Nasal carriage of methicillin-resistant coagulase-positive staphylococci among cats and dogs hospitalized in the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine – Technical University of Lisbon, Portugal

Dissertação de Mestrado Integrado em Medicina VeterináriaMethicillin-resistant coagulase-positive staphylococci (MRCPS) colonization in companion animals is an emerging and significant problem in public and animal health. During one year, nasal swabs were obtained from 40 cats and 146 dogs admitted to the Teaching Hospital of the Faculty of Veterinary Medicine - Technical University of Lisbon. MRCPS colonization was screened by plating enrichment cultures on a selective medium, Chrom MRSA ID. Bacterial species and mecA were confirmed by PCR. Clonality of the isolates was assessed by pulsed-field gel electrophoresis (PFGE). All isolates were subjected to spa and SCCmec typing. They were also tested by PCR for the lukF/lukS genes encoding Panton-Valentine leukocidin (PVL) in Staphylococcus aureus and Luk-I and Staphylococcus intermedius exfoliative toxin (SIET) in Staphylococcus pseudintermedius. Methicillin-resistant S. aureus (MRSA) was found in two cats (5 %) and one dog (0.6 %). Isolates were spa type t032, SCCmec IV and shared identical PFGE profiles. These were similar to the EMRSA-15 human clone. Strains were PVL-negative. Nine dogs carried methicillin-resistant S. pseudintermedius (MRSP) (6 %), whereas none of the cats was positive. The PFGE type A strain (n=1) showed identical characteristics as the American MRSP clone strains (CC68-MRSP-V), while PFGE cluster B grouped European MRSP isolates (CC71-MRSP-III) (n=8). All isolates were SIETnegative. The 8 European MRSP isolates were positive for the lukF/lukS genes and the American MRSP isolate was negative for both genes. Strains were multidrug-resistant, which represents a major challenge for veterinarians in terms of antibiotic therapy.RESUMO - Frequência de colonização por staphylococci coagulase-positivo meticilinaresistente em cães e gatos internados no hospital escolar da Faculdade de Medicina Veterinária – Universidade Técnica de Lisboa, Portugal - A colonização por staphylococci coagulase-positivo meticilina-resistente (MRCPS) é um problema emergente e de grande importância em termos de saúde animal e pública. Durante um ano, zaragatoas nasais de 146 cães e 40 de gatos foram obtidas de animais internados no Hospital Escolar da Faculdade de Medicina Veterinária - Universidade Técnica de Lisboa. A colonização por MRCPSfoi pesquisada por inoculação de culturas de enriquecimento num meio selectivo, Chrom MRSA ID. As espécies de MRCPS e a amplificação do gene mecA por feita por PCR. A clonalidade dos isolados foi confirmada por PFGE. Todos os isolados foram sujeitos a tipagem spa e SCCmec. Os isolados de S. aureus meticilina-resistente (MRSA) e de S. pseudintermedius meticilina-resistente (MRSP) foram testados por PCR para a presença dos genes lukF/lukS que codificam, respectivamente, a leucocidina Panton-Valentine (PVL) e a leucocidina-I (Luk-I). Os isolados de MRSP foram ainda testados para a presença da toxina exfoliativa do S. intermedius (SIET). Nesta amostra, 0,6 % (n=1) dos cães testados e 5 % (n=2) dos gatos apresentaram MRSA. Os isolados de MRSA eram spa tipo t032, SCCmec IV e partilhavam padrões idênticos de PFGE. As estirpes eram idênticas ao clone humano EMRSA-15. Os 3 isolados eram PVL negativos. Nove cães apresentaram MRSP (6 %), enquanto nenhum dos gatos foi positivo. PFGE tipo A mostrou características idênticas ao clone americano de MRSP (CC68-MRSP-V) e PFGE tipo B agrupou os isolados europeus de MRSP (CC71-MRSP-III) (n=8). Os isolados de MRSP PFGE tipo B eram Luk-I positivos mas SIET negativos. Todas as estirpes de MRSP eram multirresistentes a várias classes de antibióticos, o que representa um desafio para os médicos veterinários em termos de estratégias de antibioterapia

Insights into the dynamics of methicillin-resistant staphylococci in animals : a focus on Staphylococcus pseudintermedius in dogs

Tese especialmente elaborada para a obtenção do grau de Doutor em Ciências Veterinárias, especialidade de ClínicaStaphylococci are a group of bacteria with clinical, agricultural, and economic importance because of their wide range of virulence factors and ability to become resistant to antimicrobials. This thesis has pursued three main objectives: I. Determine the frequency of methicillin-resistant S. aureus (MRSA) strains in several animal species, identify the characteristics of strains present in animals and comparison with human strains MRSA nasal screening was performed in 71 horses and 307 calves, and the observed frequencies were 3% and 2%, respectively. Seventy-four MRSA isolated from 2001 to 2014 were characterized: fourteen spa types, three SCCmec types and three clonal complexes (CC) 5, CC22 and CC398, were found. Most isolates were multidrug-resistant. Fourteen MRSA CC398 strains had qac genes (13 qacG and 1 qacJ), while 4 isolates (three CC5 and one CC22) had insertions in the norA promoter gene. MRSA linages from pets (CC5 and CC22) harboured specific sets of virulence genes and a lower number of resistance genes than CC398 from livestock-animals. II. Reveal antimicrobial/biocide susceptibility patterns/trends and resistance genes in methicillin-resistant staphylococci (MRS) Several antimicrobial resistance patterns and genes were found in MRS from horses. Minimum bactericidal concentrations of biocides chlorhexidine acetate, benzalkonium chloride, triclosan and glutaraldehyde were lower than the recommended in-use concentrations for veterinary medicine, although two MRS carried plasmid-borne qacA and sh-fabI or qacB and qacH-like genes. An investigation on the evolution of resistance to 38 antimicrobials, corresponding mechanisms and molecular characteristics of 644 clinical Staphylococcus spp. isolates obtained from companion animals between 1999-2014 revealed resistance to the majority of antimicrobials and the number of mecA-positive strains increased significantly over time. Considering S. pseudintermedius, the methicillin-susceptible (MSSP) were genetically more diverse than methicillin-resistant (MRSP). All MRSP and two MSSP strains were multidrug- resistant, with several antimicrobial resistance genes identified. One MSSP isolate harbored a qacA and another a qacB gene. Three biocide products had high bactericidal activity (Otodine®, Clorexyderm Spot Gel®, Dermocanis Piocure-M®), while Skingel® failed to achieve a five log reduction in the bacterial counting. III. Study of the pathogenesis of S. pseudintermedius in dogs The agr type III predominated in MRSP. Five virulence genes were found in all strains and only spsO gene was significantly associated with MSSP. MSSP produced more biofilm on BHIB and BHIB+1% glucose than MRSP isolates. Several virulence genes encoding surface proteins and toxins were highly expressed in the MRSP strain (compared to MSSP). By whole proteome characterization of S. pseudintermedius through 2DE MALDI-TOF/TOF MS approach we were able to identify 367 unique proteins, of which 39 were surface proteins. By subsequent use of the serological proteome analysis (SERPA) approach we identified 4 antigenic proteins with promising features for vaccine development. These results indicate that MRS were widely disseminated in the studied animal population, the environment and people in contact with these animals. The resistant trends and mechanisms detected in MRS strains are worrying and make animals a reservoir of important MRS clones and genes. Biocides are still a good therapeutic choice, even in the presence of efflux genes. Higher expression of virulence genes may play a role in the rapid and widespread of MRSP clones. Dogs are able to mount an IgG-response against S. pseudintermedius and the proteins identified by the immune system can in the future be used as vaccine candidates.RESUMO - Estudo da dinâmica de estafilococos meticilina-resistente em animais – um foco no Staphylococcus pseudintermedius em cães - Os estafilococos são um grupo de bactérias com importância clínica, agrícola e económica devido à ampla gama de fatores de virulência e pela sua capacidade de se tornarem resistentes aos antimicrobianos. Esta tese debruçou-se sobre três objetivos principais: I. Determinar a frequência de estirpes S. aureus meticilina-resistente (MRSA) em diversas espécies animais, identificar as características das estirpes presentes em animais e comparar com estirpes humanas Colhemos zaragatoas de 71 cavalos e 307 vitelos para pesquisa de MRSA, e observaramse frequências de 3% e 2%, respetivamente. Foram caracterizadas setenta e quatro estirpes MRSA isoladas entre 2001-2014: catorze tipos de spa, três tipos de SCCmec e três complexos clonais (CC) 5, CC22 e CC398, foram encontrados. A maioria das estirpes (74%) eram multirresistentes. Catorze estirpes de MRSA CC398 tinha genes qac (13 qacG e 1 qacJ), enquanto 4 (três CC5 e um CC22) tinham inserções no gene promotor norA. As linhagens de MRSA de animais de estimação (CC5 e CC22) tinham conjuntos específicos de genes de virulência e um menor número de genes de resistência do que as linhagens associadas aos animais de produção (CC398). II. Revelar padrões/ tendências de suscetibilidade antimicrobiana/biocida e genes de resistência em estafilococos meticilina-resistente (MRS) Foram encontrados vários padrões e genes de resistência antimicrobiana em MRS de cavalos. As concentrações bactericidas mínimas dos biocidas acetato de clorhexidina, cloreto de benzalcónio, triclosan e glutaraldeído foram menores do que as recomendadas em medicina veterinária, embora dois MRS tivessem os genes plasmídicos qacA e sh-fabI ou qacB e um qacH-semelhante. Uma investigação sobre a evolução da resistência a 38 antimicrobianos, mecanismos correspondentes e características moleculares de 644 Staphylococcus spp. clínicos obtidos de animais de companhia entre 1999-2014 revelou resistência à maioria dos antimicrobianos. O número de estirpes mecA-positivo aumentou significativamente ao longo do tempo. Quanto aos S. pseudintermedius, os meticilina-suscetível (MSSP) eram geneticamente mais diversos do que os meticilina-resistente (MRSP). Todos os MRSP e 2 MSSP eram multirresistentes, com vários genes de resistência identificados. Um MSSP tinha um gene qacA e outro um qacB. Três produtos biocidas tinham elevada atividade bactericida (Otodine®, Clorexyderm Spot Gel®, Dermocanis Piocure-M®), enquanto Skingel® não conseguiu atingir uma redução de 5 log na contagem bacteriana. III. Estudo da patogenicidade de S. pseudintermedius em cães O tipo III agr predominou nos MRSP. Cinco genes de virulência foram encontrados em todas as estirpes e só o gene spsO foi significativamente associado com MSSP. MSSP produziu mais biofilme em BHIB e BHIB + 1% glucose que as estirpes de MRSP. Vários genes de virulência que codificam proteínas e toxinas de superfície foram altamente expressos na estirpe MRSP (em comparação com MSSP). Através da caracterização do proteoma total de S. pseudintermedius pela abordagem 2DE MALDI-TOF/TOF MS fomos capazes de identificar 367 proteínas únicas, das quais 39 eram proteínas de superfície. Posteriormente utilizámos a análise do proteoma serológico (SERPA) que identificou quatro proteínas antigénicas com características promissoras para o desenvolvimento de vacinas. Estes resultados indicam que MRS estavam amplamente disseminados na população animal estudada, no ambiente e nas pessoas em contato com esses animais. As tendências de resistência e os mecanismos detetados em estirpes MRS são preocupantes tornando os animais um reservatório de clones MRS e genes. Os biocidas ainda são uma boa opção terapêutica, mesmo na presença de bombas de efluxo. Uma maior expressão de genes de virulência pode desempenhar um papel na rápida expansão de clones de MRSP. Os cães foram capazes de montar uma resposta IgG contra S. pseudintermedius e as proteínas identificadas pelo sistema imunológico podem, no futuro, ser utilizadas como candidatos vacinais

Future potential of metagenomics in clinical laboratories

INTRODUCTION: Rapid and sensitive diagnostic strategies are necessary for patient care and public health. Most of the current conventional microbiological assays detect only a restricted panel of pathogens at a time or require a microbe to be successfully cultured from a sample. Clinical metagenomics next-generation sequencing (mNGS) has the potential to unbiasedly detect all pathogens in a sample, increasing the sensitivity for detection and enabling the discovery of unknown infectious agents. AREAS COVERED: High expectations have been built around mNGS; however, this technique is far from widely available. This review highlights the advances and currently available options in terms of costs, turnaround time, sensitivity, specificity, validation, and reproducibility of mNGS as a diagnostic tool in clinical microbiology laboratories. EXPERT OPINION: The need for a novel diagnostic tool to increase the sensitivity of microbial diagnostics is clear. mNGS has the potential to revolutionise clinical microbiology. However, its role as a diagnostic tool has yet to be widely established, which is crucial for successfully implementing the technique. A clear definition of diagnostic algorithms that include mNGS is vital to show clinical utility. Similarly to real-time PCR, mNGS will one day become a vital tool in any testing algorithm

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics

The emergence of nosocomial infections by multidrug-resistantStaphylococcus haemolyticusisolates has been reported in several European countries. Here, we report the first two complete genome sequences ofS. haemolyticussequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics

The emergence of nosocomial infections by multidrug-resistantStaphylococcus haemolyticusisolates has been reported in several European countries. Here, we report the first two complete genome sequences ofS. haemolyticussequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics

Presječno istraživanje prevalencije koagulaza negativnih izolata safilokoka, otpornih na meticilin, izdvojenih iz pasa u Litvi.

The aim of this study was to investigate the presence and frequency of methicillin-resistant coagulase negative staphylococci in dogs in Lithuania and to characterize them regarding antimicrobial resistance. In 2012-2013 clinical material was collected from 400 dogs. Three hundred samples from diseased dogs with different clinical conditions (dermatitis, otitis, wound infections, gastrointestinal and respiratory tract infections) as well as 100 samples from pure-breed bitches with reproductive disorders (pyometritis, metritis, partus praematurus), used as breeding animals in kennels, were selected. Twenty MRCNS isolates were obtained from 18 dogs out of 400 tested. All isolates harboured the mecA gene while the mecC (mecLGA251) gene was not found. Ten isolates were detected in vaginal samples of the bitches within 3 large kennels. The prevalence of MRCNS in dogs kept in households was 3.3 % i.e. significantly lower (P<0.01) than in dogs kept in large kennels (10 %). Ten different MRCNS species were detected with the highest prevalence for Staphylococcus haemolyticus. MRCNS isolates were resistant to macrolides (75 %) due to erm(C) and msrA genes, and to tetracycline (65 %) due to tet(K) and/or tet(M) genes. The rate of resistance to gentamicin was 50 % (attributed to aac(6′)-Ieaph(2”)-Ia, aph(3′)-IIIa), and to co-trimoxazole - 40 % (dfrG gene). One isolate of S. lentus harboured the dfrK gene. All isolates were susceptible to linezolid, daptomycin and vancomycin. This study revealed that breeding kennels might be a reservoir of MRCNS strains and may pose a risk for the spread of such strains during mating. The focus on the possible spread of multi-resistant S. haemolyticus between companion-animals and humans should be foreseen, as this species plays an important role in human infections as well.Cilj ovog istraživanja bio je ustanoviti prisutnost i učestalost koagulaza negativnih stafilokoka otpornih na meticilin (MRKNS) izdvojenih iz pasa u Litvi te odrediti njihovu otpornost na antimikrobne tvari. Klinički materijal bio je prikupljen iz 400 pasa 2012. i 2013. godine. Tri stotine uzoraka bilo je uzeto iz bolesnih pasa s različitim kliničkim znakovima (dermatitis, otitis, infekcije rana, infekcije probavnog i dišnog sustava) te 100 uzoraka iz kuja čistih pasmina s reprodukcijskim poremećajima (pyometritis, metritis, partus praematurus) upotrebljavanih za rasplod u štenarama. Od 400 pretraženih, 20 koagulaza negativnih izolata stafilokoka otpornih na meticilin bilo je izdvojeno iz 18 pasa. Svi izolati imali su gen mecA, dok gen mecC (mecLGA251) nije bio dokazan. Deset izolata bilo je izdvojeno iz uzoraka rodnice kuja iz triju velikih uzgoja. Prevalencija MRKNS u pasa držanih u domaćinstvima iznosila je 3,3 %, tj. bila je značajno manja (P<0,01) nego u pasa držanih u velikim uzgajivačnicama (10 %). Dokazano je 10 različitih vrsta koagulaza negativnih stafilokoka otpornih na meticilin s najvećom prevalencijom za vrstu Staphylococcus haemolyticus. MRKNS izolati bili su otporni na makrolide (75 %) zbog erm(C) i msrA gena i tetraciklin (65 %) zbog posjedovanja tet(K) i/ili tet(M) gena. Stopa otpornosti na gentamicin bila je 50 % (što se pripisuje genima aac(6′)-Ie-aph(2”)-Ia, aph(3′)-IIIa) i na ko-trimoksal – 40 % (gen dfrG). Jedan izolat vrste S. lentus imao je gen dfrK. Svi izolati bili su osjetljivi na linezolid, daptomicin i vankomicin. Ovo istraživanje pokazuje da uzgojne štenare mogu biti rezervoar sojeva MRKNS i mogu predstavljati rizik za širenje takvih sojeva za vrijeme parenja. Treba se usredotočiti na mogući prijenos višestruko otpornih sojeva vrste S. haemolyticus s kućnih ljubimaca na čovjeka s obzirom na to da ta vrsta ima važnu ulogu kao uzročnik infekcija u ljudi

Host DNA depletion can increase the sensitivity of Mycobacterium spp. detection through shotgun metagenomics in sputum

Identification and phenotypic drug-susceptibility testing for mycobacteria are time-consuming and challenging but essential for managing mycobacterial infections. Next-generation sequencing (NGS) technologies can increase diagnostic speed and quality, but standardization is still lacking for many aspects (e.g., unbiased extraction, host depletion, bioinformatic analysis). Targeted PCR approaches directly on sample material are limited by the number of targets that can be included. Unbiased shotgun metagenomics on direct material is hampered by the massive amount of host DNA, which should be removed to improve the microbial detection sensitivity. For this reason, we developed a method for NGS-based diagnosis of mycobacteria directly from patient material. As a model, we used the non-tuberculous mycobacterium (NTM) Mycobacterium abscessus. We first compared the efficiency of three different DNA extraction kits for isolating DNA (quality and concentration). The two most efficient kits were then used in a follow-up study using artificial sputum. Finally, one extraction kit was selected and further evaluated for DNA isolation from a patients’ sputum mixture spiked with M. abscessus at three concentrations (final concentrations 108, 107, 106 CFU/ml). The spiked sputum samples were processed with and without saponin treatment (ST) in combination with DNAse treatment prior to bacterial DNA extraction to evaluate the recovery of bacteria and depletion of host DNA by PCR and Illumina sequencing. While Ct values of the qPCR targeting mycobacterial ITS DNA remained rather stable, Ct values in the qPCR targeting the human β-actin gene increased by five Ct values in ST samples. In subsequent Illumina sequencing, a decrease of 89% of reads mapped to the human genome was observed in ST samples. The percentage of reads mapped to M. abscessus (108 CFU/ml) increased by 89%, and the sequencing depth increased two times when undergoing ST. In conclusion, the sensitivity of M. abscessus detection in artificial sputum was increased using a saponin pre-treatment step. The saponin followed by the DNase I treatment approach could be efficiently applied to detect and characterize mycobacterial infections, including tuberculosis, directly from sputum

Clonal diversity, virulence patterns and antimicrobial and biocide susceptibility among human, animal and environmental MRSA in Portugal

Objectives: The objective of this study was to identify the Staphylococcus aureus clonal types currently circulating in animals, humans in contact with animals and the environment in Portugal based on genetic relatedness, virulence potential and antimicrobial/biocide susceptibility. Methods: Seventy-four S. aureus isolates from pets, livestock, the environment and humans in contact with animalswere characterized by SCCmec typing, spa typing, PFGE and CC398-specific PCR, by antimicrobial and biocide susceptibility testing and by detection of resistance genes and genes for efflux pumps. Representative strains were analysed by DNA microarray and MLST. Results: The S. aureus isolates represented 13 spa types and 3 SCCmec types and belonged to three clonal complexes (CC5, CC22 and CC398). Most of the isolates were multiresistant and harboured the resistance genes that explained the resistance phenotype. The qacG and qacJ genes for biocide resistance were detected in 14 isolates (all MRSA CC398), while 4 isolates (3 CC5 and 1 CC22) had insertions in the 210 motif of the norA promoter. Isolates of the clonal lineages associated with pets (CC5 and CC22) harboured specific sets of virulence genes and often a lower number of resistance genes than isolates of the clonal lineage associated with livestock animals (CC398). Conclusions:We found, for the first time in animals in Portugal, four strains belonging to CC5, including ST105-II, a lineage that has been previously reported as vancomycin-resistant S. aureus in Portugal. Moreover, for the first time the qacG and qacJ genes were detected in MRSA CC398 strains. Active surveillance programmes detecting MRSA not only in livestock animals but also in companion animals are urgently needed