Minimum Inhibitory Concentration (MIC) Analysis and Susceptibility Testing of MRSA

BackgroundMethicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen associated with nosocomial and community infections. Panton Valentine leukocidin (PVL) is considered one of the important virulence factors of S. aureus responsible for destruction of white blood cells, necrosis and apoptosis and as a marker of community acquired MRSA. This study was aimed to determine the prevalence of PVL genes among MRSA isolates and to check the reliability of PVL as marker of community acquired MRSA isolates from Western Nepal.MethodsA total of 400 strains of S. aureus were collected from clinical specimens and various units (Operation Theater, Intensive Care Units) of the hospital and 139 of these had been confirmed as MRSA by previous study. Multiplex PCR was used to detect mecA and PVL genes. Clinical data as well as antimicrobial susceptibility data was analyzed and compared among PVL positive and negative MRSA isolates.ResultsOut of 139 MRSA isolates, 79 (56.8 %) were PVL positive. The majority of the community acquired MRSA (90.4 %) were PVL positive (Positive predictive value: 94.9 % and negative predictive value: 86.6 %), while PVL was detected only in 4 (7.1 %) hospital associated MRSA strains. None of the MRSA isolates from hospital environment was found positive for the PVL genes. The majority of the PVL positive strains (75.5 %) were isolated from pus samples. Antibiotic resistance among PVL negative MRSA isolates was found higher as compared to PVL positive MRSA.ConclusionOur study showed high prevalence of PVL among community acquired MRSA isolates. Absence of PVL among MRSA isolates from hospital environment indicates its poor association with hospital acquired MRSA and therefore, PVL may be used a marker for community acquired MRSA. This is first study from Nepal, to test PVL among MRSA isolates from hospital environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1531-1) contains supplementary material, which is available to authorized users.

Antimicrobial Resistance in the Intensive Care Unit: Mechanisms, Epidemiology, and Management of Specific Resistant Pathogens

Antibiotic resistance is a cause of concern worldwide. Community settings are important reservoir of drug resistant microorganisms like Staphylococcus aureus. The present study was to determine the prevalence, phenotypic and genotypic antibiotic resistance pattern of S. aureus isolated from different community settings of university campus. A total of 300 swab samples were collected for isolation of S. aureus from different community settings at university campus of Guru Angad Dev Veterinary and Animal Sciences University and Punjab Agriculture University, Ludhiana, India. Confirmed S. aureus isolates were further subjected to antibiotic sensitivity by Epsilometer test (E-test) and detection of antibiotic resistance genes. The prevalence of S. aureus in the community samples was 12% (36/300). Methicillin Resistant S. aureus (MRSA) contamination among community was 3.33% (10/300). Among S. aureus isolates from community samples 63.8% (23/36) and all the MRSA isolates were multidrug resistant (MDR). Five out of 10 MRSA carried SCCmec type IVa, and 4 were pvl positive gene, therefore, designated as community associated MRSA (CA-MRSA). Phenotypic resistance to antibiotics ciprofloxacin, chloramphenicol, ceftriaxone clindamycin and trimethoprim-sulfamethoxazole was 69.4% (MIC ≥32 μg/ml), 63.9% (MIC 32 μg/ ml), 16.7% (MIC 16–64 μg/ml), 16.7% (MIC 256 μg/ml) and 8.3% (MIC 12–64 μg/ml), respectively. Resistance genes blaZ, mecA, tetK, tetM, ermB and aacA-aphD were present. Presence of MRSA and MDR variant in community settings is a public health concern, as cell phone, offices telephone, computer keyboard and tap faucet are commonly shared or touched by people. Therefore, have potential to disseminate widely, not only in the community settings but also in hospitals environment, complicating treatment.

Trends in Methicillin resistant staphylococcus aureus (MRSA) minimal inhibitory concentration (MIC) to Vancomycin over a 2 year period in a community based hospital

A comparison of previous antibiotic therapy following isolation of MRSA versus MSSA in nursing home residents: a preliminary investigation.

357: MRSA Contamination of Emergency Department Ultrasonography Probes

Introduction: Staphylococcus aureus infections in current times have become challenging to treat because of advent of Methicillin Resistant Staphylococcus aureus (MRSA) strains which are concurrently resistant to a wide panel of drugs and posing a threat to clinicians and microbiologists globally. The optimal drug for treatment of such MRSA infections is vancomycin but strains with augmented Minimum Inhibitory concentration (MIC) for this drug also have surfaced.Objectives: To know the frequency of MRSA isolates in various clinical samples with their antimicrobial sensitivity patterns and to equate agar dilution and E-test methods for MIC determination of vancomycin to MRSA strains.Materials and Methods: A total of 50 non repeat clinical isolates of staphylococcus aureus isolates were collected from various clinical specimens and were tested for methicillin resistance using the cefoxitin disc diffusion test (30mg). All MRSA isolates were tested for specific MIC by agar dilution and E-test methods. Results: 29 (58%) isolates were resistant to cefoxitin (MRSA). 13.8% isolates had MIC of 4mg/ml for vancomycin (VISA) by both agar dilution and E-test methods. However by agar dilution method 25 (86.2%) isolates exhibited vancomycin MIC of ? 2 mg/ml and by E-test 68.9% of the isolates showed MIC ? 2 mg/ml.Conclusion: Multidrug resistant MRSA strains are on the rise and alternate drug of choice for these infections; vancomycin also is showing increased MIC so prudent use of this drug is advocated. E-test can detect MRSA strains with intermediate MIC values useful for detection of MIC creep so that vancomycin can be used rationally.

Synergism Effects of Vancomycin and Zinc Oxide Nanoparticles on Methicillin Resistance Staphylococcus aureus (MRSA) and Lung CancerBackground: Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen and a historically emerging zoonotic pathogen of public health and veterinary importance.It can cause severe chronic infections.The morbidity of MRSA infections has increased worldwide and is of great concern.Nevertheless, a change in treatment strategies, including the use of new antibiotics or combination therapy, is necessary for the treatment of this infection.The research investigated the synergistic effects of vancomycin and zinc oxide on MRSA and the viability of the lung cancer cell line A549 and the normal cell line BEAS. Materials and Methods:In this study, the minimum inhibitory concentration (MIC) of zinc oxide nanoparticles (ZnO-NPs) and vancomycin was determined using the microdilution method.The fractional inhibitory concentration index (FICI) was calculated using the checkerboard method to evaluate the synergistic effect of ZnO-NPs and vancomycin.The effect of the combination of ZnO-NPs and vancomycin on the viability of lung cancer cell line A549 was also tested by MTT assay. Results:The MIC values showed that all isolates were sensitive to vancomycin with the exception ofexcept for one isolate with an MIC of 2 g/mL.The synergistic effect of the combination of ZnO NPs and vancomycin was observed in two MRSA isolates and one MSSA strain using the checkerboard methodUsing the checkerboard method, the synergistic effect of the combination of ZnO-NPs and vancomycin was observed in two MRSA isolates and one MSSA strain.The combination of vancomycin and ZnO NPs caused less viability in the A549 lung cancer cell line (25.7%)than in BEAS (90%). Conclusion:Combining vancomycin and ZnO-NPs at appropriate dosage intervals may be beneficial in treating MRSA.The combination of vancomycin and ZnO-NPs may also play a dual role in lung cancer patients with evidence of resistance to MRSA by reducing cancer cell survival.

To identify the staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus (MRSA) isolated from bovine milk, and examine the genetic relatedness between MRSA from bovine milk and MRSA from human isolates. Antimicrobial susceptibility tests were performed on MRSA isolated from bovine milk. PCR and sequencing analysis were performed to determine the SCCmec type of MRSA, and to confirm their toxin carriage. Genetic relatedness among the bovine isolates and between bovine and human isolates was detected with PFGE and multilocus sequence typing (MLST). Fourteen MRSA and a silent mecA-carrying methicillin-susceptible S. aureus (smMSSA) were isolated from the milk of cows with an isolation ratio of 0.18%. SCCmec of 14 MRSA strains were designated as new subtype IVg, and one smMSSA strain was not classified. All 14 MRSA strains shared Panton-Valentine leucocidin (PVL) and staphylococcal enterotoxin D (SED), SEI and SEJ; the smMSSA strain had only PVL. All MRSA and smMSSA isolates showed no multidrug resistance and had community-acquired MRSA (CA-MRSA) characteristics. PFGE revealed that all isolates except the smMSSA belonged to the same genetic lineage, and MLST analysis showed that they had no genetic relatedness with CA-MRSA which had caused human infection in Korea. MRSA isolated from bovine milk harboured a unique SCCmec subtype, and they may not be correlated with the emergence of CA-MRSA in human infection in Korea.

BackgroundAs part of active surveillance in our NICU for methicillin-resistant Staphylococcus aureus (MRSA), two isolates representing modified S. aureus (MODSA), which are Methicillin resistant but lacking mecA or C were identified. Our current microbiology laboratory workflow for screening for MRSA involves plating isolates on chromoID agar (bioMérieux, Marcy-l’Etoile, France) as well as on sheep blood agar (SBA). Β hemolytic colonies on SBA that are catalase and coagulase positive are set up for confirmation and antimicrobial susceptibility testing on the Vitek 2 (bioMérieux, Marcy-l’Etoile, France).MethodsThese 2 isolates (from Baby 1 and Baby 2) tested positive for green colonies on the chromoagar plates. The Vitek 2 subsequently identified both these isolates as MRSA. However, for research purposes, all positive NICU MRSA isolates are tested via whole-genome sequencing (WGS). Both isolates were identified by WGS as methicillin-susceptible Staphylococcus aureus (MSSA). We subsequently went back and performed additional workup on these isolates. Isolates were plated on SBA and chromagar again and incubated for 24 hours. 2 colonies of different morphologies from the chromagar plates and 3 from the SBA were randomly selected and subcultured to chromagar and SBA for a total of 5 subcultures. Each of the subcultures was tested using staphaurex, mannitol salt agar and the Cepheid Xpert MRSA assay and all were confirmed to be Staphylococcus aureus.ResultsAll 10 isolates tested negative by the Cepheid Xpert MRSA assay for MRSA. Phenotypic testing was set up again for all ten isolates using the vitek GP panel, as well as cefoxitin disk, and oxacillin E test using Mueller–Hinton agar supplemented with 2% NaCl as per CLSI methods. See table attached for results.ConclusionIn conclusion, these two cases highlight the difficulty in identifying non-MecA, non-MecC-mediated MRSA isolates in the clinical microbiology laboratory. This is particularly important as more laboratories rely on testing for MecA by PCR for surveillance testing. These 2 cases were further complicated by heterogeneous sub-populations of Staphylococcus aureus. Failure to recognize these variant forms of MRSA can lead to difficulties in implementing appropriate therapy and infection control measures. Improved methodologies are needed.DisclosuresAll authors: No reported disclosures.

To provide evidence of the cross-contamination of emerging pathogenic microbes in a local network between long-term care facilities (LTCFs) and hospitals, this study emphasizes the molecular typing, the prevalence of virulence genes, and the antibiotic resistance pattern of methicillin-resistant Staphylococcus aureus. MRSA isolates were characterized from 246 samples collected from LTCFs, medical tubes of LTCF residents, and hospital environments of two cities, Chiayi and Changhua. Species identification, molecular characterization, and drug resistance analysis were performed. Hospital environments had a higher MRSA detection rate than that of LTCF environments, where moist samples are a hotspot of MRSA habitats, including tube samples from LTCF residents. All MRSA isolates in this study carried the exfoliative toxin eta gene (100%). The majority of MRSA isolates were resistant to erythromycin (76.7%), gentamicin (60%), and ciprofloxacin (55%). The percentage of multidrug-resistant MRSA isolates was approximately 50%. The enterobacterial repetitive intergenic consensus polymerase chain reaction results showed that 18 MRSA isolates belonged to a specific cluster. This implied that genetically similar isolates were spread between hospitals and LTCFs in Changhua city. This study highlights the threat to the health of LTCFs’ residents posed by hospital contact with MRSA.

Methicillin resistance in Staphylococcus aureus is associated with multidrug resistance, an aggressive course, increased mortality and morbidity in both community and health care facilities. Monitoring of newly emerging and prevalent Methicillin Resistant Staphylococcus aureus (MRSA) strains for their resistance patterns to conventional as well as novel drugs, are essential for infection control. To study the changing trends in resistance patterns of MRSA at our hospital. This cross sectional study was carried out in a 750 bed tertiary care hospital in south India. One hundred and two clinical isolates of MRSA which were obtained in 2004-2011 were identified by using oxacillin, cefoxitin disc diffusion test and oxacillin screening agar test. Antibiotic susceptibility test was done for commonly used non beta lactam anti-Staphylococcal drugs, as well as for anti-MRSA drugs like vancomycin, linezolid, mupirocin and rifampicin. Minimum inhibitory concentration (MIC) of vancomycin was determined by using Vancomycin HiComb strip (Himedia, Mumbai, India). Statistical Analysis which was done: Chi-square test and proportions were used to compare the two groups. MRSA isolates showed high resistance to co-trimoxazole (82.3%), ciprofloxacin (76.4%), gentamicin (64.7%) and tetracycline (49%) as compared to other drugs. High prevalence of ciprofloxacin resistance was detected, particularly among outpatients. Multi resistant MRSA with a ≥ 3 non-beta lactam agent resistance was 79%. All MRSA isolates were sensitive to vancomycin, linezolid, mupirocin and rifampicin. MRSA had displayed increase in resistance to most antibiotics except tetracycline in recent years. Taking into consideration the prevalence of multidrug resistance in MRSA, resistance patterns should be evaluated periodically and antibiotic therapy should be guided by susceptibility testing.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) are pathogens that have major negative impact in all aspects of patient care and are increasingly being recognized as troublesome pathogens in the community. The MRSA are multi-drug resistant bacteria responsible for higher morbidity, mortality and cost of treatment in the affected patients. Research on MRSA antimicrobial susceptibility and resistance in developing countries is limited; hence accurate burden is not certain. Objective: To determine the prevalence and susceptibility pattern of MRSA in S. aureus isolates from patients treated in various units at Moi Teaching and Referral Hospital. Study Design: Cross-sectional design was used to conduct the study. Methods and Materials: Staphylococcus aureus stored isolates from patients at various units at Moi Teaching and Referral Hospital (MTRH) were identified by laboratory conventional methods. Methicillin resistant Staphylococcus aureus was determined using cefoxitin and oxacillin, discs and confirmed by Penicillin Binding Protein (PBP2a’) latex agglutination test. Susceptibility testing was performed according to the Clinical Laboratory Standards Institute procedures. Control strains used were: ATCC 43300 for Methicillin resistant S. aureus (MRSA) strains and ATCC 29213 for methicillin susceptible S. aureus (MSSA) strains. Nine anti-microbials (Erythromycin, Gentamycin, Tetracycline, Vancomycin, Rifampin, Linezolid, Ciprofloxacin, Clindamycin and Fucidicacid) were used to test the anti-microbial susceptibility patterns. Control organisms were run along with the isolates for quality Assurance. The data was analyzed using STATA version 11. Categorical variables were summarized as frequencies and the corresponding percentages. Results were presented using tables and graphs. Results: A total of 107 isolates of S. aureus were obtained, of which 39 (37%) were MRSA. Most of the MRSA, 13 (33%) and 7 (17%), were found in pus and tracheal aspirate samples respectively. Majority of the MRSA isolates were from surgical wards and intensive care unit. The MRSA isolates were highly resistant to erythromycin (92%; 36/39) and tetracycline (92% 36/39) and moderately susceptible to linezolid (77% 30/39), Vancomycin (75% 29/39) and fucidic acid (67% 26/39). A total of 28 (74.4%) MRSA isolates were Clindamycin inducible resistant. Conclusions and Recommendations: This study showed that MRSA is a significant pathogen at MTRH. The MRSA were most detected in pus specimens and least in peritoneal aspirates. Routine Screening of S. aureus isolates for MRSA strains at MTRH is therefore imperative. Infection control measures should be put in place to reduce prevalence and prevent the spread of MRSA at MTRH.

Impact of vancomycin MIC creep on patients with methicillin-resistant Staphylococcus aureus bacteremia

Simple SummaryThe increasing observation of methicillin-resistant Staphylococcus aureus (MRSA) in wildlife species has raised the concern of its impact on the animal health and the potential of zoonotic transmission. The molecular types of MRSA and S. aureus in wild animals has been found to mix with several molecular types from humans and livestock, indicating a dynamics of S. aureus transmission between these animals and humans. Thus, it is important to investigate the epidemiological relationship of MRSA between zoo animals and humans. To investigate the prevalence and transmission of S. aureus in non-human primates and their living environment, we conducted this study on all the non-human primates in a zoo park located in eastern China. Fecal samples from non-human primates and samples from their living environments were selectively enriched and conducted for S. aureus isolation. The whole genome analysis showed that these two MRSA t034/CC398 isolates were found from a monkey fecal sample and its living environment. The virulence factor analysis showed that these two MRSA isolates carried virulence factors relating to the cell adherence, biofilm formation, toxins, and human-associated immune evasion cluster. The further phylogenetic analysis revealed that these two MRSA isolates have a close genomic relationship with the human-associated clinical isolates from China, which indicates a potential risk of bidirectional transfer of MRSA between monkeys and humans.Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important antibiotic resistant pathogens causing infections in humans and animals. The increasing observation of MRSA in wildlife species has raised the concern of its impact on animal health and the potential of zoonotic transmission. This study investigated the prevalence of S. aureus in fecal samples from non-human primates in a zoo located in Jiangsu, China, in which 6 out of 31 (19.4%) fecal samples, and 2 out of 14 (14.3%) indoor room floor swab samples were S. aureus-positive. The antibiotic susceptibility tests of the eight isolates showed that the two isolates were resistant to both penicillin and cefoxitin, the three isolates were resistant only to penicillin, while three isolates were susceptible to all detected antibiotics. The two isolates resistant to cefoxitin were further identified as MRSA by the presence of mecA. Five different spa types were identified including t034 of two MRSA isolates from Trachypithecus francoisi, t189 of two methicillin-susceptible S. aureus (MSSA) isolates from Rhinopithecus roxellana, t377 of two MSSA isolates from Colobus guereza, and two novel spa types t19488 and t19499 from Papio anubis. Whole genome sequencing analysis showed that MRSA t034 isolates belonged to ST398 clustered in clonal complex 398 (CC398) and carried the type B ΦSa3 prophage. The phylogenetic analysis showed that the two MRSA t034/ST398 isolates were closely related to the human-associated MSSA in China. Moreover, two MRSA isolates contained the virulence genes relating to the cell adherence, biofilm formation, toxins, and the human-associated immune evasion cluster, which indicated the potential of bidirectional transfer of MRSA between monkeys and humans. This study is the first to report MRSA CC398 from monkey feces in China, indicating that MRSA CC398 could colonize in monkey and have the risk of transmission between humans and monkeys.