Occurrence, genetic diversity, and antimicrobial resistance of methicillin-resistant Staphylococcus spp. in hospitalized and non-hospitalized cats in Brazil.

Methicillin-resistant Staphylococci (MRS) cause infections at various sites and exhibit multidrug resistance. Despite their importance in veterinary medicine, only little is known about Staphylococcus spp. colonizing and infecting cats. Therefore, in this study, we aimed to isolate and identify Staphylococcus spp. colonizing hospitalized and non-hospitalized domestic cats and analyze their antimicrobial resistance profiles, genetic diversity, and risk factors associated with MRS colonization. A total of 218 oral and axillary swabs were obtained from 109 cats, including 77 non-hospitalized and 32 hospitalized cats. After plating on selective media, the isolates were identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and rpoB and 16S rRNA gene sequencing. Subsequently, antimicrobial sensitivity of the strains was assessed, and they were screened for mecA gene. Methicillin-resistant S. haemolyticus (MRSH) isolates were subjected to multilocus sequence typing, whereas methicillin-resistant S. pseudintermedius (MRSP) and S. felis isolates were subjected to whole genome sequencing. S. felis was most commonly isolated from non-hospitalized cats (28.1%), whereas S. pseudintermedius and MRS were commonly isolated from hospitalized cats (25%). MRSH isolates from hospitalized animals were classified as ST3. The identified MRSP strains belonged to two well-known sequence types, ST551 and ST71. Moreover, antimicrobial use (p = 0.0001), hospitalization (p = 0.0141), and comorbidities (p = 0.002) were associated with increased MRS prevalence in cats.

Antimicrobial potential of a biosurfactant gel for the prevention of mixed biofilms formed by fluconazole-resistant C. albicans and methicillin-resistant S. aureus in catheters.

Dual-species biofilms formed by Candida albicans and Staphylococcus aureus have high virulence and drug resistance. In this context, biosurfactants produced by Pseudomonas aeruginosa have been widely studied, of which a new derivative (RLmix_Arg) stands out for possible application in formulations. The objective of this study was to evaluate the antibiofilm activity of RLmix_Arg, both alone and incorporated in a gel prepared with Pluronic F-127, against dual-species biofilms of fluconazole-resistant C. albicans (FRCA) and methicillin-resistant S. aureus (MRSA) in impregnated catheters. Broth microdilution tests, MTT reduction assays of mature biofilms, impregnation of RLmix_Arg and its gel in peripheral venous catheters, durability tests and scanning electron microscopy (SEM) were performed. RLmix_Arg showed antimicrobial activity against Candida spp. and S. aureus, by reducing the cell viability of mixed biofilms of FRCA and MRSA, and preventing their formation in a peripheral venous catheter. The incorporation of this biosurfactant in the Pluronic F-127 gel considerably enhanced its antibiofilm activity. Thus, RLmix_Arg has potential application in gels for impregnation in peripheral venous catheters, helping to prevent development of dual-species biofilms of FRCA and MRSA.

Methicillin-resistant Staphylococcus spp. investigation in hospitalized horses and contacting personnel in a teaching veterinary hospital.

Staphylococci are well-known opportunistic pathogens associated with suppurative diseases in humans and animals. Antimicrobial resistance is an emergent threat to humans and animals worldwide. This study investigated the prevalence of methicillin-resistant Staphylococcus spp. (MRS) in hospitalized horses and contacting personnel (veterinarians and staff), and assessed possible interspecies transmission in a teaching veterinary hospital. Nasal swabs from horses (n = 131) and humans (n = 35) were collected. The microorganisms were identified by traditional biochemical tests and genotypic methods, i.e., PCR, internal transcript spacer PCR (ITS-PCR), and gene sequencing. Staphylococcal species were isolated in 18% (23/131) of the horses, of which 8% (11/131) were S. hyicus, 4 % (5/131) were S. aureus, 4% (5/131) were S. pseudintermedius, and 2% (2/131) were S. schleiferi subsp. coagulans. The mecA gene was detected in an S. pseudintermedius isolate. Staphylococcus spp. was isolated in 40% (14/35) of the human samples, all of which were S. aureus. In four samples of S. aureus, the clonal profile ST398 was identified; among them, a clonal similarity of 98.1% was observed between a horse and a contacting human. This finding supports the need for biosecurity measures to avoid the spread of multidrug-resistant staphylococci in humans and horses.

Antimicrobial activity of hydralazine against methicillin-resistant and methicillin-susceptible Staphylococcus aureus.

Background: Staphylococcus aureus is a human pathogen responsible for high mortality rates. The development of new antimicrobials is urgent. Materials & methods: The authors evaluated the activity of hydralazine along with its synergism with other drugs and action on biofilms. With regard to action mechanisms, the authors evaluated cell viability, DNA damage and molecular docking. Results: MIC and minimum bactericidal concentration values ranged from 128 to 2048 µg/ml. There was synergism with oxacillin (50%) and vancomycin (25%). Hydralazine reduced the viability of biofilms by 50%. After exposure to hydralazine 2× MIC, 58.78% of the cells were unviable, 62.07% were TUNEL positive and 27.03% presented damage in the comet assay (p < 0.05). Hydralazine showed affinity for DNA gyrase and TyrRS. Conclusion: Hydralazine is a potential antibacterial.

Staphylococcus aureus is a bacterium that can cause infection. Infections of S. aureus are becoming difficult to treat, but developing new drugs is a challenge. Repurposing them may be easier. This study looks at the possibility of using hydralazine, a type of medicine used to treat high blood pressure, against S. aureus. The authors found that hydralazine can kill S. aureus and can be used with other antibiotics, including oxacillin and vancomycin. Hydralazine interferes with important processes for the multiplication and survival of this bacterium. These results are preliminary but encouraging. Further studies are needed to confirm the use of hydralazine as a new treatment for S. aureus infections.

Epidemiologic case investigation on the zoonotic transmission of Methicillin-resistant Staphylococcus pseudintermedius among dogs and their owners.

Dogs often carry methicillin-resistant Staphylococci asymptomatically. These bacteria are frequently linked to conditions such as canine pyoderma and otitis. Close interaction between dogs and humans can facilitate the exchange of resistant strains, particularly Methicillin-resistant Staphylococcus pseudintermedius (MRSP). This represents a public health issue, since these strains, in addition to occasionally causing infections in humans, can also serve as a source of resistance and virulence genes for strains of greater importance in human medicine, such as Staphylococcus aureus. Furthermore, MRSP strains are often multidrug resistant, which ends up compromising the treatment of infections. This study aimed to assess the potential transmission of Staphylococcus pseudintermedius among dogs and their owners. We examined a total of one hundred canine samples collected from cases of pyoderma and otitis to detect the presence of staphylococci. Simultaneously, we conducted evaluations on all dog owners. Staphylococci strains were identified using MALDI-TOF MS and PCR targeting the nuc gene. Methicillin resistance screening was also performed by detecting the mecA gene using PCR. Among the sampled dogs, 64 carried S. pseudintermedius. Nine were identified as MRSP. In six instances, dogs and their owners exhibited S. pseudintermedius. These samples underwent genome sequencing and were screened for antimicrobial resistance genes, SCCmec typing, MLST characterization, and Single Nucleotide Polymorphisms (SNP) analyses. The results of the phylogenetic analysis revealed that in three cases, dogs and owners had closely related isolates, suggesting interspecies transmission. Two of these cases involved MRSP and one MSSP. Moreover, in the two MRSP cases, the same SCCmec type (type V) was detected. Additionally, the sequence type was consistent across all three cases involving dogs and owners (MSSP ST2277, MRSP ST2282, and ST2286). These findings strongly indicate a transmission event. Since Staphylococcus pseudintermedius is primarily isolated from canine samples, it is plausible that dogs may have acted as a potential source. In the remaining three cases, despite identifying the same species in both samples, they had notable phylogenetic differences.

Investigation of antimicrobial susceptibility and genetic diversity among Staphylococcus pseudintermedius isolated from dogs in Rio de Janeiro.

Staphylococcus pseudintermedius is an opportunistic pathogen causing a variety of infections that are difficult to treat, especially because of the development of antimicrobial resistance. It has a clonal distribution around the world. To have a better understanding of the MRSP population, we search the presence of MRSP in colonized or infected dogs. Samples from 99 dogs with infections and 35 from asymptomatic dogs were collected. Isolates were identified by mass spectrometry and Multiplex-PCR. The mecA gene was confirmed by conventional PCR. MRSP strains were analyzed by whole-genome sequencing. 75 S. pseudintermedius were identified, most from infection cases. The species were isolated from 70 out of the 135 dogs. Penicillin and Trimethoprim/Sulfamethoxazole presented higher resistance rates. Forty-seven strains were classified as multi-drug resistant (MDR), and were more isolated from dogs with infection (P < 0.05). Eighteen samples were classified as MRSP, representing 24.0% of the population. Six of 16 MRSP sequenced samples belonged to the world spread clone ST71; others belonged to unknown clones. Most samples carried the SCCmec type IIIA. Twenty-one different genetic resistance determinants were found among MRPS strains. MRSP is circulating among infected and colonized dogs in Rio de Janeiro, Brazil.

Biogenic silver nanoparticles: in vitro activity against Staphylococcus aureus methicillin-resistant (MRSA) and multidrug-resistant coagulase-negative Staphylococcus (CoNS).

Multidrug-resistant (MDR) bacteria are one problem in health since the therapeutic alternative are reduced. For this, the application of nanotechnology through functionalized nanoparticles, like a biogenic silver nanoparticle (Bio-AgNP), obtained by biological synthesis, emerges as a possible alternative against the MDR bacteria. This study aimed to evaluate the antibacterial and antibiofilm activity of Bio-AgNP obtained for biological synthesis by Fusarium oxysporum strain 551 against methicillin-resistant Staphylococcus aureus (MRSA) and MDR coagulase-negative Staphylococcus (CoNS) isolates. Bio-AgNP has activity against S. aureus ATCC 25904, Staphylococcus epidermidis ATCC 35984, and MDR isolates, with minimal inhibitory concentration (MIC) ranging from 3.75 to 15 µg.mL-1 and minimal bactericidal concentration (MBC) from 7.5 to 30 µg.mL-1. In the membrane leakage assay, it was observed that all concentrations tested led to proteins release from the cellular content dose-dependently, where the highest concentrations led to higher protein in the supernatant. The 2×MIC of Bio-AgNP killed ATCC 35984 after 6h of treatment, and ATCC 25904 and S. aureus (SA3) strains after 24h of treatment. The 4×MIC was bactericidal in 6h of treatment for all strains in the study. The biofilm of MDR isolates was inhibited in 80.94 to 100% and eradicated in 60 to 94%. The confocal laser scanning microscopy (CLSM) analysis demonstrated similar results to the antibiofilm assays. The Bio-AgNP has antibacterial and antibiofilm activity and can be a promising therapeutic alternative against MDR bacteria.

Virulent potential of methicillin-resistant and methicillin-susceptible Staphylococcus pseudintermedius in dogs.

Staphylococcus pseudintermedius is a zoonotic pathogen responsible for several infectious diseases in pet animals, yet its pathogenic potential is not fully understood. Thus, this study aims to unravel the virulence profile of S. pseudintermedius from canine origin. Methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains were isolated from different infection sites and their genotypic and phenotypic features were compared to determine the clinical implications of MRSP and MSSP strains. Bacterial identification was performed using MALDI-TOF and 16S-rDNA sequencing. In addition, we used multilocus sequence typing (MLST) for strains' sequence type (ST) determination and phylogenetic relationship. The strains were screened for toxin genes, including cytotoxins (lukS, lukF), exfoliative toxin (siet), enterotoxins (sea, seb, sec, secCanine, sel, sem, and seq) and toxic shock syndrome toxin (tst-1). In vitro phenotypic analyses assessing antimicrobial susceptibility profile, biofilm formation ability, and expression of extracellular matrix components were performed. The investigated S. pseudintermedius strains belong to 17 unique ST, most of which were classified as ST71. MSSP and MRSP strains shared siet, lukS, and lukF virulence markers. Our findings showed that some MSSP strains also harbored sel, seq, and sem enterotoxin genes, suggesting a more diverse virulence profile. All MRSP strains and 77% of MSSP strains were classified as multidrug resistant (MDR). Moreover, all investigated S. pseudintermedius strains showed strong biofilm formation ability. In summary, our findings highlight the wide spread of highly virulent and drug-resistant zoonotic S. pseudintermedius strains, being a potential concern for One Health issues.

Genomic analysis of a multidrug methicillin-resistant staphylococcus epidermidis recovered from the urine of a guinea pig (Cavia porcellus) with suspected pyelonephritis.

Staphylococcus epidermidis is an emerging pathogen causing infant pyelonephritis. There is a lack of genomic data on Staphylococcus epidermidis as the etiology of pyelonephritis and its resistant determinants. In this study, we have conducted a genomic and microbiologic investigation of an S. epidermidis recovered from the urine of a guinea pig with suspected pyelonephritis in Brazil. The genome was sequenced using the Illumina MiSeq platform and de novo assembled using SPades. Resistome, virulome, and plasmidome were in silico predicted using bioinformatics tools. Data analysis revealed that S. epidermidis USP-LZB-G06 belonged to sequence type ST332. Strikingly, a broad resistome (antibiotics, hazardous heavy metals, and biocides) was predicted, including the presence of the clinically relevant mecA, blaZ, and qacA efflux pump genes. SNP-based analysis revealed that strain USP-LZB-G06 was clustered along mecA positive S. epidermidis strains of ST332 isolated between 2008 and 2016 from humans in Australia and the United States of America. Our results indicate that the detection of this microorganism should be considered as a urinary tract infection agent in exotic pets, particularly guinea pigs. In addition, there is an urgent need to update veterinarians regarding the detection and therapeutic management of these microorganisms.

Vancomycin heteroresistance among methicillin-resistant clinical isolates S. haemolyticus, S. hominis, S. simulans, and S. warneri.

Besides being an essential part of the skin microbiome, coagulase-negative staphylococci are the etiological factors of serious infections. The aim of the study was to evaluate the heteroresistance to vancomycin and the potential antimicrobial efficacy of teicoplanin and daptomycin against the multiresistant strains of S. haemolyticus, S. hominis, S. warneri, and S. simulans. The study covered 80 clinical coagulase-negative staphylococci. Teicoplanin, vancomycin, and daptomycin MICs for the tested strains were determined according to EUCAST recommendation. The vanA and vanB genes were searched. The brain heart infusion screen agar method detected vancomycin heteroresistance. The population analysis profile method and analysis of autolytic activity were applied for the strains growing on BHI containing 4 mg/L vancomycin. Seven S. haemolyticus, two S. hominis, and two S. warneri strains presented a heterogeneous resistance to vancomycin. Their subpopulations were able to grow on a medium containing 4-12 mg/L of vancomycin. Monitoring heteroresistance to peptide antibiotics, which are often the last resort in staphylococcal infections, is essential due to the severe crisis in antibiotic therapy and the lack of alternatives to treat infections with multiresistant strains. Our work highlights the selection of resistant strains and the need for more careful use of peptide antibiotics.

Inhibition potential of Caryocar brasiliense on methicillin-resistant Staphylococcus pseudintermedius isolated from the ocular surface of dogs with ophthalmopathies / Potencial de inibição do Caryocar brasiliense sobre Staphylococcus pseudintermedius resistente à meticilina isolado da superfície ocular de cães com oftalmopatias

The indiscriminate use of antibiotics has contributed to the emergence of multiresistant bacteria. Faced with this, the search for antibiotics from plants has proven to be a promising alternative. The objective of this work was to isolate and identify Staphylococcus sp. resistant to methicillin of the ocular surface of dogs with ophthalmopathies and to evaluate its susceptibility to alcoholic extract of the bark and hexane extract of the pulp of Caryocar brasiliense. Biological material was collected from the ocular surface of 21 dogs presenting ophthalmopathies. We isolated 64 S. pseudintermedius, among these, 4 isolates were identified as methicillin-resistant S. pseudintermedius (MRSP). The alcoholic extract of C. brasiliense peel was able to inhibit the bacterial growth of MRSP isolates at a concentration of 2.2%. Thus, the extract from the C. brasiliense peel has antimicrobial potential and represents an alternative in the control of MRSP.

O uso indiscriminado de antibióticos tem contribuído para o surgimento de bactérias multirresistentes. Diante disso, a busca por antibióticos a partir de plantas tem se mostrado uma alternativa promissora. O objetivo deste trabalho foi isolar e identificar Staphylococcus sp. resistente à meticilina da superfície ocular de cães com oftalmopatias e avaliar sua susceptibilidade ao extrato alcoólico da casca e ao extrato hexânico da polpa de Caryocar brasiliense. O material biológico foi coletado da superficie ocular de 21 cães com oftalmopatia. Isolaram-se 64 S. pseudintermedius; entre esses, quatro isolados foram identificados como S. pseudintermedius resistente à meticilina (MRSP). O extrato alcoólico da casca de C. brasiliense foi capaz de inibir o crescimento bacteriano dos isolados de MRSP na concentração de 2,2%. Dessa forma, o extrato da casca de C. brasiliense possui potencial antimicrobiano e representa uma alternativa no controle de MRSP.

Sensibilidade de bactérias causadoras de mastite bovinaaextratos de plantas nativas do cerrado / Sensitivity of bacteria causing bovine mastitis toextractsofnative plants of the cerrado / Sensibilidad de bacterias causantes de mastitis bovinaaextractos de plantas nativas en cerrado

A mastite bovina é a doença mais onerosa da produção leiteira e é caracterizadapelainflamação da glândula mamária. O tratamento da doença, sem o controle adequado, geramicrorganismos resistentes. Desta forma o uso de fitoterápicos tem se tornado uma fontedepesquisa como possível alternativa, como as plantas nativas do cerrado Dedaleiro (Lafoensiapacari), Jatobá (Hymenaea sp.) e Barbatimão (Stryphnodendron adstringens). No presentetrabalho, bactérias isoladas de vacas com mastite subclínica foram identificadas por testesbioquímicos. Posteriormente foram feitos testes de antibiograma e pesquisa por genesderesistência a antibióticos, por fim foi realizado o teste para avaliação da sensibilidadeaosextratos das plantas do cerrado. Foram identificados Staphylococcus aureus (S.aureus), Enterobacter sp. e Escherichia coli (E. coli). Entre os isolados de S. aureus, foramencontrados resistentes a meticilina (MARS), bem como resistentes à vancomicina (VARS). Foram encontrados isolados produtores de beta-lactamases de espectro extendido(ESBL)para Entreobacter sp. e E. coli. Os extratos, principalmente de Stryphnodendron adstringenseLafoensia pacari, são uma alternativa para a terapêutica antimicrobiana.(AU)

Bovine mastitis is the most costly disease of dairy production and is characterizedbyinflammation of the mammary gland. The treatment of the disease, without adequate control, generates resistant microorganisms. In this way, the use of herbal medicines has becomeasource of research as a possible alternative, such as the native plants of the cerrado Dedaleiro(Lafoensia pacari), Jatobá (Hymenaea sp.) and Barbatimão (Stryphnodendron adstringens). In the present work, bacteria isolated from cows with subclinical mastitis were identifiedthrough biochemical tests. Subsequently, antibiogram tests and research for antibioticresistance genes were carried out, finally the test was carried out to evaluate the sensitivitytoextracts of cerrado plants. Staphylococcus aureus (S.aureus), Enterobacter sp. andEscherichia coli (E. coli). Among the S. aureus isolates, methicillin resistant (MARS) as well as vancomycin resistant (VARS) were found. Extended spectrum beta-lactamases (ESBL)producing isolates were found for Entreobacter sp. and E. coli. The extracts, mainly from Stryphnodendron adstringens and Lafoensia pacari, are an alternative for antimicrobial therapy.(AU)

La mastitis bovina es la enfermedad más costosa de la producción lechera y se caracterizaporla inflamación de la glándula mamaria. El tratamiento de la enfermedad, sin uncontrol adecuado, genera microorganismos resistentes. De esta forma, el uso de fitoterápicos sehaconvertido en fuente de investigación como posible alternativa, como las plantas nativas del cerrado Dedaleiro (Lafoensia pacari), Jatobá (Hymenaea sp.) y Barbatimão (Stryphnodendronadstringens). En el presente trabajo se identificaron mediante pruebas bioquímicas bacteriasaisladas de vacas con mastitis subclínica. Posteriormente se realizaron pruebas deantibiograma e investigación de genes de resistencia a antibióticos, finalmente se realizólaprueba para evaluar la sensibilidad a extractos de plantas de cerrado. Staphylococcus aureus(S. aureus), Enterobacter sp. y Escherichia coli (E. coli). Entre los aislamientos de S. aureus, se encontraron resistentes a la meticilina (MARS) y resistentes a la vancomicina (VARS). Seencontraron aislamientos productores de betalactamasas de espectro extendido (ESBL) paraEntreobacter sp. y E. coli. Los extractos, principalmente de Stryphnodendron adstringensyLafoensia pacari, son una alternativa para la terapia antimicrobiana.(AU)

Detection of the international lineage ST71 of methicillin-resistant Staphylococcus pseudintermedius in two cities in Rio de Janeiro State.

Staphylococcus pseudintermedius is the main coagulase-positive staphylococci associated with canine skin/soft tissue infections (SSTI), otitis externa, and surgical site infections. The international spread of an epidemic and multiresistant lineage of methicillin-resistant Staphylococcus pseudintermedius (MRSP), the so-called European clone-displaying sequence type (ST) 71-requires attention. The first isolation of an MRSP ST71 isolate in South America was reported in Rio de Janeiro city, in 2010; however, a limited number of canine isolates were analyzed. Thus, to have a better panel of the MRSP spread in this city, we were stimulated to continue this study and search for the presence of MRSP in 282 colonized or infected dogs in the city of Rio de Janeiro. Among the MRSP isolates collected (N = 17; 6.1%), the pulsed-field gel electrophoresis (PFGE) patterns were similar to those of European clone. All 17 isolates were classified as ST71 by multilocus sequence typing (MLST). In order to assess whether isolates of MRSP ST71 may have also spread to the Rio de Janeiro state countryside, we collected samples from 124 infected dogs in the city of Campos dos Goytacazes (232 km away from Rio de Janeiro city). Our data showed the presence of ST71 lineage in one isolate among three MRSP detected. S. pseudintermedius was isolated from 40.6% of the clinical samples (N = 165/406). A relatively high incidence of methicillin resistance, detected by a PCR-based method, was found in 12.1% of the S. pseudintermedius recovered from animals (N = 20/165). The resistance profile of these isolates was similar to that described for the international ST71 strains whose genomes are publicly available in the GenBank. The prospect of ST71 isolates being resistant to virtually all antimicrobials used in veterinary medicine is alarming and should be considered a central issue considering that MRSP ST71 spreads over large geographic distances and its transmission from animals to humans.

Methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates from skin and nares of Brazilian children with atopic dermatitis demonstrate high level of clonal diversity.

BACKGROUND: Atopic dermatitis (AD) primarily affects the pediatric population, which is highly colonized by S. aureus. However, little is known about the genetic features of this microorganism and other staphylococcal species that colonize AD patients. OBJECTIVE: This study aimed to characterize Staphylococcus spp. isolated from the nares and skin (with and without lesion) of 30 AD and 12 non-AD Brazilian children. METHODS: Skin and nasal swabs were cultured onto mannitol salt agar, and bacterial colonies were counted and identified by matrix assisted laser desorption ionization time of flight mass spectrometry and polymerase chain reaction (PCR). Antimicrobial susceptibility was evaluated by phenotypic and genotypic tests. In S. aureus isolates, Panton-Valentine leukocidin genes were detected by PCR, and their clonality was assessed by pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS: S. aureus was more prevalent in the nares (P = 0.005) and lesional skin (P = 0.0002) of children with AD, while S. hominis was more frequent in the skin of non-AD children (P < 0.0001). All children in the study, except one from each group, were colonized by methicillin-resistant coagulase-negative Staphylococcus and 24% by methicillin-resistant S. aureus. Despite the great clonal diversity of S. aureus (18 sequence types identified), most AD children (74.1%) were colonized by the same genotype in both niches. CONCLUSION: High colonization by polyclonal S. aureus isolates was found among children with AD, while S. hominis was more frequent among non-AD children. The high prevalence of methicillin-resistant staphylococcal isolates highlights the importance of continued surveillance, especially when considering empiric antibiotic therapy for the treatment of skin infections in these patients.

Occurrence and characterization of methicillin-resistant Staphylococcus spp. in diseased dogs in Brazil.

Staphylococcus pseudintermedius is a major commensal bacterium of the skin and mucosae of dogs and an opportunistic agent responsible for several clinical infections, such as pyoderma, otitis, and surgical wound infections. The emergence of methicillin-resistant S. pseudintermedius (MRSP) has become a problem of great concern in veterinary and human medicine because it is multidrug resistant (MDR) and can also infect humans. This study aimed to identify the occurrence of Staphylococcus spp. in infected patients and investigate the antimicrobial resistance profiles and molecular structure of MRSP isolates. Samples were obtained from two different veterinary clinics; suggestive colonies were submitted to matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry and confirmed at the species level by polymerase chain reaction (PCR). Sequencing of the 16S rRNA and rpoB genes were used in selected samples that were not identified by MALDI-ToF and by the species-specific PCR. Antimicrobial susceptibility and PCR detection of mecA were performed. MRSP isolates were subjected to multilocus sequence typing. Of all the clinical staphylococci (n = 131), 98 (74.8%) were identified as S. pseudintermedius. Multidrug resistance (resistance to ≥3 classes of antimicrobials) was observed in 63.2% of S. pseudintermedius isolates, and 24.5% of S. pseudintermedius isolates were methicillin-resistant. Half of the MRSP isolates were isolated from surgical site infections. Among the ten sequence types (ST) identified, nine were novel. ST71 was the most prevalent and associated with resistance to fluoroquinolones. Prior antimicrobial therapy, hospitalization, and surgical site infections seemed to be risk factors for MRSP acquisition. The present study showed a high rate of MDR staphylococci in infected dogs. MRSP was isolated from different clinical conditions, mainly surgical site infections. Additionally, this is the first study to extensively investigate the population structure of MRSP in Brazil, which revealed the dispersion of CC71 and nine novel ST. These findings raise concerns for both animal and human health due to the zoonotic potential of this species and limited therapeutic options available for MRSP infections.

Persistence of a multidrug-resistant worldwide-disseminated methicillin-resistant Staphylococcus epidermidis clone harbouring the cfr linezolid resistance gene in a French hospital with evidence of interspecies transfer to several Staphylococcus aureus lineages.

BACKGROUND: Resistance to linezolid has become a worldwide concern since it is one of the last-resort antibiotics to treat multidrug-resistant staphylococcal and enterococcal infections. OBJECTIVES: We investigated staphylococcal infections caused by 16 cfr-positive linezolid-resistant Staphylococcus epidermidis and Staphylococcus aureus isolates in a French university hospital from 2015 to 2018. METHODS: Antimicrobial susceptibility of isolates was tested by broth microdilution and gradient strips. Genetic determinants of linezolid resistance (including cfr gene and 23S rRNA mutations) were assessed by PCR and WGS; the latter was also used to characterize the cfr-carrying plasmids in S. epidermidis and S. aureus, and to explore the clonal relationship of isolates. RESULTS: All linezolid-resistant staphylococcal isolates harboured the same cfr-carrying plasmid, sharing 99% identity with the previously described pSA737. The three S. aureus isolates belonged to different STs (ST8, ST72, ST2416); the 13 methicillin-resistant S. epidermidis (MRSE) belonged to ST2 and harboured both cfr and mutations in genes encoding 23S rRNA and ribosomal proteins. Phylogenetic analysis grouped the MRSE isolates into two clusters, one of which (n = 12 isolates) belonged to the recently reported multidrug-resistant worldwide-disseminated S. epidermidis lineages. CONCLUSIONS: The results presented herein highlight the persistence and efficient spread of a cfr-carrying plasmid in a hospital related both to the dissemination of a multidrug-resistant S. epidermidis clone and the in vivo interspecies transfer of cfr between S. epidermidis and S. aureus. The emergence of linezolid-resistant strains should be closely monitored, and the mechanisms involved systematically explored in order to limit the spread of plasmid-mediated resistance.

Epigallocatechin gallate has antibacterial and antibiofilm activity in methicillin resistant and susceptible Staphylococcus aureus of different lineages in non-cytotoxic concentrations.

Staphylococcus aureus is an opportunistic agent that can cause a variety of infections, both hospital and community-acquired. Epigallocatechin gallate (EGCG), a flavonoid present in the leaves of Camellia sinensis, has different biological activities, including antimicrobial potential. Here we evaluate the antibacterial and antibiofilm potential of EGCG in nine clinical strains of S. aureus with different genetic profile and antimicrobial susceptibilities. The minimum inhibitory concentrations (MIC) of EGCG ranged from 7.81 to 62.5 µg/mL, and bactericidal activity was observed at 4 times the MIC. Sub-inhibitory concentrations were able to inhibit biofilm production. Concentrations ≤62.5 µg/mL of EGCG were non-cytotoxic for murine macrophages. EGCG significantly reduced the mortality of infected Galleria mellonella larvae with the S. aureus, having shown relevant antibiofilm properties and efficacy in inhibiting the growth of different clinical isolates of S. aureus, thus being a promising substance for the treatment of infections caused by this agent.

Molecular epidemiology of methicillin resistant Staphylococcus species in healthcare workers of a blood bank in the Brazilian Amazon.

BACKGROUND: Healthcare workers are susceptible to colonization by multiresistant bacteria, which can increase the risk of outbreaks. METHODS: Samples were collected from the nasopharynx, hands, and lab coats of healthcare workers. The phenotypic identification was carried out using a VITEK®2 rapid test system. PCR tests for the mecA gene and the sequencing of the amplicons were performed. Staphylococcus epidermidis and Staphylococcus aureus phylogenies were reconstructed using the Bayesian inference. RESULTS: A total of 225 healthcare workers participated in this study. Of these, 21.3% were male and 78.7% female. S. epidermidis and S.aureus showed high levels of resistance to penicillin, ampicillin, erythromycin, tetracycline and cefoxitin. The prevalence of methicillin resistant S. aureus was 3.16% and methicillin resistant S. epidermidis was 100%. Multilocus sequence typing identified 23 new S. epidermidis sequence types, and one new allele and sequence type for S. aureus. The frequency of methicillin-resistant S. epidermidis in nursing and hemotherapy technicians as a percentage of the total number of healthcare workers was 5.8-3.1%, while the frequency of methicillin resistant S. aureus in hemotherapy technicians and biomedics, as a percentage of the total number of healthcare workers was 4.2-8.9%%. CONCLUSIONS: The healthcare workers at the city's blood bank, even when taking the necessary care with their hands, body and clothes, harbour methicillin-resistant S. aureus and S. epidermidis sequence types, which, as a potential source of multidrug resistant bacteria, can contribute to nosocomial infections among hematological patients.

Effectiveness of vancomycin plus cloxacillin compared with vancomycin, cloxacillin and daptomycin single therapies in the treatment of methicillin-resistant and methicillin-susceptible Staphylococcus aureus in a rabbit model of experimental endocarditis.

OBJECTIVES: To investigate if the addition of cloxacillin to vancomycin enhances the activity of both monotherapies for treating MSSA and MRSA experimental endocarditis (EE) in rabbits. METHODS: Vancomycin plus cloxacillin was compared with the respective monotherapies and daptomycin. In vitro time-kill studies were performed using standard (105 cfu) and high (108 cfu) inocula of five MRSA, one glycopeptide-intermediate (GISA) and five MSSA strains. One MSSA (MSSA-678) and one MRSA (MRSA-277) strain were selected to be used in the in vivo model. A human-like pharmacokinetics model was applied and the equivalents of cloxacillin 2 g/4 h IV and daptomycin 6 mg/kg/day IV were administered. To optimize vancomycin activity, dosage was adjusted to achieve an AUC/MIC ≥400. RESULTS: Daptomycin sterilized significantly more vegetations than cloxacillin (13/13, 100% versus 9/15, 60%; P = 0.02) and showed a trend of better activity than vancomycin (10/14, 71%; P = 0.09) and vancomycin plus cloxacillin (10/14, 71%; P = 0.09) against MSSA-678. Addition of cloxacillin to vancomycin (13/15, 87%) was significantly more effective than vancomycin (8/16, 50%; P = 0.05) and showed similar activity to daptomycin (13/18, 72%; P = 0.6) against MRSA-277. In all treatment arms, the bacterial isolates recovered from vegetations were re-tested and showed the same daptomycin susceptibility as the original strains. CONCLUSIONS: Vancomycin plus cloxacillin proved synergistic and bactericidal activity against MRSA. Daptomycin was the most efficacious option against MSSA and similar to vancomycin plus cloxacillin against MRSA. In settings with high MRSA prevalence, vancomycin plus cloxacillin might be a good alternative for empirical therapy of S. aureus IE.

Comparative genomics of ST5 and ST30 methicillin-resistant Staphylococcus aureus sequential isolates recovered from paediatric patients with cystic fibrosis.

Staphylococcus aureus chronic airway infection in patients with cystic fibrosis (CF) allows this pathogen to adapt over time in response to different selection pressures. We have previously shown that the main sequence types related to community-acquired methicillin-resistant S. aureus (MRSA) infections in Argentina - ST5 and ST30 - are also frequently isolated from the sputum of patients with CF, but in these patients they usually display multi-drug antimicrobial resistance. In this study, we sequenced the genomes of MRSA from four paediatric CF patients with the goal of identifying mutations among sequential isolates, especially those possibly related to antimicrobial resistance and virulence, which might contribute to the adaptation of the pathogen in the airways of patients with CF. Our results revealed genetic differences in sequential MRSA strains isolated from patients with CF in both their core and accessory genomes. Although the genetic adaptation of S. aureus was distinct in different hosts, we detected independent mutations in thyA, htrA, rpsJ and gyrA - which are known to have crucial roles in S. aureus virulence and antimicrobial resistance - in isolates recovered from multiple patients. Moreover, we identified allelic variants that were detected in all of the isolates recovered after a certain time point; these non-synonymous mutations were in genes associated with antimicrobial resistance, virulence, iron scavenging and oxidative stress resistance. In conclusion, our results provide evidence of genetic variability among sequential MRSA isolates that could be implicated in the adaptation of these strains during chronic CF airway infection.