Phage typing of Salmonella Typhimurium - is it still a useful tool for surveillance and outbreak investigation?

Phage typing has for decades been useful as a phenotypical, definitive method for epidemiological characterisation of Salmonella Typhimurium. The system recommended by the World Health Organization (WHO) Collaborative Centre for phage typing of Salmonella has, however, become rather complex, and the present study illustrates the challenges of sufficient standardisation of the interpretation of lysis results to make sure that the same strain is assigned to the same phage type in different laboratories. Even though molecular typing methods will replace phenotypic characterisation methods in the future, it is our opinion that phage typing will remain for some time a useful tool to strengthen global Salmonella surveillance. Therefore, improved standardisation and quality assurance is essential to obtain a robust and harmonized method that allows comparison of results between laboratories.

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.

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.

Measurement of weak electric currents in copper wire phantoms using MRI: influence of susceptibility enhancement.

The use of magnetic resonance imaging (MRI)-based methods for the direct detection of neuronal currents is a topic of intense investigation. Much experimental work has been carried out with the express aim of establishing detection thresholds and sensitivity to flowing currents. However, in most of these experiments, magnetic susceptibility enhancement was ignored. In this work, we present results that show the influence of a susceptibility artefact on the detection threshold and sensitivity. For this purpose, a novel phantom, consisting of a water-filled cylinder with two wires of different materials connected in series, was constructed. Magnitude MR images were acquired from a single slice using a gradient-echo echo planar imaging (EPI) sequence. The data show that the time course of the detected MR signal magnitude correlates very well with the waveform of the input current. The effect of the susceptibility artefacts arising from the two different wires was examined by comparing the magnitudes of the MR signals at different voxel locations. Our results indicate the following: (1) MR signal enhancement arising from the magnetic susceptibility effect influences the detection sensitivity of weak current; (2) the detection threshold and sensitivity are phantom-wire dependent; (3) sub-mu A electric current detection in a phantom is possible on a 1.5-T MR scanner in the presence of susceptibility enhancement.

Practical design of a 4 Tesla double-tuned RF surface coil for interleaved 1H and 23Na MRI of rat brain.

MRI is proving to be a very useful tool for sodium quantification in animal models of stroke, ischemia, and cancer. In this work, we present the practical design of a dual-frequency RF surface coil that provides (1)H and (23)Na images of the rat head at 4 T. The dual-frequency RF surface coil comprised of a large loop tuned to the (1)H frequency and a smaller co-planar loop tuned to the (23)Na frequency. The mutual coupling between the two loops was eliminated by the use of a trap circuit inserted in the smaller coil. This independent-loop design was versatile since it enabled a separate optimisation of the sensitivity and RF field distributions of the two coils. To allow for an easy extension of this simple double-tuned coil design to other frequencies (nuclei) and dimensions, we describe in detail the practical aspects of the workbench design and MRI testing using a phantom that mimics in vivo conditions. A comparison between our independent-loop, double-tuned coil and a single-tuned (23)Na coil of equal size obtained with a phantom matching in vivo conditions, showed a reduction of the (23)Na sensitivity (about 28 %) because of signal losses in the trap inductance. Typical congruent (1)H and (23)Na rat brain images showing good SNR ((23)Na: brain 7, ventricular cerebrospinal fluid 11) and spatial resolution ((23)Na: 1.25 x 1.25 x 5mm(3)) are also reported. The in vivo SNR values obtained with this coil were comparable to, if not better than, other contemporary designs in the literature.

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.

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.

A case-control study of risk factors for sporadic campylobacter infections in Denmark.

A case control study comprising 282 cases and 319 matched controls was conducted in Denmark during 1996-7. Two estimates of the odds ratio (OR) were determined for each risk factor with and without 'protective factors' fitted into the final model. Consumption of undercooked poultry (OR 4.5; 8.2), consumption of red meat at a barbecue (OR 2.3; 4.1), consumption of grapes (OR 1.6; 2.8) and drinking unpasteurized milk (OR 2.3; 11.8) were identified as risk factors in both models. Frequent consumption of pork chops (OR 4.4) and daily contact with domestic animals and pets were identified as risk factors in one of the two models only. Finally, foreign travel was found to be a significant risk factor (OR 2.5). Seasonal and regional interaction was observed for several risk factors and the time elapsed from interviewing of cases to interviewing of controls seemed to influence the effect of certain seasonal dependent risk factors.

Antimicrobial resistance: risk analysis methodology for the potential impact on public health of antimicrobial resistant bacteria of animal origin.

The Ad hoc Group of experts on antimicrobial resistance, appointed by the Office International des Epizooties, has developed an objective, transparent and defensible risk analysis process, providing a valid basis for risk management decisions in respect to antimicrobial resistance. The components of risk analysis and of different possible approaches in risk assessment (qualitative, semiquantitative and quantitative) are defined. The Ad hoc Group recommended the following: an independent risk assessment based on scientific data; an iterative risk analysis process; a qualitative risk assessment systematically undertaken before considering a quantitative approach; the establishment of a risk assessment policy; and the availability of technical assistance for developing countries.

Antimicrobial resistance: monitoring the quantities of antimicrobials used in animal husbandry.

This guideline, developed by the Office International des Epizooties for the monitoring of the quantities of antimicrobials used in animal husbandry, provides the methodology required to assess the amounts of antimicrobials used, to supply data to be used for risk analysis and to improve guidance on the appropriate use of antimicrobials. Information may be gathered from a number of sources, such as the competent authorities, industry and users. The usefulness of different types of information is discussed and recommendations are given on how to collect detailed information, each year, on the antimicrobial quantities used per class and active substance. Information should also be collected on the route of administration (oral and parenteral) and the animal species.

Antimicrobial resistance: standardisation and harmonisation of laboratory methodologies for the detection and quantification of antimicrobial resistance.

The Ad hoc Group of experts on antimicrobial resistance of the Office International des Epizooties has developed a guideline on the standardisation and harmonisation of laboratory methodologies used for the detection and quantification of antimicrobial resistance. The existing methods (disk diffusion [including concentration gradient strips], agar dilution and broth dilution) are reviewed, including a comparison of their advantages and disadvantages. The definitions of resistance characteristics of bacteria (susceptible, intermediate and resistant) are addressed and the criteria for the establishment of breakpoints are discussed. Due consideration has to be given to these aspects in the interpretation and comparison of resistance monitoring or surveillance data. The use of validated laboratory methods and the establishment of quality assurance (internal and external) for microbiological laboratory work and the reporting of quantitative test results is recommended. Equivalence of different methods and laboratory test results is also recommended to be established by external proficiency testing, which should be achieved by the means of a reference laboratory system. This approach allows the comparison of test results obtained using different methods generated by laboratories in different countries.

Antimicrobial resistance: harmonisation of national antimicrobial resistance monitoring and surveillance programmes in animals and in animal-derived food.

A guideline on the harmonisation of national antimicrobial resistance monitoring and surveillance programmes in animals and animal-derived foods has been developed by the Ad hoc Group of experts on antimicrobial resistance of the Office International des Epizooties. The objective of the guideline is to allow the generation of comparable data from various national surveillance and monitoring systems in order to compare the situations in different regions or countries and to consolidate results at the national, regional and international level. Definitions of surveillance and monitoring are provided. National systems should be able to detect the emergence of resistance, and to determine the prevalence of resistant bacteria. The resulting data should be used in the assessment of risks to public health and should contribute to the establishment of a risk management policy. Specific factors identified for harmonisation include the animal species, food commodities, sampling plans, bacterial species, antimicrobials to be tested, laboratory methods, data reporting, database structure and the structure of reports.

The effect of discontinuing the use of antimicrobial growth promoters on the productivity in the Danish broiler production.

On 15 February 1998, the Danish poultry industry voluntarily decided to discontinue the use of all antimicrobial growth promoters (AGPs). To investigate how the removal of AGPs influenced the broiler productivity in Denmark, data from 6815 flocks collected from November 1995 to July 1999 by the Danish Poultry Council were analysed. The three flock parameters were: kilogram broilers produced per square meter (per rotation), feed-conversion ratio (total kilogram feed used per rotation/total kilogram live weight per rotation) and total percent dead broilers ((number of dead broilers during the rotation/number of broilers put in the house per rotation)x100). Data were analysed using a mixed model, allowing the correlation structures in the data to be taken into account. The analyses showed that kilogram broilers produced per square meter and percent dead broilers in total were not affected by the discontinued use of AGPs. However, the feed-conversion ratio increased marginally 0.016 kg/kg and has remained at this level throughout the rest of the study period.

An assessment of antimicrobial consumption in food producing animals in Kenya.

Antimicrobial agents are useful for control of bacterial infections in food animals and man. Their prudent use in these animals is important to control any possible development and transfer of resistance between animals and man. The objective of this study was to generate quantitative information to evaluate antimicrobial usage patterns by animal species, route of administration, antimicrobial class and type of use from 1995 to 1999 in Kenya. Theses data are essential for risk analysis and planning and can be helpful in interpreting resistance surveillance data, and evaluating the effectiveness of prudent use efforts and antimicrobial resistance mitigation strategies. Data on quantities of active substance classes were collected from the official records of the Pharmacy and Poisons Board of the Ministry of Health and analysed in MS Excel 2000 program. The mean antimicrobial consumption for the 5-year period was 14 594 +/- 1457 kg per year. This was distributed in the various antimicrobial classes as follows: 7975 kg (54.65%) of tetracyclines, 3103.96 kg (21.27%) of sulfonamides and 954.5 kg (6.56%) of aminoglycosides, 905 kg (6.20%) of beta-lactams, 94 kg (0.64%) of quinolones, 35 kg (0.24%) of macrolides and 24 kg (0.16%) of others (tiamulin). Mean consumption per year among the various food animals was: 10 989 +/- 357 kg in large animals (cattle, sheep, pigs and goats), 2906 +/- 127 kg in poultry alone and 699 +/- 427 kg in both large animals and poultry. These quantities represented 56.56% (8255 kg) consumption per year for parenteral use, 41.79% (6098 kg) for oral use and 1.65% (241 kg) for topical use (intramammary and eye ointments) in cattle. With respect to intended use in food producing animals, the mean consumption per year was: 13 178 kg (90.30%) for therapeutic use (ST), 4 kg (0.03%) for prophylactic treatment (PT) and 1411 +/- 246 kg (9.67%) was used both for therapeutic and prophylactic purposes (GPT). The study confirmed that antimicrobials are not used for growth promotion in Kenya. There was no specific trend in the quantities of active antimicrobial classes. This study has revealed that the tetracyclines, sulfonamides and trimethoprim, nitrofurans aminoglycosides, beta-lactams and the quinolones are the most commonly used drugs in food-producing animals in Kenya. Tetracyclines contributed approximately 55% of the total consumption, and there was an increasing trend in the consumption of quinolones from 1998.

Measurement of rubidium and xenon absolute polarization at high temperatures as a means of improved production of hyperpolarized (129)Xe.

We report on a rubidium-xenon (Rb-Xe) polarization unit for the continuous production of large quantities of hyperpolarized (129)Xe. The unit includes two diagnostic systems which enable the absolute measurement of both the (85)Rb and (129)Xe polarization in situ and at high temperatures. The Rb diagnostic system allows the measurement of one- or two-dimensional images of the absolute Rb polarization and thus enables the experimental study of light penetration into the optical pumping cell. The equilibrium Xe polarization measured in the optical pumping cell and in the freezing unit is typically approximately 20%, under optimal flowing conditions, and this is much lower than the volume-averaged rubidium polarization.

Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis.

Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and SmaI pulsed-field gel electrophoresis. RiboPrinting of the 84 isolates yielded 40 types whereas PFGE-typing yielded 57 types discriminated by differences in more than three bands. By molecular typing, both clonal spread of E. faecium as well as horizontal transmission of Tn1546 between animals and humans was supported. Furthermore, it was found that the population of E. faecium spreads freely between the animal and human reservoir.

The effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in Campylobacter and Escherichia coli.

Modern food animal production depends on use of large amounts of antibiotics for disease control. This provides favourable conditions for the spread and persistence of antimicrobial-resistant zoonotic bacteria such as Campylobacter and E. coli O157. The occurrence of antimicrobial resistance to antimicrobials used in human therapy is increasing in human pathogenic Campylobacter and E. coli from animals. There is an urgent need to implement strategies for prudent use of antibiotics in food animal production to prevent further increases in the occurrence of antimicrobial resistance in food-borne human pathogenic bacteria such as Campylobacter and E. coli.

An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype typhimurium DT104.

BACKGROUND: Food-borne salmonella infections have become a major problem in industrialized countries. The strain of Salmonella enterica serotype typhimurium known as definitive phage type 104 (DT104) is usually resistant to five drugs: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. An increasing proportion of DT104 isolates also have reduced susceptibility to fluoroquinolones. METHODS: The Danish salmonella surveillance program determines the phage types of all typhimurium strains from the food chain, and in the case of suspected outbreaks, five-drug-resistant strains are characterized by molecular methods. All patients infected with five-drug-resistant typhimurium are interviewed to obtain clinical and epidemiologic data. In 1998, an outbreak of salmonella occurred, in which the strain of typhimurium DT104 was new to Denmark. We investigated this outbreak and report here our findings. RESULTS: Until 1997, DT104 infections made up less than 1 percent of all human salmonella infections. The strain isolated from patients in the first community outbreak of DT104 in Denmark, in 1998 was resistant to nalidixic acid and had reduced susceptibility to fluoroquinolones. The outbreak included 25 culture-confirmed cases. Eleven patients were hospitalized, and two died. The molecular epidemiology and data from patients indicated that the primary source was a Danish swine herd. Furthermore, the investigation suggested reduced clinical effectiveness of treatment with fluoroquinolones. CONCLUSIONS: Our investigation of an outbreak of DT104 documented the spread of quinolone-resistant bacteria from food animals to humans; this spread was associated with infections that were difficult to treat. Because of the increase in quinolone resistance in salmonella, the use of fluoroquinolones in food animals should be restricted.