Epidemiological and serological investigation of a waterborne Campylobacter jejuni outbreak in a Danish town.

Following an unusually heavy rainfall in June 2009, a community-wide outbreak of Campylobacter gastroenteritis occurred in a small Danish town. The outbreak investigation consisted of (1) a cohort study using an e-questionnaire of disease determinants, (2) microbiological study of stool samples, (3) serological study of blood samples from cases and asymptomatic members of case households, and (4) environmental analyses of the water distribution system. The questionnaire study identified 163 cases (respondent attack rate 16%). Results showed a significant dose-response relationship between consumption of tap water and risk of gastroenteritis. Campylobacter jejuni belonging to two related flaA types were isolated from stool samples. Serum antibody levels against Campylobacter were significantly higher in cases than in asymptomatic persons. Water samples were positive for coliform bacteria, and the likely mode of contamination was found to be surface water leaking into the drinking-water system. This geographically constrained outbreak presented an ideal opportunity to study the serological response in persons involved in a Campylobacter outbreak. The serology indicated that asymptomatic persons from the same household may have been exposed, during the outbreak period, to Campylobacter at doses that did not elicit symptoms or alternatively had been exposed to Campylobacter at a time prior to the outbreak, resulting in residual immunity and thus absence of clinical signs.

Seroincidence of non-typhoid Salmonella infections: convenience vs. random community-based sampling.

The incidence of reported infections of non-typhoid Salmonella is affected by biases inherent to passive laboratory surveillance, whereas analysis of blood sera may provide a less biased alternative to estimate the force of Salmonella transmission in humans. We developed a mathematical model that enabled a back-calculation of the annual seroincidence of Salmonella based on measurements of specific antibodies. The aim of the present study was to determine the seroincidence in two convenience samples from 2012 (Danish blood donors, n = 500, and pregnant women, n = 637) and a community-based sample of healthy individuals from 2006 to 2007 (n = 1780). The lowest antibody levels were measured in the samples from the community cohort and the highest in pregnant women. The annual Salmonella seroincidences were 319 infections/1000 pregnant women [90% credibility interval (CrI) 210-441], 182/1000 in blood donors (90% CrI 85-298) and 77/1000 in the community cohort (90% CrI 45-114). Although the differences between study populations decreased when accounting for different age distributions the estimates depend on the study population. It is important to be aware of this issue and define a certain population under surveillance in order to obtain consistent results in an application of serological measures for public health purposes.

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.

Trends in slaughter pig production and antimicrobial consumption in Danish slaughter pig herds, 2002-2008.

Overuse of antimicrobials in food-animal production is thought to be a major risk factor for the development of resistant bacterial populations. Data on non-human antimicrobial usage is essential for planning of intervention strategies to lower resistance levels at the country, region or herd levels. In this study we evaluated Danish national antimicrobial usage data for five antimicrobial classes used in slaughter pigs in different herd sizes and data on the number of slaughter pigs produced per herd, between 2002 and 2008, in Denmark. The objective was to ascertain if there is an association between herd size and amount of antimicrobials consumed. During this period, the overall number of herds with slaughter pigs decreased by 43%, with larger herds becoming more prevalent. The tetracycline treatment incidence (TI) rate increased from 0·28 to 0·70 animal-defined daily dose (ADD)/100 slaughter pig-days at risk while macrolide TI presented a more moderate increase, from 0·40 to 0·44 ADD/100 slaughter pig-days at risk during the study period. Linear regression analyses revealed a significant association between herd size and TI rates for tetracyclines, macrolides, sulfonamides/trimethoprim and cephalosporins, with small herds presenting significantly higher TI than moderate, large and the largest herds. This study highlights the importance of establishing an antimicrobial consumption monitoring programme, integrated with comprehensive food-animal production surveillance. Further research should be performed to address the potential causes of the detected associations between herd sizes and antimicrobial consumption in pigs.

Spatial scan statistics to assess sampling strategy of antimicrobial resistance monitoring program.

The collection and analysis of data on antimicrobial resistance in human and animal populations are important for establishing a baseline of the occurrence of resistance and for determining trends over time. In animals, targeted monitoring with a stratified sampling plan is normally used. However, to our knowledge it has not previously been analyzed whether animals have a random chance of being sampled by these programs, regardless of their spatial distribution. In this study, we used spatial scan statistics, based on a Poisson model, as a tool to evaluate the geographical distribution of animals sampled by the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP), by identifying spatial clusters of samples and detecting areas with significantly high or low sampling rates. These analyses were performed for each year and for the total 5-year study period for all collected and susceptibility tested pig samples in Denmark between 2002 and 2006. For the yearly analysis, both high and low sampling rates areas were significant, with two clusters in 2002 (relative risk [RR]: 2.91, p < 0.01 and RR: 0.06, p < 0.01) and one in 2005 (RR: < 0.01, p < 0.01). For the 5-year analysis, one high sampling rate cluster was detected (RR: 2.56, p = 0.01). These findings allowed subsequent investigation to clarify the source of the sampling clusters. Overall, the detected clusters presented different spatial locations over the years and we can conclude that they were more associated to temporary sampling problems than to a failure in the sampling strategy adopted by the monitoring program. Spatial scan statistics proved to be a useful tool for assessment of the randomness of the sampling distribution, which is important when evaluating the validity of the results obtained by an antimicrobial monitoring program.

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.

Using data on resistance prevalence per sample in the surveillance of antimicrobial resistance.

OBJECTIVES: In most existing antimicrobial resistance monitoring programmes, one single bacterial colony from each collected sample is susceptibility tested against a panel of antimicrobials. Detecting the proportion of colonies resistant to different antimicrobials in each sample can provide quantitative data on antimicrobial resistance (resistance prevalence per sample). METHODS: In this study, a total of 98 faecal samples from slaughter pigs were tested for tetracycline and sulphonamide resistance in Escherichia coli using the single colony method, and these results were compared with the results obtained using the resistance prevalence per sample method. RESULTS: The results obtained by the resistance prevalence per sample method showed a lower occurrence of resistance. Tetracycline resistance in E. coli was found in 36.7% of the samples using the single colony method, while the mean tetracycline resistance prevalence was 22.5% using the resistance prevalence per sample method. Similarly, sulphonamide resistance was 32.7% using the single colony method and 19.6% when using the resistance prevalence per sample method. Although different estimates were obtained by each method, the correlation test and the regression model demonstrated that there is a significant association between the results obtained using both methods (P value <0.01) for both antimicrobials tested. CONCLUSIONS: To support risk assessment and analysis of the association between consumption of antimicrobials and occurrence of resistance, there is a need to move towards a more quantitative approach when dealing with antimicrobial resistance in a population, and the resistance prevalence per sample method can provide some of this additional information.

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.

Relations between the occurrence of resistance to antimicrobial growth promoters among Enterococcus faecium isolated from broilers and broiler meat.

From 1995 to 2001, Enterococcus faecium isolates were collected from broiler flocks at slaughter and broiler meat products at retail outlets and were tested for susceptibility to classes of antimicrobials used for growth promotion in broilers in Denmark, namely: evernimicin, glycopeptide, macrolide and streptogramin. By February 1998, all antimicrobial growth promoters (AGPs) were withdrawn from the Danish broiler production. The present study investigates, by logistic regression analyses, the (1) changes in the occurrence of AGP resistance among E. faecium from broilers and broiler meat from the fourth quarter of 1995 to the fourth quarter of 2001 and (2) relations between the occurrence of AGP resistance among E. faecium isolates from Danish broilers and AGP resistance among E. faecium isolates from the broiler meat of Danish and unknown origin collected in the same quarter within the year. In the present study, we showed that after the AGP withdrawal, a significant decline in resistance to avilamycin, erythromycin, vancomycin and virginiamycin was observed among E. faecium from broilers and broiler meat. In addition, a decline in the occurrence of AGP resistance among E. faecium from Danish broilers was associated with a decrease in the predicted probability of isolating an AGP-resistant E. faecium isolate from a randomly selected broiler meat product. In the analyses "relations between the occurrence of AGP resistance among E. faecium isolated from broilers and broiler meat collected in the same quarter" errors in the explanatory variable were expected. Therefore, a simulation study was performed to validate the results from logistic regression analyses. The results obtained by the two methods were similar.

Space-time clustering of ampicillin resistant Escherichia coli isolated from Danish pigs at slaughter between 1997 and 2005.

In Denmark, antimicrobial resistance in bacteria in animals, animal products and humans, is routinely monitored. This study aimed at determining whether the observed variations in the prevalence of ampicillin resistant Escherichia coli isolated from healthy pigs at slaughter were random or clustered in space and time. Data on E. coli isolates between 1997 and 2005 were obtained from the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) whereas data on the quantity of ampicillin consumed was obtained from the Danish Register of Veterinary Medicines (VetStat). Space-time interaction was assessed using the space-time K-function and detection and location of significant space-time clusters was done using the space-time scan statistic. The space-time K-function analysis provided evidence of space-time interactions in ampicillin resistant E. coli (AREC) isolates in both Funen and Jutland, and Zealand. Significant space-time clusters of resistant E. coli isolates were found in the north eastern part of Jutland and Funen and in the southern part of Zealand. Seasonality was found to have a highly significant effect on space-time clustering in Funen and Jutland. The clusters of ampicillin resistant E. coli appeared at the same time as the national consumption of ampicillin in pigs increased, however antimicrobial consumption at the herd level did not appear to have any effects on space-time clustering in this study. The results could serve as a platform to highlight areas where more investigations on the occurrence and spread of ampicillin resistant E. coli in pig herds should be initiated.

Relations between the consumption of antimicrobial growth promoters and the occurrence of resistance among Enterococcus faecium isolated from broilers.

The present study investigates, at farm level, the effect of the time-span between sampling and the last time a particular antimicrobial growth promoter (AGP) was included in the feed on the probability of selecting an AGP-resistant Enterococcus faecium isolate from a broiler flock. The probability that a randomly selected E. faecium isolate was resistant to avilamycin, erythromycin or virginiamycin was 0.91, 0.92 and 0.84, respectively if the isolate originated from a broiler flock fed either avilamycin- or virginiamycin-supplemented feed. As the time-span between sampling and the last AGP consumption increased, the probability of isolating an E. faecium isolate resistant to a particular AGP decreased (probability <0.2 within 3-5 years after last exposure to AGPs). The decrease in probability over time showed little farm-to-farm variation. The number of times a particular AGP was given to previous flocks reared in the same house had no effect on the probability of isolating a resistant isolate.

Effect of abolishment of the use of antimicrobial agents for growth promotion on occurrence of antimicrobial resistance in fecal enterococci from food animals in Denmark.

From 1995 to 2000, a total of 673 Enterococcus faecium and 1,088 Enterococcus faecalis isolates from pigs together with 856 E. faecium isolates from broilers were isolated and tested for susceptibility to four classes of antimicrobial agents used for growth promotion as part of the Danish program of monitoring for antimicrobial resistance. The four antimicrobials were avilamycin, erythromycin, vancomycin, and virginiamycin. Major changes in the use of antimicrobial agents for growth promotion have occurred during the last 6 years in Denmark. The government banned the use of avoparcin in 1995 and of virginiamycin in 1998. Furthermore, the producers have voluntarily stopped all use beginning in 1999. The avoparcin ban in 1995 was followed by a decrease in the occurrence of glycopeptide-resistant E. faecium (GRE) in broilers, from 72.7% in 1995 to 5.8% in 2000. The occurrence of glycopeptide resistance among isolates from pigs remained constant at around 20% from 1995 to 1997. It was shown that, in GRE from pigs, the genes encoding macrolide and glycopeptide resistance were genetically linked and that, following the decrease in the use of tylosin during 1998 and 1999, the occurrence of GRE in pigs decreased to 6.0% in 2000. From 1995 to 1997 the occurrence of erythromycin resistance among E. faecium and E. faecalis isolates from pigs was almost 90%. Use of tylosin decreased considerably during 1998 and 1999, and this decrease was followed by decreases in the occurrence of resistance to 46.7 and 28.1% among E. faecium and E. faecalis isolates from pigs, respectively. Erythromycin resistance among E. faecium isolates from broilers reached a maximum of 76.3% in 1997 but decreased to 12.7% in 2000 concomitantly with more limited use of virginiamycin. Use of virginiamycin increased from 1995 to 1997 and was followed by an increased occurrence of virginiamycin resistance among E. faecium isolates in broilers, from 27.3% in 1995 to 66.2% in 1997. In January 1998 the use of virginiamycin was banned in Denmark, and the occurrence of virginiamycin resistance decreased to 33.9% in 2000. Use of avilamycin increased from 1995 to 1996 and was followed by an increase in avilamycin resistance among E. faecium isolates from broilers, from 63.6% in 1995 to 77.4% in 1996. Since 1996 avilamycin usage has decreased, followed by a decrease in resistance to 4.8% in 2000. Our observations show that it is possible to reduce the occurrence of antimicrobial resistance in a national population of food animals when the selective pressure is removed. Cases in which resistance to vancomycin was linked to resistance to erythromycin were exceptions. In such cases resistance did not decrease until the use of both avoparcin and tylosin was limited.