Helle Unnerstad

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.

Lessons from an outbreak of listeriosis related to vacuum-packed gravad and cold-smoked fish.

The first lesson learned from this outbreak was that vacuum-packed rainbow trout is not only an excellent medium for the growth of Listeria monocytogenes, but may also cause human listeriosis. Another lesson is that one single fish processing plant may spread multiple clonal types of L. monocytogenes by selling contaminated products to consumers. Thus, when investigating fish-borne outbreaks of listeriosis one should identify and type several isolates of L. monocytogenes from each food and environmental sample, since multiple clonal types might be present. The outbreak described in this paper involved at least eight human cases, three clonal types of L. monocytogenes, and lasted for 11 months. During the outbreak investigation, L. monocytogenes was also isolated from another brand of rainbow trout found in the refrigerator of one of the patients. These latter isolates belonged to a clonal type not associated with the outbreak. However, this clonal type is of considerable interest since it has been associated with foodborne outbreaks of listeriosis in several countries, and is also the second most common clonal type among human clinical isolates of L. monocytogenes in Sweden. Besides the described outbreak, it is likely that vacuum-packed, cold-smoked and gravad rainbow trout have been involved in additional cases of foodborne listeriosis in Sweden.

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.

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.

Pyrosequencing as a Method for Grouping of Listeria monocytogenes Strains on the Basis of Single-Nucleotide Polymorphisms in the inlB Gene

ABSTRACT By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.

Molecular grouping of Listeria monocytogenes based on the sequence of the inIB gene.

The major part of the gene inlB was sequenced in 24 strains of Listeria monocytogenes belonging to serovars 1/2a, 1/2b, 1/2c, 3b and 4b. A phylogenetic analysis based on the inlB nucleotide sequences showed that strains of serovars 1/2a and 1/2c were closely related, as well as those of serovars 1/2b and 3b. Strains sharing serovar 4b could be divided into two distinct groups. There were differences in amino-acid sequence between all serovars except between serovars 1/2b and 3b. Differences in amino-acid sequence were also seen within each of the serovars 1/2a and 4b. The data presented indicate that the inlB gene may be useful for typing purposes as an alternative or complement to serotyping.

Methicillin-resistant Staphylococcus aureus containing mecC in Swedish dairy cows

BackgroundHitherto, methicillin-resistant Staphylococcus aureus (MRSA) has not been detected in Swedish cattle. However, due to the report of mecC, a novel homologue to the mecA gene, there was reason to re-evaluate susceptibility results from strain collections of Staphylococcus aureus and test suspected isolates for the presence of mecC.FindingsBovine isolates of S. aureus with elevated minimum inhibitory concentrations of beta-lactams were retrospectively tested for presence of mecC. In four of the isolates mecC was detected.ConclusionIn Sweden, this is the first finding of MRSA in cattle and the first detection of MRSA harbouring mecC of domestic animal origin. MRSA in animal populations has implications as a potential reservoir with risk for spread to humans. Occurrence of MRSA among Swedish cattle appears still very limited.

Risk factors for antimicrobial resistance in fecal Escherichia coli from preweaned dairy calves.

The primary objective of this study was to investigate calf and farm factors associated with antimicrobial-resistant Escherichia coli in the feces of preweaned dairy calves in Sweden. In particular, we investigated the effects of feeding calves colostrum and milk from cows treated with antimicrobials. The secondary objective was to describe the prevalence of resistant E. coli in feces of preweaned dairy calves in Sweden. Fecal samples from 3 calves, aged 7 to 28d, from 243 farms were analyzed for the within-sample prevalence of E. coli resistant to nalidixic acid, streptomycin, and cefotaxime using selective agars supplemented with antimicrobials. In addition, resistance to 12 antimicrobials was tested in one randomly selected E. coli isolate per calf. Information was collected from the farmers via questionnaires regarding the use of colostrum and milk from cows treated with antimicrobials as calf feed and other uses of antimicrobials in the herd. Multivariable zero-inflated negative binomial and logistic regression models were used to assess the effect of various risk factors for shedding of resistant E. coli. Escherichia coli resistant to streptomycin, nalidixic acid, or cefotaxime were isolated from 90, 49, and 11% of the calves, respectively. Resistance to at least one antimicrobial was found in a random isolate of E. coli from 48% of the calves. Feeding colostrum from cows treated with antimicrobials at drying off did not affect the prevalence of resistant E. coli. In contrast, feeding milk from cows treated with antimicrobials during lactation resulted in significantly more nalidixic acid- and streptomycin-resistant E. coli than when such milk was discarded; no significant effect was seen for other resistance traits. Furthermore, an interaction was found between feeding milk from cows treated with antimicrobials and use of fluoroquinolones in cows. In general, the prevalence of resistance was lower for older calves and calves on small farms. Other factors that were associated with the shedding of resistant E. coli were administration of oral dihydrostreptomycin to calves, administration of systemic tetracycline and ceftiofur to cows and calves, housing of the calves, predominant breed of the herd, and geographic location of the farm. The presence of resistant E. coli in calves was clearly due to multiple factors, but minimizing the feeding of milk from cows treated with antimicrobials during lactation should lower the prevalence of resistant E. coli in the gastrointestinal tract of the calves.

Associations between bacterial genotype and outcome of bovine clinical Staphylococcus aureus mastitis

BackgroundStaphylococcus aureus is an important cause of clinical mastitis in dairy cows worldwide. The cure rate after antimicrobial treatment of clinical S. aureus mastitis is very variable due to both cow and bacterial factors. Studies have shown that bacterial genotype might affect short-term bacteriological and clinical cure, but the long-term outcome has been less studied. The objectives of this study were to investigate associations between bacterial genotype and long-term outcome of veterinary-treated clinical mastitis (VTCM) caused by S. aureus during a follow-up period of 120 days and to study genotype variation among Swedish S. aureus isolates. S. aureus isolates from cases of VTCM were genotyped by pulsed-field gel electrophoresis. Long-term outcome measurements used were somatic cell count (SCC), additional diagnoses of VTCM, milk yield and culling. Isolates were classified into clusters (>80% similarity) and pulsotypes (100% similarity). Clusters and pulsotypes were grouped according to occurrence. Multivariable mixed-effect linear regression models including cow and bacterial factors with possible influence on SCC or milk yield were used to calculate differences in SCC or milk yield between groups. Additional outcome measures were calculated using a test of proportions.ResultsThe isolates (n = 185) were divided into 18 clusters and 29 pulsotypes. Two pulsotypes were classified as common, and were found in 64% of the cases of VTCM. Remaining isolates were classified as less common or rare pulsotypes. The distribution was similar at cluster level. Outcome was calculated from follow-up data on 111 cows. Significantly lower SCC during the follow-up period was found in cows infected with common clusters compared to in cows infected with less common/rare clusters. The proportion of cows with SCC <200 000 cells/ml during the whole follow-up period was significantly higher in the group common clusters than in the group less common/rare clusters. Bacterial genotype did not influence the other outcome parameters.ConclusionsIn Sweden, two S. aureus pulsotypes, identified in about 64% of clinical S. aureus cases, were widespread. Cows infected with the common genotypes had significantly lower SCC during 120 days after treatment compared to cows infected with less common or rare genotypes.

Farming practices in Sweden related to feeding milk and colostrum from cows treated with antimicrobials to dairy calves

BackgroundMilk produced by cows in receipt of antimicrobial therapy may contain antimicrobial residues. Such antimicrobial-containing waste milk must be withdrawn from human consumption and is therefore sometimes used as calf feed. Unfortunately, this approach might promote selection of antimicrobial resistant bacteria in the calves’ intestinal microbiota. The objectives of this study were therefore to obtain an overview of waste milk feeding practices on Swedish dairy farms and to investigate if these practices were associated with certain farm characteristics. A representative group of 457 Swedish dairy farmers participated in a web-based survey with questions about the use of colostrum and milk from cows treated with antimicrobials at dry off or during lactation, respectively, as calf feed.ResultsColostrum (milk from the first milking after calving) and transition milk (milk from the second milking to the fourth day after calving) from cows treated with antimicrobials at dry off was fed to calves on 89% and 85% of the farms in the study, respectively. When antimicrobial therapy was given to cows during lactation, 56% of the farms fed milk that was produced during the course of treatment to calves, whereas milk that was produced during the subsequent withdrawal period was fed to calves on 79% of the farms. Surveyed farmers were less prone to feed such milk if the antimicrobial therapy was due to mastitis than other infections. In Sweden, a majority of antimicrobial treatments during lactation are systemic administration of benzylpenicillin and thus, the bulk of waste milk in Sweden is likely to contain residues of this drug. Feeding waste milk to calves was more common on non-organic farms, and on farms located in Southern Sweden, and was less common on farms with cows housed in cold free stalls barns.ConclusionsWaste milk that may contain antimicrobial residues is, at least occasionally, used as feed for calves on a majority of surveyed Swedish dairy farms. Future work should focus on the effect of waste milk feeding on the occurrence of antimicrobial resistant bacteria in the calves’ intestinal microbiota.