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Resistentieontwikkeling verscherpt de aandacht voor het antibioticumgebruik in de veehouderij. Het project ‘Transparant en efficiënt medicijngebruik’ brengt antibioticumgebruik in kaart via aantal dagdoseringen. Dat getal varieert van bedrijf tot bedrijf.

In de dierhouderij worden antibiotica veelvuldig gebruikt, waardoor antibioticaresistentie toeneemt, zowel bij zoönotische als bij commensale bacteriën. Dit heeft geleid tot bezorgdheid over de risico’s van overdracht van resistente zoönotische bacteriën en resistentiegenen van voedselproducerende dieren naar de mens en de mogelijke gevolgen daarvan voor de volksgezondheid en de gezondheidszorg. Dit rapport bevat een risicoprofiel, hetgeen bedoeld is om risicomanagers te informeren over de beschikbare kennis met betrekking tot dit potentiële gezondheidsrisico, als een eerste stap in het proces van risicomanagement. 3 gevaren dienen als voorbeeld in dit risicoprofiel: 1. quinolone-resistente Campylobacter jejuni; 2. veegerelateerde MRSA (v-MRSA); 3. ESBL-producerende bacteriën.

In de dierhouderij worden antibiotica veelvuldig gebruikt, waardoor antibioticaresistentie toeneemt, zowel bij zoönotische als bij commensale bacteriën. Dit heeft geleid tot bezorgdheid over de risico’s van overdracht van resistente zoönotische bacteriën en resistentiegenen van voedselproducerende dieren naar de mens en de mogelijke gevolgen daarvan voor de volksgezondheid en de gezondheidszorg. Dit rapport bevat een risicoprofiel, hetgeen bedoeld is om risicomanagers te informeren over de beschikbare kennis met betrekking tot dit potentiële gezondheidsrisico, als een eerste stap in het proces van risicomanagement. 3 gevaren dienen als voorbeeld in dit risicoprofiel: 1. quinolone-resistente Campylobacter jejuni; 2. veegerelateerde MRSA (v-MRSA); 3. ESBL-producerende bacteriën.

In het kader van een overeenkomst tussen Stichting Kennisontwikkeling Kennisoverdracht Bodem (SKB) en een breed consortium en klankbordgroep is een pilotstudie uitgevoerd naar veterinaire antibiotica in de bodem en het grondwater.

De overheid en andere belanghebbenden willen meer inzicht in het gebruik van antibiotica in de veehouderij. Antibioticagebruik bij voedselproducerende dieren draagt namelijk bij aan resistentieontwikkeling en vormt daarmee een risico voor de volksgezondheid. Om die reden verplicht de Europese commissie lidstaten tot bewaking van antimicrobiële resistentie en is ook gebruiksmonitoring van evident belang. Dit rapport brengt het antibioticagebruik in Nederland en in andere landen in beeld. Ook wordt inzicht gegeven in mogelijke oorzaken.

Terwijl antibioticum-resistentie in ziekenhuizen en landbouwhuisdieren relatief goed onderzocht is, is veel minder bekend over het optreden van resistentie in het milieu. Een mogelijk belangrijke bron van resistentie in het milieu is het uitrijden van mest van intensief gehouden landbouwhuisdieren op landbouwgronden. In deze studie werd een pilot-onderzoek verricht naar de rol van bemesting met varkensdrijfmest voor resistentie in zandige landbouwgronden. Er werd gekozen voor een veld-benadering, waarbij drie bedrijven tijdens de periode van bemesting regelmatig werden bemonstert (vlak voor, en een dag, een week en vier weken na de bemesting). Op elk bedrijf werd in de nabijheid van de veldlocatie ook een referentielocatie gekozen, om de lange termijn effecten van bemesting te kunnen vergelijken met een soortgelijke, maar niet bemestte grond.

In het kader van een overeenkomst tussen Stichting Kennisontwikkeling Kennisoverdracht Bodem (SKB) en een breed consortium en klankbordgroep is een pilotstudie uitgevoerd naar veterinaire antibiotica in de bodem en het grondwater.

De overheid en andere belanghebbenden willen meer inzicht in het gebruik van antibiotica in de veehouderij. Antibioticagebruik bij voedselproducerende dieren draagt namelijk bij aan resistentieontwikkeling en vormt daarmee een risico voor de volksgezondheid. Om die reden verplicht de Europese commissie lidstaten tot bewaking van antimicrobiële resistentie en is ook gebruiksmonitoring van evident belang. Dit rapport brengt het antibioticagebruik in Nederland en in andere landen in beeld. Ook wordt inzicht gegeven in mogelijke oorzaken.

Onderzoek naar de mogelijkheden van registratie van diergeneesmiddelengebruik op basis van private initiatieven. Dit houdt een evaluatie in van de initiatieven voor registratie vanuit vier verschillende sectoren (varkens, vleeskuikens, vleeskalveren, rundvee) en de stichting VETbase. Investigation into the possibilities of registering the use of veterinary medicines using private initiatives. The report comprises an evaluation of the initiatives for registration from the standpoint of four different sectors (pigs, broilers, veal calves and cattle) and the VETbase Foundation.

Onderzoek naar de mogelijkheden van registratie van diergeneesmiddelengebruik op basis van private initiatieven. Dit houdt een evaluatie in van de initiatieven voor registratie vanuit vier verschillende sectoren (varkens, vleeskuikens, vleeskalveren, rundvee) en de stichting VETbase. Investigation into the possibilities of registering the use of veterinary medicines using private initiatives. The report comprises an evaluation of the initiatives for registration from the standpoint of four different sectors (pigs, broilers, veal calves and cattle) and the VETbase Foundation.

Terwijl antibioticum-resistentie in ziekenhuizen en landbouwhuisdieren relatief goed onderzocht is, is veel minder bekend over het optreden van resistentie in het milieu. Een mogelijk belangrijke bron van resistentie in het milieu is het uitrijden van mest van intensief gehouden landbouwhuisdieren op landbouwgronden. In deze studie werd een pilot-onderzoek verricht naar de rol van bemesting met varkensdrijfmest voor resistentie in zandige landbouwgronden. Er werd gekozen voor een veld-benadering, waarbij drie bedrijven tijdens de periode van bemesting regelmatig werden bemonstert (vlak voor, en een dag, een week en vier weken na de bemesting). Op elk bedrijf werd in de nabijheid van de veldlocatie ook een referentielocatie gekozen, om de lange termijn effecten van bemesting te kunnen vergelijken met een soortgelijke, maar niet bemestte grond.

The Infectious Diseases Surveillance Information system that collects data of medical microbiology laboratories in the Netherlands (ISIS-MML) is not suitable for early warning of infectious diseases outbreaks. However, the system is appropriate for monitoring trends in antimicrobial resistance. These are the main outcomes of an evaluation described in this report to evaluate technical problems and doubts about the performance and usefulness of the system. ISIS-MML was developed ten years ago by RIVM to provide current information on the occurrence of infectious diseases via Internet. Important goals were to observe outbreaks in time, prevent further spread of disease, and to limit the negative consequences of these outbreaks. For this purpose all 85 medical microbiology laboratories in the Netherlands had to send in data on a daily basis. The evaluation showed that only eighteen laboratories are connected to the system; this led to a lack of national coverage by the system, causing outbreaks to be missed. For example, the outbreak of Clostridium difficile-associated disease of ribotype 027 was missed in June 2005. Furthermore, the system regularly causes false alarms since not all increases in incidence of micro-organisms require control measures. That does not mean that ISIS-MML is useless and needs to be stopped. The system is the most important database for antimicrobial resistance in the Netherlands. The European Union strongly recommends surveillance of antimicrobial resistance since it an increasing problem. Therefore RIVM advises adaptation of the current system (in which hardware and software are outdated) to an antimicrobial surveillance system.

The Infectious Diseases Surveillance Information system that collects data of medical microbiology laboratories in the Netherlands (ISIS-MML) is not suitable for early warning of infectious diseases outbreaks. However, the system is appropriate for monitoring trends in antimicrobial resistance. These are the main outcomes of an evaluation described in this report to evaluate technical problems and doubts about the performance and usefulness of the system. ISIS-MML was developed ten years ago by RIVM to provide current information on the occurrence of infectious diseases via Internet. Important goals were to observe outbreaks in time, prevent further spread of disease, and to limit the negative consequences of these outbreaks. For this purpose all 85 medical microbiology laboratories in the Netherlands had to send in data on a daily basis. The evaluation showed that only eighteen laboratories are connected to the system; this led to a lack of national coverage by the system, causing outbreaks to be missed. For example, the outbreak of Clostridium difficile-associated disease of ribotype 027 was missed in June 2005. Furthermore, the system regularly causes false alarms since not all increases in incidence of micro-organisms require control measures. That does not mean that ISIS-MML is useless and needs to be stopped. The system is the most important database for antimicrobial resistance in the Netherlands. The European Union strongly recommends surveillance of antimicrobial resistance since it an increasing problem. Therefore RIVM advises adaptation of the current system (in which hardware and software are outdated) to an antimicrobial surveillance system.

TEM-1 ß-lactamase is one of the most notorious antibiotic resistance enzymes around. It exists at high frequencies in antibiotic-resistant bacteria around the world and confers resistance to ß-lactam antibiotics, including penicillins (e.g. ampicillin) and cephalosporins. The enzyme displays a remarkable phenotypic plasticity in response to the introduction of new drugs; within a few years after the clinical debut of most new ß-lactam antibiotics resistance conferring variants of TEM-1 are isolated. Such a shift in resistance phenotype is typically caused by just a few amino acid substitutions. Until today, more than 150 variants of TEM-1 with a unique amino acid sequence have been identified. Because of the clear link between genotype and phenotype (i.e. level of resistance or fitness) and because of the ease of selecting for increased antibiotic resistance, TEM-1 has been used as a model in studies that seek new methods to optimize proteins. These studies combine the power of in vitro mutagenesis and in vivo selection and have resulted in a wealth of information about which mutations can increase resistance when the enzyme is exposed to an antibiotic that it initially hydrolyzes inefficiently. At a later stage, these techniques were adopted and used to repeat and predict the natural evolution of TEM-1 under various selective conditions. Recently, TEM-1 is increasingly being used as an experimental model for the study of fundamental evolutionary questions, particularly those that benefit from the direct relationship between genotype and phenotype. In this thesis, both the natural and laboratory evolution of TEM-1 are studied. The aim of the laboratory work is to increase our understanding of the way in which adaptive mutations interact. For this purpose, TEM-1 is mutagenized using error-prone PCR, which creates variation in the resulting copies of the TEM-1 gene. Mutated gene-copies are placed in bacteria which are subsequently selected for increased resistance to cefotaxime (an antibiotic that TEM-1 hydrolyzes poorly). By repeating this process multiple times in independent experiments, the mutations and mutational trajectories involved in the increase of cefotaxime resistance are studied. At a fundamental level, this has lead to a better understanding of the nature of mutation interaction and its consequences for evolutionary contingency and constraint. Evidence indicating that certain ‘silent’ mutations (i.e. mutations that alter the codon sequence but not the amino acid that the respective codon encodes) can also play a role in increased resistance was found in these data as well. A phylogenetic study of the sequences of the ~150 different TEM-alleles that have been isolated in hospitals and clinics so far indicates that recombination has played a significant role in the evolution of TEM-alleles, contrary to what is often assumed. Furthermore, amino acid substitutions present in these clinical isolates are compared to those found in laboratory evolution studies of TEM-1, in order to investigate to what extent laboratory evolution can be used as a predictive tool for the natural evolution of antibiotic resistance genes. This overview indicates that laboratory evolution very accurately repeats the natural evolution of TEM-1. Based on these findings, predictions are made about substitutions that may appear in future clinical TEM-isolates, and directions are given how laboratory evolution can be exploited as a predictive tool most efficiently.

TEM-1 ß-lactamase is one of the most notorious antibiotic resistance enzymes around. It exists at high frequencies in antibiotic-resistant bacteria around the world and confers resistance to ß-lactam antibiotics, including penicillins (e.g. ampicillin) and cephalosporins. The enzyme displays a remarkable phenotypic plasticity in response to the introduction of new drugs; within a few years after the clinical debut of most new ß-lactam antibiotics resistance conferring variants of TEM-1 are isolated. Such a shift in resistance phenotype is typically caused by just a few amino acid substitutions. Until today, more than 150 variants of TEM-1 with a unique amino acid sequence have been identified. Because of the clear link between genotype and phenotype (i.e. level of resistance or fitness) and because of the ease of selecting for increased antibiotic resistance, TEM-1 has been used as a model in studies that seek new methods to optimize proteins. These studies combine the power of in vitro mutagenesis and in vivo selection and have resulted in a wealth of information about which mutations can increase resistance when the enzyme is exposed to an antibiotic that it initially hydrolyzes inefficiently. At a later stage, these techniques were adopted and used to repeat and predict the natural evolution of TEM-1 under various selective conditions. Recently, TEM-1 is increasingly being used as an experimental model for the study of fundamental evolutionary questions, particularly those that benefit from the direct relationship between genotype and phenotype. In this thesis, both the natural and laboratory evolution of TEM-1 are studied. The aim of the laboratory work is to increase our understanding of the way in which adaptive mutations interact. For this purpose, TEM-1 is mutagenized using error-prone PCR, which creates variation in the resulting copies of the TEM-1 gene. Mutated gene-copies are placed in bacteria which are subsequently selected for increased resistance to cefotaxime (an antibiotic that TEM-1 hydrolyzes poorly). By repeating this process multiple times in independent experiments, the mutations and mutational trajectories involved in the increase of cefotaxime resistance are studied. At a fundamental level, this has lead to a better understanding of the nature of mutation interaction and its consequences for evolutionary contingency and constraint. Evidence indicating that certain ‘silent’ mutations (i.e. mutations that alter the codon sequence but not the amino acid that the respective codon encodes) can also play a role in increased resistance was found in these data as well. A phylogenetic study of the sequences of the ~150 different TEM-alleles that have been isolated in hospitals and clinics so far indicates that recombination has played a significant role in the evolution of TEM-alleles, contrary to what is often assumed. Furthermore, amino acid substitutions present in these clinical isolates are compared to those found in laboratory evolution studies of TEM-1, in order to investigate to what extent laboratory evolution can be used as a predictive tool for the natural evolution of antibiotic resistance genes. This overview indicates that laboratory evolution very accurately repeats the natural evolution of TEM-1. Based on these findings, predictions are made about substitutions that may appear in future clinical TEM-isolates, and directions are given how laboratory evolution can be exploited as a predictive tool most efficiently.

Salmonella is present in different food products. In this research it is concluded that Salmonella, which can survive the stomach of humans better (acid resistant bacteria), have a higher probability of causing an infection than Salmonella strains that are less able to survive the stomach (acid sensitive bacteria). The probability of such an infection is determined using different model systems. Effects on an infection with cultured human and rat intestinal cells were comparable to results obtained with a rat model. The probability of infection calculated with the results from both model systems agree with data from human experiments conducted in the fifties and with epidemiological dose response data. If these results can be founded by additional research, with different Salmonella species, it could be possible to reduce the amount of animal experiments in the future.

Salmonella is present in different food products. In this research it is concluded that Salmonella, which can survive the stomach of humans better (acid resistant bacteria), have a higher probability of causing an infection than Salmonella strains that are less able to survive the stomach (acid sensitive bacteria). The probability of such an infection is determined using different model systems. Effects on an infection with cultured human and rat intestinal cells were comparable to results obtained with a rat model. The probability of infection calculated with the results from both model systems agree with data from human experiments conducted in the fifties and with epidemiological dose response data. If these results can be founded by additional research, with different Salmonella species, it could be possible to reduce the amount of animal experiments in the future.