Antibiotic resistance in porcine pathogenic bacteria and relation to antibiotic usage.

BACKGROUND: Optimal treatment and prudent use of antimicrobials for pigs is imperative to secure animal health and prevent development of critical resistance. An important step in this one-health context is to monitor resistance patterns of important animal pathogens. The aim of this study was to investigate the antimicrobial resistance patterns of five major pathogens in Danish pigs during a period from 2004 to 2017 and elucidate any developments or associations between resistance and usage of antibiotics. RESULTS: The minimum inhibitory concentration (MIC) for Escherichia coli, Actinobacillus pleuropneumoniae, Streptococcus suis, Bordetella bronchiseptica, and Staphylococcus hyicus was determined to representatives of antibiotic classes relevant for treatment or surveillance. Escherichia coli isolates were mostly sensitive to fluoroquinolones and colistin, whereas high levels of resistance were observed to ampicillin, spectinomycin, streptomycin, sulfonamides and tetracycline. While resistance levels to most compounds remained relatively stable during the period, resistance to florfenicol increased from 2.1% in 2004 to 18.1% in 2017, likely in response to a concurrent increase in usage. A temporal association between resistance and usage was also observed for neomycin. E. coli serovars O138 and O149 were generally more resistant than O139. For A. pleuropneumoniae, the resistance pattern was homogenous and predictable throughout the study period, displaying high MIC values only to erythromycin whereas almost all isolates were susceptible to all other compounds. Most S. suis isolates were sensitive to penicillin whereas high resistance levels to erythromycin and tetracycline were recorded, and resistance to erythromycin and trimethoprim increasing over time. For S. hyicus, sensitivity to the majority of the antimicrobials tested was observed. However, penicillin resistance was recorded in 69.4-88.9% of the isolates. All B. bronchiseptica isolates were resistant to ampicillin, whereas all but two isolates were sensitive to florfenicol. The data obtained have served as background for a recent formulation of evidence-based treatment guidelines for pigs. CONCLUSIONS: Antibiotic resistance varied for some pathogens over time and in response to usage. Resistance to critically important compounds was low. The results emphasize the need for continuous surveillance of resistance patterns also in pig pathogenic bacteria.

A cross-sectional field study on potential associations between feed quality measures and usage of antimicrobials in commercial mink (Neovison vison).

Feed quality is generally assumed to affect health status in animal production. In previous studies, the feed producer has been found to affect the occurrence of gastrointestinal disease and antimicrobial use in Mink (Neovison vison). Mink are fed with moist, freshly produced feed, based on perishable ingredients. The objective of this study was to investigate the potential effect of specific feed parameters on antimicrobial use on herd level. The study was cross-sectional, including 1472 mink herds, responsible for 97% of oral antimicrobials prescribed for Danish mink during the study period, 2012-2014. Data were obtained from the national veterinary prescription database (VetStat), Kopenhagen Fur database, and the Voluntary Feed Control (Mink producers Organization). All feed batches subject to feed control were included. A multi-variable variance analysis was carried out analysing the effect of the feed parameters total volatile nitrogen, dry matter, crude protein and fat; total bacterial count (21°C), and counts of sulphite producing bacteria (21°C), Clostridium spp., faecal cocci (FC) (44°C), yeast, and mould; presence of Salmonella spp. and Clostridium perfringens (dichotome). Three outcome variables were applied: prescription of oral antimicrobial on herd level within time slots of 3, 5 or 7days after feeding of an included batch. Two binomial models were developed, adjusting for significant effects (p<0.0001) of Ps. aeruginosa infection, herd size, month (season) and year. Antimicrobial prescription was significantly (p<0.0001) associated with FC (all time slots, both models). A negative association (p<0.0001) with crude protein on antimicrobial prescription within a 7day slot suggested an association between low content of crude protein and antimicrobial use. The associations need to be confirmed in controlled studies, and ideally, potential causalities should be investigated. The perspective of such findings could be the development of tests for control of feed ingredients prior to use in the feed production.

Factors associated with usage of antimicrobials in commercial mink (Neovison vison) production in Denmark.

The American mink (Neovison vison) is used for commercial fur production in Denmark. In recent years, antimicrobial prescription for Danish mink has been increasing. In this study, the patterns and trends in antimicrobial use in mink were described and a multi-variable variance analysis was carried out with the objective of identifying risk factors for antimicrobial use on herd level. The study was based on register data for 2007-2012. Information on antimicrobial use was obtained from the national database VetStat, monitoring all medicinal products used for animals on prescription level. Data on microbiological feed quality was obtained from the Voluntary Feed Control under the Mink producers Organization, and data on herd size and the relation between farm and feed producer was obtained from the registers at Kopenhagen Fur, based on yearly reporting from the mink producers. Descriptive analysis showed a clear significant effect of season on antimicrobial use, with a peak in "treatment proportions", TP (defined daily doses per kg biomass-days) in May, around the time of whelping, and a high level in the following months. In autumn, a minor peak in antimicrobial use occurred throughout the study period. From 2007 to 2011, a 102% increase in annual antimicrobial TP was noted; on herd level, the increase was associated with an increasing frequency of prescription, and a decrease in the amounts prescribed in months with prescription. A binomial model showed that on herd level, the annual number of months with antimicrobial prescription was significantly (p<0.01) affected by feed producer, veterinarian, disease (specific laboratory diagnosis) infection, herd size and year, with an interaction between feed producer and year. A log-normal model showed that in months with antimicrobial use, the TP on herd level was significantly (p<0.001) affected by year, month (season), feed producer, feed quality score, veterinarian, herd size and laboratory confirmed diagnosis of specific infections; additionally the interaction terms year×feed producer and herd size×month were significant (p<0.001). In conclusion, antimicrobial use on herd level was significantly associated with the microbiological food quality, the feed producer, and the veterinarian. The prescription patterns varied significantly between veterinarians, and some veterinarians were associated with both larger and more frequent prescriptions of antimicrobials at herd level. Herd size is associated with different prescription patterns. Finally, infection with Pseudomonas aeruginosa, astrovirus, influenza virus and Salmonella spp. was associated with an increase in antimicrobial use.

The use of third and fourth generation cephalosporins affects the occurrence of extended-spectrum cephalosporinase-producing Escherichia coli in Danish pig herds.

Extended-spectrum cephalosporinase resistance is currently the fastest emerging antimicrobial resistance problem worldwide; however, evidence documenting the effect of potential risk factors is limited. The main objective of this study was to investigate the effect of using third and fourth generation cephalosporins on the occurrence of extended-spectrum cephalosporinase-producing Escherichia coli (ESC-Ec) in Danish pig herds. Conventional, integrated, medium to large herds were selected based on information from the Danish Central Husbandry Register and two groups were formed based on the use of third and fourth generation cephalosporins within a specified period, namely, 20 herds with no cephalosporin use (non-exposed) and 19 herds with frequent use (exposed). Data on prescribed antimicrobials were obtained from the National database (VetStat). Management data were obtained through a questionnaire. At the herd level, three pooled faecal samples were collected from sows with their piglets (farrowing pens), weaners, and finishers. ESC-Ec were then identified using selective enrichment. Because several of the herds only had a low number of weaners and/or finishers, analysis was only performed on samples from the farrowing pens. Logistic regression showed a significant effect of using cephalosporins-III/IV on the occurrence of ESC-Ec in the farrowing pens, even when adjusted for use of other antimicrobials 1 year prior to sampling. No confounding effect was identified in relation to management data. The relative risk ESC-Ec in exposed compared to non-exposed was 4.7 (95% confidence interval 2.0-11.5), confirming that regular use of cephalosporins-III/IV was a significant risk factor for the occurrence of ESC-Ec.

Temporal relationship between decrease in antimicrobial prescription for Danish pigs and the "Yellow Card" legal intervention directed at reduction of antimicrobial use.

The potential effects of the "Yellow Card" intervention, enforced by Danish authorities in December 2010, on the antimicrobial prescription in the Danish pig production were investigated. Data on antimicrobial prescription for pigs during 2002-2012 was obtained from the national database on veterinary prescribed medicines, VetStat. Descriptive analysis of temporal trends in quantitative antimicrobial prescription for pigs on national level was performed for each administration route, age group and disease group. In addition, prescription patterns of the three most prescribed antimicrobial classes (tetracyclines, macrolides and pleuromutilins) for weaners and finishers were studied at herd level. A 25% decline in the total antimicrobial use per pig produced occurred between 2009 and 2011. A decline was observed both in sows and piglets (31%), weaners (34%) and finishers (19%). Reduced prescription of tetracycline, macrolides and pleuromutilins for oral use, mainly for gastrointestinal disease (GI) in weaners and finishers, explained 76% of the total reduction. In 2012, the overall antimicrobial use increased by 10%, as a partial reversal of the preceding changes in prescription pattern. On herd level, the decline and subsequent increase was mainly related to changes in number of herds receiving regular monthly prescriptions. This study demonstrated that the steep decrease in antimicrobial use in the Danish pig production was temporally related with the announcement and introduction of the Yellow Card intervention.

Effect of insulin-induced hypoglycaemia on the peripheral nervous system: focus on adaptive mechanisms, pathogenesis and histopathological changes.

Insulin-induced hypoglycaemia (IIH) is a common acute side effect in type 1 and type 2 diabetic patients, especially during intensive insulin therapy. The peripheral nervous system (PNS) depends on glucose as its primary energy source during normoglycaemia and, consequently, it may be particularly susceptible to IIH damage. Possible mechanisms for adaption of the PNS to IIH include increased glucose uptake, utilisation of alternative energy substrates and the use of Schwann cell glycogen as a local glucose reserve. However, these potential adaptive mechanisms become insufficient when the hypoglycaemic state exceeds a certain level of severity and duration, resulting in a sensory-motor neuropathy with associated skeletal muscle atrophy. Large myelinated motor fibres appear to be particularly vulnerable. Thus, although the PNS is not an obligate glucose consumer, as is the brain, it appears to be more prone to IIH than the central nervous system when hypoglycaemia is not severe (blood glucose level ≤ 2 mm), possibly reflecting a preferential protection of the brain during periods of inadequate glucose availability. With a primary focus on evidence from experimental animal studies investigating nondiabetic IIH, the present review discusses the effect of IIH on the PNS with a focus on adaptive mechanisms, pathogenesis and histological changes.

Effect of insulin-induced hypoglycaemia on the central nervous system: evidence from experimental studies.

Insulin-induced hypoglycaemia (IIH) is a major acute complication in type 1 as well as in type 2 diabetes, particularly during intensive insulin therapy. The brain plays a central role in the counter-regulatory response by eliciting parasympathetic and sympathetic hormone responses to restore normoglycaemia. Brain glucose concentrations, being approximately 15-20% of the blood glucose concentration in humans, are rigorously maintained during hypoglycaemia through adaptions such as increased cerebral glucose transport, decreased cerebral glucose utilisation and, possibly, by using central nervous system glycogen as a glucose reserve. However, during sustained hypoglycaemia, the brain cannot maintain a sufficient glucose influx and, as the cerebral hypoglycaemia becomes severe, electroencephalogram changes, oxidative stress and regional neuronal death ensues. With particular focus on evidence from experimental studies on nondiabetic IIH, this review outlines the central mechanisms behind the counter-regulatory response to IIH, as well as cerebral adaption to avoid sequelae of cerebral neuroglycopaenia, including seizures and coma.

Indications and patterns of therapeutic use of antimicrobial agents in the Danish pig production from 2002 to 2008.

This study describes trends in the use and indications for prescriptions of antimicrobial agents in the Danish pig production in the period between 2002 and 2008 and is the first description of a complete prescription pattern for one animal species in an entire country. Data on all prescription for pigs in Denmark were retrieved from the VetStat database. Antimicrobial use was measured in defined animal daily doses (ADD) for the specific age-group and in ADD(kg) as a measure of amounts used. According to the results of the ADD(kg) data, 26% of all antimicrobials were prescribed for sows, 38% for weaner pigs, and 33% for finisher pigs. In weaner and finisher pigs, gastrointestinal infections accounted for 74-83% and 56-65% of the use, while respiratory infections accounted for 9-17% and 18-24%, respectively. From 2002 to 2008, prescription for respiratory disease increased by 145% for sows/piglets, by 141% for weaning pigs, and by 81% for finisher pig. The most commonly used class of antibiotics was tetracycline for all age-groups, replacing the previously used macrolide/lincosamide group. The use of pleuromutilin increased in 2008 to the level of macrolides. In sow/piglets, the second most used class was penicillins. The switch in choice of antimicrobial classes prescribed seems to be related primarily to changes in the price of the drugs.

Regional, seasonal, and temporal variations in the prevalence of antimicrobial-resistant Escherichia coli isolated from pigs at slaughter in Denmark (1997-2005).

The aim of this study was to analyze and discuss regional, seasonal, and temporal trends in the occurrence of antimicrobial-resistant Escherichia coli isolated from pigs at slaughter in Denmark between 1997 and 2005. Data on antimicrobial-resistant E. coli were obtained from the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme database. The Cochran-Armitage trend test was used to detect the presence and evaluate the significance of regional, seasonal, and annual trends in the occurrence of antimicrobial-resistant E. coli for four drugs. Associations between resistance and explanatory variables region, season, and the year of isolate sampling were analyzed using a logistic regression model. The Cochran-Armitage test provided evidence of significant temporal trends for ampicillin-resistant E. coli (an increasing trend, p < 0.0001) and streptomycin-resistant E. coli (a decreasing trend, p < 0.0001). The prevalence of ampicillin-resistant E. coli increased over time for all seasons (p < 0.001) except for winter when no significant variations in prevalence of resistant E. coli were captured over time. On the other hand, a significant decreasing trend in prevalence of streptomycin-resistant E. coli was observed for the spring, summer, and winter months (p < 0.001); however, there were no statistically significant trends for the autumn months (p > 0.05). The prevalence of ampicillin-resistant E. coli was observed to increase over time for the various regions, whereas that for streptomycin-resistant E. coli presented an overall significant decrease over time. The estimated odds ratios from the logistic regression model indicated varying risks for the occurrence of resistance by season and by region. The winter months were associated with an increased risk for the occurrence of resistant E. coli as compared to the other seasons of the year. Our study provides evidence of statistically significant regional, seasonal, and temporal variations for ampicillin- and streptomycin-resistant E. coli isolated from pigs at slaughter in Denmark between 1997 and 2005.

Gentamicin susceptibility in Escherichia coli related to the genetic background: problems with breakpoints.

In total, 120 Escherichia coli isolates positive for one of the gentamicin resistance (GEN(R)) genes aac(3)-II, aac(3)-IV or ant(2'')-I were tested for gentamicin susceptibility by the agar dilution method. Isolates positive for aac(3)-IV or ant(2'')-I had an MIC distribution of 8-64 mg/L, whereas isolates positive for aac(3)-II had MICs of 32 to >512 mg/L, suggesting a relationship between the distribution of MICs and the specific GEN(R) mechanism. The MIC distribution, regardless of the GEN(R) mechanism, was 8 - >512 mg/L, which supports the clinical breakpoint of MIC >4 mg/L suggested by EUCAST and questions the breakpoint recommended by the CLSI (> or =16 mg/L).

Tetracycline consumption and occurrence of tetracycline resistance in Salmonella typhimurium phage types from Danish pigs.

The aims of the present study were to investigate at the farm-owner level the effect of prescribed tetracycline consumption in pigs and different Salmonella Typhimurium phage types on the probability that the S. Typhimurium was resistant to tetracycline. In this study, 1,307 isolates were included, originating from 877 farm owners, and data were analyzed using logistic regression. The analysis showed that both the S. Typhimurium phage type (p < 0.0001) and an increase in tetracycline consumption (p = 0.0007) were significantly associated with tetracycline resistance. In particular, the phage type was strongly associated with tetracycline resistance. A further analysis of data from the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) indicates that the tetracycline-susceptible phage types only slowly become tetracycline resistant, although tetracycline consumption more than doubled at the national level from 12,000-13,000 kg of active compound in 1996-1998 to 29,000 kg of active compound in 2004. Instead, tetracycline-resistant S. Typhimurium phage types became more prevalent. This suggests that the spread of already established or new resistant clones, rather than conversion of "old" well-established susceptible clones to resistant clones by uptake of resistance genes, explains most of the increased levels of tetracycline resistance in S. Typhimurium in Danish swine production in response to increased tetracycline consumption.

Veterinary antimicrobial-usage statistics based on standardized measures of dosage.

In human medicine, the defined daily dose is used as a technical measure of drug usage, which is independent of the variations in the potency of the active compound and the formulation of the pharmaceutical product--therefore providing a measure of the relative importance of different drugs. A national system of animal defined daily doses (ADD) for each age-group and species has been defined in VetStat (the Danish national system monitoring veterinary therapeutic drug use). The usage is further standardized according to the number of animals in the target population, acquired from production data on the national level or on herd size by species and age in the Danish central husbandry register (CHR). Statistics based on standardized measures of VetStat data can be used for comparison of drug usage between different herds, veterinary practices, or geographic regions (allowing subdivision by animal species and animal production class, route of administration, disease categories, season and geographic location). Individual statistics are available as interactive reports to the control authorities, farmers and veterinary practitioners by a secure access to the database. The ADD also is used in pharmaco-epidemiogical research and to assist in the interpretation of resistance-surveillance data.

Asymptomatic radiographic disappearance of calcified intervertebral disc material in the Dachshund.

The occurrence of asymptomatic disappearance of intervertebral disc calcification was evaluated in a radiographic prospective longitudinal study of 40 Dachshunds followed from 6 or 12 months of age to 2 years of age. Radiographic follow-up was performed at 3 to 4 years of age in 12 dogs. Disappearance of calcified disc material was observed in 9 thoracic and 1 lumbar intervertebral discs. The crude incidence rate tended to increase with age from 1 year of age. The features of the disappearing calcifications involved indicate that the phenomenon is part of the progressive degenerative process. A possible pathogenesis includes tearing of the annulus fibrosus due to the severe degeneration of the disc, followed by an inflammatory response to nuclear material and phagocytic resorption of calcified material.

Development of intervertebral disk calcification in the dachshund: a prospective longitudinal radiographic study.

Plain spinal radiography was performed in 40 dachshunds at regular intervals from 6 or 12 months of age to 2 years of age. A follow-up study at 3 to 4 years of age included 12 dogs. High incidence rates of intervertebral disk calcification were seen at 6 to 18 months of age. The number of dogs affected and number of calcified disks seemed to reach a steady level or a maximum at about 24 to 27 months of age. Dissolution of previously calcified disks without clinical signs was demonstrated, causing decreasing numbers of visibly calcified disks after 2 years of age. Radiographic examination for calcified intervertebral disks in the dachshund is recommended at 24 to 30 months of age for heritability studies and selective breeding.

Inheritance of disc calcification in the dachshund.

The occurrence of intervertebral disc calcification was investigated by conventional spinal radiography in eight families of wirehaired dachshunds, with each family comprising one sire, two dams and one litter from each dam. Each offspring was examined radiographically once at 24-35 months of age. The occurrence of disc calcification was rated according to four different scales. A strong correlation was found in the occurrence of disc calcification between offspring and mean parent (P < 0.001) and between offspring and dams (P < 0.005) on an either/or scale. Statistically significant estimates of heritability of 0.60 and 0.87 were found based on the offspring-sire relationship using the total score and three-class scale, respectively. Higher correlation estimates were found based on the dam-offspring relationship than based on the sire-offspring relationship, suggesting an effect of maternal environmental factors.

Mechanical factors affecting the occurrence of intervertebral disc calcification in the dachshund--a population study.

In a population sample comprising 48 standard wirehaired dachshunds, the occurrence of intervertebral disc calcification was determined by plain spinal radiography. Body dimensions of the dogs were measured and information obtained from owners about exercise patterns, including stair climbing, and the relation of these variables to the number of calcified discs was analysed by logistic regression. In the Danish population of wirehaired dachshunds, the prevalence of disc calcification was estimated at 77%. The spinal distribution of calcified discs is similar amongst different populations, while the prevalence varies significantly. Most types of exercise included in the variable 'duration of exercise' as well as moderate stair climbing seemed to reduce the rate of occurrence of disc calcification. The effects of moderate stair climbing (OR = 0.34) and duration of exercise (OR = 0.52) were statistically significant and the effects appear to be additive. Running next to a bicycle was the only type of exercise with a positive association with the number of calcified discs. This was, however, not significant. Effects of body conformation, including absolute and relative body measurements, were not found.

Inactivation of virus during anaerobic digestion of manure in laboratory scale biogas reactors.

Reduction of porcine parvovirus, bovine enterovirus and faecal enterococci were measured in biogas reactors continuously run on manure and manure supplemented with household waste at 35 degrees C and 55 degrees C and in batch test run at 70 degrees C. The aim of the experiments was to study the sanitation effect of anaerobic digestion and to evaluate the use of faecal enterococci as an indicator of sanitation. Parallel studies on the reduction of virus and faecal enterococci were done in physiological saline solution. Heat inactivation was found to be an important factor in thermophilic biogas plants and the overall dominant factor at 70 degrees C. However, other environmental factors with a substantial virucidal and bactericidal effect were involved in inactivation. The death rates for faecal enterococci were generally higher than for porcine parvovirus and lower than for bovine enterovirus. For faecal enterococci, a logarithmic reduction of 4 (corresponding to the recommended minimum guaranteed retention time) was obtained after 300 hours at 35 degrees C and after 1-2 hours at 55 degrees C. In continuously-fed reactors, a high reduction rate was initially seen for the virus tested, followed by a reduction in the rate. For porcine parvovirus, a minimum guaranteed retention time of 11-12 hours is necessary at 55 degrees C in the initial phase (0-4 hours) and 54 hours hereafter (4-48 h). Correspondingly, for bovine enterovirus a MGRT of 23 hours was necessary at 35 degrees C and < 0.5 hours at 55 degrees C. The data indicate that faecal enterococci measurements give a good indication of inactivation of enterovirus and other more heat sensitive virus, especially under thermophilic conditions. Parvovirus is very suitable for comparative investigations on inactivation in the temperature range of 50-80 degrees C, due to the extreme thermal resistance of this virus. However, in stipulating sanitation demands for biogas reactors it seems more reasonable to use less resistant virus, such as a reovirus or picornavirus, which better represents the pathogenic animal virus.