Suspected transmission and subsequent spread of MRSA from farmer to dairy cows.

In the present study we describe an outbreak where PVL positive MRSA belonging to spa-type t002 and multi-locus sequence type ST2659 persisted in a Swedish dairy herd for at least two years, despite efforts to hinder transmission between animals and between the farmer and his animals. This is the first description of persistence and spread of MRSA in a dairy herd in Sweden. Sampling of animals in the herd was initiated by the finding of MRSA in the farmer and was performed at eight occasions from November 2012 to September 2014. In total, MRSA was detected in 25 animals and in 16 of these MRSA was detected in milk samples. In addition, MRSA was also detected in bulk milk samples. Whole genome sequencing (WGS) of twelve isolates from farmer (n = 1), animals (n = 9) and bulk milk (n = 2) revealed high relatedness, implying a common source. MRSA may initially have been transmitted from humans to cows with further spread within the herd. WGS showed minor differences in one isolate (loss of phage ΦN315) which could indicate adaption of the strain to an animal host.

High occurrence of mecC-MRSA in wild hedgehogs (Erinaceus europaeus) in Sweden.

We investigated the occurrence of mecC-MRSA in wild hedgehogs (Erinaceus europaeus) in Sweden and characterized the obtained isolates. Samples from 55 hedgehogs from five counties of Sweden were cultivated selectively for MRSA and putative isolates were confirmed by real-time PCR detecting mecA, mecC, nuc and PVL genes. mecC-MRSA was confirmed in 35 (64%) animals from three geographically separated counties. Confirmed isolates were spa-typed and tested for antimicrobial susceptibility by broth microdilution. Eight different spa-types were identified (t843, t978, t3391, t9111, t10751, t10893, t11015, t15312) of which t843 (49%) was the most common. The spa-types t843, t3391 and t978 were found in isolates from two counties. The study shows that mecC-MRSA is common in wild hedgehogs in two counties of Sweden but occurs in hedgehogs also in other parts of the country. Our findings suggest that hedgehogs could be a reservoir for mecC-MRSA. In addition, similar spa-types of isolates from hedgehogs and isolates previously described in domesticated animals and in humans indicates transfer between these populations.

Methicillin-resistant Staphylococcus aureus not detected in Swedish nucleus and multiplying pig herds.

Introduction: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged among pigs in many countries. MRSA in the pig population constitute a reservoir with risk for transmission to humans in close contact with pigs. Absence of MRSA in the top of the breeding pyramid would prevent spread to the rest of the pig population. The aim of this study was to investigate the occurrence of MRSA in nucleus and multiplying pig herds in Sweden. Materials and methods: All nucleus and multiplying pig herds in Sweden present in 2011 (n = 53) and 2014 (n = 39) were sampled for MRSA. Results and discussion: MRSA was not detected either in 2011 or in 2014. That MRSA was not detected in the top of the breeding pyramid indicates a favourable MRSA situation in the Swedish pig population. Abbreviations: MRSA: methicillin-resistant Staphylococcus aureus; LA-MRSA: livestock-associated MRSA; CC: clonal complex.

A longitudinal cohort study of acute puerperal metritis cases in Swedish dairy cows.

BACKGROUND: Acute puerperal metritis affects cows during the early postpartum period and causes fever, fetid vaginal discharge and general depression. The disease is severe and treatment with antimicrobials is often required. This study followed 79 Swedish dairy cows with acute puerperal metritis with registered treatment and outcome in terms of recovery. Bacteria isolated from the uterus and their susceptibility to penicillin were studied. Clinical cases were assigned by participating practitioners who examined the cows, performed uterine swab sampling, decided treatment and provided information about cow health and calving conditions. Fertility and culling data were collected from the official Swedish milk and health recording scheme. Recovery from disease was defined in four levels; as a cow that survived 1 or 4 months, was inseminated and subsequently became pregnant. Intervals from dates of first and latest calving to insemination date were studied. RESULTS: The most common bacterial findings were a mixed culture of Escherichia coli and bacteria such as Gram positive cocci, Fusobacterium necrophorum, Clostridium spp. or Trueperella pyogenes. The Gram positive cocci, Pasteurella spp. and F. necrophorum were generally susceptible to penicillin. The majority of cows (70%) were treated with penicillin in accordance with the Swedish policy on treatment of metritis while 19% were treated with tetracycline and 8% were not treated with antimicrobials. Recovery rates were similar between treatments. Besides "calving to last insemination" interval (CLI) that was 5 days shorter than the national mean, fertility was slightly reduced compared to national means. "Calving to first insemination" interval (CFI) was 4 days longer than national mean and number of inseminations/cow increased from 1.9 to 2.1. Escherichia coli culture positive cows did not become pregnant to the same extent as cows without E. coli in the uterus (P = 0.046). Twin births resulted in a longer CFI (P = 0.034). The culling rate was generally high (42% within 300 days after occurrence of metritis), though death associated with acute disease was low (6%). CONCLUSIONS: Escherichia coli was the most common bacterial pathogen isolated from cases of acute puerperal metritis in the present study. This bacterium is inherently resistant to penicillin, but although most cows were treated with penicillin, death due to acute disease was low and recovery and final fertility results were acceptable. In times of emerging antimicrobial resistance and demand for prudent antimicrobial use, we suggest that penicillin is a "good enough" choice if antimicrobial treatment of acute puerperal metritis is needed.

Risk factors for quinolone-resistant Escherichia coli in feces from preweaned dairy calves and postpartum dairy cows.

Quinolone resistance may emerge in gut bacteria (e.g., in Escherichia coli) of animals. Such bacteria could cause infections in the animal itself or be transmitted to humans via the food chain. Quinolone resistance is also observed in fecal E. coli of healthy dairy cattle, but the prevalence varies between farms, not solely as a result of varying degree of fluoroquinolone exposure. The objective of this study was to identify risk factors for the fecal shedding of quinolone-resistant E. coli (QREC) from dairy calves and postpartum cows. Rectal swabs from 15 preweaned calves and 5 postpartum cows per farm were collected on 23 Swedish dairy farms to determine the prevalence of QREC. Risk factors for the shedding of QREC were investigated using multivariable statistical models. Quinolone-resistant E. coli were found on all but one farm. Factors associated with QREC shedding by calves were being younger than 18 d, being fed milk from cows treated with antimicrobials, recent use of fluoroquinolones in the herd, carriage of QREC by postpartum cows, and using the calving area never or rarely as a sick pen compared with often. Factors associated with QREC shedding by cows were calving in group pens or freestalls compared with single pens or tiestalls, purchasing cattle, sharing animal transports with other farmers, and poor farm hygiene. Proper biosecurity and improved hygiene, as well as minimizing fluoroquinolone exposure and waste milk feeding, may be important factors to reduce the burden of QREC on dairy farms.

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.

Prevalence of bacterial genotypes and outcome of bovine clinical mastitis due to Streptococcus dysgalactiae and Streptococcus uberis.

BACKGROUND: Streptococcus dysgalactiae and Streptococcus uberis are common causes of clinical mastitis (CM) in dairy cows. In the present study genotype variation of S. dysgalactiae and S. uberis was investigated, as well as the influence of bacterial species, or genotype within species, on the outcome of veterinary-treated CM (VTCM). Isolates of S. dysgalactiae (n = 132) and S. uberis (n = 97) were genotyped using pulsed-field gel electrophoresis. Identical banding patterns were called pulsotypes. Outcome measurements used were cow composite SCC, milk yield, additional registered VTCMs and culling rate during a four-month follow-up period. RESULTS: In total, 71 S. dysgalactiae pulsotypes were identified. Nineteen of the pulsotypes were isolated from more than one herd; the remaining pulsotypes were only found once each in the material. All S. uberis isolates were of different pulsotypes. During the follow-up period, the SCC of S. dysgalactiae-cows was significantly lower than the SCC of S. uberis-cows (P <0.05). Median SCC of S. dysgalactiae-cows was 71 500 cells/ml and of S. uberis-cows 108 000 cells/ml. No other differences in outcome parameters could be identified between species or genotypes. CONCLUSIONS: Identical S. dysgalactiae genotypes were isolated from more than one herd, suggesting some spread of this pathogen between Swedish dairy herds. The genetic variation among S. uberis isolates was substantial, and we found no evidence of spread of this pathogen between herds. The milk SCC was lower during the follow-up period if S. dysgalactiae rather than S. uberis was isolated from the case, indicating differences in treatment response between bacterial species.

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

BACKGROUND: Staphylococcus 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. RESULTS: The 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. CONCLUSIONS: In 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.

BACKGROUND: Milk 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. RESULTS: Colostrum (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. CONCLUSIONS: Waste 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.

Methicillin-resistant Staphylococcus aureus containing mecC in Swedish dairy cows.

BACKGROUND: Hitherto, 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. FINDINGS: Bovine 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. CONCLUSION: In 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.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The 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 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.