the AMR debate. Sanitation In addition, 1 billion people openly defecate around the world. Of these, 60% live in India, a country with highly resistant microbes. Conclusion A One Health approach integrating human, animal and environmental whole-genome sequencing surveillance data is essential to getting to the root of AMR and developing effective prevention and control strategies.

Due to Australia’s management of antimicrobial use in poultry, particularly the discontinued use of avoparcin for nearly 20 years, it is hypothesized that vancomycin-resistant enterococci associated with human disease are not derived from poultry isolates. This study evaluated antimicrobial resistance (AMR) of five enterococcal species isolated from Australian meat chickens, genomic features of Enterococcus faecium and Enterococcus faecalis, and the phylogenetic relationship of the poultry-derived E. faecium with isolates from human sepsis cases. All enterococcal isolates from chicken ceca were subjected to antimicrobial susceptibility testing. E. faecium and E. faecalis underwent whole-genome sequencing. E. faecium was compared at the core genome level to a collection of human isolates (n = 677) obtained from cases of sepsis over a 2-year period spanning 2015 to 2016. Overall, 205 enterococci were isolated consisting of five different species. E. faecium was the most frequently isolated species (37.6%), followed by E. durans (29.7%), E. faecalis (20%), E. hirae (12.2%), and E. gallinarum (0.5%). All isolates were susceptible to vancomycin and gentamicin, while one isolate was linezolid resistant (MIC 16 mg/liter). Core genome analysis of the E. faecium demonstrated two clades consisting predominantly of human or chicken isolates in each clade, with minimal overlap. Principal component analysis for total gene content revealed three clusters comprised of vanA-positive, vanB-positive, and both vanA- and vanB-negative E. faecium populations. The results of this study provide strong evidence that Australian chicken E. faecium isolates are unlikely to be precursor strains to the currently circulating vancomycin-resistant strains being isolated in Australian hospitals.

Vancomycin-resistant enterococcus (VRE) is a global threat to public health. Knowledge about the occurrence of vanA-carrying enterococci in broiler and environmental samples is important as antibiotic resistance can be transferred to human bacteria. The aim of this study was to investigate the presence of VRE in broiler cloacal and environmental (house) samples and to genotype the isolates. In this study, 350 swabs were collected from broiler farms. All samples were plated onto enterococcus selective agar containing 6 mg/L vancomycin and 64 mg/L ceftazidime. Minimum inhibitory concentration (MIC) values were determined for vancomycin and teicoplanin. Vancomycin-resistant Enterococcus faecium (VREfm) was isolated from 6 out of 300 (2%) broiler cloacal samples and 13 out of 50 (26%) house samples. All E. faecium isolates had vanA genes. All VREfm isolates (19 isolates) were confirmed to be 95% similar to each other. In conclusion, although 20 years have passed since the ban on avoparcin in Turkey, the present study shows that VREfm isolates are still present in broiler production and especially in broiler houses, and most importantly, a major VREfm clone was isolated from broiler cloacal and house samples.

Several works of literature research for the contribution of Antibiotic-Resistant Enterococci (ARE) and, especially Vancomycin Resistant Enterococci (VRE) which entered into the food chain has gained importance with the increasing significance of VREs in hospital infections. Various studies conducted in Europe, United States of America (USA) and the Middle East were evaluated in terms of prevalence, epidemiology and risk factors of foodborne enterococci in VRE infections. VRE epidemiology has shown some distinctions in Europe and USA. VRE was generally isolated from animals in Europe, which was connected to the extensive/massive use of “avoparcin” as a growth promoter in animal feed in the agriculture sector. Animals fed with this feed act as reservoirs of transferable vanA type resistance. On the other hand, since “avoparcin” was not used in the USA, VRE could not be isolated in animals and healthy humans. However, hospital-acquired VRE infections are more showed in the USA than in European countries. According to numerous studies, since enterococci are used as starter culture and probiotic culture, they have no relationship genetically with the strains which include vancomycin/resistant to antibiotic/or having resistance and virulence genes. In this chapter, important features of enterococci, the role of food chain for ARE especially, VRE infections in community including strategies for future solutions about the problem are summarised.

Vancomycin-resistant Enterococcus spp. is a major health problem worldwide and livestock have been implicated in constituting a reservoir for the transmission of vancomycin resistance to zoonotic pathogens. Vancomycin resistance determinants can be situated on mobile genetic elements and transferred between bacterial species The livestock reservoir must therefore be included in a risk assessment of the vancomycin resistance burden. Avoparcin, a vancomycin analogue, has not been used in Danish pig production for over 20 years and vancomycin has never been used. The objective of this study was to screen faecal samples from Danish pig farms for nine selected vancomycin resistance determinants. We found at least four different vancomycin resistance determinants in all screened Danish pig farms (665 finisher farms and 78 sow farms). The vancomycin resistance determinants present in vanB or vanG clusters were found at significantly different levels in sow and finisher farms. However, vanA was not detected in any of the farms. In conclusion, vancomycin resistance determinants are still present in Danish pig production 25 years after the ban on avoparcin use.

The occurrence and diversity of vancomycin-resistant Enterococcus faecium (VREF) were investigated in 100 Danish broiler flocks 15 years after the avoparcin ban. VREF occurred in 47 flocks at low fecal concentrations detectable only by selective enrichment. Vancomycin resistance was prevalently associated with a transferable nontypeable plasmid lineage occurring in multiple E. faecium clones. Coselection of sequence type 842 by tetracycline use only partly explained the persistence of vancomycin resistance in the absence of detectable plasmid coresistance and toxin-antitoxin systems.

Vancomycin resistant enterococci (VRE) belong to the most common causes of nosocomial infections worldwide. It has been reported that use of the glycopeptide growth promoter avoparcin selected for a significant livestock-reservoir of VRE in many European countries, including Norway. However, although avoparcin was banned as a feed-additive in 1995, VRE have for unknown reasons consistently been reported in samples from Norwegian broilers. When avoparcin was banned, broiler-feed was supplemented with the polyether ionophore narasin in order to control the diseases coccidiosis and the frequent sequela necrotic enteritis. A potential link between transferrable vancomycin resistance and reduced susceptibility to narasin was recently reported. The use of narasin as a feed additive was abolished by the Norwegian broiler industry in 2016 and since then, broilers have been reared without in-feed antibacterial supplements. In this study, we demonstrate that all VRE isolates from Norwegian broilers collected in 2006–2014 displayed reduced susceptibility to narasin. Surveillance data collected two years after the narasin abolishment show a significant reduction in VRE, below the detection limit of the surveillance method, and a concurrent marked reduction in Enterococcus faecium with reduced susceptibility to narasin. The significant decline of E. faecium with reduced susceptibility to these antimicrobial compounds also coincided with an increased focus on cleaning and disinfection between broiler flocks. Furthermore, data from a controlled in vivo experiment using Ross 308 broilers indicate that the proportion of E. faecium with reduced susceptibility to narasin was heavily reduced in broilers fed a narasin-free diet compared to a diet supplemented with narasin. Our results are consistent with that the abolishment of this feed additive, possibly in combination with the increased focus on cleaning and disinfection, has had a substantial impact on the occurrence of VRE in the Norwegian broiler population.

Well-characterized pure-substance reference materials for the use as calibrants are essential to establish the metrological traceability of the results of chemical measurements. Normally, the characterization of this type of reference material is conducted through a thorough purity assessment of the compound concerned. For this reason, studies on purity assessment, especially for neat organic compounds, continues as an important part of work being undertaken by metrological institutions around the world. Among others, the need for certified pure reference standards continues to increase for residues analysis in foods, particularly for those compounds which have been banned for food safety reasons, but their residues in foods are still monitored under food surveillance program in many countries. In this respect, avoparcin serves as a very good example where testing laboratories have difficulties in obtaining traceable and comparable results on determination of avoparcin in food matrix samples due, in part, to the unavailability of certified pure-substance reference material as calibrant. In this study, it was attempted to assess the purity of a commercially available test material of avoparcin using the mass balance approach. The objective of this paper is to share the difficulties encountered during the course of purity assessment and how they were addressed. As expected, the most challenging part of work was to identify and estimate the amount of unknown impurities, both organic and inorganic-related ones, given the chemical structure and properties of avoparcin. For instance, avoparcin exists in two forms in the test material, i.e., α- and β-avoparcin, and they were found to be susceptible to hydrolysis under certain conditions.

One hundred and 26 faecal samples from healthy dogs (2009) and 157 faecal samples from healthy humans (2007) from La Rioja region (Spain) were tested to know the carriage of vancomycin-resistant enterococci (VRE). VRE with intrinsic resistance (vanC) were found in 12% of healthy dogs and humans (29 Enterococcus gallinarum and four Enterococcus casseliflavus). Nevertheless, VRE with acquired mechanism of resistance were not detected among these samples. Four Enterococcus faecalis isolates with vancomycin MIC of 8-16 mg L(-1) were recovered in human samples, but no single organism with known mechanism of acquired resistance could be identified. These 37 VRE isolates (33 E. gallinarum/E. casseliflavus and four E. faecalis) of dog and human origin were further characterized (PCR detection of antibiotic resistance, virulence and bacteriocin genes). High prevalence of tetracycline resistance was identified (70%), especially among dog isolates harbouring tet(M) ± tet(L) genes; erythromycin resistance was also higher among isolates from dogs and they harboured the erm(B) gene, associated with erm(A) gene in one case. Virulence genes were only identified among E. faecalis isolates of human origin (agg, cpd and/or gelE) and never among E. gallinarum/E. casseliflavus of human or dog origin. Five E. gallinarum isolates of dog and three E. faecalis of human origin expressed bacteriocin activity; among them, only one E. faecalis presented activity against Listeria monocytogenes. The bacteriocin structural gene ef1097 was identified in 3 bacteriocin-producing E. faecalis isolates, associated with ent1071 in one of them.

Objectives: This study investigated the prevalence of vancomycin-resistant enterococci (VRE) in the broiler production environment after the avoparcin ban and their epidemiological relationship with human clinical VRE from the same geographical regions in Greece. Methods: Caecal contents from broilers (n ¼500) from eight livestock farms and faecal samples from poultry slaughterers (n¼ 50), all collected in two slaughterhouses during 2005 –08, were analysed for species and vancomycin resistance gene identification using multiplex PCR. Sixty-three human clinical vancomycin-resistant Enterococcus faecium (VREF) isolates, obtained during 2006– 09, were also examined. Discriminant analysis (DA) was used to establish the relationship of antimicrobial resistance profiles (ARPs) among broiler, poultry slaughterer and human clinical VREF. PFGE was conducted to study the genetic relatedness among VREF from the different sources. Results: A total of 120 VRE were recovered from 113 (22.6%) broiler samples. VREF carrying the vanA gene were predominant, being recovered from 72 (14.4%) samples from five (62.5%) broiler farms. Concerning poultry slaughterers, VREF were recovered from 10 (20%) samples. Susceptibility testing revealed that broiler VREF were consistently resistant to tetracycline, whereas 93.7% of clinical VREF were resistant to ampicillin. Furthermore, 92.1% of clinical VREF compared with 54.4% of broiler VREF were multiresistant (resistant to at least five antimicrobial classes). DA classified broiler and human clinical VREF into their corresponding source with high classification rates (100% and 85.7%, respectively), while the classification rate of poultry slaughterer VREF was relatively low (50%), with 40% of them classified closely to broiler VREF. PFGE patterns were clearly related to the source of the VREF, with broiler isolates being clustered distinctly from all human isolates. Conclusions: A remarkable persistence of VREF was observed in the broiler production environment even .10 years after the avoparcin ban. Human and broiler VREF belonged to clearly unrelated populations, strongly indicating no clonal spread of VREF among the different sources, even between broilers and poultry slaughterers, despite them sharing common ARPs, as also supported by DA.

The isolation rate of high-level vancomycin-resistant enterococci (VRE) from poultry samples in Japan has increased in recent years. As this raises concerns for the potential spread of genes encoding vancomycin resistance, poultry is routinely screened for VRE. Here, we report the isolation and characterization of a vanA genotype vancomycin-resistant Enterococcus cecorum strain (E. cecorum IPHa84) from retail domestic poultry in September 2009. The species identification was performed by biochemical testing and sequencing of the 16S rRNA and manganese-dependent superoxide dismutase genes. The vancomycin and teicoplanin susceptibility tests showed that E. cecorum IPHa84 was resistant to vancomycin and susceptible to teicoplanin, demonstrating that this isolate was VanB phenotype-vanA genotype VRE. Moreover, a vanA gene cluster was found in a chromosomally encoded Tn1546-related element, which exhibited the characteristic structure of the prototype Tn1546 element, but contained eight point mutations. The vanS sequence of E. cecorum IPHa84 contained three point mutations and was 100% identical to those of VRE isolated from different broiler droppings in Japan prior to the banning of avoparcin, indicating that the Tn1546-related element may be stable in poultry production environments, even in the absence of selective pressure. The isolation of a novel enterococcal species harboring the vanA gene reconfirms that poultry can serve as a reservoir of VanA-type VRE or vancomycin resistance genes, and suggests that the transmission of these risk factors from poultry to humans through the food chain remains a potential threat in Japan.

Enterococcus faecium and Enterococcus faecalis belong to the gastrointestinal flora of humans and animals. Although normally regarded harmless commensals, enterococci may cause a range of different infections in humans, including urinary tract infections, sepsis, and endocarditis. The use of avoparcin, gentamicin, and virginiamycin for growth promotion and therapy in food animals has lead to the emergence of vancomycin- and gentamicin-resistant enterococci and quinupristin/dalfopristin-resistant E. faecium in animals and meat. This implies a potential risk for transfer of resistance genes or resistant bacteria from food animals to humans. The genes encoding resistance to vancomycin, gentamicin, and quinupristin/dalfopristin have been found in E. faecium of human and animal origin; meanwhile, certain clones of E. faecium are found more frequently in samples from human patients, while other clones predominate in certain animal species. This may suggest that antimicrobial-resistant E. faecium from animals could be regarded less hazardous to humans; however, due to their excellent ability to acquire and transfer resistance genes, E. faecium of animal origin may act as donors of antimicrobial resistance genes for other more virulent enterococci. For E. faecalis, the situation appears different, as similar clones of, for example, vancomycin- and gentamicin-resistant E. faecalis have been obtained from animals and from human patients. Continuous surveillance of antimicrobial resistance in enterococci from humans and animals is essential to follow trends and detect emerging resistance.

The aim of this study was to estimate the occurrence of vancomycin-resistant enterococci (VRE) in fecal samples of ostriches from a farm of Southern Portugal, the mechanisms implicated, and the associated virulence factors, 13 years after the banning of the glycopeptide avoparcin as animal growth promoter in the European Union. Fifty-four fecal samples of ostriches were inoculated in Slanetz-Bartley supplemented with vancomycin (4 microg/mL) for VRE recovery. Susceptibility to 11 antibiotics was performed by disk-diffusion agar method in recovered VRE isolates. The mechanism of resistance to vancomycin and to other antibiotics and the presence of the esp and hyl virulence genes were determined by polymerase chain reaction and sequencing. VRE were detected in 7 of the 54 ostrich fecal samples (13%); Enterococcus durans isolates with the vanA genotype were found in 4 of the 54 fecal samples (7.4%), and Enterococcus gallinarum with the intrinsic vanC1 genotype in the remaining three VRE-positive samples. All vanA-containing E. durans isolates showed resistance to tetracycline and erythromycin, and one of them also to ciprofloxacin; they harbored the erm(B) and tet(M) genes, as well as the specific sequences of Tn916 and Tn5397 transposons, but not the esp or hyl virulence genes. Two of the three vanC1 isolates showed resistance to tetracycline [with the tet(M) gene] and one to erythromycin [with the erm(B) gene], and all three contained the hyl gene. Fecal samples of ostriches represent a reservoir of vanA-containing enterococci that could be transmitted to humans through the food chain.

Summary A quantitative study was made of the resistance and cross-resistance to antimicrobial agents of enterococci and pharyngeal staphylococci from 9-days-old chicks given 10 or 100 ppm. avoparcin in the diet for 55 days. Examination of multi-replicate samples indicated wide fluctuations in both total bacterial count and prevalence of antimicrobial resistance during the period of the experiment. There was no cross-resistance in either the enterococci or the pharyngeal staphylococci between avoparcin and any of the other drugs tested. Evidence was also obtained of instability of induced avoparcin resistance in faecal streptococci. Zusammenfassung Die Auswirkung von Avoparcin als Futterzusatz auf die Eigenschaften der Antibiotika-Resistenz bei Bakterien des Darmes und des Pharyvon Broilern Eine quantitative Studie wurde durchgefuhrt zur Erfassung der Resistenz und Kreuzresistenz gegenuber bakteriellen Substanzen bei Enterokokken und Staphylokokken des Pharynx von Mastkuken, an die ab dem 9. Lebenstag 55 Tage lang 10 oder 100 ppm Avoparcin mit dem Futter verabreicht wurde. Die mehrfach wiederholte Untersuchung von Proben lies eine weite Fluktuation in der Gesamtkeimzahl der Bakterien und des Hervortretens von Antibiotika-Resistenz bei beiden Dosierungen wahrend der Dauer des Experiments erkennen. Jedoch trat eine Kreuzresistenz weder bei Enterokokken noch bei den Staphylokokken des Pharynx zwischen Avoparcin und Chemotherapeutika auf, gegen die getestet wurde. Auch konnte der Nachweis der induzierten Avoparcin-Resistenz bei Fekalstreptokokken erbracht werden. Resume Action de l'avoparcine comme additif alimentaire sur les proprietes de resistance aux antibiotiques chez des bacteries de l'intestin et du pharynx de poulets Une etude quantitative a ete faite pour determiner la resistance et la resistance croisee vis-a-vis de substances bacteriennes chez des enteŕocoques et des staphylocoques du pharync de poulets d'engrais qui avaient recu 10 ou 100 ppm d'avoparcine dans l'aliment des le 9e jour apres l'eclosion et jusqu'a l'âge de 55 jours. L'examen plusieurs fois repete d'echantillons a montre une large fluctuation dans le nombre total des bacteries et l'apparition de resistance aux antibiotiques avec les deux dosages pendant la duree de l'experience. Il n'y a pas eu de resistance croisee chez les enterocoques et les staphylocoques du pharynx entre l'avoparcine et les chimiotherapies testees. La mise en evidence d'une resistance induite a l'avoparcine chez des streptocoques fecaux a pu etre rejetee. Resumen La repercusion de Avoparcina como medicacion de piensos sobre las propiedas de la resistencia frente a los antibioticos en bacterias intestinales y faringeas en broilers Se llevo a cabo un estudio cuantitativo para abarcar la resistencia y la resistencia cruzada frente a substancias bacterianas en enterococos y estafilococos faringeos de pollitos de engorde, a los que se habia administrado a partir del dia 9 de vida, durante 55 dias, 10 o 100 ppm. de Avoparcina con el pienso. El analisis, repetido varias veces, de muestras permitio reconocer una fluctuacion amplia en la cifra total de germenes de las bacterias y de la emersion de resistencia a los antibioticos en ambas dosificaciones durante el tiempo que duro el experimento. No aparecio, sin embargo, ninguna resistencia cruzada ni en los enterococos ni en los estafilococos faringeos entre Avoporcina y los quimioterapicos, frente a los que se contrasto. Tambien se pudo aportar la comprobacion de la resistencia inducida contra la Avoporcina en los estreptococos fecales.

A total of 1024 enterococci were recovered from faeces of healthy animals from 178 cattle, 178 pig and 156 broiler farms. Enterococcus faecium was the predominant species recovered (35.8%), followed by E. faecalis (31.3%) and E. hirae (25.6%). Oxytetracycline resistance was most frequently found among E. faecalis (85.9%), E. faecium (58.8%) and E. hirae (48.1%). Resistance rates to almost all antimicrobials were higher in E. faecalis than E. faecium and E. hirae. Isolates from cattle were more susceptible to the antimicrobials studied than those from pigs and broilers. VanA- or VanB-type vancomycin-resistant enterococci have not been found since the ban of avoparcin use 5 years ago.

Zusammenfassung Der Einflus von Monensin und Avoparcin auf die Verdaulichkeit und Pansenfermentation Der Einflus von 24 und 29 ppm Monensin und 2 Konzentrationen Avoparcin (24 bis 31 und 61 bis 64 ppm) auf die Verdaulichkeit und die molare Konzentration fluchtiger Fettsauren wurde untersucht mit Hammeln. Die Rationen waren zusammengesetzt aus 85% Maissilage und 15% Kraftfutter. Monensin bewirkte eine signifikant geringere Verdaulichkeit der Trockensubstanz und Rohfaser. Der Einflus auf die Verdaulichkeit des Fettes und der NFE war unterschledlich, wahrend die Verdaulichkeit der organischen Substanz und des Rohproteins nicht signifikant beeinflust wurde. Etwa 30 ppm Avoparcin hatte keinen Einflus auf die Verdaulichkeit der organischen Substanz, von Rohprotein und NFE. Die Verdaulichkeit von Trockensubstanz, Fett und Rohfaser war unterschiedlich. Hohere Konzentrationen Avoparcin verminderten die Verdaulichkeit der Trockensubstanz, der Rohfaser und der NFE im Vergleich mit der Kontrolle, Monensin, und etwa 30 ppm Avoparcin. Der Einflus auf die organische Substanz und das Rohprotein waren verschieden zwischen den Versuchen. Monensin und Avoparcin bewirkten eine Verminderung der Azetatkonzentration und einen Anstieg der Propionatkonzentration, obwohl dieser Effekt nur in einem der zwei Versuche mit 30 ppm Avoparcin gefunden wurde. Avoparcin erzeugte das engste Azetat: Propionat Verhaltnis im Vergleich mit der Kontrolle und Monensin.

The persistence or loss of acquired antimicrobial-drug resistance in bacterial populations previously exposed to drug-selective pressure depends on several biological processes. We review mechanisms promoting or preventing the loss of resistance, including rates of reacquisition, effects of resistance traits on bacterial fitness, linked selection, and segregational stability of resistance determinants. As a case study, we discuss the persistence of glycopeptide-resistant enterococci in Norwegian and Danish poultry farms 12 years after the ban of the animal growth promoter avoparcin. We conclude that complete eradication of antimicrobial resistance in bacterial populations following relaxed drug-selective pressures is not straightforward. Resistance determinants may persist at low, but detectable, levels for many years in the absence of the corresponding drugs.

The aim of this paper was to describe an increased occurrence of vancomycin-resistant enterococci (VRE) in Swedish broilers since 2000 and to investigate the genetic relatedness of isolates. Caecal content from slaughtered broilers was cultured for VRE on medium supplemented with vancomycin (16 mg/L). Species identification, antibiotic susceptibility determination, vancomycin resistance genotyping, multilocus sequence typing (MLST) and characterization of Tn1546 were performed. The proportion of VRE-positive samples increased gradually from

In this report we examined the glycopeptide susceptibility of enterococci, isolated in 2005, from slaughtered animals, within the confines of Hungarian Antibiotic Resistance Monitoring System. We determined the presence of the van genes as well as their genetic relatedness in enterococci from poultry. Enterococcus sp. strains (n=175) were collected from intestinal samples of slaughtered poultry in 2005. The origin of the samples was registered at county level. After screening the strains with 30 mg vancomycin disc 19 (86%) intermediate resistant and 4 (3%) fully resistant strains were found. The distribution of minimum inhibitory concentration (MIC)-values among 23 enterococcus strains which were intermediate or resistant to vancomycin were 0.25 mg/L (4.4%), 2 mg/L (8.6%), 4 mg/L (8.6%), 8 mg/L (61%), 16 mg/L (8.6%) and 256 mg/L (8.6%). The MICs of teicoplanin were 0.25 mg/L (4.3%), 1 (8.6%), 4 mg/L (78.3%), 16 mg/L (4.3%) and 256 mg/L (4.3%). The two most vancomycin-resistant strains were vanA carriers (1 E. faecalis and 1 E. faeciuum). The farms that produced these strains can be reservoirs of VRE and the affected farms should change the technology of disinfection and breeding in order to prevent the emergence of high numbers of human VRE isolates in Hungary.

Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

Objectives Glycopeptide-resistant enterococci are still present within the broiler sector, despite the EU ban of avoparcin more than a decade ago. In the present study, we have developed a rapid method for screening the flanking regions at the integration point of Tn1546 in glycopeptide-resistant Enterococcus faecium isolated from broiler farms. Methods Total DNA was digested, ligated and amplified using primers from inside Tn1546. The resulting amplicons were purified and sequenced. Two new primers were designed based on obtained sequences. Results Two main insertion points have been repeatedly found in isolates from the UK (n = 150). The first insertion point revealed that 25 isolates harboured Tn1546 positioned in a sequence with 96% homology to a streptomycin adenyltransferase gene (AY604739) from a Staphylococcus intermedius plasmid. At this insertion point, a direct repeat (GTCCT) was duplicated as previously described, indicating transposition at the target site. Furthermore, this 'hot spot' was also detected in isolates from Norway (2/8) and Denmark (17/20). The second insertion point detected in 45 isolates from the UK revealed integration into an Inc18-like plasmid, most likely by a process of target site recombination. Conclusions The presence of a common insertion point for isolates from different geographical areas could suggest the insertion of Tn1546 by transposition in a plasmid-specific site, followed by genetic rearrangement both inside the transposon and in the flanking regions.

The issue of antimicrobial use in animals used as food is of global concern. Antimicrobials are used in animal agriculture to improve health and welfare of animals, meat quality, the economic efficiency of growth and production and public health by decreasing shedding of zoonotic pathogens. However, large quantities are often used without professional supervision. The growth-promotant (now reclassified as zootechnical feed additives) effect of low levels of antibiotics in animal feeds was first described in the late 1940s. Already in 1969 the Swann Committee recommended that use of antibiotics as a supplement in animal feedstuff should be restricted to those with little or no application as therapeutic agents for humans and animals, which would not impair the efficacy of therapeutic antibiotics through the development of resistant strains of organisms. Antimicrobials like avoparcin, ardacin, zinc bacitracin, virginiamycin, tylosin, spriramycin, carbadox and olaquindox were withdrawn within the period 1997-1999. Four others (monensin sodium, salinomycin sodium, avilamycin and flavophospholipol) were still permitted for use as growth promoters in animal feed to animals marketed in the European Union (EU). Since January 2006, they have been banned as well. This review focuses on the analytical methods developed to be an effective tool for monitoring compliance with the ban.

The glycopeptide avoparcin, bacitracin, the macrolides spiramycin and tylosin, and the streptogramin virginiamycin were withdrawn as growth promoters in the European Union between 1995 and 1999 on the basis of the Precautionary Principle. Relevant resistance thereupon diminished among enterococci (the indicator organisms) isolated from animal and human faeces. However, animal enterococci were shown to differ from those that caused human infections, although their resistance genes were sometimes indistinguishable and thus probably have a common origin. Before the ban, human clinical isolates of enterococci resistant to vancomycin or teicoplanin were uncommon in many, but not all, parts of Europe and resistance to quinupristin/dalfopristin in the case of Enterococcus faecium was very rare. After the ban, these resistances increased in prevalence almost universally, to the detriment of human health. Campylobacters, normally susceptible to macrolides, increased in prevalence before and after the ban. Analyses suggest that the added risk to human health from resistance among enterococci and campylobacters selected by growth promoter use is small, whilst the benefit to human health from their use, hitherto largely ignored, might more than counterbalance this.

Vancomycin-resistant enterococci (VRE) in healthy people and in food-producing animals seems to be quite common in Europe. The existence of this community reservoir of VRE has been associated with the massive use of avoparcin in animal husbandry. Eight years after the avoparcin ban in Europe, we investigated the incidence of VanA enterococci, their resistance patterns, and the mobility of their glycopeptide-resistance determinants in a sampling of animal rectal swabs and clinical specimens. A total of 259 enterococci isolated from equine, swine, and clinical samples were subcultured on KF-streptococcus agar (Difco Laboratories, Detroit, MI) supplemented with vancomycin and teicoplanin; 7 (6.7%), 10 (16%), and 8 (8.6%) respectively were found to be glycopeptides resistant (VanA phenotype). Slight differences in antimicrobial resistance patterns resulted among VRE recovered from the different sources. Polymerase chain reaction amplification demonstrated the presence of the vanA gene cluster and its extrachromosomal location in VRE plasmid DNA. VanA resistance was transferred in 7 out of 25 mating experiments, 4 with clinical, 2 with swine, and only 1 with equine donors. The conjugative plasmids of animal strains showed a high homology in the restriction profiles, unlike plasmids of clinical microrganisms. Our observations confirmed the possible horizontal transfer of VanA plasmids across different strains and, consequently, the diffusion of the vancomycin-resistance determinants.

Objectives: VanA glycopeptide resistance has persisted on broiler farms in the UK despite the absence of the antimicrobial selective pressure, avoparcin. This study aimed to investigate the contribution of horizontal gene transfer of Tn1546 versus clonal spread in the dissemination of the resistance. Methods and results: One hundred and one vancomycin-resistant Enterococcus faecium isolated from 19 unrelated farms have been investigated. Tn1546 characterization by long PCR and ClaI-digestions of amplicons showed a very low diversity of Tn types (n 5 4) in comparison to the high genotypic diversity demonstrated by PFGE (n 5 62). Conjugation experiments were carried out to assess the transfer of vancomycin resistance. Co-transfer of vanA together with erm(B) positioned on the same conjugative plasmid containing a replicon similar to pRE25 was demonstrated and also the presence of different plasmid replicons, associated with antimicrobial resistance on several unrelated farms. Conclusions: Horizontal transfer of vancomycin resistance may play a more important role in the persistence of antimicrobial resistance than clonal spread. The presence of different plasmid replicons, associated with antimicrobial resistance on several unrelated farms, illustrates the ability of these enterococci to acquire and disseminate mobile genetic elements within integrated livestock systems.

Summary Avoparcin, a vancomycin analogue, was banned as a feed additive in Taiwan in 2000. A nationwide surveillance was conducted to study the prevalence of vancomycin-resistant enterococci (VRE) on chicken farms between 2000 and 2003. Among the 1021 E. faecalis and 967 E. faecium isolates studied, resistance to tetracycline, erythromycin, high-level aminoglycosides, ciprofloxacin and chloramphenicol either increased or remained high except vancomycin. The proportion of VRE decreased, between 2000 and 2003, from 13.7% (22/161) to 3.7% (11/299) for E. faecalis, and 3.4% (4/119) to 0% (0/300) for E. faecium. Only 8.8% (7/80) of the chicken farms surveyed harboured VRE in 2003 compared with 25% (15/60) in 2000. All VRE were resistant to tetracycline and erythromycin. All VRE possess the vanA gene but nearly all (79 of 83 isolates) were susceptible to teicoplanin, indicating VanB phenotype. Some clones were detected from different farms in various regions over the years. We conclude that the frequency of VRE in chicken farms decreased in association with a ban on avoparcin; and the continued presence of VRE may be due to the ability of some strains to persist in the farms, transfer of vancomycin resistance determinants or co-selection by the continued use of other antibiotics.

International audience; Antibiotics, prebiotics and probiotics are widely used as growth promoters in agriculture. In the 1940s, use of Strepromyces aureofaciens probiotics resulted in weight gain in animals, which led to the discovery of chlortetracycline. Tetracyclines, macrolides, avoparcin and penicillins have been commonly used in livestock agriculture to promote growth through increased food intake, weight gain, and improved herd health. Prebiotic supplements including oligosaccharides, fructooligosaccharides, and galactosyl-lactose improve the growth performance of animals. Probiotics used in animal feed are mainly bacterial strains of Gram-positive bacteria and have been effectively used for weight gain in chickens, pigs, ruminants and in aquaculture. Antibiotics, prebiotics and probiotics all modify the gut microbiota and the effect of a probiotic species on the digestive flora is probably determined by bacteriocin production. Regulations governing the introduction of novel probiotics and prebiotics vary by geographical region and bias is very common in industry-funded studies. Probiotic and prebiotic foods have been consumed for centuries, either as natural components of food, or as fermented foods and it is possible to cause the same weight gain effects in humans as in animals. This review presents the use of growth promoters in food-producing animals to influence food intake and weight gain. (C) 2016 Elsevier Ltd. All rights reserved.

The search for new and effective chiral selectors capable of separating a wide variety of enantiomeric compounds is an ongoing process. In the past decade, macrocyclic antibiotics have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance by means of HPLC, TLC and electrophoresis. More chiral analytes have been resolved through the use of glycopeptides than with all the other macrocyclic antibiotics combined (ansamycins, thiostrepton, aminoglycosides, etc.). The glycopeptides avoparcin, teicoplanin, ristocetin A and vancomycin have been extensively used as chiral selectors in the form of chiral bonded phases in HPLC, and HPLC stationary phases based on these glycopeptides have been commercialized. Teicoplanin, vancomycin, their analogs and ristocetin A seem to be the most useful glycopeptide HPLC bonded phases for the enantioseparation of proteins and unusal native and derivatized amino acids. In fact, the macrocyclic glycopeptides are to some extent complementary to one another: where partial enantioresolution is obtained with one glycopeptide, there is a high probability that baseline or better separation can be obtained with another. This review sets out to characterize the physicochemical properties of these antibiotics and their application in the enantioseparations of amino acids. The mechanism of separation, the sequence of elution of the stereoisomers and the relation to the absolute configuration are also discussed.

Prevalence of vancomycin-resistant enterococci (VRE) was investigated in Korean livestock 4 years after the ban of avoparcin in feed additives. VRE were isolated from approximately 16.7% of the chicken samples (57 strains from 342 meat samples) and 1.9% of the pig samples (4 from 214 fecal samples). No VRE, however, was isolated from 110 bovine fecal samples. All the 61 VRE isolates were vanA-type Enterococcus faecium expressing a high-level resistance to vancomycin, and showed resistance to teicoplanin as well except two poultry isolates. In addition, the VRE isolates had heterogeneous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, although identical or closely related profiles were observed among strains isolated from the same farm. Although the chicken isolates were all poultry type with G at position 8,234 of the vanX gene, the pig isolates were all swine type with T at position 8,234 of the vanX gene.

Environmental reservoirs of glycopeptide-resistant enterococci (GRE) in Norway have been linked to former growth promoting use of the glycopeptide avoparcin in poultry production. We have examined the prevalence of fecal GRE in poultry and poultry farmers 3 to 8 years after the Norwegian avoparcin ban in 1995 and performed molecular analyses of the GRE population. Fecal samples from poultry farmers and their flocks on 29 previously avoparcin-exposed farms were collected on five occasions during the study period (1998 to 2003). All flocks (100%) were GRE positive in 1998. Throughout the study period, 78.5% of the poultry samples were GRE positive. Glycopeptide-resistant Enterococcus faecium (GREF) was isolated from 27.6% of the farmer samples in 1998 and from 27.8% of the samples collected between 1998 and 2003. The prevalence of fecal GRE in poultry declined significantly during the study period, but prevalence in samples from the farmers did not decline. PCR analysis revealed a specific Tn 1546 -plasmid junction fragment in 93.9% of E. faecium isolates. A putative postsegregation killing (PSK) system linked to Tn 1546 was detected in 97.1% of the isolates examined. Multilocus sequence typing of glycopeptide-susceptible ( n = 10) and -resistant ( n = 10) E. faecium isolates from humans ( n = 10) and poultry ( n = 10) on two farms displayed 17 different sequence types. The study confirms the continuing persistence of a widespread common plasmid-mediated vanA -pRE25-PSK element within a heterogeneous GRE population on Norwegian poultry farms 8 years after the avoparcin ban. Moreover, it suggests an important role of PSK systems in the maintenance of antimicrobial resistance determinants in reservoirs without apparent antimicrobial selection.

In the recent past years, some relevant changes have been introduced in the nutrition of farmed animals. To meet safety issues induced by Bovine Spongiform Encephalopathy (BSE) and ‘dioxins’ crises, new kinds of vegetal feed materials have been introduced in the diet, to replace animal proteins and fats. Moreover, the progressive withdrawal of some feed additives with anti-microbial activity, such as avoparcin, bacitracin, virginiamycin, tylosin, spiramycin, carbadox and olaquindox due to a renewed analysis about their toxicological and pharmacoresistance risk, could prompt the market to propose some other alternatives to farmers, such as the administration of immuno-stimulants (i.e. colostrum) and/or natural substances having a direct or indirect anti-bacterial effect (i.e. prebiotics, probiotics, herbal extracts). Last but not least, one of the fastest growing areas is represented by the study of feed formulations able to produce nutraceuticals and dietary foods of animal origin on large scale, thus boosting consumers perception about their overall quality, such as polyunsaturated fatty acids and Vitamin E enriched eggs, via the use of selected materials in feeds. This paper aims to illustrate some examples of such innovative trends in animal feeding, along with the discussion on the analytical challenges useful for a reliable assessment of a possible new risks/benefits ratio along the food chain.

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF ( n = 136) and glycopeptide-susceptible ( n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA -containing Tn 1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn 1546 -plasmid junction fragment strongly indicated the presence of a common single Tn 1546 -plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn 1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA -containing element within a polyclonal GREF population that changes over time. In addition, we propose that “plasmid addiction systems” may contribute to the persistence of GREF in nonselective environments.

Vancomycin-resistant enterococci (VRE) have emerged as important nosocomial pathogens, initially in the USA, but now also in Europe, where hospital outbreaks are being reported with increasing frequency, although the incidence of VRE infections remains extremely low in most European countries. The recently demonstrated in-human transmission of vancomycin resistance from VRE to methicillin-resistant Staphylococcus aureus (MRSA) in two American patients underscores the potential danger of a coexisting reservoir of both pathogens. As MRSA is already endemic in many European hospital settings, prevention of endemicity with VRE seems relevant, but should be balanced against the costs associated with the implementation of effective strategies. The presence of a large community reservoir of VRE in Europe could hamper the feasibility of infection control strategies. Although the prevalence of colonisation amongst healthy subjects has apparently decreased after the ban on avoparcin use in the agricultural industry, a large proportion of admitted patients are still potential sources of VRE transmission. With no risk profile available to identify these carriers, effective screening, followed by barrier precautions for carriers, seems to be impossible. Recent studies, however, have suggested that hospital outbreaks are almost exclusively caused by specific genogroups of VRE that can be characterised phenotypically and genotypically (e.g., co-resistance to ampicillin and the presence of the variant esp gene). Based on our own experience, we propose that VRE infection control programmes should be restricted to patients colonised with these VRE strains. If such a strain is cultured from a clinical sample, surveillance amongst contact patients is recommended and barrier precautions should be implemented in the case of documented spread.

Avoparcin, a glycopeptide antimicrobial agent related to vancomycin, has been used extensively as a growth promoter in animal feeds for more than 2 decades, and evidence has shown that such use contributed to the development of vancomycin-resistant enterococci. A cluster that includes three genes, vanH, vanA, and vanX, is required for high-level resistance to glycopeptides. In the vancomycin producer Amycolatopsis orientalis C329.2, homologs of these genes are present, suggesting an origin for the cluster. We found substantial bacterial DNA contamination in animal-feed-grade avoparcin. Furthermore, nucleotide sequences related to the cluster vanHAX are present in this DNA, suggesting that the prolonged use of avoparcin in agriculture led to the uptake of glycopeptide resistance genes by animal commensal bacteria, which were subsequently transferred to humans.

When avoparcin was prohibited for use as feed additive in poultry in Norway on 31 May 1995, an increased incidence of Clostridium perfringens-associated necrotic enteritis (NE) and an increase in the use of antibacterial (AB) drug therapy in meat-type poultry was expected. The consumption of AB drugs for use against NE in poultry in the period 1990-2001 was investigated by use of sales statistics at the drug-wholesaler level. Defined daily dose (DDD) per kg live weight poultry was the unit of measurement for drug use (to correct for differences in the dosages). Sales figures of the AB drugs were converted to number of DDDpoultry sold for the numbers of broilers at risk (broilers were 97% of the slaughter poultry). Estimated annual percentages of the broilers treated against NE increased abruptly after the avoparcin ban--but in 1996, this figure declined to the same level as before the ban and has remained at that low level since then. In November 1995, narasin was approved temporarily as an ionophore feed additive (IFA) in broilers. The usage patterns of IFAs in broilers were measured as the weight of feed to which an IFA was added per broiler chicken produced. In 1996-2001, the IFAs used in broilers were predominantly narasin. We note that the temporary increase in NE after the avoparcin ban coincide with the period before narasin became available. The increase in the consumption of AB drugs for the treatment of NE in poultry following the avoparcin ban has been negligible.

Enterococci are the second to third most important bacterial genus in hospital infections. Especially Enterococcus (E.) faecium possesses a broad spectrum of natural and acquired antibiotic resistances which are presented in detail in this paper. From medical point of view, the transferable resistances to glycopeptides (e.g., vancomycin, VAN, or teicoplanin, TPL) and streptogramins (e.g., quinupristin/dalfopristin, Q/D) in enterococci are of special interest. The VanA type of enterococcal glycopeptide resistance is the most important one (VAN-r, TPL-r); its main reservoir is E. faecium. Glycopeptide-resistant E. faecium (GREF) can be found in hospitals and outside of them, namely in European commercial animal husbandry in which the glycopeptide avoparcin (AVO) was used as growth promoter in the past. There are identical types of the vanA gene clusters in enterococci from different ecological origins (faecal samples of animals, animal feed, patients in hospitals, persons in the community, waste water samples). Obviously, across the food chain (by GREF-contaminated meat products), these multiple-resistant bacteria or their vanA gene clusters can reach humans. In hospital infections, widespread epidemic-virulent E. faecium isolates of the same clone with or without glycopeptide resistance can occur; these strains often harbour different plasmids and the esp gene. This indicates that hospital-adapted epidemic-virulent E. faecium strains have picked up the vanA gene cluster after they were already widely spread. The streptogramin virginiamycin was also used as feed additive in commercial animal husbandry in Europe for more than 20 years, and it created reservoirs for streptogramin-resistant E. faecium (SREF). In 1998/1999, SREF could be isolated in Germany from waste water of sewage treatment plants, from faecal samples and meat products of animals that were fed virginiamycin (cross resistance to Q/D), from stools of humans in the community, and from clinical samples. These isolations of SREF occurred in a time before the streptogramin combination Q/D was introduced for therapeutic purposes in German hospitals in May 2000, while other streptogramins were not used in German clinics. This seems to indicate that the origin of these SREF or their streptogramin resistance gene(s) originated from other sources outside the hospitals, probably from commercial animal husbandry. In order to prevent the dissemination of multiple antibiotic-resistant enterococci or their transferable resistance genes, a prudent use of antibiotics is necessary in human and veterinary medicine, and in animal husbandry.

Animals and humans constitute overlapping reservoirs of resistance, and consequently use of antimicrobials in animals can impact on public health. For example, the occurrence of vancomycin-resistant enterococci in food-animals is associated with the use of avoparcin, a glycopeptide antibiotic used as a feed additive for the growth promotion of animals. Vancomycin-resistant enterococci and vancomycin resistance determinants can therefore spread from animals to humans. The bans on avoparcin and other antibiotics as growth promoters in the EU have provided scientists with a unique opportunity to investigate the effects of the withdrawal of a major antimicrobial selective pressure on the occurrence and spread of antimicrobial resistance. The data shows that although the levels of resistance in animals and food, and consequently in humans, has been markedly reduced after the termination of use, the effects on animal health and productivity have been very minor.

Glycopeptide resistant enterococci (GRE) isolated from animals and humans were characterised using both AFPL typing and genetic characterisation of the glycopeptide resistance transposon Tn1546. All isolates were collected in 1997 when the glycopeptide avoparcin was still being used as growth promoter. All investigated animal isolates were from mixed pig and poultry farms in the Netherlands and the human isolated from the farmers of these farms. A total of 24 isolates were investigated. AFLP and Tn1546 typing revealed that both pig and poultry related enterococcal and vanA transposon genotypes were found among the human isolates indicating spread of glycopeptide resistance from both pig and poultry to the farmers. These findings contradict previous finding that showed that GRE recovered from the general population were genotypically undistinguishable from GRE isolated from pigs but are in line with other studies that demonstrated spread of GRE from poultry to farmers in poultry farms.

A trial was conducted with 32 indigenous goat kids (Capra prisca) 8,5 weeks old to examine the effectiveness of dietary avoparcin as growth promoting factor. The kids were randomly allocated into two groups (control- and avoparcin group with initial mean body weight of 11,61 ±1,16 kg and 11,57±1,47 kg, respectively) comprising of 16 kids (8 male and 8 female) each. For a period of 12 weeks, kids in the control group were given lucerne (alfalfa) hay (mean daily consumption 170 g/head) and an appropriate compound diet (given ad libitum), while kids in the avoparcin group were fed the same diet except that the compound diet was supplemented with avoparcin (20 mg/kg). Avoparcin supplementation significantly (P0.05) were noticed neither in carcass yield and carcass chemical composition nor in percentages of small intestine-, major omentum-, lung-,heart-, liver- and spleen weight in the body weight.

The glycopeptide growth promoter avoparcin was banned from animal production in the EU in 1997 due to concern for the spread of vancomycin-resistant enterococci (VRE) from food animals to humans. In recent Norwegian and Danish studies, extensive occurrence of VRE on broiler farms and in broiler flocks after the avoparcin ban has been reported. The present study was undertaken to investigate the epidemiology of VRE on broiler farms in the absence of the selective pressure exerted by avoparcin. Environmental samples were obtained from five broiler houses after depopulation, cleaning, and disinfection of the houses between rotations, and two consecutive broiler flocks from each house were sampled by taking cloacal swabs from the broilers at the time of slaughter. A total of 69 vancomycin-resistant Enterococcus faecium isolates obtained from broiler flocks and broiler houses were subjected to molecular typing by pulsed-field gel electrophoresis (PFGE). Forty-one PFGE-profiles were observed. VRE with indistinguishable or highly similar PFGE profiles were isolated from consecutive broiler flocks and from environmental samples from the houses in which the flocks were reared, whereas VRE-isolates from different broiler houses and from flocks reared in different houses appeared to be genetically unrelated. These findings indicated that VRE was transmitted between consecutive broiler flocks by clones of resistant bacteria surviving in the broiler houses despite cleaning and disinfection between rotations. Thus, the extensive occurrence of VRE in broiler flocks after the avoparcin ban may be explained by persistence of VRE in the broiler house environment.

Three plate systems (combinations of indicator organism and growth medium) were evaluated for the detection of analytical standards of the banned feed additives avoparcin, bacitracin zinc, spiramycin, tylosin and virginiamycin. When authorized in the EU, the previously recommended minimum inclusion rate (MIR) for each compound was 5 mg kg(-1). One of the plate systems (Micrococcus luteus ATCC 10240, nutrient agar) detected all five additives. This plate was used in a further study that evaluated the suitability of accelerated solvent extraction (ASE) as a first step in the development of a rapid single-plate screening assay. A drug-free (negative control) feedingstuff was fortified with the compounds (0-50 mg kg(-1)), extracted by ASE and the extracts applied to the plate at each of three pH ranges - unadjusted extract (pH 5.7-5.9), pH 6.5 and 8.0. At pH 6.5, sub-MIR concentrations of virginiamycin and tylosin were detectable. Avoparcin was detectable at 6.3 mg kg(-1). The detection of zinc bacitracin was#10; pH-independent (10 mg kg(-1)). At pH 8.0, spiramycin was detectable at 5.4 mg kg(-1). Mean +/- SD analytical recoveries from fortified feedingstuffs (n = 10) ranged from 57 +/- 1.5% for avoparcin to 96 +/- 4% for virginiamycin. The five additives were also detectable following ASE extraction from a range of different feedingstuffs fortified with each of the drugs. A further 24 compounds permitted for use in animal feeds were tested. Of these, nine were detectable at their recommended MIR. It is concluded that ASE is a versatile technique suitable for the automated extraction of a range of antimicrobials from animal feedingstuffs. Employing ASE with this single-plate detection system permits the rapid antimicrobial screening of animal feedingstuffs and allows the detection of the banned additives. Whilst the method is applicable as a screening test, more specific postscreening methods would be necessary for subsequent identification (and quantification) of antimicrobials in screening-positive samples.

In Europe the use of the growth promoter avoparcin is considered to have selected for vancomycin-resistant enterococci (VRE). Sweden ceased using avoparcin in 1986, and only occasional cases of VRE from hospitals have been reported since 1995. Within the framework of a European study, samples from urban raw sewage, treated sewage, surface water, and hospital sewage in Sweden ( n = 118) were screened for VRE. Surprisingly, VRE were isolated from 21 of 35 untreated sewage samples (60%), from 5 of 14 hospital sewage samples (36%), from 6 of 32 treated sewage samples (19%), and from 1 of 37 surface water samples. Thirty-five isolates from 33 samples were further characterized by geno- and phenotyping, MIC determination, and PCR analysis. Most isolates (30 of 35) carried the vanA gene, and the majority (24 of 35) of the isolates were Enterococcus faecium . Most of the VRE were multiresistant. The typing revealed high diversity of the isolates. However, one major cluster with seven identical or similar isolates was found. These isolates came from three different sewage treatment plants and were collected at different occasions during 1 year. All VRE from hospital sewage originated from one of the two hospitals studied. That hospital also had vancomycin consumption that was 10-fold that of the other. We conclude that VRE were commonly found in sewage samples in Sweden. The origin might be both healthy individuals and individuals in hospitals. Possibly, antimicrobial drugs or chemicals released into the sewage system may sustain VRE in the system.

A new approach is presented for the determination of avoparcin in tissue. Complete recovery from spiked swine kidney was achieved with hot water modified with 30% ethanol (v/v). The samples were extracted at 75 “C and 50 atm by accelerated solvent extraction. In situ sample clean-up was achieved by using matrix solid-phase dispersion utilizing the acrylic polymer XAD-7 HP, and by adding triethylammonium phosphate (TEAP) to the extraction solvent. The aqueous extracts were concentrated by solid-phase extraction (SPE) on the hydrophilic interaction chromatography (HILIC) material polybydroxyethyl aspartamide. Complete analyte retention was possible during SPE when the kidney extracts were modified with 70% ethanol. A 200 A, 5 km HILIC column with W,,, detection was used for the separation of avoparcin. The retention time was less than 15 min with 47% aqueous component in acetonitrile and 15 mM TEAP as eluent. The average recovery of avoparcin from kidney samples was 108%. Published by Elsevier Science B.V.

To compare the differential antimicrobial susceptibilities of Enterococcus faecium from humans and whole chicken carcasses, MICs of 12 antimicrobial agents were determined for 54 clinical-isolates (31 vancomycin-resistant [VREF]) and 60 chicken-isolates (29 VREF). Chicken VREF were slightly but consistently more resistant to vancomycin, teicoplanin and avoparcin, compared with human VREF (P0.01). MICs of LY333328 wereor = 2 mg/l. All human VREF were resistant to erythromycin and tylosin, compared with only 58.6% of chicken VREF (P0.01). Streptogramins were active against all isolates except four chicken strains. MIC(90s) of amoxycillin and gentamicin for human E. faecium were 8-16-fold higher than chicken isolates. Chicken VREF were significantly more resistant to tetracycline but more susceptible to chloramphenicol than human VREF (P0.001).

The glycopeptide and lipoglycopeptide antibiotics are natural antibiotics obtained from cultures of certain microorganisms. These antibiotics inhibit bacterial cell wall peptidoglycan synthesis but cannot cross the cell wall of gram-negative bacteria. Their antibacterial spectrum includes aerobic and anaerobic gram-positive bacteria. The glycopeptides may be divided into at least five groups on the basis of their chemical structures. Due to their large mass, glycopeptides cannot cross the lipoprotein cell wall of gram-negative bacteria and are therefore totally inactive against them. The molecules belonging to group II (Avoparcin Type) are mainly actinodin, avoparcin, chloropolysporin, galacardin, helvecardin, and synmonicin. Resistance to vancomycin and teicoplanin is due to synthesis of modified precursors that display decreased affinity for both compounds. Six types of resistance have been reported to date: VanA, VanB, VanC, VanD, VanE, and VanG. The combination of a glycopeptide with an aminoglycoside is synergistic against the majority of strains of staphylococci and enterococci. The in vitro activity of dalbavancin was investigated by following NCCLS-recommended methods and comparatively with vancomycin, linezolid, and quinuprisitin-dalfoprisitin (Q-D).

Summary Vancomcyin-resistant enterococci (VRE) have emerged as nosocomial pathogens in the past 10 years, causing epidemiological controversy. In the USA, colonisation with VRE is endemic in many hospitals and increasingly causes infection, but colonisation is absent in healthy people. In Europe, outbreaks still happen sporadically, usually with few serious infections, but colonisation seems to be endemic in healthy people and farm animals. Vancomycin use has been much higher in the USA, where emergence of ampicillin-resistant enterococci preceded emergence of VRE, making them very susceptible to the selective effects of antibiotics. In Europe, avoparcin, a vancomycin-like glycopeptide, has been widely used in the agricultural industry, explaining the community reservoir in European animals. Avoparcin has not been used in the USA, which is consistent with the absence of colonisation in healthy people. From the European animal reservoir, VRE and resistance genes have spread to healthy human beings and hospitalised patients. However, certain genogroups of enterococci in both continents seem to be more capable of causing hospital outbreaks, perhaps because of the presence of a specific virulence factor, the variant esp gene. By contrast with the evidence of a direct link between European animal and human reservoirs, the origin of American resistance genes remains to be established. Considering the spread of antibioticresistant bacteria and resistance genes, the emergence of VRE has emphasised the non-existence of boundaries between hospitals, between people and animals, between countries, and probably between continents.