Prevalence and Genetic Characteristics of Staphylococcus aureus CC398 Isolates From Invasive Infections in Spanish Hospitals, Focusing on the Livestock-Independent CC398-MSSA Clade

Background: Livestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported. Objectives: To determine the prevalence of Staphylococcus aureus (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates. Methods: One thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6–12 months in 17 Spanish hospitals (2018–2019) were studied. CC398 lineage identification, detection of spa-types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing. Results: Forty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven spa-types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent spa-types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin–inducible-resistant/erm(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km2) (p = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately spa-t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD (p < 0.005). Conclusion: CC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers.Agencia Estatal de Investigación (AEI) (project SAF2016-76571- R) and by the Fondo Europeo de Desarrollo Regional (FEDER

Staphylococcus spp. from animals intended for human consumption, animalderived food and humans in Spain and Senegal: genetic lineages, antibioresistance and virulence

El primer informe global de la Organización Mundial de la Salud (OMS) sobre la situación de la resistencia a antibióticos, publicado en 2014, destacaba la necesidad de abordar ese problema con un enfoque One Health, centrándose en las interacciones entre los humanos, los animales y el medio ambiente. Las especies del género Staphylococcus, tanto coagulasa-positivo (SCoP) como coagulasa-negativo (SCoN), son comensales de piel y mucosas de personas y animales sanos, siendo a su vez patógenos oportunistas que pueden causar infecciones de piel y partes blandas, mastitis, intoxicaciones alimentarias, bacteriemias, etc. El objetivo de esta tesis fue analizar en primer lugar la diversidad de especies estafilocócicas en distintos nichos ecológicos incluyendo los animales salvajes y los de granja destinados al consumo humano, los animales de producción y las personas. Posteriormente, determinar el fenotipo/genotipo de resistencia a antibióticos de los aislados detectados, y evaluar la prevalencia de las líneas asociadas al ganado entre los aislados de S. aureus. En el primer capítulo, se estudió la epidemiología molecular de Staphylococcus spp. en especies animales destinadas al consumo humano minoritarias como son los jabalíes y los caballos en España. Se observó una gran diversidad estafilocócica en muestras nasales de jabalíes, siendo S. aureus (76.1%) y S. hyicus (16.4%) las especies predominantes entre los SCoP, y S. sciuri (39.7%) y S. xylosus (13%) las predominantes entre los SCoN. En los caballos, las especies predominantes fueron: S. aureus (37%), S. delphini (21%) y S. sciuri (21%). La mayoría de los aislados de jabalíes fueron pan sensibles (73,1% de los SCoP y 77,6% de los SCoN). Sin embargo, se detectaron entre los aislados SCoN, genes inusuales codificantes de la resistencia a macrólidos, lincosamides y fenicoles. En cuanto a los aislados de caballos, un 88,7% de los SCoP fueron pan sensibles mientras que un 48,6% de los SCoN mostraron resistencia a al menos un antibiótico testado. Se observaron fenotipos de multirresistencia solo en aislados SCoN en ambas especies animales. Entre los S. aureus de jabalíes, el linaje predominante fue el t3750-ST2328/CC133 (29,4%). También se detectó un aislado t011 del clon asociado a ganado SARM-CC398. ST1640 fue la línea genética más frecuente entre los S. aureus de caballos (61,7%). Todos los aislados S. aureus de caballos, excepto los del linaje ST1640 portaban los genes codificantes de la leucocidina específica de équidos (LukPQ) y del bloqueador de la activación del sistema complementario equino (eqSCIN). El segundo capítulo se centró en estafilococos de animales de producción de dos países con diferentes políticas de uso de antibióticos (ternero, cordero y cabra (España); vaca y pollo (Senegal)). En España, la prevalencia de estafilococos fue más elevada en muestras de ovino (54%) y caprino (50%), en comparación con las de vacuno (21%). En Senegal, se detectó una prevalencia mucho más alta en vacuno (26,8%) que en pollo (3%). La línea genética de S. aureus más frecuente en los aislados de España, fue el CC133 mientras que el ST291 fue el más abundante entre los aislados de Senegal. Se identificaron seis especies SCoN en cada uno de los países, siendo S. sciuri y S. simulans las más frecuentes en ambos países. Los aislados SCoP fueron mayoritariamente pan sensibles (España) o resistentes a penicilina (Senegal). En ambos países, un porcentaje relativamente elevado de los aislados SCoN presentaron resistencia a al menos uno de los antibióticos testados (32,5% (Senegal) y 47% (España)). Entre los SCoN de ambos países se detectaron aislados resistentes a penicilina, tetraciclina y SXT. Además, se observó resistencia a meticilina, estreptomicina, clindamicina, eritromicina, cloranfenicol y tobramicina entre los aislados de España. En aislados de ambos países, se detectaron los genes codificantes de la leucocidina de Panton-Valentine y se identificó el clon USA300 entre los aislados de España. En los capítulos tres y cuatro, se analizó la presencia del linaje CC398, por un lado entre aislados S. aureus de productos derivados de cerdo, y por otro lado entre aislados S. aureus de hemocultivos de pacientes de hospitales españoles (estudio multicéntrico) situados en provincias con diferentes niveles de densidad porcina. El linaje CC398 fue predominante entre los aislados S. aureus de productos derivados de cerdo (64,1%; n=23). La prevalencia de S. aureus resistente a meticilina (SARM)-CC398 fue del 20,8% (en productos con piel (oreja/morro, 76,6%); y sin piel (filete, 3,3%; carne picada, 14,8%); siendo este clon mayoritariamente asociado al spa-tipo t011. Todos los aislados MRSA-CC398 fueron mecA-positivos, tetraciclina-resistentes (tet(M) y tet(K)+/- tet(L)) y scn-negativo. El 82,6% de estos aislados fueron multirresistentes y el 17,4% portaban genes de virulencia. Se detectaron dos aislados sensibles a meticilina (SASM)-CC398. Ambos aislados fueron resistentes a penicilina y eritromicina/clindamicina-inducible (erm(T)), scn-positivos y tipados como t5452. En cuanto a los aislados clínicos, el 4,3% de los aislados S. aureus pertenecían al linaje CC398, siendo el 90,2% de ellos SASM, lo que representa un 5,2% y 3,9% de los aislados SASM y S. aureus, respectivamente. Los spa-tipos t571 y t1451 fueron predominantes entre los aislados SASM-CC398. La resistencia a eritromicina/clindamicina-inducible mediada por el gen erm(T) fue observada solo en aislados SASM-CC398 (72,9%). Todos los aislados SASM-CC398, excepto tres, fueron scn-positivos. No se observó una relación clara entre la prevalencia de SASM-CC398 en los hospitales y el nivel de densidad porcina de la zona correspondiente. Los aislados LA-SARM-CC398 fueron infrecuentes y detectados en hospitales situados en provincias de alta densidad porcina.The first antimicrobial resistance global report on surveillance of the World Health Organization (WHO) in 2014 pointed out the need to deal with the problem under a One Health approach which focusses on the interactions between humans, animals and their environments, in order to enhance health and welfare. Staphylococcal species (both coagulase-positive (CoPS) and -negative (CoNS)) are normal colonizers of skin and mucous of healthy humans and animals, but also opportunistic pathogens, that can cause skin and soft tissues infections, mastitis, food intoxication, bacteraemia etc. This thesis aimed to analyse the diversity of staphylococcal species in different niches, including wild and farm animals destined for human consumption, animal-derived food and humans, to determine the antimicrobial resistance phenotype/genotype of recovered isolates, and to evaluate the occurrence of livestock-associated lineages among S. aureus isolates. The first chapter studied the molecular epidemiology of staphylococcal species in nasal and/or faecal samples of non-conventional food animals such as wild boar and horses in Spain. A great diversity of staphylococcal species was observed in wild boar, being S. aureus (76.1%) and S. hyicus (16.4%) the predominant CoPS, and S. sciuri (39.7%) and S. xylosus (13%) the predominant CoNS. In horses, the predominant species were S. aureus (37%), S. delphini (21%) and S. sciuri (21%). Most isolates recovered from wild boar were pan susceptible (73.1% of CoPS and 77.6% of CoNS). However unusual genes involved in macrolide, lincosamide and phenicol resistance were detected among CoNS. Regarding horses isolates, 88.7% of CoPS were pan susceptible while 48.6% of CoNS showed resistance to at least one of the antimicrobial agents. A multidrug resistance phenotype (MDR) was only observed among CoNS from both animal species. The predominant S. aureus lineage in wild boar was t3750-ST2328/CC133 (29.4%) and one isolate of the livestock-associated methicillin resistant S. aureus lineage (LA-MRSACC398) (t011-ST398) was identified. S. aureus lineage ST1640 (61.7% of S. aureus), is associated with horses for the first time in this study. All S. aureus isolates detected from horses, except those of lineage ST1460, carried the genes encoding the equid-adapted leukocidin (LukPQ) and the blocker of equine complement system activation (eqSCIN). The second chapter focussed on staphylococci from common food-producing animals from two countries with different antibiotic use policy (calve, lamb, goat (Spain); cow and chicken (Senegal)). In Spain, the prevalence of staphylococci was higher in lamb (54%) and goat (50%) than in cow (21%) samples. In Senegal, staphylococci prevalence was higher in cow (26.8%) than in chicken (3%). In Spain, the most frequent lineage was CC133 while the ST291 was dominant in Senegal isolates. Six species were identified among CoNS in each country, with prevalent species being S. sciuri and S. simulans. CoPS isolates from Spain were mostly pan susceptible, while those from Senegal were mostly penicillin resistant. In both countries, a relatively high percentage of CoNS isolates (32.5% (Senegal), 47% (Spain)) was resistant to at least one of the antimicrobials tested. Resistance to penicillin, tetracycline and SXT was observed among CoNS isolates of both countries. Additionally, resistance to methicillin, streptomycin, clindamycin, erythromycin, chloramphenicol and tobramycin was observed among Spain isolates. The Panton-Valentine Leukocidin (PVL) encoding genes were present in isolates of both countries, and the human virulent clone USA300 was identified among Spain isolates. In the chapters three and four, the lineage CC398 was screened for pig-derived food S. aureus isolates on one hand, and among patients blood culture S. aureus isolates from Spanish hospitals located in regions with different pig density degrees, on the other hand (multicentre study). CC398 was the predominant lineage among S. aureus isolates of pig products (64.1%; n=23). The prevalence of MRSA-CC398 was 20.8%, observing differences in products with skin (76.5% (ear/snout)) and without skin (3.3% (fillet), 14.8% (chopped meat)); this clone was mostly typed as t011. All MRSA-CC398 isolates were mecA-positive, tetracycline resistant (tet(M) and tet(K)+/- tet(L)) and scn-negative. 82.6% were MDR and 17.4% harboured virulence genes. Two methicillin susceptible S. aureus (MSSA)-CC398 isolates were detected; both being resistant to penicillin and erythromycin/clindamycininducible (erm(T) gene). They were typed as t5452 and were scn-positive. Concerning the clinical isolates, the prevalence of CC398 isolates among S. aureus was 4.3%. MSSA-CC398 represented 90.2% of total CC398 isolates, corresponding to 5.2% of MSSA and 3.9% of S. aureus. t571 and t1451 were the predominant spa-types among MSSA-CC398 isolates. Resistance to erythromycin/clindamycin-inducible, mediated by erm(T) gene, was only observed among MSSA-CC398 isolates (72.9%). All MSSA-CC398 isolates, but three, were scn-positive. No obvious correlation was observed between MSSA-CC398 prevalence at hospital level and the pig density degree in surrounding areas. LA-MRSA-CC398 and LA-MRSA-CC1 were detected in hospitals located in high pig density areas

Antimicrobial resistance, virulence, and genetic lineages of staphylococci from horses destined for human consumption: High detection of S. aureus isolates of lineage ST1640 and those carrying the lukPQ gene

This work aimed to determine the frequency and diversity of Staphylococcus species carriage in horses intended for human consumption, as well as their resistance and virulence determinants. Eighty samples (30 nasal; 50 faecal) were recovered from 73 healthy horses in a Spanish slaughterhouse. The samples were cultured for staphylococci and methicillin-resistant staphylococci (MRS) recovery. The phenotype/genotype of antimicrobial resistance was analysed for all isolates. The spa-type and sequence-type (ST) were determined in Staphylococcus aureus strains; moreover, the presence of virulence and host-adaptation genes (tst, eta, etb, pvl, lukPQ, scn-eq, and scn) was studied by PCR. Staphylococcus species were detected in 27/30 (90%) and 33/50 (66%) of nasal and faecal samples, respectively. Ninety isolates belonging to eight species were recovered, with predominance of S. aureus (n = 34), Staphylococcus delphini (n = 19), and Staphylococcus sciuri (n = 19). S. aureus strains were all methicillin-susceptible (MSSA), 28/34 were susceptible to all the antibiotics tested, and the remaining six showed resistance to (gene-detected) streptomycin (ant (6)-Ia), penicillin (blaZ), and trimetroprim/sulphametoxazole (SXT) (dfrA, dfrG). The lineage ST1640/t2559 was predominant (n = 21). The genes lukPQ and scn-eq were present in all but the ST1640 isolates. Three S. sciuri isolates were multidrug-resistant. Healthy horses in Spain seem to be a reservoir for virulent MSSA and the lineage ST1640, although the presence of the latter in horses is described for the first time in this study. Moreover, the equine-adapted leukocidin gene lukPQ is frequent among S. aureus strains. A large variety of staphylococcal species with low antibiotic resistance rate were also observed.ISSN:2076-261

High frequency of coagulase-positive staphylococci carriage in healthy wild boar with detection of MRSA of lineage ST398-t011

The objective of this study was to determine the frequency and diversity of coagulase-positive staphylococci (CoPS) in nasal samples of healthy wild boar, to study their resistance phenotypes/genotypes and to check the occurrence of the MRSA-ST398. Nasal samples of 371 wild boars were collected in Spain for staphylococci and MRSA recovery. Staphylococci identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). The susceptibility to 11 antimicrobials was tested by disc-diffusion and the presence of resistance genes by PCR. Molecular typing and virulence factors determination were carried out by PCR and sequencing. The rate of CoPS carriage (Staphylococcus aureus, Staphylococcus hyicus and Staphylococcus pseudintermedius) in wild boar was of 17.8% (13.7%, 2.7% and 1.6%, respectively). Susceptibility to all tested antimicrobials was shown in 74.5% of S. aureus and one strain was MRSA [lineage ST398-t011-agrI, carrying blaZ, mecA, tet(M) and tet(K) genes]. A total of 22 spa-types and 17 STs were detected among S. aureus, including: ST398/CC398 (n = 1), ST2328-ST133/CC133 (n = 20), ST425/CC425 (n = 7), ST5/CC5 (n = 5), ST1/CC1 (n = 3), ST130/CC130 (n = 2) and ST88/CC88 (n = 1). Two spa-types (t02, t15) and four STs (ST455, ST796, ST797, ST798) were detected among the six S. pseudintermedius isolates recovered, and all of them carried the lukF/S-I and siet virulence genes. All S. hyicus isolates were susceptible to antimicrobials tested.This work was supported by project SAF2016-76571-R from the Ministerio de Economía y Competitividad (MINECO) of Spain and FEDER. OMM has a pre-doctoral fellowship of Mujeres por África-Universidad de La Rioja (Spain), and LRR a pre-doctoral fellowship of the Universidad de La Rioja (Spain).Peer reviewe

High diversity of coagulase negative staphylococci species in wild boars, with low antimicrobial resistance rates but detection of relevant resistance genes

This work was focused to determine the prevalence and the species diversity of coagulase-negative staphylococci (CoNS) in wild boars, and to study their antimicrobial resistance phenotype and genotype. Nasal samples of 371 wild boars from six Spanish regions were collected for CoNS recovery. The identification was performed by MALDI-TOF mass-spectrometry. Antimicrobial susceptibility for eight antimicrobial agents was studied by disc-diffusion method and the presence of 31 antimicrobial resistance genes by PCR. CoNS were detected in nasal samples of 136/371 animals tested (36.6%), and 161 isolates were obtained (1–3/animal); a high diversity of species was found (n = 17), with predominance of S. sciuri (n = 64), S. xylosus (n = 21) and S. chromogenes (n = 17). Among CoNS isolates, 22.4% showed resistance to at least one antimicrobial tested. Tetracycline-resistance phenotype was the most frequently detected (10.5%), generally mediated by tet(K) gene [associated or not with tet(L)]. Other relevant resistance genes were identified including unusual ones [mecA, erm(B), erm(F), mphC, erm(43), msr(A)/msr(B), lnu(A), dfrG, fexA, and catpC221]. This is the first study in which CoNS isolates from wild boars are analysed. The knowledge of antimicrobial phenotype and genotype of CoNS in natural ecosystems is highly important since these staphylococcal species can act as vectors of relevant antimicrobial resistance mechanisms.This work was supported by project SAF2016-76571-R from the Agencia Española de Investigación (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER). Olouwafemi Mistourath Mama has a predoctoral fellowship of Mujeres por África-Universidad de La Rioja (Spain). Laura Ruiz-Ripa has a predoctoral FPI fellowship of the Universidad de La Rioja (Spain). David González-Barrio is granted by the Spanish Ministry of Science through a Juan de la Cierva postdoctoral fellowship (FJCI-2016-27875).Peer reviewe

Prevalence and Genetic Characteristics of Staphylococcus aureus CC398 Isolates From Invasive Infections in Spanish Hospitals, Focusing on the Livestock-Independent CC398-MSSA Clade

Livestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported. To determine the prevalence of Staphylococcus aureus (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates. One thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6-12 months in 17 Spanish hospitals (2018-2019) were studied. CC398 lineage identification, detection of spa-types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing. Forty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven spa-types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent spa-types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin-inducible-resistant/erm(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km2) (p = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately spa-t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD (p CC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers