Methicillin-resistant Staphylococcus Aureus Phenotype Research Articles

Population Analysis of Daptomycin-non-Susceptible Methicillin-Resistant Staphylococcus aureus Reveals the Presence of Variants That Contribute to Daptomycin Resistance.

In this study, population analysis (PA) of methicillin-resistant Staphylococcus aureus (MRSA), before and after long-duration daptomycin (DAP) treatment, was used to detect subpopulations with different susceptibilities to DAP and to verify the changes in the number of resistant cells. Furthermore, we aimed to characterize the bacteriology of the variants present in the non-susceptible cell subpopulation. A DAP non-susceptible (NS) MRSA phenotype (D2) that emerged from a DAP- susceptible MRSA phenotype (D1) during treatment of an open wound, was used for testing. We performed bacteriological and genetic analyses of cryptic DAP-NS MRSA variants detected by PA to study the variants present in the resistant cell subpopulation. PA results suggest that MRSA adapted to survival in the presence of DAP are selected leading to reduced susceptibility. Within the cell population growing in media containing 2.0mg/L of DAP, three variants with different pigment production and colony size were detected. Variant 3 was an orange colony due to enhanced production of staphyloxanthin. Our results revealed that the DAP minimum inhibitory concentration (MIC) value increased two-fold (4mg/L) in variant 3, in which pigment production was most enhanced, compared to the parental strain D2. In conclusion, our results indicate that long-duration DAP treatment can lead to the emergence and increased proportion of DAP-NS subpopulations. Furthermore, slow-growing variants that can be detected only under antimicrobial selective pressure are present among DAP-NS cells, suggesting that these variants may also contribute to the development of DAP resistance.

PHENOTYPIC DETERMINATION OF INDUCIBLE CLINDAMYCIN RESISTANT AND METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS FROM CLINICAL ISOLATES OF KHYBER TEACHING HOSPITAL, PESHAWAR

Methicillin Resistant Staphylococcus aureus (MRSA) is a major pathogen involved in nosocomial infections and to some extent, in community acquired infections. Among Macrolide Lincosamine Streptogramin B (MLSB) class of drugs, Clindamycin was vigorously preferred for treating staphylococcal infections in the past few decades but, some genetic factors i.e. erm and msrA genes contribute in developing Inducible Clindamycin Resistance (iCR). Sensitivity tests performed on a routine basis cannot detect inducible resistance and may result in the failure of Clindamycin to be used as an effective medication. This study aimed to detect the phenotype of MRSA and iCR S. aureus from the clinical samples of Khyber Teaching Hospital, Peshawar. A total of 204 samples were collected randomly from each gender, 130 (63.72%) samples were isolated as S. aureus, while 74 (36.27%) were other bacterial species. Double disk diffusion (D-test) was performed to detect iCR phenotype, and 80 (61.5%) isolates showed iCR, while 50 (38.4%) were negative in this regard. MRSA phenotype was determined by strains conferring resistance to Cefoxitin antibiotic, which resulted in 84 (64.6%) isolates of MRSA and 46 (35.3%) of Methicillin Sensitive Staphylococcus aureus (MSSA). Antibiogram analysis showed efficient antimicrobial activity by Tigecycline 129 (99.2%), Fusidic Acid 126 (96.9%), and Doxycycline 124 (95.3%), while the highest resistance pattern was recorded against Ciprofloxacin 31(23.8%) and Clindamycin 28(21.5%). Our study concludes that misuse of antibiotics should be avoided to inhibit the spread of MRSA, and implementation of D-test regularly in hospitals is crucial.

Identification of Staphylococcus aureus Penicillin Binding Protein 4 (PBP4) Inhibitors.

Methicillin-resistant Staphylococcus aureus (MRSA) is a global healthcare concern. Such resistance has historically been attributed to the acquisition of mecA (or mecC), which encodes an alternative penicillin binding protein, PBP2a, with low β-lactam affinity. However, recent studies have indicated that penicillin binding protein 4 (PBP4) is also a critical determinant of S. aureus methicillin resistance, particularly among community-acquired MRSA strains. Thus, PBP4 has been considered an intriguing therapeutic target as corresponding inhibitors may restore MRSA β-lactam susceptibility. In addition to its role in antibiotic resistance, PBP4 has also recently been shown to be required for S. aureus cortical bone osteocyte lacuno-canalicular network (OLCN) invasion and colonization, providing the organism with a niche for re-occurring bone infection. From these perspectives, the development of PBP4 inhibitors may have tremendous impact as agents that both reverse methicillin resistance and inhibit the organism’s ability to cause chronic osteomyelitis. Accordingly, using a whole-cell high-throughput screen of a 30,000-member small molecule chemical library and secondary assays we identified putative S. aureus PBP4 inhibitors. Quantitative reverse transcriptase mediated PCR and PBP4 binding assays revealed that hits could be further distinguished as compounds that reduce PBP4 expression versus compounds that are likely to affect the protein’s function. We also showed that 6.25 µM (2.5 µg/mL) of the lead candidate, 9314848, reverses the organism’s PBP4-dependent MRSA phenotype and inhibits its ability to traverse Microfluidic-Silicon Membrane-Canalicular Arrays (µSiM-CA) that model the OLCN orifice. Collectively, these molecules may represent promising potential as PBP4-inhibitors that can be further developed as adjuvants for the treatment of MRSA infections and/or osteomyelitis prophylactics.

Impacts of NaHCO3 on β-Lactam Binding to PBP2a Protein Variants Associated with the NaHCO3-Responsive versus NaHCO3-Non-Responsive Phenotypes.

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to β-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many β-lactam antibiotics with less affinity than PBPs which are predominant in methicillin-susceptible (MSSA) strains. A novel, rather frequent in vitro phenotype was recently identified among clinical MRSA bloodstream isolates, termed “NaHCO3-responsiveness”. This phenotype features β-lactam susceptibility of certain MRSA strains only in the presence of NaHCO3. Two distinct PBP2a variants, 246G and 246E, have been linked to the NaHCO3-responsive and NaHCO3-non-responsive MRSA phenotypes, respectively. To determine the mechanistic impact of PBP2a variants on β-lactam susceptibility, binding profiles of a fluorescent penicillin probe (Bocillin-FL) to each purified PBP2a variant were assessed and compared to whole-cell binding profiles characterized by flow cytometry in the presence vs. absence of NaHCO3. These investigations revealed that NaHCO3 differentially influenced the binding of the fluorescent penicillin, Bocillin-FL, to the PBP2a variants, with binding intensity and rate of binding significantly enhanced in the 246G compared to the 246E variant. Of note, the NaHCO3-β-lactam (oxacillin)-responsive JE2 strain, which natively harbors the 246G variant, had enhanced Bocillin-FL whole-cell binding following exposure to NaHCO3. This NaHCO3-mediated increase in whole-cell Bocillin-FL binding was not observed in the NaHCO3-non-responsive parental strain, COL, which contains the 246E PBP2a variant. Surprisingly, genetic swaps of the mecA coding sites between JE2 and COL did not alter the NaHCO3-enhanced binding seen in JE2 vs. COL. These data suggest that the non-coding regions of mecA may be involved in NaHCO3-responsiveness. This investigation also provides strong evidence that the NaHCO3-responsive phenotype in MRSA may involve NaHCO3-mediated increases in both initial cell surface β-lactam binding, as well as ultimate PBP2a binding of β-lactams.

Levonadifloxacin

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major public health concerns in India, MRSA infections continue to be therapeutically challenging both in hospitals and in the community. The currently available therapeutic options for MRSA are unable to provide safe and efficacious treatment with an option of oral switchover in the treatment of MRSA infections, especially in the community. Levonadifloxacin (LND) is a broad-spectrum benzoquinolizine fluoroquinolone with potent activity against quinolone-resistant S. aureus and MRSA phenotypes developed by an Indian company Wockhardt. LND and its oral prodrug alalevonadifloxacin have been recently approved in India for the treatment of acute bacterial skin and skin structure infections with concurrent bacteraemia and diabetic foot infections.

Molecular detection of methicillin resistant Staphylococcus aureus associated with mastitis in goats

Antimicrobial resistance is an emerging concern in the world while combating with a wide range of pathogen associated with various disease conditions. It is considered as a silent pandemic nowadays. A constant monitoring and measures for controlling emergence of antimicrobial resistance are crucial in the current situation. The present study was conducted with the objective of detection of methicillin resistant Staphylococcus aureus (MRSA) associated with mastitis in goats. Milk samples were collected from 66 goats suffering from mastitis and screened for the presence of S. aureus. Conventional microbiological tests and species specific PCR were employed for the species confirmation. A total of 10 samples (15.15%) were found to be positive for S. aurues. Among the ten isolates of S. aureus obtained two isolates (3.03%) were found to harbour mecA gene suggestive of the presence of MRSA phenotype. All the isolates were negative for the presence of pvl gene which is present in highly virulent strains. In short, even though the study demonstrated the presence of MRSA at a lower rate, the results are of a marked public health significance due to the zoonotic significance of the pathogen.

Antibacterial Activity of Selected Essential Oil Compounds Alone and in Combination with β-Lactam Antibiotics Against MRSA Strains.

This study aimed to determine the effect of selected essential oil compounds (EOCs) on the antibacterial activity of β-lactam antibiotics (βLAs) against methicillin-resistant Staphylococcus aureus (MRSA) strains. The following parameters were studied: antibiotic susceptibility testing, detection of mecA gene and evaluation of genotypic relativity of isolates using molecular techniques, analysis of chemical composition applying Fourier-transform infrared (FTIR) spectroscopy, and determination of antibacterial activity of EOCs alone and in combination with βLAs against MRSA strains using microdilution and checkerboard methods. It was found that all isolates expressed MRSA and resistance phenotypes for macrolides, lincosamides, and streptogramins B. All isolates harbored the mecA gene and belonged to three distinct genotypes. Eight of the 10 EOCs showed efficient antimicrobial activity against the MRSA reference strain. The analysis of interaction between EOCs and βLAs against the MRSA reference strain revealed a synergistic and additive effect of the following combinations: methicillin (Met)-linalyl acetate (LinAc), penicillin G (Pen)-1,8-cineole (Cin), and Pen-LinAc. Analysis of EOC-βLA interactions showed a synergistic and additive effect in the following combinations: Met-LinAc (against low- and high-level βLAs resistance strains), Pen-Cin, and Pen-LinAc (against low-level βLAs resistance strains). It was also confirmed that changes in phosphodiester, -OH, -CH2 and -CH3 groups may change the interactions with βLAs. Moreover, the presence of two CH3O- moieties in the Met molecule could also play a key role in the synergistic and additive mechanism of LinAc action with Met against MRSA strains. Direct therapy using a Met-LinAc combination may become an alternative treatment method for staphylococcal infections caused by MRSA. However, this unconventional therapy must be preceded by numerous cytotoxicity tests.

Occurrence of Methicillin-Resistant Staphylococcus aureus in Ready-to-Eat Raw Fish from Japanese Cuisine Restaurants in Salvador, Brazil

The presence of methicillin-resistant Staphylococcus aureus (MRSA) strains in food products is a major issue for food safety. The present study was conducted to evaluate the occurrence and antimicrobial resistance profile of S. aureus, focusing on MRSA isolates, in ready-to-eat sashimi from Japanese restaurants in Salvador, Brazil. A total of 127 sashimi samples were collected directly from the take-out service in 16 restaurants. The staphylococcal isolates were identified morphologically and biochemically with standard laboratory procedures. S. aureus isolates were tested with a disk diffusion assay against seven antibiotics, and the cefoxitin and oxacillin were used to identify MRSA strains. Isolates with the MRSA phenotype were confirmed with a PCR assay. S. aureus was found in 73% of the sashimi samples, including sashimi from tuna (75.5% of samples) and salmon (72.5% of samples). Among those positive samples, 37% were contaminated with MRSA strains, found among 38.8% of salmon sashimi and 34.0% of tuna sashimi. Penicillin resistance was the most common type of antimicrobial resistance, found in 65.5% of the sashimi samples, followed by resistance to tetracycline (22.5%), erythromycin (16.0%), and ciprofloxacin (3.2%). Only two S. aureus isolates collected from different fish samples and restaurants had presumed resistance to vancomycin. The high prevalence of S. aureus and MRSA in these sashimi samples indicates a potential risk for foodborne disease, especially MRSA, spreading in the community.

Bactericidal and antibiotic-modulation activities of methanol crude extracts of Ligustrum lucidum and Lobelia inflata against MRSA phenotypes: Molecular docking studies of some isolated compounds from both plants against DNA gyrase A

The present study evaluated antibacterial, plasmid conjugal transfer inhibition, growth kinetics and bacterial resistance modifying activities of methanol crude extracts from the fruits of Ligustrum lucidum (LLFE) and the leaves of Lobelia inflata (LILE) against a panel of methicillin-resistant Staphylococcus aureus (MRSA) expressing active efflux pumps. The results showed that both extracts had weak antibacterial activity against the MRSA strains to have MIC > 512 mg/L. The combination of extracts with standard antibiotics was synergistic (2–64 fold potentiation) and bactericidal (≤ 3log10 CFU/mL) on more than 70% of the bacteria. LLFE displayed a toxic effect on HepG2 cells having IC50 27.61 mg/L while LILE demonstrated moderate toxicity (IC50 125.06 mg/L). LILE extract exhibited a weak inhibition of transfer conjugation mediated by PKM 101 (30%) and promising inhibition against TP114 (74%), while LLFE enhanced conjugal transfer to both plasmids. The chromatogram peaks showed that both extracts contain rutin and gallic acid with a retention time corresponding to the standards at 330 nm. Molecular docking of lobelanidine showed that it was the safest and most potent inhibitor of DNA-gyrase A of Staphylococcus aureus when compared with the standard norfloxacin. Our findings suggest that Ligustrum lucidum and Lobelia inflata are potential pharmacological agents in the future design of combination anti-infective therapies against multidrug-resistant strains.

Non detection of <i>mecA</i> gene in methicillin resistant <i>Staphylococcus aureus</i> isolates from pigs

Background: Methicillin resistant Staphylococcus aureus (MRSA) have become a global health problem causing infections in both humans and livestock, ranging from skin and soft tissue to life threatening blood stream infections. The mecA gene is known to confer resistance to MRSA isolates. This study investigated the carriage of mecA gene by MRSA isolates from pigs.Methods: One hundred non duplicate staphylococcal isolates recovered from blood samples of pigs in Bariga district of Lagos State at the Molecular Biology and Biotechnology unit of the Nigerian Institute of Medical Research were used in the study. S. aureus was identified by cultural characteristics, and positive catalase, coagulase and deoxyribonuclease tests. Phenotypic methicillin resistance was determined by the modified Kirby Bauer disk diffusion method and mecA gene was detected by conventional polymerase chain reaction (PCR) assay.Results: Twenty-five S. aureus were identified, of which 11 (44%) were MRSA by phenotypic method. All the isolates were mecA negative on PCR.Conclusion: The MRSA phenotype observed in the pig isolates in this study appears not to be the classical mecA mediated resistance. There may be alternative mechanisms of resistance in MRSA isolates in pigs.Keywords: MRSA, phenotypic, mecA gene, PCR, pigs

Methicillin-Resistant Staphylococcus aureus Carriage in Hemodialysis Vicinity: Prevalence and Decolonization Approach.

Hemodialysis (HD) patients are at risk for developing serious infections. Methicillin- resistant Staphylococcus aureus (MRSA) is one of the most prevalent pathogens in healthcare facilities with a major threat to the medical community. We aimed to determine the prevalence of MRSA colonization among patients and medical staff members in a HD Unit and determine efficacy of mupirocin as a decolonizing agent. This cross-sectional study enrolled 250 patients and 35 health care providers of a HD unit. Nasal and hand swabs were collected to assess the prevalence of MRSA carriage. Those exhibiting MRSA phenotype were subjected to conventional Polymerase chain reaction (PCR) assay for detection of mecA gene. Colonized patients and medical personnel with MRSA were prescribed mupirocin ointment (2%) for decolonization. The screening approach identified 54/285 (18.9%) nasal MRSA carriers (41/250 of HD patients and 13/35 of the medical staff members). Concomitant extranasal MRSA colonization of the hands was observed in 10 (18.5%) of these 54 MRSA carriers. In relation to PCR results the sensitivity, specificity, and diagnostic accuracy of cefoxitin disk test were 98.2%, 75%, and 93.9% respectively and for MRSA Select II agar screening method, the sensitivity, specificity, and diagnostic accuracy were 92.6%, 66.7%, and 87.9% respectively. Decolonization approach using mupirocin ointment revealed an overall success rate up to 77.8% (42/54) and failure rate of 16.7% (9/54), while 5.6% (3/54) of decolonized carriers showed recolonization. There is still high prevalence of MRSA colonization in HD vicinity. Implementation of strict infection control measures is essential in dialysis units to avoid MRSA cross-transmission and invasive infections.

The Molecular Detection of mecA Genes of Staphylococcus Aureus

Antibiotic resistance is common among pathogenic bacteria associated with community acquired and nosocomial infections. Methicillin-resistant Staphylococcus aureus (MRSA) infections have become a global health problem particularly in hospital setup causing simple skin infections to life threatening infections. The present study aimed to investigate the presence of mecA genes in MRSA from pigs, using Polymerase Chain Reaction. One hundred S. aureus isolates of blood samples from Pigs in Bariga, Lagos State were collected from Molecular Biology and Biotechnology Unit, Nigeria Institute of Medical Research. Methicillin resistance was determined by Kirby-Bauer’s disc diffusion method. The PCR was used for mecA gene detection from MRSA strains. Twenty-five pure Staphylococcus aureus isolates were identified based on cultural characteristics, biochemical reactions and positive slide coagulase test. Out of these, 11 (44%) strains were MRSA by phenotypic method. Amplification of mecA gene for all the 11 MRSA isolates was negative when visualized on 2% agarose gel electrophoresis. Eleven strains of MRSA were found among Staphylococcus aureus isolates of blood samples from Pigs. The MRSA phenotype observed in the isolates was not the classical mecA mediated resistance. Hence, it is highly recommended to consider alternative mechanisms for β-lactams resistance that may compete with mecA gene in the emergence of MRSA phenomenon in Nigeria.

Synergy between Ursolic and Oleanolic Acids from Vitellaria paradoxa Leaf Extract and β-Lactams against Methicillin-Resistant Staphylococcus aureus: In Vitro and In Vivo Activity and Underlying Mechanisms.

Combining antibiotics with resistance reversing agents is a key strategy to overcome bacterial resistance. Upon screening antimicrobial activities of plants used in traditional medicine, we found that a leaf dichloromethane extract from the shea butter tree (Vitellaria paradoxa) had antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) with further evidence of synergy when combined with β-lactams. Using HPLC-MS, we identified ursolic (UA) and oleanolic acids (OA) in leaves and twigs of this species, and quantified them by HPLC-UV as the major constituents in leaf extracts (21% and 6% respectively). Both pure triterpenic acids showed antimicrobial activity against reference and clinical strains of MRSA, with MICs ranging from 8–16 mg/L for UA to 32–128 mg/L for OA. They were highly synergistic with β-lactams (ampicillin and oxacillin) at subMIC concentrations. Reversion of MRSA phenotype was attributed to their capacity to delocalize PBP2 from the septal division site, as observed by fluorescence microscopy, and to disturb thereby peptidoglycan synthesis. Moreover, both compounds also inhibited β-lactamases activity of living bacteria (as assessed by inhibition of nitrocefin hydrolysis), but not in bacterial lysates, suggesting an indirect mechanism for this inhibition. In a murine model of subcutaneous MRSA infection, local administration of UA was synergistic with nafcillin to reduce lesion size and inflammatory cytokine (IL-1β) production. Thus, these data highlight the potential interest of triterpenic acids as resistance reversing agents in combination with β-lactams against MRSA.

High frequency of methicillin-resistant Staphylococcus aureus (MRSA) with SCCmec type III and spa type t030 in Karaj's teaching hospitals, Iran.

Methicillin-resistant Staphylococcus aureus (MRSA) has been one of the most important antibiotic-resistant pathogen in many parts of the world over the past decades. This cross-sectional study was conducted to investigate MRSA isolated between July 2013 and July 2014 in Karaj, Iran. All tested isolates were collected in teaching hospitals from personnel, patients, and surfaces and each MRSA was analyzed by SCCmec and spa typing. Antibiotic susceptibility testing was accomplished by disk diffusion method. Out of 49 MRSA isolates from the Karaj's teaching hospitals, 82%, 10%, and 6% of the isolates were SCCmec types III, II, and I, respectively. The main spa type in this study was spa t030 with frequency as high as 75.5% from intensive care unit (ICU) of the hospitals and high rate of resistance to rifampicin (53%) was found in MRSA isolates. In conclusion, high frequency of spa t030 with SCCmec type III and MRSA phenotype illustrated circulating of one of the antibiotic-resistant strains in ICU of Karaj's teaching hospitals and emphasizes the need for ongoing molecular surveillance, antibiotic susceptibility monitoring, and infection control.

Population Dynamics of Staphylococcus aureus in Cystic Fibrosis Patients To Determine Transmission Events by Use of Whole-Genome Sequencing.

Strict infection control practices have been implemented for health care visits by cystic fibrosis (CF) patients in an attempt to prevent transmission of important pathogens. This study used whole-genome sequencing (WGS) to determine strain relatedness and assess population dynamics of Staphylococcus aureus isolates from a cohort of CF patients as assessed by strain relatedness. A total of 311 S. aureus isolates were collected from respiratory cultures of 115 CF patients during a 22-month study period. Whole-genome sequencing was performed, and using single nucleotide polymorphism (SNP) analysis, phylogenetic trees were assembled to determine relatedness between isolates. Methicillin-resistant Staphylococcus aureus (MRSA) phenotypes were predicted using PPFS2 and compared to the observed phenotype. The accumulation of SNPs in multiple isolates obtained over time from the same patient was examined to determine if a genomic molecular clock could be calculated. Pairs of isolates with ≤71 SNP differences were considered to be the "same" strain. All of the "same" strain isolates were either from the same patient or siblings pairs. There were 47 examples of patients being superinfected with an unrelated strain. The predicted MRSA phenotype was accurate in all but three isolates. Mutation rates were unable to be determined because the branching order in the phylogenetic tree was inconsistent with the order of isolation. The observation that transmissions were identified between sibling patients shows that WGS is an effective tool for determining transmission between patients. The observation that transmission only occurred between siblings suggests that Staphylococcus aureus acquisition in our CF population occurred outside the hospital environment and indicates that current infection prevention efforts appear effective.

StaphylococcalCassette Chromosomemec(SCCmec) Typing of Methicillin-ResistantStaphylococcus aureusStrains Isolated from Community- and Hospital-Acquired Infections

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for the growing number of hospital- and community-acquired infections. Objectives: In this study, we aimed to determine the prevalence of MRSA, its antimicrobial resistance profile, and molecular typing of strains isolated from different infections in Iran. Methods: A total of 100S. aureus strains were isolated from various clinical specimens from Al-Zahra Hospital of Isfahan, Iran during January-June, 2015. Antimicrobial susceptibility test was performed, using the disk diffusion method. For identifying the MRSA phenotype, oxacillin agar screening was performed. Detection of mecA gene among the isolates was performed via polymerase chain reaction (PCR) using specific primers, followed by multiplex PCR for SCCmec typing of MRSA isolates. Results: In the present study, 54 (54%) isolates were identified as MRSA. Overall, 12 (22.23%) and 42 (77.7%) isolates were obtained from community- and hospital-acquired infections, respectively. SCCmec typing among MRSA isolates showed that 19 (35.18%), 13 (24.07%), 6 (11.11%), 5 (9.25%), and 3 (5.55%) isolates contained SCCmec type III, type I, type IV, type II, and type V, respectively; however, 8 (14.81%) isolates were nontypable. Conclusions: In the current study, SCCmec type III isolates were the most common among 54 MRSA isolates in a teaching hospital in the center of Iran. This finding might be attributed to antibiotic pressure, facilitating clonal selection.

Optimizing the detection of methicillin-resistant Staphylococcus aureus with elevated vancomycin minimum inhibitory concentrations within the susceptible range.

BackgroundDetermination of vancomycin minimum inhibitory concentration (MIC) can influence the agent used to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. We studied diagnostic accuracy using E-test and VITEK® 2 against a gold standard broth microdilution (BMD) methodology, the correlation between methods, and associations between vancomycin MIC and MRSA phenotype from clinical isolates.MethodsMRSA isolates were obtained from April 2012 to December 2013. Vancomycin MIC values were determined prospectively on all isolates by gradient diffusion E-test and automated VITEK® 2. The Clinical and Laboratory Standards Institute reference BMD method was performed retrospectively on thawed frozen isolates. Diagnostic accuracy for detecting less susceptible strains was calculated at each MIC cutoff point for E-Test and VITEK® 2 using BMD ≥1 µg/mL as a standard. The correlation between methods was assessed using Spearman’s rho (ρ). The association between MRSA phenotype and MIC for the three methods was assessed using Fisher’s exact test.ResultsOf 148 MRSA isolates, all except one (E-test =3 µg/mL) were susceptible to vancomycin (MIC of ≤2 µg/mL) irrespective of methodology. MICs were ≥1.0 µg/mL for 9.5% of BMD, 50.0% for VITEK® 2, and 27.7% for E-test. Spearman’s ρ showed weak correlations between methods: 0.29 E-test vs VITEK® 2 (P=0.003), 0.27 E-test vs BMD (P=0.001), and 0.31 VITEK® 2 vs BMD (P=0.002). The optimal cutoff points for detecting BMD-defined less susceptible strains were ≥1.0 µg/mL for E-test and VITEK® 2. E-test sensitivity at this cutoff point was 0.85 and specificity 0.29, while VITEK® 2 sensitivity and specificity were 0.62 and 0.51, respectively. Multiresistant MRSA strains tended to have higher MIC values compared to nonmultiresistant MRSA or epidemic MRSA 15 phenotypes by E-test (Fisher’s exact P<0.001) and VITEK® 2 (Fisher’s exact P<0.001).ConclusionOverall diagnostic accuracy and correlations between MIC methods used in routine diagnostic laboratories and the gold standard BMD showed limited overall agreement. This study helps optimize guidance on the effective use of vancomycin.

Oritavancin Combinations with β-Lactams against Multidrug-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococci.

Oritavancin possesses activity against vancomycin-resistant enterococci (VRE) and methicillin-resistantStaphylococcus aureus(MRSA).In vitrodata suggest synergy between beta-lactams (BLs) and vancomycin or daptomycin, agents similar to oritavancin. We evaluated the activities of BLs combined with oritavancin against MRSA and VRE. Oritavancin MICs were determined for 30 strains, 5 each of MRSA, daptomycin-nonsusceptible (DNS) MRSA, vancomycin-intermediate MRSA (VISA), heteroresistant VISA (hVISA), vancomycin-resistantEnterococcus faecalis, and vancomycin-resistantEnterococcus faecium Oritavancin MICs were determined in the presence of subinhibitory concentrations of BLs. Oritavancin combined with ceftaroline, cefazolin, or nafcillin was evaluated for lethal synergy against MRSA, and oritavancin combined with ceftaroline, ampicillin, or ertapenem was evaluated for lethal synergy against VRE in 24-h time-kill assays. Oritavancin at 0.5× the MIC was combined with BLs at 0.5× the MIC or the biological free peak concentration, whichever one was lower. Synergy was defined as a ≥2-log10-CFU/ml difference between the killing achieved with the combination and that achieved with the most active single agent at 24 h. Oritavancin MICs were ≤0.125 μg/ml for all MRSA isolates except three VISA isolates with MICs of 0.25 μg/ml. Oritavancin MICs for VRE ranged from 0.03 to 0.125 μg/ml. Oritavancin in combination with ceftaroline was synergistic against all MRSA phenotypes and statistically superior to all other combinations against DNS MRSA, hVISA, and MRSA isolates (P< 0.02). Oritavancin in combination with cefazolin and oritavancin in combination with nafcillin were also synergistic against all MRSA strains. Synergy between oritavancin and all BLs was revealed against VRE strain 8019, while synergy between oritavancin and ampicillin or ertapenem but not ceftaroline was demonstrated against VRE strain R7164. The data support the potential use of oritavancin in combination with BLs, especially oritavancin in combination with ceftaroline, for the treatment of infections caused by MRSA. The data from the present study are not as strong for oritavancin in combination with BLs for VRE. Further study of both MRSA and VRE in more complex models is warranted.

Distribution of erm genes among Staphylococcus aureus isolates with inducible resistance to clindamycin in Isfahan, Iran.

Background:The rising frequency of methicillin resistant Staphylococcus aureus (MRSA) has led to an increased use of antibiotics such as macrolide, lincosamide, streptogramin B (MLSB) for the treatment of S. aureus infections. Resistance to MLSB in S. aureus is commonly encoded by erm genes, which can be constitutive MLSB (cMLSB) or inducible MLSB (iMLSB). The purpose of this study was to determine the frequency of cMLSB, iMLSB, and MS phenotypes using D-test and polymerase chain reaction (PCR) methods.Materials and Methods:A total of 215 isolates of S. aureus were collected from January 2010 to May 2012 from Al-Zahra Hospital in Isfahan. PCR was performed for detection of mecA gene on all isolates using specific primers. The frequency of MLSB-resistant isolates was determined using D-test, and then a multiplex PCR was performed for detection of ermA, ermB, and ermC genes.Results:Among 215 S. aureus isolates examined, 82 (40.9%) were MRSA, and iMLSB, cMLSB, and MS resistance phenotypes had a frequency of 9 (4.18%), 58 (26.9%), and 11 (5.1%), respectively. Among nine isolates with iMLSB resistance phenotype, four isolates contained ermC gene, two isolates ermB gene, and one isolate ermA gene. Two isolates did not have any erm gene.Conclusion:In the current study, cMLSB was the most frequent phenotype and ermC was the most common gene in iMLSB resistant phenotypes.

Does resistance in severe infections caused by methicillin-resistant Staphylococcus aureus give you the ‘creeps’?

The clinical implications of reduced vancomycin susceptibility amongst methicillin-resistant Staphylococcus aureus (MRSA) are controversial, and crossresistance to daptomycin amongst such strains has been reported. As a consequence of 'MIC creep', higher trough levels were recommended for serious infections. This review focusses on the new data published in the past 18 months that pertain to these issues. Heteroresistant vancomycin-intermediate Staphylococcus aureus reduces the clinical response rates to vancomycin in bacteraemic MRSA patients without impacting on mortality as opposed to 'MIC creep' with vancomycin minimum inhibitory concentration (MIC) levels of ≥1.5 mg/l that are significantly associated with mortality. Although daptomycin resistance is rare, 'concomitant MIC creep' amongst MRSA isolates with increasing vancomycin MICs may occur or exist concurrently amongst such strains. The aggressive vancomycin dosing regimens are still associated with unacceptable high microbiological failure rates and it is not currently possible to achieve probability of target attainment at higher vancomycin MICs of 2 mg/l. The nephrotoxic impact of high-dose vancomycin therapy has been confirmed. Continued monitoring of patients on aggressive vancomycin dosing schedules is advised. Unless alternative dosing strategies prove otherwise efficacious, an alternative antibiotic should be considered for severe MRSA infections with vancomycin MICs greater than 1 mg/l. The utility of vancomycin may be waning but will depend on the prevalence of resistant MRSA phenotypes in a specific ICU.