Metabolic remodeling by RNA polymerase gene mutations is associated with reduced ß-lactam susceptibility in oxacillin-susceptible MRSA.

The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to ß-lactam antibiotics, these strains can easily acquire reduced ß-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced ß-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. IMPORTANCE: The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to ß-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced ß-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Phenotypic and genotypic characteristics of mecA - positive oxacillin-sensitive Staphylococcus aureus isolated from patients with bloodstream infection in a tertiary hospital in Southern Brazil.

Staphylococcus aureus are extremely important microorganisms, either from an epidemiological point of view or as a pathogen, responsible for causing a series of infectious processes, whether simple, restricted to the skin, or invasive infections such as bacteremia. The emergence of Oxacillin Sensitive-Methicillin Resistant S.aureus (OS-MRSA) isolates has imposed difficulties in the treatment of patients with staphylococcal infection, as such isolates can be mistakenly classified as sensitive and lead to failure of the therapy used. Thus, the objective of this study is to evaluate the prevalence, and genotypic and phenotypic characteristics, of OS-MRSA isolates, from bloodstream infections, collected from patients admitted to a hospital in southern Brazil, as well as to evaluate the treatment used. For this, 801 unique isolates of S. aureus, collected from blood cultures, between January 2011 and December 2020 were evaluated. Of these, 96 isolates were classified as sensitive to oxacillin. The isolates were identified and had their sensitivity profile performed by manual and automated methods. The minimum inhibitory concentration for vancomycin, daptomycin, oxacillin, linezolid and teicoplanine was performed by e-test. The mecA, vanA genes, typing of the SCCmec elements, as well as the search for the icaA, tst-1 and pvl virulence genes were performed by PCR. Biofilm formation was performed using the crystal violet technique. The Sequence Type (ST), as well as the Clonal Complex (CC) of the isolates was evaluated by the RTq -PCR. The clinical characteristics of the patients were evaluated through an active search in medical records. After investigating the mecA gene, 27.1% (26/96) of the isolates were considered OS-MRSA, with SCCmec type I being the most prevalent, 46.1% (12/26). Among the evaluated isolates, 41% (9/22) were included in CC5 and ST9. As for virulence, all isolates were positive for the icaA gene and characterized as strong biofilm formers. The pvl gene was found in 92.3% (24/26) of the isolates and the toxic shock syndrome toxin was present in 61.5% of the isolates (16/26). All isolates were negative for the presence of the van A gene. As for the clinical outcome, 73% (19/26) of the patients were discharged from the hospital and 27% (7/26) died. It was possible to observe a high frequency of OS-MRSA isolates causing bloodstream infections. Furthermore, such isolates contain several virulence genes, which may contribute to a worse clinical outcome.

Phenotypic and genomic characteristics of oxacillin-susceptible mecA-positive Staphylococcus aureus, rapid selection of high-level resistance to beta-lactams.

The aim of this study is to describe the phenotypic and genetic properties of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) isolates and their beta-lactam resistant derivatives obtained after selection with oxacillin. A collection of hospital- (HA-) and community-acquired (CA-) MRSA was screened for oxacillin susceptibility. Antibiotic susceptibility testing, population analysis profile (PAP), mecA expression analysis, and whole genome sequencing (WGS) were performed for 60 mecA-positive OS-MRSA isolates. Twelve high-level beta-lactam resistant derivatives selected during PAP were also subjected to WGS. OS-MRSA were more prevalent among CA-MRSA (49/205, 24%) than among HA-MRSA (11/575, 2%). OS-MRSA isolates belonged to twelve sequence types (ST), with a predominance of ST22-t223-SCCmec IVc and ST59-t1950-SCCmec V lineages. OS-MRSA were characterized by mecA promoter mutations at - 33 (C→T) or - 7 (G→T/A) along with PBP2a substitutions (S225R or E246G). The basal and oxacillin-induced levels of mecA expression in OS-MRSA isolates were significantly lower than those in control ST8-HA-MRSA isolates. Most of the OS-MRSA isolates were heteroresistant to oxacillin. High-level beta-lactam resistant OS-MRSA derivatives selected with oxacillin carried mutations in mecA auxiliary factors: relA (metabolism of purines), tyrS, cysS (metabolism of tRNAs), aroK, cysE (metabolism of amino acids and glycolysis). Cefoxitin-based tests demonstrated high specificity for OS-MRSA detection. The highest positive predictive values (PPV > 0.95) were observed for broth microdilution, the VITEK® 2 automatic system, and chromogenic media. Susceptibility testing of CA-MRSA requires special attention due to the high prevalence of difficult-to-detect OS-MRSA among them. Mis-prescription of beta-lactams for the treatment of OS-MRSA may lead to selection of high-level resistance and treatment failures.

Occurrence of antimicrobial-resistant Staphylococcus aureus in a Brazilian veterinary hospital environment.

Antimicrobial resistance is a threat to public health. The emergence of antibiotic-resistant Staphylococcus aureus represents a priority for the implementation of preventive measures. The objective was to isolate S. aureus in humans, animals, and animal health care environment, and to characterize the genotypic and phenotypic profile of antimicrobial resistance in these isolates. We isolated S. aureus from staff, animals, and environment of a veterinary hospital, and identified their antimicrobial resistance profiles. Samples were collected from 20 humans, 13 animals, 14 surfaces, 8 mobile phones, and 7 veterinarians' stethoscopes by using sterile swabs. S. aureus was isolated by culturing on mannitol salt agar and preliminary identification was done by Gram staining and catalase test. Subsequently, a polymerase chain reaction was performed for species confirmation and investigating their antimicrobial-resistant genotypic profiles. Phenotypic profiles of resistant isolates were determined using the disk-diffusion technique. Ten S. aureus isolates were recovered from 5/20 humans (25%), it was also recovered from 2/13 animals (15.38%), including 1 dog and 1 cat, and from 1/14 of surfaces (7.14%). The oxacillin-susceptible mecA-positive Staphylococcus aureus phenotype was identified in a feline. Most of the isolates carried at least two resistance genes of different antimicrobial classes, with 90% (9/10) presenting the gene blaZ, with 10% (1/10) presenting the gene mecA, 20% (2/10) presenting tet38, 10% (1/10) presenting tetM, 90% (9/10) presenting norA, 50% (5/10) presenting norC, 10% (1/10) presenting ermA, and 60% (6/10) presenting ermB. In antibiograms, resistance to penicillin was identified in all the isolates, resistance to erythromycin was identified in 80% (8/10), and all the isolate's resistance to erythromycin presented erythromycin-induced resistance to clindamycin. Antimicrobial resistance in the veterinary hospital requires attention due to the risk of interspecies transmission, gene transfer between bacteria that colonize companion animals and humans and, can make antimicrobial therapy difficult.

Phenotypic and genotypic characterization of oxacillin-susceptible and mecA positive Staphylococcus aureus strains isolated in Uruguay / Caracterización fenotípica y genotípica de cepas de Staphylococcus aureus sensibles a oxacilina y mecA positivas aisladas en Uruguay

Abstract The aim of this study was to characterize phenotypically and genotypically 27 mecApositive Staphylococcus aureus strains with oxacillin MICs of ≤2 g/ml by Vitek 2, isolated indifferent regions of Uruguay. Susceptibility to oxacillin and cefoxitin was studied by gradient dif-fusion, disk diffusion to cefoxitin, and Phoenix and MicroScan systems. PBP2a was determined.SCCmec typing was performed and the isolates were compared by PFGE. Twenty-six isolateswere susceptible to oxacillin; one strain was susceptible to cefoxitin by disk diffusion and 3strains by gradient diffusion. Phoenix and MicroScan panels detected methicillin resistance in25 and 27 strains, respectively. Twenty-six strains tested positive for PBP2a. Twenty-six strainscarried SCCmec V and 24 belonged to pulsotype A. One strain carried SCCmec IV and did notbelong to pulsotype A. Cefoxitin disk diffusion test and PBP2a detection correctly identified 26of these 27 strains as MRSA. PFGE results suggest the dissemination of a cluster of MRSA carryingSCCmec V.

Resumen El objetivo de este estudio fue caracterizar fenotípicamente y genotípicamente 27 cepas de Staphylococcus aureus positivas para mecA y con CIM de oxacilina <2 pg/ml según Vitek 2, obtenidas en diferentes regiones del país. La sensibilidad frente a la oxacilina y la cefoxitina se estudió por difusión en gradiente, por disco-difusión (cefoxitina) y por los sistemas Phoenix y MicroScan. Se analizó la portación de PBP2a, se realizó la tipificación de SCCmec y las cepas se compararon mediante PFGE. Resultaron sensibles a oxacilina por difusión en gradiente 26 cepas; una fue sensible a cefoxitina por disco-difusión y 3 lo fueron por difusión en gradiente. Los sistemas Phoenix y MicroScan detectaron resistencia a meticilina en 25 y 27 cepas, respectivamente. Asimismo, 26 cepas portaban PBP2a y 26 cepas mostraron presencia de SCCmec V, 24 correspondieron al pulsotipo A. Una portaba SCCmec IV y no perteneció al pulsotipo A. La prueba de disco-difusión con cefoxitina y la detección de PBP2a identificaron 26 de 27 cepas como MRSA. La PFGE sugiere la diseminación de un grupo MRSA con SCCmec V. © 2022 Asociación Argentina de Microbiología. Publicado por Elsevier Espana, S.L.U. Este es un artículo Open Access bajo la licencia CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Phenotypic and genotypic characterization of oxacillin-susceptible and mecA positive Staphylococcus aureus strains isolated in Uruguay.

The aim of this study was to characterize phenotypically and genotypically 27 mecA positive Staphylococcus aureus strains with oxacillin MICs of ≤2µg/ml by Vitek 2, isolated in different regions of Uruguay. Susceptibility to oxacillin and cefoxitin was studied by gradient diffusion, disk diffusion to cefoxitin, and Phoenix and MicroScan systems. PBP2a was determined. SCCmec typing was performed and the isolates were compared by PFGE. Twenty-six isolates were susceptible to oxacillin; one strain was susceptible to cefoxitin by disk diffusion and 3 strains by gradient diffusion. Phoenix and MicroScan panels detected methicillin resistance in 25 and 27 strains, respectively. Twenty-six strains tested positive for PBP2a. Twenty-six strains carried SCCmec V and 24 belonged to pulsotype A. One strain carried SCCmec IV and did not belong to pulsotype A. Cefoxitin disk diffusion test and PBP2a detection correctly identified 26 of these 27 strains as MRSA. PFGE results suggest the dissemination of a cluster of MRSA carrying SCCmec V.

First Report of Oxacillin Susceptible mecA-Positive Staphylococcus aureus in a Children's Hospital in Kunming, China.

PURPOSE: The present study investigated the prevalence characteristics of oxacillin susceptible mecA-positive Staphylococcus aureus (OS-MRSA) in a children's hospital in Kunming from January 2019 to December 2020. METHODS: A total of 499 S. aureus strains were included in the study and tested for oxacillin susceptibility using the VITEK 2 Compact automated antimicrobial susceptibility test system. All oxacillin-susceptible strains were detected mecA and mecC by polymerase chain reaction (PCR). E-test was used to compare the minimum inhibitory concentration (MIC) values of methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and OS-MRSA for oxacillin, cefoxitin, penicillin, vancomycin, erythromycin, and clindamycin. Molecular typing of OS-MRSA was performed by MLST and SCCmec typing. Toxin genes were detected by PCR. RESULTS: Forty-five OS-MRSA strains were detected, for an overall rate of 9.02% (45/499). The MICs of MSSA, OS-MRSA, and MRSA against oxacillin were concentrated at 0.38, 0.38, and 12 µg/mL, respectively; the cefoxitin MICs of MSSA and MRSA were concentrated at 2 and 32 µg/mL respectively; and MICs of OS-MRSA were concentrated at 2 and 8 µg/mL; penicillin, vancomycin and erythromycin MICs against MSSA, OS-MRSA, and MRSA showed same centralized points and were 32, 1, and 256 µg/mL, respectively; the MICs of clindamycin against MSSA were 0.5 µg/mL, while that against OS-MRSA and MRSA were concentrated at 256 µg/mL. Molecular typing of OS-MRSA was dominated by ST59-SCCmec IV. The carrier rates of hemolysin genes (hl-a, hl-d) and fibrinogen-binding clumping factor genes (clfA, clfB) were 100% in OS-MRSA, followed by 40% (18/45) for enterotoxin genes (sea, seb). CONCLUSION: OS-MRSA has a high detection rate in children, and main molecular typing is ST59-SCCmecIV in Kunming. The identification ability of automated antibacterial drug sensitivity test detection systems for OS-MRSA is very limited. A combination of phenotypic analysis and molecular detection should be used to improve OS-MRSA identification.

Comparison of Phenotypic MRSA Detection Methods with PCR for mecA Gene in the Background of Emergence of Oxacillin-Susceptible MRSA.

Background: Phenotypic methods for detection of methicillin resistance in Staphylococcus aureus (MRSA) can be inaccurate due to heterogeneous expression of resistance and due to environmental factors that influence the expression of resistance. This study aims to compare various phenotypic methods of detection of methicillin resistance with polymerase chain reaction (PCR) for mecA gene and to detect the presence of oxacillin-susceptible MRSA (OS-MRSA). Materials and Methods: A total of 150 S. aureus isolates were tested using cefoxitin disk diffusion, oxacillin salt agar (OSA), latex agglutination test for penicillin binding protein 2a antigen, chromogenic MRSA ID agar, and mecA PCR. Results: Using PCR as the gold standard, 91 (60.66%) of 150 clinical S. aureus strains were identified as MRSA. Three oxacillin-susceptible (minimum inhibitory concentration ≤2 µg/mL) mecA-positive isolates were classified as OS-MRSA. Among the different phenotypic MRSA detection methods studied, latex agglutination had the highest sensitivity and specificity (98.9% and 98.3%), followed by cefoxitin disk diffusion (95.6% and 98.3%), MRSA ID (97.8% and 83.05%), and OSA (86.81% and 94.92%). Conclusion: The sensitivity of cefoxitin disk diffusion method may be reduced in areas with a high prevalence of OS-MRSA where a combination of cefoxitin disk diffusion test with MRSA ID agar or latex agglutination is recommended.

Current status of oxacillin-susceptible mecA-positive Staphylococcus aureus infection in Shanghai, China: A multicenter study.

BACKGROUND: Oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) represents an important issue, as its oxacillin susceptibility has contributed to misidentification by conventional susceptibility tests and consequently potential therapeutic failure, but limited data on the current status of OS-MRSA infection in Chinese hospitals are available. METHODS: This multicenter study performed a battery of susceptibility tests and diagnostic tests for 956 S. aureus isolates from 10 hospitals, including automated susceptibility testing on VITEK 2, broth microdilution, disk diffusion, and detection of PBB2a, mecA gene and mecC gene. For all identified OS-MRSA, multi-locus sequence typing (MLST), together with spa typing, SCCmec typing and PVL detecting, was carried out. RESULTS: OS-MRSA, most of which were from pediatric inpatients, represented 1.8% (17/956) of total isolates. Of these 17 OS-MRSA, 10 were ST59, followed by ST965 (3/17), and 11 carried SCCmec type IV, while 5 carried SCCmec type V, but only one was Panton-Valentine leucocidin (PVL)-positive, also, 16 had one or two point mutations within mecA promoter. OS-MRSA had inducible oxacillin resistance and significantly lower MDR (Multi-Drug Resistant) rate. We observed that the VITEK 2 system exhibited some deficiency in OS-MRSA detection, whereas cefoxitin disk diffusion was shown to be a reliable and cost-saving alternative and should be supplemented in detecting S. aureus with borderline oxacillin susceptible MICs. CONCLUSION: This study has characterized phenotypically and molecularly OS-MRSA in China, and provided insights into more effective management of OS-MRSA.

Characterization of Oxacillin-Susceptible mecA-Positive Staphylococcus aureus from Food Poisoning Outbreaks and Retail Foods in China.

In this study, we explored the prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) in staphylococcal food poisoning outbreak isolates and foodborne isolates, and then investigated their molecular characteristics, classical staphylococcal enterotoxins (SEs), and drug resistance. Eight (2.9%) of 275 isolates from food poisoning outbreaks and 7 (3.8%) of 184 isolates from retail foods were identified as OS-MRSA isolates. Among the 15 OS-MRSA isolates, the most frequently detected toxin genes were hld (100%), hla (93.3%), pvl (80.0%), and hlb (46.7%) followed by seg and seq (33.3%, each), hlg (26.7%), seb and hlgv (20.0%, each), sec, seh, sel, sep, and tst (13.3%, each), and sei, sem, sen, and seo (6.7%, each). None of isolates carried other tested virulence genes. The most frequently detected classical SEs were SEB and SEC (26.7%, each), followed by SEA and SEE (20.0%, each), and SED (6.7%). Resistance was most frequently observed in ampicillin, penicillin, and cefoxitin (100%, each), followed by trimethoprim/sulfamethoxazole (93.3%), erythromycin (73.3%), amoxicillin/clavulanic acid (46.7%), tetracyclines (26.7%), and ciprofloxacin (6.7%). All isolates were susceptible to other tested antibiotics. A dominant molecular type belonged to ST398-IVa-t034 (26.7%), followed by ST59-IVa-t437 (20.0%), ST88-III-t14340 and ST1-IVa-t114 (13.3%, each), and ST5-II-t002, ST630-t4549, ST5-II, and ST4495-t10738 (6.7%, each). Our findings indicated that OS-MRSA strains had a low prevalence rate among outbreak strains and foodborne strains, which frequently harbored SCCmec IVa, and carried a variety of toxin genes, and also expressed numerous classical SEs. In addition, all OS-MRSA isolates were susceptible to the majority of antibacterial agents except ß-lactam. Our study is the first to report that OS-MRSA isolates are associated with food poisoning outbreaks worldwide.

Dissemination Of t437-SCCmecIV And Coagulase-Negative t037-SCCmecIII Types Among Borderline Oxacillin-Resistant Staphylococcus aureus Isolated From Skin Infections And Diabetic Foot Ulcers.

BACKGROUND: In a recent decade, the occurrence of S. aureus isolates with low-level oxacillin resistance, have been reported increasingly. The aim of this study was to estimate the prevalence of S. aureus with low-level of oxacillin resistance and to determine their molecular characteristics, including spa types, SCCmec types and presence of toxin genes. METHODS: A total of 249 S. aureus strains were analyzed. Antimicrobial susceptibility was preliminarily tested by the disk diffusion method, and further was verified with the E-test and agar dilution methods. All borderline oxacillin-resistant strains (BORSA) were screened for the mecA gene and virulence factors, including Panton-Valentine leukocidin (PVL). Staphylococcal cassette chromosome mec (SCCmec) typing and spa typing were also carried out. RESULTS: Twelve (4.8%) borderline oxacillin-resistant strains with MIC ≤4 µg/mL were identified. Almost all strains (11/12) were oxacillin-susceptible methicillin resistant S. aureus carrying mecA gene (OS-MRSA). Among the 12 bordeline strains, five spa types (t437, t037, t015, t216, t267) and two SCCmec types (III, IV) were identified, with the most prevalent being t437-SCCmecIV pvl-positive. The second most frequent spa type, t037-SCCmecIII, was sea-positive and did not produce coagulase. The majority of borderline strains originated from skin infections and diabetic foot ulcers and were multidrug-resistant (macrolides, lincosamides and chloramphenicol). CONCLUSION: This study demonstrated that S. aureus with borderline resistance to oxacillin represented primarily SCCmecIV spa type t437 and coagulase-negative SCCmecIII spa type t037 and were isolated from skin infections and diabetic foot ulcers.

Oxacillin-susceptible mecA-positive Staphylococcus aureus associated with processed food in Europe.

We report for the first time an oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with a processed food product in Europe. One isolate (MRSA-ST5-type V SCCmec) was found in cheese among 600 food samples confiscated from air passengers from international flights in Vienna Airport (Austria). Type V SCCmec strains do not harbor functional mecI-mecR1 genes and in such strains mecA expression is regulated by the bla system (blaI-blaR1-blaZ). It has been recently reported that malfunctions in the bla system lead to the constitutive expression of mecA. The OS-MRSA reported in this study harbored the bla system on a plasmid and one deletion occurred in the blaR1 gene causing a frameshift variant that lead to an incomplete BlaR1 protein. This finding highlights the potential role of food as a neglected route of dissemination of emerging MRSA variants.

Fatal sepsis caused by mecA-positive oxacillin-susceptible Staphylococcus aureus: First report in a tertiary hospital of southern Brazil.

mecA-positive oxacillin phenotypically susceptible Staphylococcus aureus (OS-MRSA) is increasingly reported worldwide. This bacterium poses a therapeutic threat, as it can be misidentified as an oxacillin-susceptible organism by phenotypic methods that are routinely used in the majority of clinical microbiology laboratories. Herein, we report the first case of fatal sepsis in a 43-year-old female patient caused by an OS-MRSA SCCmec type IVa/ST1/CC1 in a tertiary hospital in southern Brazil, which highlights the difficulties involved in diagnosing this bacterium. Blood cultures and phenotypic susceptibility tests on admission yielded a penicillin-resistant S. aureus. Although vancomycin therapy was initiated, this antibacterial was replaced by oxacillin, based on the susceptibility result. However, the clinical conditions of the patient deteriorated rapidly evolving to fatal septic shock. Clinical microbiology laboratories should consider the use of additional tests to accurately distinguish between various antimicrobial phenotypes of S. aureus.

Short communication: Outbreak of methicillin-resistant Staphylococcus aureus (MRSA)-associated mastitis in a closed dairy herd.

Cows are probably the main source of contamination of raw milk with Staphylococcus aureus. Mammary glands with subclinical mastitis can shed large numbers of Staph. aureus in milk. Because of the risk of this pathogen to human health as well as animal health, the aim of this paper was to describe an outbreak of mastitis caused by methicillin-resistant Staph. aureus (MRSA), oxacillin-susceptible mecA-positive Staph. aureus (OS-MRSA), and methicillin-susceptible Staph. aureus (MSSA) on a dairy farm. Milk samples were obtained from all quarters, showing an elevated somatic cell count by the California Mastitis Test. The isolates were identified by phenotypic and genotypic methods. Staphylococcus spp. were isolated from 53% (61/115) of the milk samples, with 60 isolates identified as Staph. aureus (98.4%) and 1 isolate identified as Staphylococcus epidermidis (1.6%). The presence of the mecA gene was verified in 48.3% of Staph. aureus isolates. Of the Staph. aureus isolates, 23.3% were MRSA and 25.0% were OS-MRSA. The total of mastitis cases infected with MRSA was 12.2%. The detection of this large percentage of mastitis cases caused by MRSA and OS-MRSA is of great concern for the animals' health, because ß-lactams are still the most important antimicrobials used to treat mastitis. In addition, Staph. aureus isolates causing bovine mastitis represent a public health risk.

Methicillin-resistant Staphylococcus aureus isolates with SCCmec type V and spa types t437 or t1081 associated to discordant susceptibility results between oxacillin and cefoxitin, Central Taiwan.

Staphylococcus aureus isolates with discordant susceptibility results between oxacillin and cefoxitin obtained using automated microbiology systems are infrequently observed. From April 2013 to December 2014, 1956 methicillin-resistant S. aureus (MRSA) and 1761 methicillin-susceptible S. aureus isolates were obtained from different patients. Forty isolates (1.1% and 2% in case of S. aureus and MRSA, respectively) with discordant susceptibility results (oxacillin susceptible and cefoxitin resistant) and carrying mecA gene were obtained. Except 2 SCCmec type IV isolates, 38 MRSA isolates were all SCCmec type V (VT or non-VT), which were further divided into VT (n=22) and non-VT (n=16). The most common spa type in VT and non-VT isolates were t437 (n=19) and t1081 (n=13), respectively. Only 55% of patients received effective antimicrobial agents; 2 mortalities were not attributable to MRSA infection. Using standard agar dilution, 17 MRSA isolates (0.46% and 0.87% in case of S. aureus and MRSA, respectively) had oxacillin MIC in the susceptible ranges (oxacillin-susceptible MRSA [OS-MRSA]); all carried SCCmec type V (VT, n=8; non-VT, n=9). The most common spa-MLST types of OS-MRSA in VT and non-VT were t437-ST59 (n=4) and t1081-ST45 (n=7), respectively. Concomitant testing by both cefoxitin- and oxacillin-based methods is a practical strategy for OS-MRSA detection in the clinical laboratories. Continuous monitoring of OS-MRSA isolates is necessary to elucidate their impact in clinical infectious diseases.

Clonal diversity and epidemiological characteristics of Staphylococcus aureus: high prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with clinical isolates in Brazil.

BACKGROUND: Staphylococcus aureus is the major cause of global and nosocomial infections with a significant impact in hospitals worldwide. Our objective was to investigate clinical and molecular characteristics of S. aureus isolates causing infections in patients admitted to hospitals from Recife city, Brazil, and investigate the prevalence of oxacillin-susceptible mecA-positive S. aureus (OS-MRSA) in the region, as well as genetically characterize the isolates and compare with epidemic clones. RESULTS: We characterized 89 isolates in total, 31 clinical methicillin-resistant S. aureus (MRSA) and 58 methicillin-sensitive (MSSA) isolates by PFGE, MLST, spa typing and SCCmec genotyping. Isolates belonging to international MRSA clones were present: Brazilian epidemic clone (BEC) (61 % of MRSA isolates), Paediatric (36 %), New York/Japan (3 %). Some MSSA isolates were related to MRSA clones: USA400-related (10 % of MSSA isolates), Berlin clone (2 %), Paediatric (14 %), New York/Japan (2 %) and Southwest Pacific clone (17 %). MLST revealed new sequence types (ST's): ST2381, ST2382, and ST2383 and new spa types: 10548 and 10550. Among isolates phenotypically identified as MSSA by antimicrobial susceptibility assays, we verified 30 oxacillin-susceptible isolates, which exhibited the mecA gene, without mec complex amplification and were thus classified as OS-MRSA. We observed clonal spread of MRSA and MSSA, including OS-MRSA, within several areas of the main hospital investigated and closely related isolates between hospitals analyzed. CONCLUSIONS: The results of this study suggest a possible spread of the strains in hospital environment that could be responsible for nosocomial infections. We documented the presence of several MRSA clones, as well as new MLST and spa types, that were responsible for severe infections in hospitalized patients. The finding of OS-MRSA isolates could have implications for therapy, because testing for mecA and PBP2a is not a routine procedure performed by clinical microbiology laboratories in Brazil and, as consequence, these isolates could be misclassified as MSSA. Our data alert to the necessity to develop more effective strategies for epidemiological control of S. aureus in order to avoid an increase of hospital infections provoked by this pathogen. We reinforce the use of genetic methods, in addition to phenotypic tests, for a precise identification of MRSA.