Metisilliiniresistentti staphylococcus pseudintermedius (MRSP) ja Yliopistollisen eläinsairaalan MRSP-epidemia 2010 - 2011

Metisilliiniresistentin Staphylococcus pseudintermedius -bakteerin (MRSP) leviäminen on 2000-luvulla yleistynyt ongelma pieneläinlääketieteessä. Se aiheuttaa ongelmia hankalahoitoisten infektioiden ja lisääntyneiden hoitokustannusten kautta. Bakteeri kykenee lisäksi aiheuttamaan sairaalaepidemioita. Sellainen koettiin Yliopistollisen eläinsairaalan Pieneläinsairaalassa vuosina 2010 – 2011. Tämän lisensiaatin tutkielma on kaksiosainen. Kirjallisuusosiossa annetaan tietoa MRSP:stä, sen kyvystä tulla resistentiksi tavallisimmille mikrobilääkkeille, sekä MRSP-infektion hoidosta. Tutkimusosiossa kuvaillaan Pieneläinsairaalan MRSP-epidemian vaiheet ja vastustustoimenpiteet, sekä selvitetään tartunnalle altistaneita tekijöitä tapaus-verrokkitutkimuksella. Marraskuussa 2010 Pieneläinsairaalassa tehtiin ensimmäinen moniresistentti MRSP-löydös. Tavattu kanta oli vastustuskykyinen erytromysiinille, klindamysiinille, sulfa-trimetopriimille, enrofloksasiinille, gentamisiinille, sekä kloramfenikolille. Kanta oli herkkä vain fusidiinihapolle ja amikasiinille. Seurantajakson (1.11.2010 – 31.10.2011) aikana epidemiakanta eristettiin kaikkiaan 55 potilaalta, joilta 24:llä MRSP löydettiin infektiopesäkkeestä. 15 tapauksessa kyseessä oli leikkaushaavainfektio. Loput löydökset (n=31) tehtiin seulontanäytteistä. Pieneläinsairaalan tapauskertymä osastopotilailla oli 2,2 % (55/2483). Riskitekijöiden selvittämistä varten kerättiin tiedot MRSP-tapauspotilaista, sekä 213 kontrollipotilaasta, joilta oli otettu MRSP-negatiivinen seulontanäyte seurantajakson aikana. Tutkitut muuttujat liittyivät potilaan seurantajakson aikana saamaan lääkehoitoon, potilaalle tehtyihin toimenpiteisiin, sekä potilaan käynteihin eri osastoilla. Tilastollisesti merkittäviä, MRSP-kantajuudelle tai infektiolle altistavia tekijöitä, olivat ihorikko (OR 12,9; p: <0,0001), kirurginen toimenpide (OR 9,8, p: <0,0001), mikrobilääketerapia (OR 11,1; p: <0,0001), sekä teho-osastohoito (OR 2,0; p: 0,036). Se oliko kirurginen toimenpide ortopedinen vai ei (OR 1,8; p: 0,13), tai oliko se tehty päivystyskirurgiana (OR 1,0; p: 1,0) ei ollut tilastollisesti merkittävä tekijä. Mahahapposalpaajahoito ei altistanut tartunnalle (OR 1,6; p: 0,15). Tulokset ovat alustavia ja niistä suoritetaan vielä lisätutkimuksia sekoittavien tekijöiden hallitsemiseksi. Pieneläinsairaalassa tehtiin epidemia-aikana mittavia vastustustoimenpiteitä. Riskipotilaita seulottiin aktiivisella näytteenotolla ja Pieneläinsairaalaan perustettiin kohorttiosasto tunnettujen kantajapotilaiden hoitoa varten. Sairaala jaettiin lisäksi eri alueisiin joihin eri riskiluokan potilaat ohjattiin. Sairaalassa otettiin myös lukuisia ympäristönäytteitä. Vastustustoimilla uusien tautitapausten esiintyminen väheni. Helmikuussa 2012 tilanne näyttää rauhoittuneen. Saadut tulokset tukevat ja täydentävät aiempia tutkimuksia, ja antavat lisätietoa riskipotilaiden tunnistamisen helpottamiseksi. Vaikka lisää tutkimuksia riskitekijöistä vielä tarvitaan, niiden tunteminen mahdollistaa torjuntakeinojen oikean kohdentamisen ja epidemiapurkausten ehkäisemisen

Epidemiology of methicillin-resistant Staphylococcus pseudintermedius in Finland

Staphylococcus pseudintermedius (SP) is part of the normal microbiota of dogs and cats. Since the mid-1980s, an ever-increasing number of methicillin-resistant SP (MRSP) isolates have been reported. In the mid-2000s, two predominant MRSP clones, ST71 (sequence type 71) and ST68, spread through Europe and North America, respectively. MRSP isolates are commonly multidrug resistant (MDR), and are thus capable of causing infections that do not respond to routinely used antimicrobials. MRSP appeared in the small animal population of Finland in the late 2000s, also causing numerous infections at the Veterinary Teaching Hospital (VTH) of the University of Helsinki. No data on the epidemiology of MRSP in Finland have been published. This thesis study aimed to explore the epidemiology of MRSP in the Finnish small animal population. This was done by investigating and describing the MRSP outbreak at the VTH, and investigating risk factors for patients being colonized or infected by MRSP in the hospital during the outbreak. The prevalence of MRSP and the risk factors for MRSP carriage were investigated in a canine subpopulation at the Guide Dog School for the Visually Impaired. The susceptibility of SP isolates to antimicrobials in 2011–2015 was also investigated using data from the Clinical Microbiology Laboratory (CML) of the Faculty of Veterinary Medicine, University of Helsinki. Risk factors for an SP isolate being MRSP, as well as for a screening specimen revealing MRPS, were also investigated. Furthermore, the molecular epidemiology of all MRSP isolates stored in 2010–2014 was investigated using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. Antimicrobial therapy, whether previous or ongoing during sampling, was a risk factor for MRSP in all studies. Furthermore, a prolonged hospital stay and veterinary visits were risk factors among guide dogs. An SP isolate originating from a private clinic (versus the VTH) was a significant risk factor for MRSP among clinical specimens. The same could be seen among screening specimens from patients with risk factors for MRSP. In addition, it was noted that a sizeable proportion (~20–60%) of animals in the studies had been or were being treated with antimicrobials. During the outbreak at the VTH, rigorous hygiene and barrier measures were necessary to achieve control. ST71, the MRSP clone that caused the outbreak, was the predominant clone in 2010–2011, accounting for over 50% of MRSP isolates, even among non-outbreak-related isolates. By 2014 the situation had changed, as ST71 represented only ~10% of MRSP isolates. MRSP clones belonging to CC45 (clonal complex 45) and CC258, and other unrelated STs, dominated the MRSP population by that time. SCCmec type IV was detected in a majority of different STs, indicating the horizontal spread of resistance genes. The prevalence of MRSP was only 3% among guide dogs. The proportion of clinical specimens from small animals that revealed MRSP was similar, 2.5%. However, 9% of screening specimens from high-risk patients revealed MRSP. Overall, 14% of SP isolates were MRSP. Roughly 30–40% of isolates were not susceptible to alternative antimicrobials, such as lincosamides, macrolides, or tetracyclines. MRSP in feline specimens was rare (<1% in all specimens after 2011). Our results give further credence to the hypothesis that antimicrobial therapy and contact with the veterinary environment are risk factors for MRSP in small animals. However, cats do not appear to be a significant source of MRSP. Our data suggest that the epidemiology of MRSP has changed from a predominantly clonal spread to a mix of clonal spread and the spread of genetic elements. The resistance rates among SP are at an alarming level. Decisive action, including the use of non-antimicrobial treatments whenever feasible and more prudent use of antimicrobials, is required to improve the situation.Staphylococcus pseudintermedius -bakteeri on osa koirien ja kissojen normaalimikrobistoa ja yleinen ihotulehduksen aiheuttaja koirilla. Metisilliinille resistenttejä eli vastustuskykyisiä S. pseudintermedius (MRSP) -kantoja alkoi esiintyä jo 1980-luvulla, mutta niiden määrä on lisääntynyt voimakkaasti vuodesta 2005 alkaen. Vastustuskyky metisilliinille aikaansaa vastustuskyvyn kaikille beetalaktaamiantibiooteille (ml. penisilliinit ja kefalosporiinit), joita käytetään yleisesti pieneläinten stafylokokkitulehdusten hoitoon. Nämä bakteerit ovat lisäksi usein moniresistenttejä, eli vastustuskykyisiä myös muille antibioottiryhmille, mikä vaikeuttaa tulehdusten hoitoa entisestään. Vuonna 2010 MRSP-bakteeri aiheutti laajan epidemian Yliopistollisessa eläinsairaalassa (YES). MRSP-bakteerin epidemiologiaa Suomessa ei ole aiemmin tutkittu. Tämän väitöstutkimuksen tavoite oli selvittää MRSP-bakteerin epidemiologiaa Suomessa tutkimalla YES:n epidemiaa, tartunnalle altistavia tekijöitä, bakteerin esiintyvyyttä Suomen Opaskoirakoulun koirilla, sekä bakteerikantojen sukulaisuutta. Lisäksi tarkasteltiin bakteerin herkkyyttä eri antibiooteille. Tutkimuksessa hyödynnettiin Eläinlääketieteellisen tiedekunnan kliinisen mikrobiologian laboratorion aineistoa. YES:n MRSP-epidemian aikana yli 60 potilasta sai tartunnan ja tilanne saatiin hallintaan vasta tiukkojen hygieniatoimien avulla. Riski saada MRSP -tartunta oli suurempi niillä potilailla, joilla oli ihorikko (esim. leikkaushaava), jotka olivat saaneet antibioottihoitoa, tai jotka olivat olleet hoidossa teho-osastolla. Opaskoirilla riski olla MRSP kantaja oli suurempi, jos koiraa oli viimeisimmän vuoden aikana hoidettu antibiooteilla tai koira oli käynyt eläinklinikalla. Myös bakteeriviljelynäytteestä löydetty S. pseudintermedius oli todennäköisemmin MRSP, mikäli potilas oli antibioottilääkityksellä näytteenoton aikana. MRSP -bakteeria esiintyi vain kolmella prosentilla opaskoirista. Saman verran MRSP:tä todettiin myös muista laboratorioon saapuneista bakteeriviljelynäytteistä. Bakteerin varalta seulotuilla potilailla, joilla oli tartunnalle altistava tekijä, kuten runsas antibioottikulutus, esiintyvyys oli korkeampi – yhdeksän prosenttia. Kissojen näytteissä MRSP -esiintyvyys oli kuitenkin hyvin matala (<1 %). YES:n taudinpurkauksen aiheuttanut bakteeriklooni oli Suomen yleisin MRSP -klooni vielä vuosina 2010-2011. Vuonna 2014 muut bakteerikloonit olivat kuitenkin syrjäyttäneet sen. Tämä viittaa bakteerin leviämistavan muuttumiseen – bakteerisukupolvelta toiselle siirtyvän resistenssin ohella resistenssi siirtyy nykyään myös bakteerikantojen välillä. Kaikista bakteeriviljelynäytteistä eristetyistä S. pseudintermedius -bakteereista 14 % oli MRSP -kantoja. Lisäksi 30-40 %:iin kannoista eivät tehonneet useat vaihtoehtoiset antibiootit, kuten makrolidit, linkosamidit ja tetrasykliinit. Tutkimuksen tulokset osoittavat, että antibioottien käyttö ja eläinklinikoilla tai -sairaaloissa käynti lisää lemmikin riskiä olla MRSP-kantaja. Kissat toimivat bakteerin kantajina kuitenkin vain harvoin. Tulokset viittaavat myös siihen, että metisilliiniresistenssin leviäminen S. pseudintermedius -bakteerin joukossa on muuttunut monimuotoisemmaksi, mikä vaikeuttaa bakteerin leviämisen vastustusta. Lisäksi tutkimuksessa kävi ilmi, että S. pseudintermedius -bakteerin vastustuskyky eri antibiooteille on huolestuttavalla tasolla. Ongelman ratkaiseminen vaatii määrätietoisia päätöksiä, kuten paikallishoidon suosimista ja antibioottien käytön rajoittamista

Ex-Vivo Adhesion of Enterococcus faecalis and Enterococcus faecium to the Intestinal Mucosa of Healthy Beagles

Some Enterococcus faecalis and E. faecium strains are used as probiotics or feed additives. Adherence to the intestinal mucosa is considered a crucial step for intestinal bacteria to colonize and further interact with the host epithelium and the immune system. In dogs, there are no studies investigating the adhesion of E. faecalis and E. faecium to paraffin-embedded intestinal mucosa. Therefore, we aimed to investigate the adhesion of E. faecalis and E. faecium to the intestinal mucosa of six healthy beagles using bacteria derived from dogs and chickens. In addition, we aimed to validate a method to test the adhesion of Alexa Fluor-labeled bacteria to paraffin-embedded canine intestinal mucosa. The results of our study show that both canine- and chicken-derived E. faecalis strains adhered significantly better than E. faecium to the duodenal mucosa of healthy beagles (p = 0.002). In addition, canine E. faecalis and E. faecium adhered in higher numbers to canine duodenal mucosa, compared to chicken-derived strains of the same species (p = 0.015 for E. faecalis and p = 0.002 for E. faecium). The determination of the hydrophobicity of bacteria revealed that canine E. faecalis had the highest hydrophobicity level (36.6%), followed by chicken E. faecalis (20.4%), while canine E. faecium (5.7%) and chicken E. faecium (4.5%) had the lowest levels. Our results suggest that both the bacterial species and the host origin of the strain may influence mucosal adhesion

Ex-Vivo Adhesion of Enterococcus faecalis and Enterococcus faecium to the Intestinal Mucosa of Healthy Beagles

Some Enterococcus faecalis and E. faecium strains are used as probiotics or feed additives. Adherence to the intestinal mucosa is considered a crucial step for intestinal bacteria to colonize and further interact with the host epithelium and the immune system. In dogs, there are no studies investigating the adhesion of E. faecalis and E. faecium to paraffin-embedded intestinal mucosa. Therefore, we aimed to investigate the adhesion of E. faecalis and E. faecium to the intestinal mucosa of six healthy beagles using bacteria derived from dogs and chickens. In addition, we aimed to validate a method to test the adhesion of Alexa Fluor-labeled bacteria to paraffin-embedded canine intestinal mucosa. The results of our study show that both canine- and chicken-derived E. faecalis strains adhered significantly better than E. faecium to the duodenal mucosa of healthy beagles (p = 0.002). In addition, canine E. faecalis and E. faecium adhered in higher numbers to canine duodenal mucosa, compared to chicken-derived strains of the same species (p = 0.015 for E. faecalis and p = 0.002 for E. faecium). The determination of the hydrophobicity of bacteria revealed that canine E. faecalis had the highest hydrophobicity level (36.6%), followed by chicken E. faecalis (20.4%), while canine E. faecium (5.7%) and chicken E. faecium (4.5%) had the lowest levels. Our results suggest that both the bacterial species and the host origin of the strain may influence mucosal adhesion

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

Sharing more than friendship – transmission of NDM-5 ST167 and CTX-M-9 ST69 Escherichia coli between dogs and humans in a family, Finland, 2015

Peer reviewe

Streptococcus halichoeri : Comparative Genomics of an Emerging Pathogen

Streptococcus halichoeri is an emerging pathogen with a variety of host species and zoonotic potential. It has been isolated from grey seals and other marine mammals as well as from human infections. Beginning in 2010, two concurrent epidemics were identified in Finland, in fur animals and domestic dogs, respectively. The fur animals suffered from a new disease fur animal epidemic necrotic pyoderma (FENP) and the dogs presented with ear infections with poor treatment response. S. halichoeri was isolated in both studies, albeit among other pathogens, indicating a possible role in the disease etiologies. The aim was to find a possible common origin of the fur animal and dog isolates and study the virulence factors to assess pathogenic potential. Isolates from seal, human, dogs, and fur animals were obtained for comparison. The whole genomes were sequenced from 20 different strains using the Illumina MiSeq platform and annotated using an automatic annotation pipeline RAST. The core and pangenomes were formed by comparing the genomes against each other in an all-against-all comparison. A phylogenetic tree was constructed using the genes of the core genome. Virulence factors were assessed using the Virulence Factor Database (VFDB) concentrating on the previously confirmed streptococcal factors. A core genome was formed which encompassed approximately half of the genes in Streptococcus halichoeri. The resulting core was nearly saturated and would not change significantly by adding more genomes. The remaining genes formed the pangenome which was highly variable and would still evolve after additional genomes. The results highlight the great adaptability of this bacterium possibly explaining the ease at which it switches hosts and environments. Virulence factors were also analyzed and were found primarily in the core genome. They represented many classes and functions, but the largest single category was adhesins which again supports the marine origin of this species.Peer reviewe

Comparison of Streptococcus halichoeri isolates from canine and fur animal infections : biochemical patterns, molecular characteristics and genetic relatedness

Background Streptococcus halichoeri infections have been reported in grey seals, a European badger, a Stellar sea lion and humans, but its presence in companion and fur animals is unknown. Since 2010, S. halichoeri-like bacteria (SHL) have been isolated from fur animals and dogs in Finland. Our aim was to retrospectively investigate laboratory records for SHL from canine and fur animal infections, characterize the isolates and compare their genetic relatedness in relation to three reference strains: CCUG 48324(T), originating from a grey seal, and strains 67100 and 61265, originally isolated from humans. Results A total of 138 and 36 SHLs from canine and fur animal infections, respectively, were identified in the laboratory records. SHL was commonly associated with skin infections, but rarely as the only species. A set of 49 canine and 23 fur animal SHLs were further characterized. MALDI-TOF confirmed them as being S. halichoeri. The growth characteristics were consistent with the original findings, but isolates were catalase positive. In total, 17 distinct API 20 Strep patterns were recorded among all 75 isolates tested, of which pattern 5563100 was the most common (n = 30). Antimicrobial resistance to erythromycin and clindamycin was common in canine isolates, but rare in fur animal isolates. Three clusters were observed by PFGE, and 16S rRNA sequencing revealed 98.1-100% similarities with the human strains and 98.1-99.5% with the seal strain. A phylogenetic tree of concatenated 16S rRNA and rpoB revealed closely related isolates with two clades. Fifteen canine isolates were identical to the human strains based on concatenated 16S rRNA and rpoB sequencing. Conclusions Streptococcus halichoeri appears to be quite a common bacterial species in the skin of dogs and fur animals. The clinical significance of S. halichoeri is uncertain, as it was rarely isolated as a monoculture. No apparent temporal or spatial clustering was detected, but isolates from different sources were genetically very similar. Because many canine isolates were genetically similar to the human reference strains, transmission between dogs and humans may be possible. WGS sequencing of strains from different sources is needed to further investigate the epidemiology and virulence of S. halichoeri.Peer reviewe

Epidemiology of methicillin resistant Staphylococcus pseudintermedius in guide dogs in Finland

Background: Methicillin resistant Staphylococcus pseudintermedius (MRSP) and Staphylococcus aureus (MRSA) are common multi-drug resistant (MDR) bacteria in dogs. In 2012-2013 three dogs of the Guide Dog School of the Finnish Federation of the Visually Impaired were found to be MRSP positive. Guide dogs have regular contact with each other during their first year of life and prolonged contact when in training. Since dogs are placed in different parts of Finland after training, there is a risk for national spread of MDR bacteria. In this study the prevalence of MRSP and MRSA, as well as the risk factors for MRSP were determined in the Finnish guide dog population. MRSP isolates were investigated using molecular methods and compared to the earlier isolates. Results: Out of 132 tested dogs 4 were MRSP positive thus giving the prevalence estimate of 3% (95% CI: 1-8%) for MRSP in the target population. MRSA was not detected (prevalence estimate 0%, 95% CI: 0-3%). Risk factors associated with MRSP were being a breeding bitch (OR = 8.4; 95% CI: 1.1-64.1, P = 0.012), the number of veterinary visits (OR = 1.23; 95% CI: 1.0-1.5, P = 0.025) and number of antimicrobial courses (OR = 1.63; 95% CI: 1.0-2.55; P = 0.035). Identified MRSP isolates belonged to five different sequence types (ST45, 71, 402, 403 and 404). All ST71 isolates carried SCCmec II-III, while the SCCmec type of the ST45 and ST402 (a single locus variant of ST45) isolates were non-typeable with the method used. Conclusions: MRSP and MRSA had low prevalence in the studied dog population despite the close contact between dogs, and the MRSP population was heterogenic. Antimicrobial therapy and veterinary visits are risk factors for MRSP even among a small case group.Peer reviewe

The Composition of the Microbiota in the Full-Term Fetal Gut and Amniotic Fluid : A Bovine Cesarean Section Study

The development of a healthy intestinal immune system requires early microbial exposure. However, it remains unclear whether microbial exposure already begins at the prenatal stage. Analysis of such low microbial biomass environments are challenging due to contamination issues. The aims of the current study were to assess the bacterial load and characterize the bacterial composition of the amniotic fluid and meconium of full-term calves, leading to a better knowledge of prenatal bacterial seeding of the fetal intestine. Amniotic fluid and rectal meconium samples were collected during and immediately after elective cesarean section, performed in 25 Belgian Blue cow-calf couples. The samples were analyzed by qPCR, bacterial culture using GAM agar and 16S rRNA gene amplicon sequencing. To minimize the effects of contaminants, we included multiple technical controls and stringently filtered the 16S rRNA gene sequencing data to exclude putative contaminant sequences. The meconium samples contained a significantly higher amount of bacterial DNA than the negative controls and 5 of 24 samples contained culturable bacteria. In the amniotic fluid, the amount of bacterial DNA was not significantly different from the negative controls and all samples were culture negative. Bacterial sequences were identified in both sample types and were primarily of phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, with some individual variation. We conclude that most calves encounter in utero maternal-fetal transmission of bacterial DNA, but the amount of bacterial DNA is low and viable bacteria are rare.Peer reviewe