Livestock-associated methicillin-resistant Staphylococcus aureus – studies on prevention of zoonotic transmission

Abstract

Antimicrobial resistance is a major global concern driven especially by the misuse and overuse of antimicrobials both in humans and in animals. Methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) are major causes of nosocomial infections. As these multiresistant bacteria are increasingly found in livestock, the role of livestock as a reservoir for human infections deserves attention. MRSA has been causing healthcare-related infections since the 1960s and community-associated infections since the 1990s. In 2004, new MRSA strains were detected in humans in Europe that were shown to be related to contact with pigs. These strains were later shown to belong to MRSA clonal complex 398 and are called livestock-associated MRSA (LA-MRSA). Prevalence of LA-MRSA in Europe is highest in pigs and veal calves, but it has also been found in other livestock, horses and companion animals. From asymptomatic, colonized animals, LA-MRSA may be transmitted especially to people working in close contact with livestock such as farmers and veterinarians, who in turn may transmit it further to healthcare facilities. ESBL-PE species are found in livestock, horses and companion animals. In Europe, prevalence is highest in pigs and broilers. Evidence on the zoonotic transmission of ESBL-PE is contradictory. People working on European pig and broiler farms have in some studies been shown to have higher ESBL-/AmpC-producing Escherichia coli prevalence rates than the general population. Infection prevention and control (IPC) practices including proper hand hygiene and the use of personal protective equipment are important to prevent the spread of multiresistant bacteria and other pathogens in healthcare settings. Studies on IPC practices in the veterinary setting have mostly concentrated on companion animal clinics. Ambulatory veterinarians work on several farms a day and transport their equipment and medications from farm to farm. Hand hygiene facilities are offered by the farm or stable owner. There are several studies on the eradication of LA-MRSA from pig farms. A less studied approach is the use of bacteriophages, viruses that infect bacteria, which have been studied as an alternative to antimicrobial agents. Studies on bacteriophage treatment against S. aureus infections in livestock are few and the results have been inconclusive. The aim of this study was to study 1) the carriage of multiresistant zoonotic bacteria in veterinarians in Finland, 2) LA-MRSA colonization and genomic diversity in Finnish pigs, 3) the practices of ambulatory livestock and equine veterinarians to prevent zoonotic transmission, and 4) bacteriophage treatment as a possible way to eradicate LA-MRSA from colonized pigs. MRSA and ESBL-PE prevalence was studied in veterinarians in Finland using both phenotypic and genotypic methods to characterize the isolates. A questionnaire was used to collect important background information on risk factors. To study the adherence of ambulatory livestock and/or equine veterinarians to IPC measures, the replies to the questionnaire of a subset of the respondents were analysed using statistical methods. Quantity and colonization patterns of LA-MRSA in Finnish pigs were studied on two farms – farm 1 and farm 2 (10 pigs per farm) – and in a controlled facility (9 pigs). The strains were further compared with sequences from Finnish Food Authority surveillance and infection samples using core genome multilocus sequence typing (cgMLST). The suitability of bacteriophages for the eradication of LA-MRSA from healthy carrier pigs was investigated in an open, controlled study. A cocktail containing three Staphylococcus bacteriophages was administered to the study group pigs (n=10) and a placebo solution to the control group pigs (n=9) three times. Phage and MRSA levels were observed for 22 days. In addition, phage antibodies in pig sera were monitored. Prevalence of LA-MRSA in veterinarians in Finland was 0.3% (1/320) and the strain found belonged to spa type t011 and multilocus sequence type (ST)398. Virulence genes and resistance genes were typical for LA-MRSA strains found in Finnish pigs. ESBL-producing E. coli were carried by 3.0% of veterinarians (9/297) and one of the strains was also an AmpC producer. Several STs, blaESBL/AmpC genes and plasmid types were found, with ST131 being the most prevalent ST. Travelling and use of antibiotics during the past 12 months was as common as among the average Finnish population. Ambulatory livestock and equine veterinarians (n=129) did not conform adequately to hygiene guidelines. Handwashing facilities were often adequate on farms according to 66.9% of the veterinarians but in stables only according to 21.4% (p<.001). When moving to the next farm or stable, 75.0% always washed their hands or used hand sanitizer in livestock practice but only 42.5% in equine practice (p<.001). In livestock practice a protective coat or overalls were always used by 91.6%, whereas in equine practice only by 27.7% (p<0.001). Altogether, 30.0% of the veterinarians reported cleaning their stethoscope less frequently than once a week. Quantities of LA-MRSA in pigs were low. On farm 1, MRSA was detected in all three samplings in the nares of all 10 pigs. On farm 2, MRSA was detected only in one sampling in three pigs. Quantities in the nasal samples were between 101 and 103 CFU/swab and in the skin samples between 101 and 102 CFU/swab. In the controlled facility, the nasal samples of all nine pigs were MRSA-positive at least once, and the trend was declining. Persistent low levels were detected on the skin. The strains and all sequences from the Finnish Food Authority but one clustered into one cluster in the cgMLST analysis. Bacteriophage treatment did not reduce LA-MRSA levels in healthy carrier pigs. Phages were detected in the samples the day following application. No side effects were observed in the pigs, and no bacteriophage antibodies were detected in the pig sera. In conclusion, prevalence of MRSA and ESBL-PE in veterinarians was low despite frequent animal contact. However, LA-MRSA was detected for the first time in a veterinarian in Finland, which, regarding the high prevalence in Finnish pigs, underlines the importance of protecting people working in close contact with pigs also in Finland. Ambulatory livestock and equine veterinarians’ adherence to IPC guidelines varied significantly between practice types. This may be partly explained by the poor availability of proper handwashing facilities in stables compared with farms as reported by the veterinarians. In addition, further education of both veterinarians and farm or stable owners is necessary. Low levels of LA-MRSA were observed in pigs on two farms and in the controlled facility. The declining trend in nasal samples in the clean environment of the controlled facility may indicate that air contamination could have been an important factor upholding the nasal carriage of LA-MRSA in pigs. The close relatedness of LA-MRSA t034 strains at the national level raises the question of which factors facilitate the spread of this successful clone. Possibly, due to the low levels of LA-MRSA in the pigs in the bacteriophage treatment trial, eradication of LA-MRSA using bacteriophages did not succeed. Further studies on the LA-MRSA t034 clone circulating in Finnish pigs as well as factors related to pig management that may explain the lower LA-MRSA prevalence in alternative farming systems may help to find ways to curb the spread of LA-MRSA in Finnish pigs.Useille antibiooteille vastustuskykyiset metisilliiniresistentti Staphylococcus aureus (MRSA) ja laajakirjoisia betalaktamaaseja (ESBL) tuottavat enterobakteerit ovat yleistyneet eläimillä ja ihmisillä. Viime vuosina niiden zoonoottinen leviäminen eläimistä ihmisiin on herättänyt kasvavaa huolta. Tuotantoeläimiin liitetty MRSA (LA-MRSA), jota Suomessa on todettu erityisesti sioilla, on uhka tuotantoeläinten kanssa työskenteleville ja voi heidän kauttaan levitä myös terveydenhuoltoon. Keski-Euroopassa sika- ja broileritiloilla työskentelevillä on todettu muuta väestöä korkeampi ESBL-bakteerien esiintyvyys. Väitöskirjan tavoitteena oli tutkia keinoja erityisesti LA-MRSA:n zoonoottisen leviämisen ehkäisemiseksi. Ensimmäisessä osatutkimuksessa selvitettiin MRSA- ja ESBL-bakteerien esiintyvyyttä eläinlääkäreillä. Esiintyvyys eläinlääkäreillä oli lukuisista eläinkontakteista huolimatta matala eikä poikennut muusta väestöstä. LA-MRSA:ta todettiin kuitenkin eläinlääkärillä Suomessa ensimmäistä kertaa. Tutkimuksen perusteella on tärkeää suojella sikojen parissa työskenteleviä tartunnalta. Talleilla ja maatiloilla eläimiä hoitavien eläinlääkärien hygieniakäytäntöjä selvittäneessä kyselytutkimuksessa todettiin, ettei erityisesti talleilla ollut riittäviä käsienpesumahdollisuuksia. Eläinlääkärit myös noudattivat hygieniasuosituksia talleilla huonommin kuin tiloilla. Havainnon perusteella eläinten omistajien ja eläinlääkärien infektiontorjunnan opastus on ensiarvoisen tärkeää zoonoottisten taudinaiheuttajien leviämisen torjunnassa. LA-MRSA-kantajuutta kartoittaneessa tutkimuksessa bakteeripitoisuudet sekä sikojen sieraimissa että iholla olivat hyvin matalia. Sioista eristettyjä kantoja verrattiin geneettisesti toisiinsa sekä Ruokaviraston sioilta seurantatutkimuksissa ja infektionäytteistä eristämiin kantoihin vuosilta 2008–2017. Tulosten perusteella suomalaisissa sioissa on kiertänyt useiden vuosien ajan sama LA-MRSA-klooni, jonka menestyksen syytä olisi tärkeä selvittää tarkemmin. LA-MRSA:n esiintyvyyttä yritettiin kokeellisesti vähentää sioissa bakteriofagien eli faagien avulla. Faagit ovat luonnossa esiintyviä bakteereja infektoivia viruksia, jotka lisääntymiskiertonsa päätteeksi tappavat bakteerin. Kokeessa faagit eivät vähentäneet LA-MRSA:n pitoisuuksia tai esiintyvyyttä tutkituissa sioissa, mikä voi selittyä oireettoman kantajuuden alhaisilla bakteeripitoisuuksilla. Tutkimuksessa saatiin kuitenkin tärkeää tietoa myöhempiä faagitutkimuksia varten. Väitöskirja tuo uutta tietoa LA-MRSA:sta ja sen torjunnasta Suomessa. Eläinten kanssa työskentelevien valistus ja LA-MRSA-kantojen tarkempi tuntemus ovat tässä avainasemassa

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