Vancomycin-intermediate S. Aureus Isolates Research Articles

Synergistic bactericidal effects of phage-enhanced antibiotic therapy against MRSA biofilms.

The prevalence of biofilm-associated medical device infections caused by methicillin-resistant Staphylococcus aureus (MRSA) presents a pressing medical challenge. The latest research demonstrates the potential of phage-antibiotic combinations (PACs) as a promising solution, notably in vitro antibiofilm efficacy. By adopting modified checkerboard and 24-h time-kill assays, the study investigated the synergistic action of phages combined with humanized-simulated doses of daptomycin (DAP) and ceftaroline (CPT). The results were promising: a combination of DAP, CPT, and either a 2-phage or 3-phage cocktail effectively exhibited bactericidal activity against both DAP non-susceptible vancomycin intermediate S. aureus MRSA and DAP-susceptible MRSA strains within 168-h biofilm models. Moreover, post-treatment evaluations revealed no discernible rise in antibiotic resistance or complete phage resistance. This pioneering work suggests the potential of PACs in addressing MRSA biofilm infections, setting the stage for further expansive research tailored to diverse bacterial strains and treatment durations.

Alpha-amyrin as an anti-biofilm agent against methicillin-resistant and vancomycin-intermediate Staphylococcus aureus

Staphylococcus aureus has caused life-threatening infections and developed resistance against conventional antimicrobials, posing a significant threat to human health worldwide. Biofilms that surround the bacteria cells act as a protective layer, allowing cells inside the biofilm to be resistant to external stresses such as antimicrobials. Therefore, biofilms further complicate treatment available for infections caused by multi-drug resistant Staphylococcus aureus. A previous study on alpha-amyrin (AM), derived from ursane, was reported to significantly reduce the biomass and inhibit the metabolic activity of reference strain methicillin-resistant and methicillin-sensitive S. aureus (MRSA and MSSA, respectively). In this study, the antibiofilm activity of AM was extended to include clinical isolates of MSSA and MRSA, and laboratory-generated vancomycin-intermediate S. aureus (VISA) collected from University Kebangsaan Malaysia Medical Center (PPUKM) and Universiti Kebangsaan Malaysia Medical Molecular Biology Institute (UMBI). Pre-formed biofilms of biofilm-forming isolates identified from the Congo Red Agar (CRA) assay were then exposed to AM, vancomycin and oxacillin, and evaluated using the crystal violet and resazurin assays. The results showed that AM reduced the biofilm biomass of three isolates of MSSA, eight isolates of MRSA and four isolates of VISA but increased the metabolic activity in certain MSSA, MRSA and VISA isolates, indicating AM may possess biofilm reduction effects but not bactericidal effects. Based on these findings, AM could be further studied and developed as a potential therapeutic agent for chronic S. aureus infections.

Phenotypic and genotypic changes of Staphylococcus aureus in the presence of the inappropriate concentration of chlorhexidine gluconate

BackgroundChlorhexidine gluconate (CHG) is a disinfectant agent with different applications in health care. Improper use of CHG causes antimicrobial resistance in bacteria as a public health threat. Since Staphylococcus aureus is a common bacteria, it is expected usually exposed to CHG in the hospital and community. The present study aimed to correlate the phenotypic and genotypic changes in a S. aureus strain upon serial adaptation with supra-inhibitory CHG concentration for 50 days.ResultsAfter in vitro serial culture of 5 × 105 CFU/ml of a clinical vancomycin-susceptible S. aureus strain (VAN-S) into brain heart infusion (BHI) broth containing CHG 1/4, 1/2, 1, and 2 × minimal inhibitory concentration (MIC) values of VAN-S in 37 °C during 50 days, we isolated a S. aureus strain (CHGVan-I) with a ≥ twofold decrease in susceptibility to CHG and vancomycin. CHG-induced CHGVan-I strain was considered as a vancomycin-intermediate S. aureus (VISA) strain with a VAN MIC of 4 μg/ml using the broth macro dilution method. However, reduced resistance was observed to tetracycline family antibiotics (doxycycline and tetracycline) using a modified Kirby-Bauer disk diffusion test. Moreover, a remarkable reduction was detected in growth rate, hemolysis activity (the lysis of human red blood cells by alpha-hemolysin), and colony pigmentation (on BHI agar plates). Biofilm formation (using the Microtiter plate method and crystal violet staining) was significantly increased upon CHG treatment. Adaptive changes in the expression of a set of common genes related to the development of VISA phenotype (graTSR, vraTSR, walKR, agr RNAIII, sceD, pbpB, and fmtA) were analyzed by Reverse Transcription quantitative PCR (RT-qPCR) experiment. Significant changes in vraTSR, agr RNAIII, sceD, and pbpB expression were observed. However, gene sequencing of the two-component system vraTSR using the Sanger sequencing method did not detect any non-synonymous substitution in CHGVan-I compared to wild-type. The clonality of VAN-S and CHGVan-I strains was verified using the pulsed-field gel electrophoresis (PFGE) method.ConclusionsThe importance of the present study should be stated in new detected mechanisms underlying VISA development. We found a link between the improper CHX use and the development of phenotypic and genotypic features, typical of VISA clinical isolates, in a CHG-induced strain.Since disruption of the cell wall biosynthesis occurs in VISA isolates, our CHG-induced VISA strain proved new insights into the role of CHG in the stimulation of the S. aureus cell wall.

Methicillin-Resistant Staphylococcus aureus (MRSA): Prevalence, Antimicrobial Susceptibility Pattern, and Detection of mecA Gene among Cardiac Patients from a Tertiary Care Heart Center in Kathmandu, Nepal.

Background:Methicillin Resistant Staphylococcus aureus (MRSA) is a significant human pathogen associated with nosocomial infections. mecA in the S. aureus is a marker of MRSA. The main objective of this study was to detect mecA and vanA genes conferring resistance in S. aureus among cardiac patients attending Sahid Gangalal National Heart Centre (SGNHC), Kathmandu, Nepal between May and November 2019.Methods:A total of 524 clinical samples (blood, urine, sputum) were collected and processed. Bacterial isolates were tested for antimicrobial susceptibility test (AST) and screening for MRSA was carried out by cefoxitin disc diffusion method. Minimum inhibitory concentration (MIC) of vancomycin for MRSA was established by agar dilution method and chromosomal DNA was extracted and used in polymerase chain reaction targeting the mecA and vanA genes.Results:Out of 524 specimens, 27.5% (144/524) showed bacterial growth. Among 144 culture positive isolates, S. aureus (27.1%; 39/144) was the predominant bacteria. Among 39 S. aureus isolates, all isolates were found resistant to penicillin followed by erythromycin (94.9%; 37/39), gentamicin (94.9%; 37/39) and cefoxitin (87.2%; 34/39). Out of 39 S. aureus, 87.2% (34/39) were MRSA. Among 34 MRSA, 8.8% (3/34) were vancomycin intermediate S. aureus (VISA). None of the MRSA was resistant to vancomycin. All of the 3 VISA isolates were obtained from inpatients. Of 39 S. aureus, 82.1% (32/39) harbored mecA gene. Similarly, the entire VISA isolates and 94.1% (32/34) of the MRSA isolates were tested positive for mecA gene.Conclusions:High prevalence of MRSA among the cardiac patients indicates the increasing burden of drug resistance among bacterial isolates. Since infection control is the crucial step in coping with the burgeoning antimicrobial resistance in the country, augmentation of diagnostic facilities with routine monitoring of drug resistance is recommended.

Global prevalence and distribution of vancomycin resistant, vancomycin intermediate and heterogeneously vancomycin intermediate Staphylococcus aureus clinical isolates: a systematic review and meta-analysis

Vancomycin-resistant Staphylococcus aureus (VRSA), Vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) are subject to vancomycin treatment failure. The aim of the present study was to determine their precise prevalence and investigate prevalence variability depending on different years and locations. Several international databases including Medline (PubMed), Embase and Web of Sciences were searched (data from 1997 to 2019) to identify studies that addressed the prevalence of VRSA, VISA and hVISA among human clinical isolates around the world. Subgroup analyses and meta-regression were conducted to indicate potential source of variation. Publication bias was assessed using Egger’s test. Statistical analyses were conducted using STATA software (version 14.0). Data analysis showed that VRSA, VISA and hVISA isolates were reported in 23, 50 and 82 studies, with an overall prevalence of 1.5% among 5855 S. aureus isolates, 1.7% among 22,277 strains and 4.6% among 47,721 strains, respectively. The overall prevalence of VRSA, VISA, and hVISA before 2010 was 1.2%, 1.2%, and 4%, respectively, while their prevalence after this year has reached 2.4%, 4.3%, and 5.3%. The results of this study showed that the frequency of VRSA, VISA and hVISA after 2010 represent a 2.0, 3.6 and 1.3-fold increase over prior years. In a subgroup analysis of different strain origins, the highest frequency of VRSA (3.6%) and hVISA (5.2%) was encountered in the USA while VISA (2.1%) was more prevalent in Asia. Meta-regression analysis showed significant increasing of VISA prevalence in recent years (p value ≤ 0.05). Based on the results of case reports (which were not included in the calculations mentioned above), the numbers of VRSA, VISA and hVISA isolates were 12, 24 and 14, respectively, among different continents. Since the prevalence of VRSA, VISA and hVISA has been increasing in recent years (especially in the Asian and American continents), rigorous monitoring of vancomycin treatment, it’s the therapeutic response and the definition of appropriate control guidelines depending on geographical regions is highly recommended and essential to prevent the further spread of vancomycin-resistant S. aureus.

Identification and evaluation of Anisochilus carnosus (L. fil.) Wall. as a novel candidate with therapeutic potential against multi-drug resistant Staphylococcus aureus

BackgroundStaphylococcus aureus has been constantly emerging with newer drug resistance patterns; vancomycin intermediate S. aureus (VISA) and vancomycin resistant S. aureus (VRSA) being recent entries. Hence the growing quest for newer antibiotics has entered the realm of the plants as they are less explored and usually have negligible side effects. Although a number of medicinal plants are being used traditionally, their spectrum and mechanism of action are not completely deciphered. ObjectivesTo evaluate the aqueous (the traditionally administered extract) and acetone/ethanol (organic solvents with the highest capacity to extract phytoconstituents) extracts of ten medicinal plants traditionally used to treat respiratory infections in south India for antibacterial activity against S. aureus. MethodsThe antibacterial activity against S. aureus standard strain was evaluated by the broth dilution method. The extracts exhibiting antibacterial activity were further validated using 50 S. aureus isolates by the broth dilution and agar diffusion methods. ResultsOnly Anisochilus carnosus acetone extract (ACace; MIC-0.33 ± 0.14 mg/mL) exhibited significant antibacterial activity, and its zone diameter break points were determined. The sensitivity of the 50 S. aureus clinical isolates to ACace (72 %) was comparable to methicillin (68 %), linezolid (94 %) and clindamycin (82 %). Additionally, ACace, inhibited majority of the MRSA (87.5 %), VISA (70 %) and VRSA (83 %) isolates. ConclusionThis is the first study to report the antibacterial potential of a novel candidate, ACace against S. aureus, evaluate its inhibitory effect on clinical isolates including drug-resistant strains and highlight the importance of zone diameter break points for the evaluation of medicinal plant extracts.

Molecular Study of Vancomycin Resistance in Hospital Acquired Staphylococcus Infection

Purpose: Vancomycin is treatment of choice in MRSA infection. In the last years there is increasing incidence of vancomycin resistance either vancomycin intermediate S. aureus (VISA) or vancomycin-resistant staphylococci (VRSA). This increasing in resistance strains is challenging problem especially in hospital acquired infection. The present study was carried out to find the incidence of(VISA)and(VRSA) among MRSA in children hospital in Egypt with correlation of molecular study for mec A gene and Vancomycin resistance gene Van A, Van B and Van C genes by multiplex PCR. Method: Out of 678 hospital acquired infection (365) staphylococcal isolates was obtained in which coagulase negative were (152) (41.6%) and positive (213) (58.4%) were isolated from various samples which confirmed to be hospital acquired infection according to CDC guidelines. All Staphylococcal aureus isolates and coagulase negative (CoNS) were suspected to antibiotic disc diffusion. MRSA strain were subjected to MIC for vancomycine and molecular study for detection mec A and Van A, Van B and Van C genes by multiplex PCR Results: MRSA was reported in (103) staphylococcal and (10) CONS mec gene was present in all MRSA stain. Vancomycine resistance gene was not found in any of CoNS. While in staphylococcal aureus vanA was present in 2 VRSA and 4 VISA isolates and van B was present in 6 VISA and in one VRSA isolates vanC gene was not found either in Staphylococcus aureus nor CoNS. Conclusion: The results of the current study illustrate the emergence of vancomycin resistance among methicillin-resistant S. aureus isolated from children with healthcare-associated infections. The majority revealed the occurrence of vanA and vanB as an accountable mechanism for this resistance.

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.

Detection of Methicillin Resistant Staphylococcus Aureus with Reduced Susceptibility to Vancomycin

Background: As there is no molecular-based assays available for the detection of hVISA and VISA. However, increasing amounts of data support a number of methods for the screening and confirmation of heterogeneous vancomycin intermediate S. aureus (hVISA) and vancomycin intermediate S. aureus (VISA) infection. The vancomycin MIC result alone is unable to accurately distinguish hVISA from VSSA isolates, and the use of MIC testingalone will fail to detect hVISA strains that are relatively common among isolates of Staphylococcus aureus (S. aureus) with broth MICs of 2 g per ml. Objective: The aim of the present work was to detect the efficacy of phenotypic and automated methods for detection of MRSA with reduced susceptibility to vancomycin. It aimed also, to determine the best MIC concentration in vancomycin screening agar for detection of VISA among MRSA isolates. Methods: One hundred MRSA isolates were obtained from 100 patients from different departments of Ain Shams University Hospitals during the period from October 2015 to the end of April 2016. They were isolated from different clinical specimens; sputum, wound swabs, blood, pus, urine, and body fluid that were referred to central microbiology laboratory for routine culture and sensitivity. Detection of S. aureus with reduced susceptibility to vancomycin was done by vancomycin screening agar with different concentrations 2,4,6 ug/ml with and without casein, MIC broth microdilution method for vancomycin according to CLSI 2015, and Vitek 2 automated system for determination of vancomycin MIC. Results: Out of 100 MRSA isolates, vancomycin screening agar 2ug/ml with casein showed highest detection rate for VISA isolates (48 %) among other screening agars. Vancomycin screening agar 6 ug/ml without casein gave the lowest detection rate (29%). So, adding casein to vancomycin screening agar did not increase detection of VISA in any of vancomycin screening agar except for that with 2ug/ml vancomycin. Vancomycin screening agar 2ug/ml with casein gave the best sensitivity among all vancomycin screening agar tested. VITEK 2 system failed to detect any isolates with reduced susceptibility to vancomycin. They were sensitive to linezolid (100%) followed by tigecyclin (99%) then Quinupristin-dalfopristin (91%). However, most of the isolates were resistant to tetracycline (85%) followed by gentamicin (80%) then ciprofloxacin (63%). Conclusion: BHI agar with 2ug/ml vancomycin and 16 g/l casein is a reliable, easy to perform, and inexpensive method to screen large number of S. aureus isolates for detection of reduced susceptibility to vancomycin on a daily basis. Applying quadruplicate technique in vancomycin screening agar may increase the yield for detection of VISA isolates. Although vancomycin screening agar 6 ug/ml is recommended by CLSI as a screening method for detection of VISA, yet it did not perform well and underestimated VISA isolates. VITEK 2 system is not an appropriate method for detection of S. aureus with reduced susceptibility to vancomycin (VISA). MRSA isolates with reduced susceptibility to vancomycin can be treated effectively with Linezolid.

Novel single-nucleotide variations associated with vancomycin resistance in vancomycin-intermediate Staphylococcus aureus.

Prolonged vancomycin usage may cause methicillin-resistant Staphylococcus aureus to become vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA). Mechanisms of vancomycin resistance of VISA and hVISA are still unclear. In this study, analyses of nucleotide sequence variations in 30 vancomycin-sensitive S. aureus (VSSA), 41 hVISA and 16 VISA isolates revealed 29 single-nucleotide variations in 12 genes (fmtC, graR, graS, htrA, mecA, pbp2, pbp4, srtA, tcaA, upps, vicK and vraR) that are related to cell wall synthesis or the two-component system. Six of these 29 single-nucleotide variations were novel and resulted in the following amino acid changes: Q692E in FmtC; T278I, P306L and I311T in HtrA; and I63V and K101E in Upps. Since P306L and I311T in HtrA and I63V in Upps were present in the majority (76.7%–86.7%) of VSSA isolates, these three amino acid variations may not be associated with vancomycin resistance. The other three amino acid variations (T278I in HtrA, K101E in Upps and Q692E in FmtC) were present in the majority (87.5%–93.8%) of hVISA and VISA isolates, but only in a small number (22.9%–25.7%) of VSSA isolates, suggesting that they are associated with vancomycin resistance.

Molecular characterization of vancomycin-intermediate Staphylococcus aureus isolates from Tehran

ObjectiveTo determine the prevalence and some genetic characteristics of clinical isolates of Staphylococcus aureus (S. aureus) with reduced susceptibility to vancomycin. MethodsA total of 414 isolates of S. aureus were collected from clinical specimens from hospitals in Tehran. Vancomycin-intermediate S. aureus (VISA) was determined by brain heart infusion agar containing 4 μg/mL vancomycin screening plate and confirmed via E-test. VISA isolates were analysed for vanA, vanB, mecA, staphylococcal cassette chromosome mec types, surface protein A (Spa) types and agr specific groups. ResultsBrain heart infusion agar containing 4 μg/mL vancomycin screening tests revealed that 17.14% (n = 71) of S. aureus isolates were VISA phenotype. Ten of the 71 isolates were confirmed by E-test method (minimal inhibitory concentration was 4 to 8 μg/mL). All VISA isolates were susceptible to linezolid and 6 isolates (60%) were methicillin-resistant S. aureus. Five isolates belonged to agr Group II, 4 belonged to agr Group I and 1 belonged to agr Group III. Spa type t030, and staphylococcal cassette chromosome mec Type III were dominant among VISA isolates. ConclusionsThis study provides further evidence of the global dissemination of VISA isolates and emphasizes to vancomycin susceptibility testing prior to antibiotic therapy.

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.

Phenotypic Characteristics of Vancomycin-Non-Susceptible Staphylococcus aureus.

Background:Staphylococcus aureus, with reduced vancomycin susceptibility, is probably under the regulation of several genes and various express phenotypes.Objectives:This study aimed to investigate the phenotypic differences between vancomycin-susceptible S. aureus (VSSA), vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA) isolates.Materials and Methods:A total of 130 methicillin-resistant S. aureus (MRSA) isolates were studied, including 49 VSSA, 28 hVISA, and 5 VISA isolates from blood cultures and 48 isolates (two VSSA, six hVISA, and 40 VISA) derived in vitro (laboratory-induced/sub-passaged). Their phenotypes were examined using a coagulase tube test, colony spreading on soft agar, and urease activity. The SCCmec and agr typing were performed using multiplex PCR.Results:Most of the MRSA isolates were SCCmec III-agr I (84.5%), followed by SCCmec II-agr II (11.8%). The average plasma coagulation time of vancomycin-non-susceptible isolates was longer than that of the susceptible isolates (12 vs. 2.6 hours). Four hVISA (P = 0.023) and nine VISA (P < 0.001) isolates yielded a negative coagulase test after 24-hour incubation. The percentage of VSSA isolates showing non-spreading colonies (accessory gene regulator (agr) dysfunction) was significantly lower than in the VISA group (P = 0.013), but no significant difference was found between VSSA and hVISA. The VISA group showed higher urease activity than that of the VSSA and hVISA groups (P = 0.002).Conclusions:There were diverse phenotypic changes among vancomycin-non-susceptible S. aureus isolates. This may be due to the variety of related regulatory systems. The diversity of phenotypic expression may result in its misidentification in routine laboratory checks.

Genome Sequence-Based Discriminator for Vancomycin-Intermediate Staphylococcus aureus

Vancomycin is the mainstay of treatment for patients with Staphylococcus aureus infections, and reduced susceptibility to vancomycin is becoming increasingly common. Accordingly, the development of rapid and accurate assays for the diagnosis of vancomycin-intermediate S. aureus (VISA) will be critical. We developed and applied a genome-based machine-learning approach for discrimination between VISA and vancomycin-susceptible S. aureus (VSSA) using 25 whole-genome sequences. The resulting machine-learning model, based on 14 gene parameters, including 3 molecular typing markers and 11 genes implicated in reduced vancomycin susceptibility, is able to unambiguously distinguish between the VISA and VSSA isolates analyzed here despite the fact that they do not form evolutionarily distinct groups. As such, the model is able to discriminate based on specific genomic markers of antibiotic susceptibility rather than overall sequence relatedness. Subsequent evaluation of the model using leave-one-out validation yielded a classification accuracy of 84%. The machine-learning approach described here provides a generalized framework for the application of genome sequence analysis to the classification of bacteria that differ with respect to clinically relevant phenotypes and should be particularly useful in defining the genomic features that underlie antibiotic resistance.

Peptide Biomarker Discovery for Identification of Methicillin-Resistant and Vancomycin-Intermediate Staphylococcus aureus Strains by MALDI-TOF

Rapid identification of community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA), hospital-associated (HA) MRSA, and vancomycin-intermediate S. aureus (VISA) is essential for proper therapy and timely intervention of outbreaks. In this study, peptide biomarkers for rapid identification of methicillin-resistant and vancomycin-intermediate S. aureus strains were discovered by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results showed that the 1774.1 and 1792.1 m/z peaks corresponding to the phenol-soluble modulin α1 and phenol-soluble modulin α2 peptides, respectively, were present in the majority (95%, 121 of 127) of SCCmec types IV and V isolates, but only in 8% (15 of 185) of SCCmec types I-III isolates. Since SCCmec types I-III isolates are recognized as HA-MRSA and most CA-MRSA isolates belong to SCCmec types IV and V, these two peptides may serve as markers for discrimination between HA-MRSA and CA-MRSA isolates. The 1835.0 and 1863.0 m/z peaks were present in 50% (4 of 8) of heterogeneous VISA and 88% (14 of 16) of VISA isolates. The peptides of these two peaks were identified as proteolytic products of the acyl carrier protein. The results of this study provide the possibility to develop methods for identification of CA-MRSA, HA-MRSA, and vancomycin-resistant S. aureus isolates based on the presence of these peptides.

Rapid detection of vancomycin-non-susceptible Staphylococcus aureus using the spiral gradient endpoint technique

Several methods have been introduced for detection of vancomycin-non-susceptible Staphylococcus aureus [heterogeneous vancomycin-intermediate S. aureus (hVISA) and vancomycin-intermediate S. aureus (VISA)]. However, the limitations of these methods can delay appropriate therapy for the patient. This study evaluated the spiral gradient endpoint (SGE) technique for detection of hVISA/VISA. The SGE method was evaluated for intra-batch, inter-batch and inter-observer reproducibility in comparison with MICs determined by agar dilution. Three media, Mueller-Hinton agar, brain heart infusion agar and brain heart infusion agar with 5% glucose, were evaluated. The SGE method was compared with agar dilution for correlation of MIC and susceptibility category using control strains, clinical isolates and induced vancomycin-non-susceptible strains. The SGE method had good reproducibility and there was excellent correlation of MICs generated by SGE using brain heart infusion agar with those by agar dilution (r(2) =0.950), with no difference in resistance categories generated by the two methods. All VISA isolates were correctly identified and the method allowed easy identification of hVISA by means of the trailing endpoint. SGE offers a simple, rapid and cost-effective alternative method for the detection of hVISA/VISA for the routine laboratory. Early recognition of vancomycin-non-susceptible strains can allow the change to appropriate antibiotics, resulting in potentially better patient outcomes.

Management of Infections Caused by Vancomycin-Resistant Gram-Positive Bacteria

For 3 decades, vancomycin has been a favored therapy for serious penicillinand cephalosporin-resistant Gram-positive bacterial infections. Development of vancomycin resistance may have been restrained by the requirement for intravenous administration, which limited its use and lessened exposure to the gastrointestinal flora. Nevertheless, physicians are increasingly encountering children and adults infected with vancomycin-resistant Gram-positive organisms. Resistance to vancomycin has been encountered in all commonly occurring, clinically significant Gram-positive bacterial species except Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae. Although therapeutic options are usually guided by the antimicrobial susceptibilities of individual isolates, recommended infection-control measures vary with the specific species and the mechanism of resistance. VANCOMYCIN-RESISTANT STAPHYLOCOCCUS AUREUS Vancomycin-resistant Staphylococcus aureus (VRSA) were first recognized in Japan in 1996; the first case of infection due to VRSA in the United States was reported in 2002. Since then, at least 8 additional cases of VRSA infection have been identified in the United States, with more than half of these cases occurring in Michigan. Isolates often seem to have evolved independently in settings where patients were previously colonized with vancomycin-resistant enterococcus (VRE) and methicillin-resistant S. aureus (MRSA). Environments that promote biofilm formation may facilitate transmission of resistance from VRE to MRSA. VRSA isolates (vancomycin MIC 16 g/mL) are fundamentally different from vancomycin-intermediate S. aureus (VISA) isolates (vancomycin MIC 4 – 8 g/mL). Resistance in VISA isolates is typically mediated via mechanisms that develop in the presence of vancomycin and are not readily transferrable to other strains. The potential for spread of these isolates is low in the absence of vancomycin therapy. In contrast, VRSA isolates uniformly contain the vanA gene derived from enterococcus (discussed below). The VanA phenotype is transferable to other MRSA strains and across microbial species, with a much greater potential for spread, even in the absence of vancomycin therapy. Thus far, VRSA outbreaks have not occurred; however, because of the potential for spread with dire consequences, a case of VRSA infection warrants a more rigorous infection-control response than a case of infection with MRSA, VISA, or VRE. Infection-control measures should be implemented urgently according to a written plan, if VRSA is suspected. The local health department and the Centers for Disease Control and Prevention should be consulted immediately. Management of contacts should occur according to contact categorization. Attempts to decolonize individuals who are found to be colonized should be considered on a case-by-case basis. Infections with S. aureus isolates that have vancomycin MICs 4 g/mL typically do not respond clinically to vancomycin therapy. To date, all VRSA have also been found to be resistant to methicillin. Fortunately, all VRSA and VISA isolates have been susceptible to one or more commercially available antibiotics. Recommended antibiotics for VRSA and VISA, according to susceptibilities and other clinical considerations, are listed in Table 1. Secondary agents include teicoplanin (not available in the United States), trimethoprim-sulfamethoxazole, tetracycline, rifampin (usually in combination therapy), and chloramphenicol.

Prevalence and accessory gene regulator (agr) analysis of vancomycin-intermediate Staphylococcus aureus among methicillin-resistant isolates in Taiwan—SMART program, 2003

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in nosocomial staphylococcal infections in Taiwan has exceeded 50% since 2000. However, little relevant data has been available concerning vancomycin-intermediate S. aureus (VISA) and heteroresistant VISA (hVISA). We collected 1,000 MRSA isolates from ten medical center hospitals in Taiwan during 2003. All were initially screened for reduced susceptibility to vancomycin on brain heart infusion (BHI) agar containing 5 mg/L vancomycin. Among 34 MRSA isolates that grew on the screening plates, two VISA isolates (0.2%) and seven hVISA isolates (0.7%) were evident. Vancomycin-resistant S. aureus was not detected. The accessory gene regulator (agr) typing of all 1,000 MRSA strains were typed by multiplex polymerase chain reaction (PCR); 919 strains (91.9%) including the VISA and hVISA isolates belonged to agr group I, 78 strains (7.8%) were agr group II, two strains (0.2%) were agr group III, and one isolate (0.1%) was agr group IV. There was no relationship between sample sites and agr typing. In 2003, the incidence of hVISA and VISA in Taiwan was low. Continued surveillance is recommended, given the implementation of new Clinical and Laboratory Standards Institute (CLSI) criteria for S. aureus and the increasing clinical use of glycopeptides.

Genetic changes associated with glycopeptide resistance in Staphylococcus aureus: predominance of amino acid substitutions in YvqF/VraSR

To further understand the mechanism of intermediate-level glycopeptide resistance, resulting from multiple endogenous mutations, in both laboratory-derived and clinically isolated Staphylococcus aureus. Laboratory-derived S. aureus strains were generated under selection using a variety of cell-wall-active antibiotics. Complete sequences of 27 genes, including 17 two-component histidine kinase sensors, were then compared with those of their susceptible parent strain. Further genetic analysis was performed on 125 clinical S. aureus isolates and 42 geographically diverse isolates of vancomycin-intermediate S. aureus (VISA). Selective pressure using imipenem resulted in single point mutations leading to amino acid substitutions in two genes: vraS, encoding a two-component histidine kinase sensor; and SA1702 (also called yvqF, located immediately upstream of vraS), encoding a conserved hypothetical protein. The accumulation of the mutation in two distinct proteins-MsrR, a peptide methionine sulphoxide reductase regulator, and TcaA, a teicoplanin-resistance-associated protein-correlated with further increases in the glycopeptide MIC. The prevalence of YvqF/VraSR mutants among 125 clinical isolates along with the corresponding teicoplanin MICs was as follows: 0% (0/39), < or =1 mg/L; 48.6% (17/35), 2 mg/L; 72.7% (24/33), 4 mg/L; 93.8% (15/16), 8 mg/L; and 100% (2/2), 16 mg/L. Genetic analysis of 42 VISA isolates also identified the predominant amino acid substitutions in YvqF/VraS: 9 isolates (21.4%) revealed mutations in YvqF, followed by 7 isolates with mutations in VraS (16.7%). Our findings provide novel insights into the high prevalence and genetic diversity of YvqF/VraSR mutants among clinical S. aureus isolates with reduced susceptibility to teicoplanin.