Vancomycin-susceptible S. Aureus Research Articles

Metabolic Profiling Reveals the Importance of Arginine Metabolism via the Arginine Deiminase Pathway in Vancomycin-intermediate S. aureus

Gene mutations located in VraSR and GraSR, two-component regulatory systems have been reported to cause vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). Subsequent comparative proteomic profiling between vancomycin-susceptible S. aureus (VSSA) and VISAs of the Mu50 genetic lineage revealed up-regulated level of catabolic ornithine carbamoyltransferase (ArcB) in the latter, suggesting a role of arginine catabolism in VISA development. This study aimed to further investigate metabolic pathways associated with VISA development using liquid chromatography mass spectrometry (LCMS)-based untargeted metabolomic profiling. Metabolite profiles were compared among 3 isogenic strains of the Mu50 lineage: Mu50Ω (VSSA), Mu50Ω-vraSm (VISA) and Mu50Ω-vraSm-graRm (VISA). Intracellular levels of α-hydroxyglutaric acid, choline, lysine, malic acid, N-acetylornithine, nicotinamide, and cystathionine were found to be significantly different between VISAs compared to VSSA. VISA cells were found to have lower levels of intracellular citrulline and extracellular arginine, with higher extracellular level of ornithine compared to VSSA. These differences in metabolite levels between VISA and VSSA suggested the importance of arginine metabolism. Activation of the arginine deiminase pathway (ADI) in VISAs of the Mu50 lineage could be further explored as a target for antibiotic development.

Genetic characterization of tetracycline-resistant Staphylococcus aureus with reduced vancomycin susceptibility using whole-genome sequencing.

This study aimed to analyze the genetic characteristics of Staphylococcus aureus with reduced vancomycin susceptibility (RVS-SA). Whole-genome sequencing was performed on 27 RVS-SA clinical isolates, and comparative genomic analysis was performed using S. aureus reference strains. Pan-genome orthologous groups (POGs) were identified that were present in RVS-SA but absent in the reference strains, but further analysis showed that the presence of these POGs was influenced by tetracycline resistance rather than vancomycin resistance. Therefore, we restricted our analysis to tetracycline-resistant (tetR) RVS-SA and tetR vancomycin-susceptible S. aureus (VSSA). Phylogenomic analysis showed them to be closely related, and further analysis revealed the presence of an uncharacterized protein SAB0394 and the absence of lytA in tetR RVS-SA, which are involved in cell wall thickening. In summary, using whole-genome sequencing we identified gain or loss of genes in tetR RVS-SA strains. These findings provide insights into the investigation of mechanisms associated with reduced vancomycin susceptibility and have the potential to contribute to the development of molecular biomarkers for the rapid and efficient detection of RVS-SA.

Comparing the Synergistic and Antagonistic Interactions of Ciprofloxacin and Levofloxacin Combined with Rifampin against Drug-Resistant Staphylococcus aureus: A Time-Kill Assay.

Treatment of device-related infections by drug-resistant Staphylococcus aureus can be challenging, and combination therapy has been proposed as a potential solution. We compared the effectiveness of levofloxacin-rifampin and ciprofloxacin-rifampin combinations in killing methicillin-resistant S. aureus (MRSA) using a time-kill assay. We randomly selected 15 vancomycin-susceptible S. aureus (VSSA) strains, 3 vancomycin-intermediate S. aureus (VISA) strains, and 12 heterogeneous VISA (hVISA) strains from the Asian Bacterial Bank. Time-kill experiments were performed in duplicate for each isolate. Viable bacterial counts were determined at 0 h, 4 h, 8 h, and 24 h for the ciprofloxacin- and levofloxacin-rifampin combinations at 1× MIC and 0.5× MIC. We compared synergistic and antagonistic interactions between the two combinations. The viable bacterial count significantly decreased after 24 h of exposure to ciprofloxacin-rifampin and levofloxacin-rifampin combinations, with synergy observed more frequently in isolates exposed to ciprofloxacin-rifampin (43.3%) than levofloxacin-rifampin (20.0%) (p = 0.0082). The synergistic interactions of both combinations were more frequently observed in resistant strains with high MICs of ciprofloxacin (≥16 mg/L) and levofloxacin (≥8 mg/L). Levofloxacin tended to exhibit more frequent antagonistic interactions with rifampin than ciprofloxacin, although there was no statistical difference in antagonism between the two combinations. Our study demonstrated that ciprofloxacin exhibits superior synergistic activity against MRSA strains, including VISA/hVISA, when combined with rifampin compared with levofloxacin. High MICs of fluoroquinolones were found to predict synergism. Our results suggest that ciprofloxacin may be a more effective choice than levofloxacin for combination therapy with rifampin in the treatment of MRSA infections.

Development of a Single-nucleotide Polymorphism Genotyping Assay for the Rapid Detection of Vancomycin-intermediate Resistance in Staphylococcus aureus Epidemic Lineage ST5.

Vancomycin is a treatment option for patients with severe methicillin-resistant Staphylococcus aureus (MRSA) infection. Unfortunately, reduced susceptibility to vancomycin in S. aureus is becoming increasingly common. We developed a method for the rapid and accurate diagnosis of vancomycin-intermediate resistant S. aureus (VISA). We performed a microbial genome-wide association study to discriminate between VISA and vancomycin-susceptible S. aureus (VSSA) using 42 whole-genome sequences. A TaqMan single-nucleotide polymorphism (SNP) genotyping assay was developed to detect target SNPs in VISA strains. Four SNPs in the VISA strains resulting in nonsynonymous amino-acid substitutions were associated with reduced susceptibility to vancomycin: SA_RS01235 (G203S), SA_RS09725 (V171A), SA_RS12250 (I48F), and SA_RS12550 (G478A). These four SNPs were mainly detected in the typical hospital-associated sequence type (ST)5 clonal lineage. The TaqMan assay successfully detected all four SNPs using as little as 0.2 ng DNA per reaction. Using 10 VSSA and VISA clinical strains each, we validated that the assay accurately discriminates between VISA and VSSA. The TaqMan SNP genotyping assay targeting four SNPs may be an alternative to current standard methods for the rapid detection of vancomycin-intermediate resistance in S. aureus epidemic lineage ST5.

Resistance and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Heterogeneous Vancomycin-Intermediate Staphylococcus aureus.

To investigate the molecular epidemiology of Staphylococcus aureus (S. aureus) isolated from patients in the Yellow River Delta region and to construct antibiotic resistance profiles in different genetic backgrounds. Antibiotics susceptibility testing, staphylococcal protein A (spa) typing and accessory gene regulator (agr) typing were performed for all the 204 strains. Isolates with the positive mecA gene and heterogeneous vancomycin-intermediate S. aureus (hVISA) were subjected to multilocus sequence typing (MLST) and Staphylococcal chromosomal cassettes mec (SCCmec) typing. Thirty-nine MRSA strains were identified by mecA gene. Twenty-two hVISA isolates including 9 MRSA and 13 methicillin-sensitive S. aureus (MSSA) strains were confirmed, the rest isolates (n = 182) were vancomycin-susceptible S. aureus (VSSA). 94.9% of MRSA and 63.6% of hVISA belonged to agrI. Fifty-seven distinct spa types including 5 novel types were mainly t309 (30.9%), t078 (11.8%) and t437 (11.8%). Fourteen sequence types (STs) containing 3 new STs were classified into 3 clone complexes (CCs) and 7 singletons among MRSA and hVISA isolates. Most MRSA isolates (87.2%) belonged to type Ⅳ SCCmec. The predominant genotype among MRSA population was ST59-t437-agrI-IVa (53.8%), followed by ST72-t2431-agrI-IVF (15.4%). ST72 and CC5 (ST5/965/7197) were the most common hVISA clones. Both CC59 (ST59/7437) and ST72 clones were resistant to erythromycin and clindamycin among MRSA population. Strains of MSSA with phenotypic hVISA (MS-hVISA) exhibited a striking genetic diversity accompanied by the diversification of drug resistance patterns.

Heterogeneous vancomycin resistance in Staphylococcus aureus does not predict development of vancomycin resistance upon vancomycin pressure

It is unclear whether Staphylococcus aureus with heterogeneous intermediate vancomycin resistance (hVISA) can develop vancomycin resistance faster than vancomycin-susceptible S. aureus (VSSA) strains. We compared the kinetics of vancomycin MIC increase for 15 days of sustained in vitro vancomycin exposure for clinical hVISA (n = 12) and VSSA (n = 24) isolates, as well as for reference strains Mu3 (hVISA) and ATCC 29213 (VSSA). Clinical isolates were categorized as hVISA using the population analysis profile method. MICs were monitored for 15 days and the rate of MIC increase under exposure, for each strain, was evaluated in a linear regression model relative to time. All isolates acquired vancomycin resistance upon exposure. Vancomycin MICs increased faster for VSSA compared with hVISA isolates (P < 0.01). The hVISA phenotype does not correspond to an enhanced adaptation potential to in vitro vancomycin pressure.

Effect of Vancomycin on Cellular Fatty Acid Profiles of Vancomycin-Susceptible and Nonsusceptible Staphylococcus aureus.

Vancomycin is widely used for treatment of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) leading to an increasing appearance of low-level vancomycin-resistant isolates called heterogeneous vancomycin-intermediate S. aureus (hVISA). The mechanism of vancomycin tolerance in hVISA is still unclear. This study aimed to investigate the fatty acid compositions of S. aureus isolates under the stress environment with vancomycin. The different responses of hVISA and vancomycin-susceptible S. aureus (VSSA) may lead to more understanding the mechanism. The bacterial lipid profiles were tested three times from three extractions of each isolate cultured on tryptic soy agar (TSA) and TSA with vancomycin. Of the 30 MRSA isolates studied, 13, 12, and 5 isolates were VSSA, hVISA, and VISA, respectively. The analysis of bacterial lipid profiles showed that under vancomycin stress, there was a reduction of straight chain fatty acids (SCFAs) in VSSA isolates but an increase in branched chain fatty acids (BCFAs). In contrast, the hVISA group exhibited an increase only in the BCFAs but not in SCFAs. Of interest, vancomycin had no effect on either BCFAs or SCFAs of the VISA cells. This study provided information of bacterial adaptation during stress with vancomycin that may be helpful to overcome the resistant bacteria.

Vancomycin Resistance Among Clinical Isolates of Staphylococcus aureus Obtained from Selected Hospitals in Sokoto Metropolis

The decreased vancomycin susceptibility and subsequent emergence of vancomycin resistant Staphylococcus aureus (VRSA) strains is a major public health problem. This study was aimed at detecting the prevalence of vancomycin resistant Staphylococcus aureus among clinical isolates obtained from patients attending Specialist Hospital Sokoto and Maryam Abacha Women and Children Hospital Sokoto. A total of 80 S. aureus clinical isolates were obtained from the medical microbiology laboratories of the selected hospitals. Antibiotic sensitivity testing of the isolates was carried out using the agar dilution method and isolates were screened for vancomycin resistance using vancomycin agar screen method. Of the 80 S. aureus isolates studied, 69 (86.0%) were identified as vancomycin susceptible S. aureus (VSSA) with MIC value of ≤2 µg/ml, 11 (13.8%) were identified as vancomycin intermediate S. aureus (VISA) and had MIC value of 4-8 µg/mL (VISA) and none of the isolates was identified as vancomycin resistant aureus (VRSA). The study detects high prevalence rate of VISA in the study area and identifies the need for increased public awareness on the danger associated with the presence of drug resistant bacteria. Emphasis should be directed at discouraging practices such as the use of over the counter medications which increase the rate of development of drug resistant organisms.&#x0D;

Protein Biomarker Discovery for Methicillin-Sensitive, Heterogeneous Vancomycin-Intermediate and Vancomycin-Intermediate Staphylococcus aureus Strains Using Label-Free Data-Independent Acquisition Proteomics.

Rapid identification of methicillin-sensitive Staphylococcus aureus (MSSA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-intermediate S. aureus (VISA) is important for accurate treatment, timely intervention, and prevention of outbreaks. Here, 90 S. aureus isolates were analyzed for protein biomarker discovery, including MSSA, vancomycin-susceptible S. aureus (VSSA), hVISA, and VISA strains. Label-free data-independent acquisition proteomics was used to identify protein biomarkers that allow for discrimination among MSSA, hVISA, and VISA strains. There were 8786 nonredundant peptides identified, corresponding to 418 different annotated nonredundant proteins. Two VISA protein biomarkers, two hVISA protein biomarkers, and one MSSA protein biomarker with high sensitivities and specificities were discovered and verified. Data are available via MassIVE with identifier MSV000085776.

Detection of heterogeneous vancomycin intermediate resistance in MRSA isolates from Latin America.

Vancomycin is a common first-line option for MRSA infections. The heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) phenotype is associated with therapeutic failure. However, hVISA isolates are usually reported as vancomycin susceptible by routine susceptibility testing procedures. To detect and characterize the hVISA phenotype in MRSA isolates causing infections in nine Latin American countries. We evaluated a total of 1189 vancomycin-susceptible MRSA isolates recovered during 2006-08 and 2011-14. After an initial screening of hVISA using glycopeptide-supplemented agar strategies, the detection of hVISA was performed by Etest (GRD) and Macro-method (MET). Isolates deemed to be hVISA were subjected to population analysis profile/AUC (PAP/AUC) and WGS for further characterization. Finally, we interrogated alterations in predicted proteins associated with the development of the VISA phenotype in both hVISA and vancomycin-susceptible S. aureus (VSSA) genomes. A total of 39 MRSA isolates (3.3%) were classified as hVISA (1.4% and 5.6% in MRSA recovered from 2006-08 and 2011-14, respectively). Most of the hVISA strains (95%) belonged to clonal complex (CC) 5. Only 6/39 hVISA isolates were categorized as hVISA by PAP/AUC, with 6 other isolates close (0.87-0.89) to the cut-off (0.9). The majority of the 39 hVISA isolates exhibited the Leu-14→Ile (90%) and VraT Glu-156→Gly (90%) amino acid substitutions in WalK. Additionally, we identified 10 substitutions present only in hVISA isolates, involving WalK, VraS, RpoB and RpoC proteins. The hVISA phenotype exhibits low frequency in Latin America. Amino acid substitutions in proteins involved in cell envelope homeostasis and RNA synthesis were commonly identified. Our results suggest that Etest-based methods are an important alternative for the detection of hVISA clinical isolates.

Reduced vancomycin susceptibility and increased macrophage survival in Staphylococcus aureus strains sequentially isolated from a bacteraemic patient during a short course of antibiotic therapy.

The purpose of the present study was to determine the relatedness of Staphylococcus aureus strains successively isolated over a 7-day period from a single bacteraemic patient undergoing antibiotic treatment with vancomycin. The S. aureus strains had been isolated and sequenced previously. Antibiotic susceptibility testing, population analysis profiling, and lysostaphin sensitivity and phagocytic killing assays were used to characterize these clonal isolates. The seven isolates (MEH1-MEH7) were determined to belong to a common multilocus sequence type (MLST) and spa type. Within the third and fifth day of vancomycin treatment, mutations were observed in the vraS and rpsU genes, respectively. Population analysis profiles revealed that the initial isolate (MEH1) was vancomycin-susceptible S. aureus (VSSA), while those isolated on day 7 were mostly heteroresistant vancomycin-intermediate S. aureus (hVISA). Supporting these findings, MEH7 was also observed to be slower in growth, to have an increase in cell wall width and to have reduced sensitivity to lysostaphin, all characteristics of VISA and hVISA strains. In addition, MEH7, although phagocytosed at numbers comparable to the initial isolate, MEH1, survived in higher numbers in RAW 264.7 macrophages. Macrophages infected with MEH7 also released more TNF-α and IFN-1β. We report an increasing resistance to vancomycin coupled with daptomycin that occurred within approximately 3 days of receiving vancomycin and steadily increased until the infection was cleared with an alternative antibiotic therapy. This study reiterates the need for rapid, efficient and accurate detection of hVISA and VISA infections, especially in high-bacterial load, metastatic infections like bacteraemia.

Vancomycin MIC Distribution among Methicillin-Resistant Staphylococcus Aureus. Is Reduced Vancomycin Susceptibility Related To MIC Creep?

AIM:To determine the distribution of vancomycin MIC and the frequency of S. aureus strains with reduced vancomycin susceptibility among Methicillin-Resistant Staphylococcus aureus (MRSA) isolates.METHODS:MRSA isolates (n = 100) were tested for reduced susceptibility to vancomycin using MIC broth microdilution method (BMD), vancomycin screening agar with different vancomycin concentrations with and without casein, and Vitek 2 system.RESULTS:BMD detected (22%) vancomycin-intermediate S. aureus (VISA) and (78%) vancomycin-susceptible S. aureus (VSSA) but couldn’t detect nine (Heterogeneous VISA) (hVISA) isolates (9%) with MIC ≤ 2 µg/ml that grew on screening agar 4 µg/ml or 6 µg/ml. Adding casein to vancomycin screening agar increased detection rate of VISA by 4.5%. Screening agar with 6 µg/ml vancomycin overall detection rate for VISA was 95.45%. Probable ‘pre-hVISA’isolates (17%) showed growth on vancomycin screening agar 2 µg/ml with casein. Vitek 2 system failed to detect any VISA isolates.CONCLUSION:Vancomycin screening agar; 2 µg/ml and (4 and 6 µg/ml) were able to detect; probable “pre hVISA and (hVISA and VISA) isolates respectively based on their BMD MIC values. Decreased vancomycin susceptibility in MRSA isolates might be related to MIC creep. Analysis of vancomycin MIC values over longer periods is recommended to further study this phenomenon and its impact on vancomycin treatment failure.

Detection of VanA type vancomycin resistance among MRSA isolates from an emergency hospital in Egypt

Resistance of methicillin-resistant Staphylococcus aureus (MRSA) strains to vancomycin is increasing and represents a major threat in the therapeutic fields. To assess susceptibility of S. aureus to vancomycin and determine the occurrence of VanA gene among MRSA isolates. Agar diffusion method using cefoxitin disc (30 μg) was used to identify MRSA among 100 isolates of S. aureus. Among MRSA, vancomycin resistance was determined by broth macrodilution and VanA gene was detected by PCR. Frequency of MRSA was 92%. Phenotypically, MRSA isolates were vancomycin-susceptible S. aureus (VSSA) (67.4%), vancomycin-intermediate S. aureus (VISA) (10.9%), and vancomycin-resistant S. aureus (VRSA) (21.7%). 17.4% of MRSA were VanA positive (75% were VRSA and 25% were VSSA). 82.6% of MRSA were VanA negative (76.3% were VSSA, 13.2% were VISA, and 10.3% were VRSA). Sixty percent of VRSA and 6.5% of VSSA were VanA gene positive but none of the detected VISA isolates was carrier of VanA gene. Percentage of MRSA, VISA, and VRSA detected in this study is alarming and highlights the need for implementation of strict infection control measures to prevent further dissemination of such resistant strains in our region.

Rapid and easy detection of low-level resistance to vancomycin in methicillin-resistant Staphylococcus aureus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Vancomycin-intermediately resistant Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are associated with treatment failure. hVISA contains only a subpopulation of cells with increased minimal inhibitory concentrations, and its detection is problematic because it is classified as vancomycin-susceptible by standard susceptibility testing and the gold-standard method for its detection is impractical in clinical microbiology laboratories. Recently, a research group developed a machine-learning classifier to distinguish VISA and hVISA from vancomycin-susceptible S. aureus (VSSA) according to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data. Nonetheless, the sensitivity of hVISA classification was found to be 76%, and the program was not completely automated with a graphical user interface. Here, we developed a more accurate machine-learning classifier for discrimination of hVISA from VSSA and VISA among MRSA isolates in Japanese hospitals by means of MALDI-TOF MS data. The classifier showed 99% sensitivity of hVISA classification. Furthermore, we clarified the procedures for preparing samples and obtaining MALDI-TOF MS data and developed all-in-one software, hVISA Classifier, with a graphical user interface that automates the classification and is easy for medical workers to use; it is publicly available at https://github.com/bioprojects/hVISAclassifier. This system is useful and practical for screening MRSA isolates for the hVISA phenotype in clinical microbiology laboratories and thus should improve treatment of MRSA infections.

Activity of maltodextrin and vancomycin against staphylococcus aureus biofilm.

We aimed to assess the anti-biofilm activity of vancomycin, maltodextrin, and their combination against vancomycin resistant Staphylococcus aureus (VRSA) and vancomycin-susceptible S. aureus (VSSA) strains based on an in vitro static model. Biofilms of 4 VSSA and 2 VRSA strains were grown in a 96-well static model. Vancomycin 2mM, maltodextrin 10 mM, and both in combination were tested using tetrazolium salt (XTT), resazurin, and cfu/well counts. The efficacy of the antimicrobial solutions was expressed as the percentage reduction in metabolic activity with each method. Overall percentage reduction in the metabolic activity of VSSA was 79.3%, 34%, and 75.7% for vancomycin, maltodextrin, and their combination (p<0.001). Overall percentage reduction in metabolic activity of VRSA was 46.7%, 27.8%, and 34.6% for vancomycin, maltodextrin, and their combination (p>0.05). Maltodextrin did not improve the anti-biofilm efficacy of vancomycin in VSSA or in VRSA biofilms. XTT cannot replace cfu counts as a means of quantifying cell viability. Futures studies are needed to assess the synergistic effects of other non-antimicrobial molecules combined with vancomycin.

Multidrug and vancomycin resistance among clinical isolates of Staphylococcus aureus from different teaching hospitals in Nigeria.

Staphylococcus aureus has emerged as a major public health concern because of the occurrence of multi-drug resistant strains. This study aimed at investigating the multi-drug and vancomycin resistance profile of S. aureus from different infection sites in some teaching hospitals in Nigeria. Swabs were collected from different infection sites from out-patients in three teaching hospitals from October 2015 to May, 2016. The antibiotic-susceptibility test was carried out with selected antibiotics usually administered anti-microbials in the treatment of infections in these hospitals. The prevalence of multi-drug and vancomycin resistance strains of S. aureus from clinical samples was determined using disk diffusion and agar dilution methods respectively. The result showed (165)82.5% of the isolates were resistant to ≥3 antibiotics tested. They were highly resistant to ceftazidime 180(90%), cloxacillin 171(85.6%) and augmentin 167(83.3%), but susceptible to ofloxacin 150(75%), gentamicin 142(71.7%), erythromycin 122(61.1%), ceftriaxone 111(55.6%) and cefuroxime 103(51.7%). All the isolates from the HVS were all multidrug resistant strains. While (56)90.16% were multidrug resistant (MDR) in urine samples, followed by (8)88.89% MDR strains in sputum, (37)88.81% MDR strains in semen, (49)71.64% MDR strains in wounds and (6)60% MDR strains in ear swabs samples. Although (147)73.5% of the isolates were vancomycin susceptible S. aureus (VSSA), (30)15% were vancomycin intermediate resistant S. aureus (VISA) and (89)44.5% of the isolates were considered vancomycin resistant S. aureus (VRSA). The high percentage of the VRSA could have resulted from compromising treatment options and inadequate antimicrobial therapy. The implication, infections caused by VRSA would be difficult to treat with vancomycin and other effective antibiotics of clinical importance. Ensuring proper monitoring of drug administration will, therefore, enhance the legitimate role of vancomycin as an empiric choice for both prophylaxis against and treatment of staphylococcal infections.

Activated ADI pathway: the initiator of intermediate vancomycin resistance in Staphylococcus aureus.

Comparative proteomic profiling between 2 vancomycin-intermediate Staphylococcus aureus (VISA) strains, Mu50Ω-vraSm and Mu50Ω-vraSm-graRm, and vancomycin-susceptible S. aureus (VSSA) strain Mu50Ω revealed upregulated levels of catabolic ornithine carbamoyltransferase (ArcB) of the arginine catabolism pathway in VISA strains. Subsequent analyses showed that the VISA strains have higher levels of cellular ATP and ammonia, which are by-products of arginine catabolism, and displayed thicker cell walls. We postulate that elevated cytoplasmic ammonia and ATP molecules, resulting from activated arginine catabolism upon acquisition of vraS and graR mutations, are important requirements facilitating cell wall biosynthesis, thereby contributing to thickened cell wall and consequently reduced vancomycin susceptibility in VISA strains.

Phenotypic characteristics and comparative proteomics of Staphylococcus aureus strains with different vancomycin-resistance levels

Reduced vancomycin susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide problem. Unfortunately, its genetic marker and molecular mechanisms remained unknown. This study investigated differential phenotypic characteristic and protein expression profiles among three groups of MRSA isolates, including vancomycin-susceptible S. aureus (VSSA), heterogeneous vancomycin-intermediate S. aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) (n = 7 isolates/group). Phenotypic characteristic revealed significant greater number of isolates with non-spreading colony in VISA as compared to both VSSA and hVISA groups. 2-DE followed by nanoLC-MS/MS analyses revealed increased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both hVISA and VISA, whereas 50S ribosomal protein L14 (RplN) and DNA-binding protein II (Hup) were increased only in VISA. The non-spreading colony and GAPDH level of MRSA may be used as the markers for differentiation of VSSA, hVISA and VISA.

In vitro synergistic effects of various combinations of vancomycin and non-beta-lactams against Staphylococcus aureus with reduced susceptibility to vancomycin

The aim of our study was to determine the potential for synergistic effect of vancomycin combined with a non–beta-lactam agent and of combinations of orally available non-beta-lactam agents against vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA). Three VISA isolates, three hVISA isolates, and two vancomycin-susceptible S. aureus (VSSA) isolates were used. The combinations included vancomycin and one of clindamycin, ciprofloxacin, gentamicin, rifampicin, or trimethoprim/sulfamethoxazole. Pairwise combinations among five non–beta-lactam agents were also tested for synergy. Synergy was determined using time-kill curves at 24h. Vancomycin combined with either ciprofloxacin or gentamicin showed synergy against some isolates of VISA, hVISA and VSSA. Vancomycin combined with trimethoprim/sulfamethoxazole showed synergy against VISA and hVISA. Among orally available non-beta-lactam agents, only ciprofloxacin combined with either clindamycin or trimethoprim/sulfamethoxazole showed synergy against one isolate of VISA.

Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus.

Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the β subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology.