Antimicrobial resistance in Staphylococcus pseudintermedius and the molecular epidemiology of methicillin-resistant S-pseudintermedius in small animals in Finland

Objectives: To investigate antimicrobial susceptibility in Staphylococcus pseudintermedius and the occurrence of methicillin-resistant S. pseudintermedius (MRSP), to explore the molecular structure of the MRSP population and to analyse risk factors for MRSP. Methods: Susceptibility data for clinical S. pseudintermedius isolates in 2011-15 were analysed using WHONET. All MRSP isolates in 2010-14 (n = 362) were typed using PFGE. Representative isolates (n = 87) of clusters were analysed using MLST and staphylococcal cassette chromosome mec (SCCmec) typing. Risk factors were analysed using logistic regression. Results: Of the clinical S. pseudintermedius (n-1958; 98% from dogs), 14% were MRSP. Resistance to other antimicrobials varied between 12% and 39%. No trends were observed over time. Among clinical specimens (from infection sites) and screening specimens (from potential carriers), respectively, 2.5% (267/10813) and 9% (211/2434) revealed MRSP. MLST revealed 42 different STs, including 19 new ones. Clonal complexes 71, 45 and 258 were the most common, but the MRSP population diversified over the years. A clinical S. pseudintermedius isolate was more likely to be MRSP if the patient was on antimicrobials at the time of sampling or was male. The presence of MRSP in screening specimens was more likely if the patient was on multiple antimicrobials at the time of sampling. Specimens from private clinics (versus the Veterinary Teaching Hospital of the University of Helsinki) had a higher likelihood of MRSP in both analyses. Conclusions: Resistance to antimicrobials among S. pseudintermedius in Finland is high, emphasizing the importance of infection control measures and susceptibility testing prior to therapy. The diverse MRSP population indicates non-clonal spread.Peer reviewe

Species-specific and Indication-based Use of Antimicrobials in Dogs, Cats, Cattle and Horses in Finland : Data collected using three different methods

Increasing antimicrobial resistance in bacteria has led to the need for better understanding of antimicrobial usage patterns. In 1999, the World Organisation for Animal Health (OIE) recommended that an international ad hoc group should be established to address human and animal health risks related to antimicrobial resistance and the contribution of antimicrobial usage in veterinary medicine. In European countries the need for continuous recording of the usage of veterinary antimicrobials as well as for animal species-specific and indication-based data on usage has been acknowledged. Finland has been among the first countries to develop prudent use guidelines in veterinary medicine, as the Ministry of Agriculture and Forestry issued the first animal species-specific indication-based recommendations for antimicrobial use in animals in 1996. These guidelines have been revised in 2003 and 2009. However, surveillance on the species-specific use of antimicrobials in animals has not been performed in Finland. This thesis provides animal species-specific information on indication-based antimicrobial usage. Different methods for data collection have been utilized. Information on antimicrobial usage in animals has been gathered in four studies (studies A-D). Material from studies A, B and C have been used in an overlapping manner in the original publications I-IV. Study A (original publications I & IV) presents a retrospective cross-sectional survey on prescriptions for small animals at the Veterinary Teaching Hospital of the University of Helsinki. Prescriptions for antimicrobial agents (n = 2281) were collected and usage patterns, such as the indication and length of treatment, were reviewed. Most of the prescriptions were for dogs (78%), and primarily for the treatment of skin and ear infections most of which were treated with cephalexin for a median period of 14 days. Prescriptions for cats (18%) were most often for the treatment of urinary tract infections with amoxicillin for a median length of 10 days. Study B (original publication II) was a retrospective cross-sectional survey where prescriptions for animals were collected from 17 University Pharmacies nationwide. Antimicrobial prescriptions (n = 1038) for mainly dogs (65%) and cats (19%) were investigated. In this study, cephalexin and amoxicillin were also the most frequently used drugs for dogs and cats, respectively. In study C (original publications III & IV), the indication-based usage of antimicrobials of practicing veterinarians was analyzed by using a prospective questionnaire. Randomly selected practicing veterinarians in Finland (n = 262) recorded all their antimicrobial usage during a 7-day study period. Cattle (46%) with mastitis were the most common patients receiving antimicrobial treatment, generally intramuscular penicillin G or intramammary treatment with ampicillin and cloxacillin. The median length of treatment was four days, regardless of the route of administration. Antimicrobial use in horses was evaluated in study D, the results of which are previously unpublished. Firstly, data collected with the prospective questionnaire from the practicing veterinarians showed that horses (n = 89) were frequently treated for skin or wound infections by using penicillin G or trimethoprim-sulfadiazine. The mean duration of treatment was five to seven days. Secondly, according to retrospective data collected from patient records, horses (n = 74) that underwent colic surgery at the Veterinary Teaching Hospital of the University of Helsinki were generally treated according to national and hospital recommendations; penicillin G and gentamicin was administered preoperatively and treatment was continued for a median of three days postoperatively. In conclusion, Finnish veterinarians followed well the national prudent use guidelines. Narrow-spectrum antimicrobials were preferred and, for instance, fluoroquinolones were used sparingly. Prescription studies seemed to give good information on antimicrobials usage, especially when combined with complementary information from patient records. A prospective questionnaire study provided a fair amount of valuable data on several animal species. Electronic surveys are worthwhile exploiting in the future.Kansainvälinen eläinten tartuntatautien järjestö OIE (World Organisation for Animal Health) sekä monet Euroopan maat ovat tiedostaneet eläimille käytettävien mikrobilääkkeiden tarkemman tiedon keruun tarpeen. Tieto on tärkeää arvioidessa eläinten lääkekäytön vaikutuksia resistenssin lisääntymiseen ihmisten ja eläinten tautia aiheuttavissa bakteereissa. Maa- ja metsätalousministeriön asettama työryhmä laati vuonna 1996 ensimmäiset mikrobilääkkeiden käyttösuositukset eläinten yleisimpiin tulehdustauteihin. Suosituksia on päivitetty 2003 ja 2009. Tutkimuksia siitä, ovatko eläinlääkärit noudattaneet annettuja suosituksia, ei kuitenkaan ole tehty. Tämä väitöskirjatutkimus perustuu eläinlaji- ja käyttöaihekohtaiseen mikrobilääkkeiden käyttöön koirilla, kissoilla, naudoilla ja hevosilla Suomessa. Tutkimuksen tarkoituksena oli selvittää, miten ministeriön suosituksia on noudatettu ja mihin suuntaan suosituksia tulisi kehittää. Tietoa mikrobilääkkeiden käytöstä koirilla ja kissoilla kerättiin Yliopistollisen eläinsairaalan potilaille määrätyistä mikrobilääkeresepteistä (n = 2281) sekä potilastiedostoista. Lisäksi käyttöä selvitettiin keräämällä tietoja kaikista Suomen Yliopiston Apteekeista kuukauden ajalta toimitetuista eläinten mikrobilääkeresepteistä (n = 1038). Suurin osa resepteistä oli määrätty koirille. Yleisin käyttöaihe oli iho- ja korvatulehdus. Kissoille määrätyissä resepteissä oli yleisimpänä käyttöaiheena virtsatietulehdus. Kefaleksiini ja amoksisilliini olivat yleisimmin käytetyt lääkeaineet. Kolmas aineisto kerättiin praktiikkaa tekeville eläinlääkäreille suunnatun kyselytutkimuksen avulla. Eläinlääkärit kirjasivat viikon ajan kaikki tiedot antamistaan tai määräämistään mikrobilääkehoidoista. Eniten mikrobilääkkeitä käytettiin naudoille, joilla yleisimmin hoidettiin utaretulehdusta, joko lihaksensisäisellä penisilliinillä tai intramammaareilla. Hevosten lääkekäytöstä koottiin kaksi erillistä aineistoa. Eläinlääkäreille suunnatun kyselytutkimuksen perusteella hevosia hoidettiin useimmiten iho- tai haavatulehduksen takia käyttäen penisilliiniä tai trimetopriimi-sulfadiatsiinia. Toinen hevosia koskeva aineisto kerättiin Yliopistollisen eläinsairaalan potilasasiakirjoista. Aineisto koostui kaikista kolmen vuoden aikana hevossairaalassa ähkyn takia leikattujen hevosten mikrobilääkkeiden käytöstä sairaalahoidon aikana. Lääkekäyttö oli pääosin suositusten mukainen, koska suurin osa potilaista sai penisilliiniä ja gentamisiinia suonensisäisesti ennen leikkausta ja hoitoa jatkettiin keskimäärin kolmen vuorokauden ajan leikkauksen jälkeen. Yhteenvetona voidaan todeta, että Suomen eläinlääkärit noudattivat Maa- ja metsätalousministeriön antamia mikrobilääkkeiden käyttösuosituksia hyvin. Kapeakirjoisia lääkeaineita suosittiin ja esimerkiksi fluorokinolonien käyttö oli maltillista ja perusteltua. Reseptitutkimukset antavat hyvää tietoa mikrobilääkkeiden eläinlaji- ja käyttöaihekohtaisesta käytöstä, erityisesti jos puuttuvia tietoja on mahdollista täydentää esimerkiksi potilasasiakirjojen avulla. Eteenpäin suuntautuva kyselytutkimus antoi runsaasti tietoa mikrobilääkkeiden käytöstä eri eläinlajeilla. Jatkossa elektronisten apuvälineiden käyttöä kyselytutkimuksissa tulee hyödyntää paremmin

Low prevalence of zoonotic multidrug-resistant bacteria in veterinarians in a country with prudent use of antimicrobials in animals

The occurrence of multidrug-resistant zoonotic bacteria in animals has been increasing worldwide. Working in close contact with livestock increases the risk of carriage of these bacteria. We investigated the occurrence of extended-spectrum beta-lactamase (ESBL) and plasmidic AmpC beta-lactamase producing Enterobacteriaceae (ESBL/pAmpC-PE) and livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in Finnish veterinarians (n = 320). In addition to microbiological samples, background information was collected. Bacterial whole genome sequencing was performed to deduce sequence types (STs), spa types and resistance genes of the isolates. In total, 3.0% (9/297) of the veterinarians carried ESBL producing Escherichia coli, with one ESBL producing E. coli isolate producing also AmpC. Seven different STs, sequences of several different plasmid groups as well as several different bla(ESBL/pAmpC )genes existed in different combinations. No carbapenemase or colistin resistance genes were detected. MRSA was detected in 0.3% (1/320) of the samples. The strain belonged to LA-MRSA clonal complex (CC) 398 (ST398, spa type 011, lacking Panton-Valentine leukocidin genes). In conclusion, this study shows low carriage of multidrug-resistant zoonotic bacteria in Finnish veterinarians. However, finding LA-MRSA for the first time in a sample from a veterinarian in a country with prudent use of animal antimicrobials and regarding the recent rise of LA-MRSA on Finnish pig farms, a strong recommendation to protect people working in close contact with animals carrying LA-MRSA CC398 is given. Further studies are needed to explain why the prevalence of LA-MRSA in veterinarians is lower in Finland than in other European countries.Peer reviewe

Circulating Liver-enriched Antimicrobial Peptide-2 Decreases During Male Puberty

Context: Circulating levels of liver-enriched antimicrobial peptide 2 (LEAP2), a ghrelin receptor antagonist, decrease under caloric restriction and increase in obesity. The role of LEAP2 in male puberty, a phase with accelerated energy demand, is unclear. Objective: This work aimed to investigate whether circulating LEAP2 levels are downregulated in boys following the onset of puberty to respond to the energy need required for growth. Methods: We determined circulating LEAP2 levels in 28 boys with constitutional delay of growth and puberty (CDGP) who participated in a randomized controlled trial (NCT017977181, and were treated with letrozole (n = 15) or intramuscular low-dose testosterone (T) (n = 13) for 6 months. Blood sampling and dual-energy x-ray absorptiometry-measured body composition were performed at 0-, 6-, and 12-month visits. Results: Serum LEAP2 levels decreased statistically significantly during pubertal progression (0-6 months: mean decrease -4.3 (10.3) ng/mL, P = .036 and 0-12 months: -3.9 19.31 ng/mL, P = .033). Between 0 and 6 months, the changes in serum LEAP2 levels correlated positively with changes in percentage of body fat (r(s) = 0.48, P = .011), and negatively with growth velocity and estradiol levels (r(s) = -0.43, P = .022, r(s) = -0.55, P = .003, respectively). In the T group only, the changes in serum LEAP2 correlated negatively with changes in T and estradiol levels. Between 0 and 12 months, the change in LEAP2 levels correlated negatively with the change in high-density lipoprotein levels (r(s) = -0.44, P = .022) and positively with the change in insulin (r(s) = 0.50, P = .009) and HOMA-IR (r(s )= 0.51, P = .007) levels. Conclusion: Circulating LEAP2 levels decreased after induction of puberty reciprocally with increased growth rate and energy demand, reflecting the metabolic state of the adolescent. Further, the results suggest that estradiol levels may have a permissive role in downregulating circulating LEAP2 levels.Peer reviewe

Biosecurity and antimicrobial use in pig production

Antimicrobial use (AMU) has led to a development of antimicrobial resistant bacteria that complicate treatment of infectious diseases in animals and humans. Majority of veterinary AMU occurs in pigs, which is known to contribute to the development of antimicrobial resistance (AMR). Especially in large pork producing countries, the majority of antimicrobials are administered as group treatments for pigs that enhances AMR development. Biosecurity means measures that prevent pathogen transmission to a herd (external biosecurity) and within a herd (internal biosecurity). Consequently, when there is a global demand to decrease AMU, biosecurity has been introduced as an alternative to that. In Finland, veterinary AMU is one of the lowest level in the EU and group treatments are not preferred. However, Finnish pig herds have been small-sized in general and disease situation has been relatively good thorough the country thus there has been no need to invest in biosecurity. Herd size is increasing and therefore farmers must adopt alternative methods to decrease the need to use antimicrobials. This study aimed to investigate current biosecurity status of Finnish pig herds and their AMU, and study associations between them. Study population consisted of ten farrow-to-finish herds whose biosecurity status was evaluated by using an international Biocheck.UGent scoring system. AMU of individual herds was collected from national Sikava register covering around 90 % of Finnish pig herds. It is therefore the best available source for AMU data and makes comparison between herds possible. Furthermore, detailed calculation of AMU at different production stages was done. The biosecurity scores of study herds varied considerably. Mean external biosecurity score was higher than internal biosecurity score (Mean ± SD; 69 ± 1.2 vs. 44 ± 5.3, P < 0.001) and better in large herds compared to small herds (LSmeans ± SE; 72 ± 1.3 vs. 66 ± 1.3, P < 0.05). AMU tended to increase with decreasing biosecurity according to the study hypothesis. AMU was highest in suckling piglets but there were no significant correlations between the total biosecurity scores and AMU at any age groups studied. Regardless of the small sample size of this study, current findings support other studies indicating the potential to improve biosecurity of Finnish herds in order to avoid increase in AMU

A value chain analysis of interventions to control production diseases in the intensive pig production sector

Value chain analysis (VCA) calculated the financial effects on food chain actors of interventions to improve animal health and welfare in the intensive pig sector. Two interventions to reduce production diseases were studied. A generic chain diagram of linkages between stakeholders and value-added dimensions was designed. Data on structure and financial performance were collected for the sector. The production parameters and financial effects of the interventions were then described to illustrate impact on the supply chain. The effects of the interventions were also assessed at market level using economic welfare analysis. The sectors in Finland and the UK are small in farm numbers and few companies produced much of the output in a largely vertically-integrated structure. The most beneficial intervention in financial terms to farmers was improved hygiene in pig fattening (around +50% in gross margin). It was calculated to reduce the consumer price for pig meat by up to 5% when applied at large, whereas for improved management measures, it would reduce consumer price by less than 0.5%. However, the latter added value also through food quality attributes. We show that good hygiene and animal care can add value. However, evaluation of the financial and social viability of the interventions is needed to decide what interventions are adopted. The structure of supply chains influences which policy measures could be applied. Of the two interventions, improved pig hygiene had the largest potential to improve efficiency and reduce costs. The studied interventions can also provide new business opportunities to farms, slaughterhouses and food sector companies. More evidence is needed to support public policies and business decision-making in the sector. For this, evidence on consumer attitudes to production diseases is needed. Nevertheless, the study makes an important contribution by showing how improvements in health and welfare benefit the whole chain

Campylobacter spp. ja Listeria monocytogenes linnulihatoodetes Eestis

A Thesis for applying for the degree of Doctor of Philosophy in Veterinary Medicine and Food Science (Food Hygiene and Food Quality)Campylobacteriosis, caused by thermotolerant Campylobacter species, is continued to be one of the most commonly reported zoonosis in humans in the European Union, and Campylobacter is recognized as the most common causative agents of bacterial gastroenteritis in developed world. Listeriosis, the disease caused by Listeria monocytogenes, is a lifethreatening disease for individuals with compromised immune systems. Consumption of different foods, including poultry products, have been associated with campylobacteriosis and listeriosis. Healthy birds may shed Campylobacter spp. and L. monocytogenes in fecal material asymptomatically. Poultry meat may become contaminated with fecal matter during slaughter or through cross-contamination during processing. The main goals of the present thesis were 1) to determine the prevalence and counts of Campylobacter spp. and the prevalence of L. monocytogenes in Estonian and imported raw poultry meat, 2) to serotype and genotype using pulsed-field gel electrophoresis both Campylobacter and L. monocytogenes isolates, and 3) to determine the antimicrobial susceptibility of the Campylobacter isolates. The studies showed that the poultry products of Estonian origin and imported to Estonia were contaminated with Campylobacter spp. and L. monocytogenes. There was high sero- and genotype diversity among Campylobacter and L. monocytogenes isolates. High level of resistance to fluoroquinolones, tetracycline, ampicillin, and erythromycin among the Campylobacter isolates were detected.Termofiilsed kampülobakterid on paljudes maades ühed põhilised inimese bakteriaalse mao-peensoolepõletiku põhjustajad ja Euroopa Liidu riikides on kampülobakterioos jätkuvalt kõige sagedamini esinev zoonoos. Listerioos on nakkushaigus, mille kutsub esile bakter Listeria monocytogenes ja mis ohustab eelkõige immuunpuudulikkusega inimesi. Haigusjuhtude põhjuseks on kas bakteriga Campylobacter spp. või bakteriga L. monocytogenes saastunud toiduainete, sh linnuliha, tarbimine. Linnud võivad olla nii kampülobakterite kui ka listeeriate asümptomaatilised kandjad. Peamiselt saastub linnuliha fekaalse saastumise teel lindude algtöötlemisel tapamajas või ristsaastumise teel linnuliha edaspidise töötlemise käigus. Käesoleva väitekirja peamised eesmärgid olid 1) määrata bakteri Campylobacter spp. levimus ja arvukus ning bakteri L. monocytogenes levimus Eestis toodetud ja Eestisse imporditud toores linnulihas, 2) serotüpiseerida ja genotüpiseerida pulseeriva välja geelelektroforeesi meetodil toorest linnulihast isoleeritud patogeenitüved ja 3) määrata bakteri Campylobacter spp. isolaatide tundlikkus antibiootikumide suhtes. Tehtud uuringud näitasid, et Eestis toodetud ja Eestisse imporditud linnuliha osutus saastunuks bakteritega Campylobacter spp. ja L. monocytogenes. Uuritud patogeenide tüpiseerimine viitas sero- ja genotüübilisele mitmekesisusele. Tuvastati isoleeritud kampülobakterite tüvede suur resistentsus fluorokinoloonide, tetratsükliini, ampitsilliini ja erütromütsiini suhtes.The publication of this dissertation is granted by the Estonian University of Life Science

Species distribution and in vitro antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitic milk

Background: Coagulase-negative staphylococci (CoNS) are the most common bovine mastitis causing bacteria in many countries. It is known that resistance for antimicrobials is in general more common in CoNS than in Staphylococcus aureus but little is known about the antimicrobial resistance of specific CoNS species. In this study, 400 CoNS isolates from bovine mastitic milk samples were identified to species level using ribotyping and MALDI-TOF MS, and their antimicrobial susceptibility was determined using a commercially available microdilution system. The results were interpreted according to the epidemiological cut-off values by the European Committee on Antimicrobial Susceptibility testing. Results: The most common CoNS species were S. simulans, S. epidermidis, S. chromogenes and S. haemolyticus. Penicillin resistance was the most common type of antimicrobial resistance. Staphylococcus epidermidis was the most resistant among the four major species. Almost one-third of our S. epidermidis isolates were resistant to >2 antimicrobials and close to 7 % were multidrug resistant. The majority of S. epidermidis isolates were resistant to benzylpenicillin. On the contrary, only few S. simulans isolates were penicillin-resistant. Phenotypic oxacillin resistance was found in all four main species, and 34 % of the isolates were oxacillin resistant. However, only 21 isolates (5 %) were positive for the mecA gene. Of these, 20 were S. epidermidis and one S. sciuri. mecC positive isolates were not found. Conclusion: Staphylococcus epidermidis differed from the three other major CoNS species as resistance to the tested antimicrobials was common, several isolates were multidrug resistant, and 19 % of the isolates carried the mecA gene encoding methicillin resistance.Peer reviewe

Effect of Farm Management Practices on Morbidity and Antibiotic Usage on Calf Rearing Farms

Antimicrobial resistance has been recognized as one of the top health threats to human society. Abundant use of antibiotics in both humans and animals has led to ever-increasing antibiotic resistance in bacteria. In food production, decreasing morbidity in beef herds would be an effective way to reduce the use of antibiotics. The objective of this retrospective observational study was to determine overall morbidity on calf rearing farms and to identify associated risk factors. Data were collected by questionnaire, meat companies’ databases and the national cattle register for 28,228 calves transported to 87 calf rearing farms. All medications given to these calves were retrospectively followed for 180 days from calf arrival to the farm. In total, 34,532 parenteral antibiotic medications were administered to the 28,228 study calves (122.3%), and 17,180 calves (60.9%) were medicated with antibiotics at least once during the follow-up. Higher numbers of calves transported to the same farm and larger age variation in calves in the same arrival batch were both associated with increased morbidity. In contrast, higher arrival age of individual calves was associated with decreased morbidity. Our study identifies several factors to consider in decreasing morbidity and antibiotic usage on calf rearing farms