Björn Bengtsson

Microbial Aetiology of Acute Clinical Mastitis and Agent-Specific Risk Factors

A nation wide study on the microbial aetiology of cases of acute clinical mastitis in Swedish dairy cows was conducted with the aim to investigate changes in the microbial panorama compared to a previous study performed 1994-1995. Another aim was to investigate some agent-specific environmental and individual risk factors. Milk samples were collected from 987 udder quarter cases from 829 cows during six 2-month periods from May 2002 to April 2003, and data on risk factors and demography were collected at sampling by means of a questionnaire. In total, 1056 bacteriological diagnoses were made. The most frequently isolated bacterial species was Staphylococcus aureus constituting 21.3% of the diagnoses, followed by Escherichia coli (15.9%), Streptococcus dysgalactiae (15.6%), Streptococcus uberis (11.1%), coagulase-negative staphylococci (6.2%), Arcanobacterium pyogenes (6.1%) and Klebsiella spp. (4.2%). Samples with no growth or contamination constituted 10.6% and 4.5% of the diagnoses, respectively. A major shift in the panorama of udder pathogens was not observed compared to the survey in 1994-1995. Isolation of Klebsiella spp. was strongly associated with the use of sawdust as bedding material. On the other hand, using sawdust as bedding reduced the risk of isolating S. uberis relative to using straw or peat. The risk of isolating E. coli increased with increasing milk yield and was higher in loose housing systems than in tie stalls. Isolation of S. aureus was associated with tie stalls, and A. pyogenes with low yielding cows and teat lesions. S. dysgalactiae infections were also associated with teat lesions.

Antimicrobial susceptibility of udder pathogens from cases of acute clinical mastitis in dairy cows.

To investigate occurrence of acquired antimicrobial resistance in udder pathogens MICs in Staphylococcus aureus (n=211), coagulase-negative staphylococci (CNS) (n=56), Streptococcus uberis (n=113), Streptococcus dysgalactiae (n=152), Streptococcus agalactiae (n=6), Escherichia coli (n=163), and Klebsiella spp. (n=42) were determined using microdilution. Isolates were from a nation wide survey employing strict inclusion criteria. Presence of acquired resistance was evaluated by species-specific epidemiological cut-off values issued by EUCAST. Penicillin or methicillin resistance in staphylococci were however evaluated by beta-lactamase production or presence of the mecA gene, respectively. Staphylococci were mostly susceptible to antimicrobials tested but 7.1% of S. aureus and 12.5% of CNS were resistant to penicillin by beta-lactamase production. Methicillin resistance was not found in S. aureus. All Streptococcus dysgalactiae and S. agalactiae were susceptible to penicillin. Bimodal MIC distributions for tetracycline in S dysgalactiae and S. uberis indicate acquired resistance in some isolates. Among E. coli 12.3% of isolates were resistant to one or more antimicrobials. Resistance to streptomycin (11.0%), sulphametoxazole (8.6%), ampicillin (7.4%), or tetracycline (4.9%) were the most common traits. Klebsiella spp. were resistant to ampicillin and some isolates also to tetracycline (7.1%) or sulphonamide (9.5%). The study shows that in Sweden bacteria associated with acute clinical mastitis for the most part are susceptible to antimicrobials used in therapy but resistance to penicillin in S. aureus is not uncommon. Penicillin is recommended for treatment of mastitis caused by gram-positive pathogens and regular monitoring of beta-lactamase production in S. aureus is therefore recommended in herds with udder health problems.

Incidence of mastitis and bacterial findings at clinical mastitis in Swedish primiparous cows - influence of breed and stage of lactation.

Mastitis is a common disease also among primiparous dairy cows. Identification of the extent and type of problem is important to initiate correct control measures. In Sweden, unique national production and disease databases are available. The main aim of the study was to investigate the occurrence of mastitis, measured by the annual incidence of veterinary-treated clinical mastitis (VTCM) and geometric mean of monthly milk somatic cell count (SCC) recordings in Swedish primiparous cows in relation to older cows during 2002-2006 with emphasis on breed differences. Other aims were to study differences between primiparous and older cows in the distribution of bacterial findings at clinical mastitis, and the occurrence of VTCM and bacterial findings in relation to stage of lactation using data from a Swedish field study performed 2002-2003. Descriptive statistics and univariable analyses were used in the investigations. During 2002-2006 approximately 10% of Swedish primiparous cows experienced VTCM each year, while the geometric mean SCC of Swedish primiparous cows was approximately 65,000 ml(-1). Both parameters were lower than in older cows. Primiparous cows of the Swedish Red (SR) breed had better udder health than cows of the Swedish Holstein (SH) breed. The overall distribution of udder pathogens was similar in primiparous and older cows. In primiparous cows, most VTCM occurred during the first week after calving, and Staphylococcus aureus and Streptococcus dysgalactiae were the most common udder pathogens during this period. Better control measures directed at these infections are warranted around calving to reduce the risk of mastitis in primiparous cows.

Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis.

In order to evaluate the usefulness of some phenotypic and genotypic methods for species identification of coagulase-negative staphylococci (CNS), isolates were obtained from bovine cases of clinical and sub-clinical mastitis from different geographical areas in Sweden. By using the Staph-Zym test, antimicrobial susceptibility testing, and sequencing of part of the CNS tuf gene and, when needed, part of the 16S rRNA gene we characterized 82 clinical isolates and 24 reference strains of 18 different species of staphylococci. The genotypic methods identified nine different species of CNS among the 82 milk isolates. A comparison with results obtained by tuf gene sequencing showed that Staph-Zym correctly identified CNS reference strains to species level more often than bovine milk CNS isolates (83% and 61%, respectively). In addition, tests supplementary to the Staph-Zym were frequently needed in both groups of isolates (50% of reference strains and 33% of milk isolates) to obtain an identification of the strain. It is notable that Staph-Zym judged two isolates as CNS, although they belonged to other species, could not give a species name in 11% of the bovine CNS isolates, and gave 28% of the isolates an incorrect species name. The present study indicates that the studied phenotypic methods are unreliable for identification of CNS from bovine intra-mammary infections.

Vertical transmission of Escherichia coli carrying plasmid-mediated AmpC (pAmpC) through the broiler production pyramid

OBJECTIVES In Sweden the prevalence of Enterobacteriaceae with transferable resistance to extended-spectrum cephalosporins (ESCs) is low. However, in broilers ESC-resistant Escherichia coli is common, with a clear dominance of blaCMY-2. Antimicrobials are rarely used in broiler production in Sweden and cephalosporins are never used. Introduction through imported breeding stock and subsequent vertical transmission of the bacteria through the production pyramid could be one explanation for this high prevalence. METHODS To test this hypothesis, paper linings from imported flocks of grandparent animals were screened for the presence of ESC-resistant E. coli and a positive flock, together with its progeny, was followed longitudinally through the production pyramid using boot swabs. The relationship of isolated ESC-resistant E. coli was investigated using multiple-locus variable number tandem repeat analysis (MLVA). RESULTS ESC-resistant E. coli carrying blaCMY-2 was isolated from six out of eight imported flocks of grandparent animals. One clone of E. coli carrying blaCMY-2 occurred in all levels of the production pyramid and in flocks of imported grandparent animals. CONCLUSIONS E. coli carrying blaCMY-2 is frequently present among grandparent animals imported to Sweden for breeding purposes. The occurrence of one clone in all levels of the production pyramid indicates that its introduction through imported breeding stock and vertical transmission through the production pyramid could be one explanation for the high proportion of Swedish broilers colonized with ESC-resistant E. coli.

Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and Brachyspira pilosicoli isolated in Sweden between 1990 and 2010

BackgroundThe anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed.MethodsThe MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum.ResultsThe compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78T (ATCC® 27164T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml.ConclusionsThe broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli.

Antimicrobial Resistance in Scandinavia after a Ban of Antimicrobial Growth Promoters

The banned use of antimicrobial growth promoters resulted in a considerably decreased use of antimicrobials in food animal production in Sweden (65%), Denmark (47%), Norway (40%) and Finland (27%). The current prevalence of antimicrobial resistance in animal bacterial populations is also considerably lower than in some other countries in the EU. In the swine production, no or limited effect was found in the finisher production (>25 to 30 kg). Temporary negative effects occurred during the post weaning period (7–30 kg). In Denmark, the cost of production from birth to slaughter per pig produced increased by approximately 1.0 €with a high variability between pig producers. In the broiler production the termination had no significant negative effect on animal health and welfare or on production economy.

Antimicrobial resistance and virulence factors in Escherichia coli from Swedish dairy calves.

BackgroundIn Sweden, knowledge about the role of enteropathogenic Escherichia coli in neonatal calf diarrhea and the occurrence of antimicrobial resistance in E. coli from young calves is largely unknown. This has therapeutic concern and such knowledge is also required for prudent use of antimicrobials.MethodsIn a case control study Esherichia coli isolated from faecal samples from dairy calves were phenotyped by biochemical fingerprinting and analyzed for virulence genes by PCR. Antimicrobial susceptibility was tested by determination of minimum inhibitory concentration (MIC). Farm management data were collected and Fishers exact test and univariable and multivariable logistic regression analysis were performed.ResultsOf 95 E. coli tested for antimicrobial susceptibility 61% were resistant to one or more substances and 28% were multi-resistant. The virulence gene F5 (K99) was not found in any isolate. In total, 21 out of 40 of the investigated virulence genes were not detected or rarely detected. The virulence genes espP, irp, and fyuA were more common in resistant E. coli than in fully susceptible isolates (P < 0.05). The virulence gene terZ was associated with calf diarrhea (P ≤ 0.01).The participating 85 herds had a median herd size of 80 lactating cows. Herds with calf diarrhea problems were larger (> 55 cows; P < 0.001), had higher calf mortality (P ≤ 0.01) and calf group feeders were more in use (P < 0.05), compared to herds without calf diarrhea problems.There was no association between calf diarrhea and diversity of enteric E. coli.ConclusionsAntimicrobial resistance was common in E. coli from pre-weaned dairy calves, occurring particularly in calves from herds experiencing calf diarrhea problems. The results indicate that more factors than use of antimicrobials influence the epidemiology of resistant E. coli.Enteropathogenic E. coli seems to be an uncommon cause of neonatal calf diarrhea in Swedish dairy herds. In practice, calf diarrhea should be regarded holistically in a context of infectious agents, calf immunity, management practices etc. We therefore advice against routine antimicrobial treatment and recommend that bacteriological cultures, followed by testing for antimicrobial susceptibility and for virulence factors, are used to guide decisions on such treatment.

Spread without known selective pressure of a vancomycin-resistant clone of Enterococcus faecium among broilers

OBJECTIVES The aim of this paper was to describe an increased occurrence of vancomycin-resistant enterococci (VRE) in Swedish broilers since 2000 and to investigate the genetic relatedness of isolates. METHODS Caecal content from slaughtered broilers was cultured for VRE on medium supplemented with vancomycin (16 mg/L). Species identification, antibiotic susceptibility determination, vancomycin resistance genotyping, multilocus sequence typing (MLST) and characterization of Tn1546 were performed. RESULTS The proportion of VRE-positive samples increased gradually from <1% in 2000 to slightly over 40% in 2005. Between 2005 and 2006, the proportion of VRE-positive samples decreased and between 2006 and 2007, it was stable at just below 30%. All isolates tested were Enterococcus faecium and carried the vanA gene. A majority of the isolates had similar antibiograms, the same MLST sequence type and Tn1546 transposon. CONCLUSIONS The proportion of VRE-positive samples from broilers has increased since 2000, and this is due to the spread of one major clone. Moreover, this has taken place in an environment without any obvious selective pressure.

Antibiotic resistance--consequences for animal health, welfare, and food production.

Abstract Most of the literature on the consequences of emergence and spread of bacteria resistant to antibiotics among animals relate to the potential impact on public health. But antibiotics are used to treat sick animals, and resistance in animal pathogens may lead to therapy failure. This has received little scientific attention, and therefore, in this article, we discuss examples that illustrate the possible impact of resistance on animal health and consequences thereof. For all animals, there may be a negative effect on health and welfare when diseases cannot be treated. Other consequences will vary depending on why and how different animal species are kept. Animals kept as companions or for sports often receive advanced care, and antibiotic resistance can lead to negative social and economic consequences for the owners. Further, spread of hospital-acquired infections can have an economic impact on the affected premises. As to animals kept for food production, antibiotics are not needed to promote growth, but, if infectious diseases cannot be treated when they occur, this can have a negative effect on the productivity and economy of affected businesses. Antibiotic resistance in animal bacteria can also have positive consequences by creating incentives for adoption of alternative regimes for treatment and prevention. It is probable that new antibiotic classes placed on the market in the future will not reach veterinary medicine, which further emphasizes the need to preserve the efficacy of currently available antibiotics through antibiotic stewardship. A cornerstone in this work is prevention, as healthy animals do not need antibiotics.