hVISA Isolates Research Articles

Presence of multiple van genes among glycopeptide non-susceptible Staphylococcus aureus exhibiting in vitro MIC creep phenomenon: A study from north-east India.

Background & objectives The global prevalence of vancomycin-resistant Staphylococcus aureus (VRSA) has increased two fold since 2010, accounting for 2.4 per cent of S. aureus infections. The emerging hVISA isolates and their increasing trends pose a serious therapeutic challenge. The present study investigated in vitro vancomycin and teicoplanin minimum inhibitory concentration (MIC) creep in S. aureus and assessed their revertants. Methods A total of 845 isolates were collected for this study, and 246 were confirmed as S. aureus. Molecular characterization of vancomycin resistance was carried out by PCR assay targeting genes types viz: vanA, vanB, vanC, vanC2/C3, vanD, vanE, and vanG. MIC was determined for vancomycin and teicoplanin by agar dilution method. MIC creep and revertant analysis were done by broth dilution method in the presence and absence of antibiotics. Results PCR assay confirmed 12 isolates were harboured vanA, followed by vanD (n=8) and vanB (n=7). The study showed 69 isolates were screened positive for glycopeptide non-susceptibility. While analyzing vancomycin MIC creep, four isolates showed a significant increase in MIC, whereas no creep phenomenon was observed for the rest. In the case of teicoplanin, seven isolates showed the MIC creep phenomenon. Revertant analysis of all the isolates that showed MIC creep phenomenon for vancomycin and teicoplanin reverted to their original MIC when the antibiotic pressure was withdrawn. Interpretation & conclusions In the present study setting, glycopeptide non-susceptibility was found in eight per cent of the isolates, and the present study found the occurrence of multiple van genes from isolates calculated from a single study center will impose a serious challenge in infection control and antibiotic policy. This study also underscores that heterogenic resistant isolates, upon exposure to vancomycin and teicoplanin at a minimum level, exhibited an increase in MIC, which will impact individuals receiving glycopeptide therapy.

ERADICATION OF HETEROGENEOUS VANCOMYCIN ITERMEDIATED STAPHYLOCOCCUS AUREUS (hVISA) USING PEGANUM HARMALA L. SEEDS EXTRACTS

The main purpose of this research is to evaluate the antibacterial essential oil of Peganum harmala L. seeds against locally isolated heterogeneous vancomycin intermediated Staphylococcus aureus (hVISA) at doses ranging from 0.03 to 200 mg/ml. The filtered essential oil of P. harmala, after extract by the 96% ethanol in a Soxhlet extraction device, was analyzed by GC/MS (gas chromatography-mass spectroscopy). The results showed that the P. harmala seeds oils contained several compounds, such as Harmine (58.26%), followed by 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (28.55%) and Hexadecanoic acid, methyl ester (4.30%), It is highly effective against targeted bacteria. The MIC method was used to investigate antibacterial activity by measuring the lowest inhibitory concentration of the extract and observed that the inhibition concentration against hVISA isolates was 0.3 mg/ml. The gene expression was detected after RNA extract from hVISA (untreated and treated) with P. harmala essential oil using the Quantitative Real-Time PCR method the results revealed that gene expression was low after bacterial with plant extract treatment. Finally, the ethanolic extract of p. harmala seeds appears to be a potential therapy choice for hVISA infection.

Global distribution of heterogeneous vancomycin-intermediate Staphylococcus aureus strains (1997–2021): a systematic review and meta-analysis

Heterogeneous vancomycin-intermediate Staphylococcus aureus is considered one of the main causes in treatment failure of vancomycin, which leads to poor clinical outcomes. Herein, we comprehensively evaluated characteristics such as global prevalence, trend, and genetic backgrounds of these strains. In this study, we conducted a meta-analysis based on PRISMA checklist 2020. In the beginning, global databases were searched to achieve the studies related to the prevalence of hVISA in clinical isolates of methicillin-resistant Staphylococcus aureus. After retrieving the eligible English studies, the prevalence of hVISA isolates and their trend changes were assessed using event rate with 95% confidence intervals. In the present study, the prevalence of 114 801 MRSA isolates (of 124 studies) was 64%. According to our results, although the frequency of infection with hVISA is increasing in recent years, there is not a significant difference between Asian countries and Europe/America (6.1% vs. 6.8%). In addition, infection with hVISA bacteria was higher in bacteraemic patients than other infections (9.4% vs. 5.5%), which increases hospitalization, treatment costs, and mortality in these patients. Isolates harbouring SCCmec types II and III are most common genotypes in hVISA strains. The prevalence of hVISA is increasing, which will reduce the effectiveness of vancomycin treatment in the coming years. The presence of hVISA stains in blood samples was higher than the other samples, which is threatening for bacteraemic patients. The results of the current study indicate a universal program to identify and control the spread of such strains in nosocomial infections.

Prevalence and molecular characteristics of heterogeneous vancomycin intermediate Staphylococcus aureus in a tertiary care center of northern China

The use of glycopeptide medications may decline in line with the annual decline in methicillin-resistant Staphylococcus aureus (MRSA) detection rates in China. The rate of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA)detection may be impacted by this. However, there is currently a dearth of information on the incidence of hVISA in China. This study aims to analyze the recent epidemiology and molecular characteristics of hVISA strains in Beijing, China. A total of 175 non-duplicate MRSA strains from various infection sites were collected from a medical center between January 2018 and May 2023 and underwent molecular typing and susceptibility testing (Vitek2). Vancomycin and teicoplanin MICs were also evaluated by standard broth microdilution method and agar dilution method, respectively. Isolates growing on screening agar (BHIV4 and BHIT5, brain heart infusion agar containing 4 μg/ml vancomycin and 5 μg/ml teicoplanin, respectively) were characterized further by analysis of macro-Etest (MET) and population analysis profiling with area under the curve (PAP-AUC). The proportion of hVISA among MRSA isolates was 8.6 %. BHIT5 could select all hVISA strains while BHIV4 and MET only selected two hVISA strains. Compared with vancomycin- susceptible Staphylococcus aureus (VSSA), hVISA isolates were less susceptible to erythromycin and clindamycin. In addition, hVISA frequency was MIC-independent despite using different detection methods. In total, 11 types of STs, 28 types of spa typing, four types of SCCmec typing, and two types of agr typing were identified and the predominant type in both MRSA and hVISA isolates was ST239-t030-SCCmecIII-agr I. The analysis of biofilm formation, growth, and virulence genes in hVISA strains revealed sparse information. The dataset presented in this study provided the prevalence and molecular characteristics of hVISA in hospital settings and the combination of BHIT5 and PAP-AUC may identify hVISA efficiently. The result of genotyping suggested the genotype of hVISA was mainly consistent with that of local MRSA. Additional studies on the characteristics of hVISA strains were necessary.

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.

Controlling the heterogeneous vancomycin intermediated Staphylococcus aureus (hVISA) through the use of Rosmarinus officinalis L. leaves extract

Treatment failure and persistent infection are two serious problems that result from Staphylococcus aureus having decreased vancomycin susceptibility. The present study investigates the effects of Rosmarinus officinalis L. leaves extract on the growth and quorum sensing (QS) of heterogeneous vancomycin-intermediate S. aureus hVISA isolates. Real-time RT-polymerase chain reaction (PCR) is used to determine the transcriptional changes of the accessory gene regulator (agr) in the (hVISA) strain after and before being treated with the R. officinalis leaves extract. The antibacterial activity of the EOs was determined through the use of a microtitre plate test (MtP); with a minimum inhibitory concentration (MIC) of 0.7 mg/ml against hVISA, a rosemary leaves extract has a high level of inhibitory activity. However, the extraction of essential oils is done in a soxhlet apparatus by using ethanol 96%. Gas chromatography and mass spectrometry (GC/MS) and revealed the most common chemicals containing verbenone, 36.20%, and 1,8-cineol (Eucalyptol), 12.14%. Finally, after treatment with sub-MIC concentration, hld gene levels significantly decreased in hVISA strains (a mean of 0.4-fold) in comparison to the control strains (1.0-fold) in late-exponential phases, regardless of whether the agr mutation is present. The down regulation of the hld gene may be a significant genetic event in the hVISA strain.

Multiomics characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates with heterogeneous intermediate resistance to vancomycin (hVISA) in Latin America.

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) compromise the clinical efficacy of vancomycin. The hVISA isolates spontaneously produce vancomycin-intermediate Staphylococcus aureus (VISA) cells generated by diverse and intriguing mechanisms. To characterize the biomolecular profile of clinical hVISA applying genomic, transcriptomic and metabolomic approaches. 39 hVISA and 305 VSSA and their genomes were included. Core genome-based Bayesian phylogenetic reconstructions were built and alterations in predicted proteins in VISA/hVISA were interrogated. Linear discriminant analysis and a Genome-Wide Association Study were performed. Differentially expressed genes were identified in hVISA-VSSA by RNA-sequencing. The undirected profiles of metabolites were determined by liquid chromatography and hydrophilic interaction in six CC5-MRSA. Genomic relatedness of MRSA associated to hVISA phenotype was not detected. The change Try38 → His in Atl (autolysin) was identified in 92% of the hVISA. We identified SNPs and k-mers associated to hVISA in 11 coding regions with predicted functions in virulence, transport systems, carbohydrate metabolism and tRNA synthesis. Further, capABCDE, sdrD, esaA, esaD, essA and ssaA genes were overexpressed in hVISA, while lacABCDEFG genes were downregulated. Additionally, valine, threonine, leucine tyrosine, FAD and NADH were more abundant in VSSA, while arginine, glycine and betaine were more abundant in hVISA. Finally, we observed altered metabolic pathways in hVISA, including purine and pyrimidine pathway, CoA biosynthesis, amino acid metabolism and aminoacyl tRNA biosynthesis. Our results show that the mechanism of hVISA involves major changes in regulatory systems, expression of virulence factors and reduction in glycolysis via TCA cycle. This work contributes to the understanding of the development of this complex resistance mechanism in regional strains.

Investigation of the VISA and hVISA Presence in Methicillin-Resistant Staphylococcus aureus Isolates by Various Methods

Staphylococcus aureus is an important human pathogen that causes community and hospital-acquired infections. The role of vancomycin in the treatment of methicillin-resistant S.aureus infections is indisputable. However, vancomycin intermediate susceptible S.aureus (VISA) and heterogeneously VISA (hVISA) isolates, that cause treatment failures during the use of vancomycin, cannot be detected by routine laboratory methods. The gold standard method for the detection of these isolates is the population profile analysis-area under the curve (PAP-AUC) method. In this study, it was aimed to determine the presence of mecA and mecC gene regions that cause methicillin resistance, the clonal relationship between isolates, and the presence of VISA and hVISA. A total 68 methicillin-resistant S.aureus (MRSA) strains were included in this study which were isolated in the microbiology laboratory of the hospital between 2015- 2020. Identification of the isolates were determined by matrix assisted laser desorption ionization-time of flight mass spectrophotometry (VITEK MS, BioMérieux, France). Methicillin resistance was investigated by disk diffusion method using cefoxitin (30 µg, Bioanalyse, Türkiye) disk and vancomycin MIC values were determined by broth microdilution method. mecA and mecC gene regions were investigated by polymerase chain reaction (PCR) method. The presence of VISA and hVISA were investigated by modified agar screening, macro gradient diffusion test and confirmated by PAP-AUC methods, and the clonal relationship between isolates were investigated by pulsed field gel electrophoresis method. The mecA gene region was determined in all isolates, but the mecC gene region was not found in any of the isolates. The MIC50 value of the isolates was determined as 1 μg/mL and the MIC90 value was determined as 2 μg/mL by broth microdilution method. Six VISA and four hVISA suspected strains were detected by a modified agar screening method. Among the isolates identified as suspicious by the modified agar screening method, one isolate was evaluated as VISA and one isolate was evaluated as hVISA by the gold standard PAP-AUC method. No dominant epidemic isolate has been identified by PFGE. As a result, VISA and hVISA were determined in the hospital. The increase in these isolates is a serious concern. For this reason, it is believed that it would be beneficial to investigate the VISA/hVISA ratios in MRSA isolates at certain periods, and to take necessary infection control measures to implement measures and practices to prevent the spread of these isolates in the community and hospital environment.

Vancomycin for methicillin-resistant staphylococcus aureus biofilm eradication is associated with the emergence of heterogeneous vancomycin intermediate staphylococcus aureus

Vancomycin is the first-line therapy for MRSA infections, even though the standard dose is inadequate for biofilm eradication. This study aimed to asses the efficacy of vancomycin in eradicating biofilms and the influence of exposure on the emergence of hVISA isolates. The biofilm formed by MRSA isolates was exposed to vancomycin concentrations of 1 times the MIC, 1,000 times the MIC, and 10,000 times the MIC; exposed continously for 24 hours vs intermittently for 6 hours/day for 3 days. Measurement of the optical density of the biofilm was carried out to determine the percentage of biofilm eradication. Biofilm specimens exposed to vancomycin were subcultured onto BHIA-VC selective media to isolate hVISA. The highest biofilm eradication effect was found in isolates exposed to vancomycin at a concentration of 10,000 times the MIC. Vancomycin exposure correlated with the emergence of hVISA isolates, especially after exposure to low concentrations of vancomycin. For optimum eradication of MRSA biofilms, vancomycin concentrations exceeding 1.000 times the MIC are required. Exposure to vancomycin at a dose equal to one-times the MIC had no effect on biofilm eradication and was associated with the emergence of MRSA isolates with decreased susceptibility to vancomycin.

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.

Detection of Heteroresistant Vancomycin-Intermediate Staphylococcus aureus among Methicillin-Resistant Staphylococcus aureus

Background: The testing of heteroresistance vancomycin-intermediate Staphylococcus aureus (hVISA) by the population analysis profile-area under the curve (PAP-AUC) is tedious and laborious. The goal of this study was to determine the prevalence of hVISA among methicillin-resistant S. aureus (MRSA) and compare two screening tests: brain heart infusion agar with vancomycin 4 mg/L (BHIV4) and macromethod E-test (MET) with PAP-AUC as the gold standard. Materials and Methods: Antibiotic sensitivity test and detection of MRSA were performed according to the CLSI guidelines. PAP-AUC, BHIV4, and MET were used to detect hVISA. Results: A total of 288 MRSA isolates were tested for hVISA, with 48 (16.66%) being detected by PAP-AUC. With PAP-AUC, BHIV4's sensitivity, specificity, positive predictive value, and negative predictive value were 70.83%, 96.66%, 80.95%, and 94.3%, respectively. The majority of hVISA isolates were found in the vancomycin minimum inhibitory concentration range 1–2 mg/L. Conclusions: hVISA was found to be 16.6% in this investigation. When compared to MET, BHIV4 had a higher sensitivity, and teicoplanin and linezolid were found to be the most effective drug against hVISA.

1249. Metabolomic Profile of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA) in Latin-American MRSA Isolates

BackgroundVancomycin (VAN) is a first-line therapeutic option in severe infections caused by MRSA in Latin-America (LA). Development of reduced susceptibility to VAN has been associated with multiple changes in genes encoding pathways for cell wall metabolism and envelope stress responses. Nevertheless, a detailed and coherent mechanistic model to explain the phenotype remains elusive. To gain further insights into the hVISA phenotype, we sought to explore the metabolomic profile of hVISA isolates from LA. MethodsThe undirected profile of intracellular S. aureus metabolites was analysed in four clinical isolates (two hVISA and two VSSA [Vancomycin susceptible S. aureus]) belonging to the Chilean/Cordobes clone-ST5 (the predominant hVISA lineage in LA), and two reference strains Mu3 and N315. The metabolites were obtained in mid-exponential growth phase in trypticase soy broth in five independent replicates. The metabolites were determined by reverse phase liquid chromatography and hydrophilic interaction. The metabolic profile was determined by variable importance in the projection score (VIP > 1). The differences between hVISA and VSSA were maximized by orthogonal partial least squares discriminant analysis (OPLS-DA) and the affected metabolic pathways were identified with MetaboAnalyst.ResultsAmong the differences identified in the metabolic profiles of hVISA respect to VSSA, 69 metabolites were relevant. Of these, 47 were fatty acids (including glycerol), 7 amino acids and 6 nucleosides (Table 1). These changes mainly impact the biosynthesis of amino acids derived from pyruvate since tyrosine, valine and leucine, had a reduction of 34%, 57% and 41% in hVISA compared to VSSA, respectively, which suggests alterations of the acid cycle tricarboxylic (TCA). Additionally, a reduction in purine and pyrimidine metabolism in hVISA was identified with reduction of nucleosides and dinucleotides derived from the pentose phosphate pathway.Table 1. Metabolites with higher VIP scores in the comparison of the hVISA and VSSA profile ConclusionWe were able to observe metabolic alterations in TCA, pentose phosphate pathway and purine intermediates in hVISA-ST5 isolates. Our results support that gluconeogenesis and biosynthesis of carbohydrates and nucleic acids are the main pathways involved in the reduced susceptibility to VAN as reported in VISA isolates.Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support) Lorena Diaz, PhD , Nothing to disclose

Synergistic effect of vancomycin combined with cefotaxime, imipenem, or meropenem against Staphylococcus aureus with reduced susceptibility to vancomycin

Background/aim We investigated the synergistic effect between vancomycin and β-lactams against vancomycin-susceptible (VSSA) and nonsusceptible MRSA isolates [heterogeneous vancomycin-intermediate S. aureus (hVISA) and VISA].Materials and methods A total of 29 MRSA, including 6 VISA, 14 hVISA, and 9 VSSA isolates, were subjected to a microbroth dilution-minimum inhibitory concentration (MIC) checkerboard using vancomycin combined with cefotaxime, imipenem, or meropenem. To confirm synergistic activity, the representative strains of VISA, hVISA, and VSSA were then selected for the time-kill curve method.Results The combination of vancomycin with imipenem, meropenem, or cefotaxime exhibited synergistic effects against 17 (2 VISA, 9 hVISA, and 6 VSSA), 14 (3 VISA, 9 hVISA and 2 VSSA), and 5 (3 VISA and 2 hVISA) isolates, respectively. Additive and indifferent effects were found in the remaining isolates, but no antagonistic effect was observed. Using time-kill assay, the vancomycin combined with either imipenem or cefotaxime demonstrated synergism against both VISA and hVISA isolates, while the synergistic effect with meropenem was obtained only in the VISA isolates.Conclusion This study demonstrated in vitro enhanced antibacterial activity of vancomycin plus β-lactams against clinical hVISA or VISA isolates. These combinations may be an alternative treatment for MRSA infections in clinical practice.

Investigation of heteroresistant vancomycin intermediate Staphylococcus aureus among MRSA isolates.

Heteroresistant vancomycin intermediate Staphylococcus aureus (hVISA) testing is recommended when therapeutic failure is suspected in the clinics. In our research, we aimed to investigate the prevalence of hVISA among methicilline-resistant S. aureus (MRSA) isolates in our university hospital and compared three methods for detection of hVISA. One hundred MRSA clinical isolates were collected in our medical microbiology laboratory between 01.04.2018 and 01.10.2019. For screening of hVISA, we used two screening agar plates and used one commercial medium; brain heart infusion agar (BHI) plates containing 4 µg/mL vancomycin and 16 g/Lt casein (BHIA-VC; Satola's test), BHI agar plates containing 4 µg/mLvancomycin (BHIAV), and commercially obtained vancomycin resistant Enterococci (VRE) agar for detetection of hVISA. Colonies which could grow on plates were counted manually at 24th and 48th hours. Among 100 MRSA isolates, 43 (43%) were found as hVISA using Satola's test. BHIAV and VRE agar screening test results were found 70% and 4%, respectively. Finally, at the step, MIC values of 20 (47%) hVISA isolates reduced to 2 µg/mL after sub culturing for the gradient test. We found higher rates of hVISA comparing other studies in Turkey. Both VRE agar and BHIAV screening test failed to detect hVISA properly. Meropenem in combination with vancomycin inhibited the growth of 90% hVISA isolates in our study.

In Vitro Activity of Dalbavancin against Refractory Multidrug-Resistant (MDR) Staphylococcus aureus Isolates.

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections, always treated with vancomycin and daptomycin, has led to the emergence of vancomycin-intermediate (VISA), heteroresistant vancomycin-intermediate (hVISA) and daptomycin non-susceptible (DNS) S. aureus. Even if glycopeptides and daptomycin remain the keystone for treatment of resistant S. aureus, the need for alternative therapies that target MRSA has now become imperative. The in vitro antibacterial and bactericidal activity of dalbavancin was evaluated against clinically relevant S. aureus showing raised antibiotic resistance levels, from methicillin-susceptible to Multidrug-Resistant (MDR) MRSA, including hVISA, DNS and rifampicin-resistant (RIF-R) strains. A total of 124 S. aureus strains were tested for dalbavancin susceptibility, by the broth microdilution method. Two VISA and 2 hVISA reference strains, as well as a vancomycin-resistant (VRSA) reference strain and a methicillin-susceptible Staphylococcus aureus (MSSA) reference strain, were included as controls. Time–kill curves were assayed to assess bactericidal activity. Dalbavancin demonstrated excellent in vitro antibacterial and bactericidal activity against all S. aureus resistance classes, including hVISA and DNS isolates. The RIF-R strains showed the highest percentage of isolates with non-susceptibility, reflecting the correlation between rpoB mutations and VISA/hVISA emergence. Our observations suggest that dalbavancin can be considered as an effective alternative for the management of severe MRSA infections also sustained by refractory phenotypes.

Vancomycin MIC and agr dysfunction in invasive MRSA infections in southern Brazil.

In methicillin-resistant Staphylococcus aureus (MRSA) treatment, the vancomycin minimum inhibitory concentration (MIC) increase, vancomycin heteroresistance (hVISA) presence, and accessory gene regulator (agr) dysfunction are predictors of vancomycin therapy failure. This study evaluated the association between vancomycin MIC (≥ 1.0μg/mL) and agr dysfunction in invasive MRSA isolates. Vancomycin MIC, hVISA phenotype, agr group, and function were determined in 171 MRSA isolates obtained between 2014 and 2019 from hospitals in Porto Alegre, Brazil. All MRSA were susceptible to vancomycin; 16.4% of these had MIC ≥ 1.0μg/mL. Seventeen MRSA isolates expressed the hVISA phenotype; 35.3% of them had MIC of 1.5μg/mL. agr groups I (40.9%) and II (47.1%) were the most found groups for MRSA and hVISA isolates, respectively. The proportion of MRSA with vancomycin MIC ≥ 1.0μg/mL in agr group II was significantly higher than in agr groups I and III (p = 0.002). agr dysfunction was observed in 4.7% (8/171) of MRSA, especially those with vancomycin MIC ≥ 1.0μg/mL (p < 0.001). In addition, six isolates (35.3%; 6/17) with hVISA phenotype presented agr dysfunction, which was significantly higher than that in non-hVISA phenotype (p < 0.001). In conclusion, agr dysfunction in MRSA is associated with vancomycin MIC ≥ 1.0μg/mL and hVISA phenotype, which suggests that agr dysfunction might confer potential advantages on MRSA to survive in invasive infections.

Bloodstream Infections Caused by S. aureus: Daptomycin Nonsusceptibility and Clinical Aspects

Background:Staphylococcus aureus is one of the leading pathogens isolated from bloodstream infections (BSIs), and vancomycin has been the main choice to treat MRSA (methicillin-resistant S. aureus) infections. Vancomycin-intermediate S. aureus (VISA) and heteroresistant-VISA (hVISA) have been described, limiting this antibiotic use. We evaluated aspects associated with the resistance and its clonality of the S. aureus isolated from BSIs, and we determined their association with clinical aspects of patients attended at Rio de Janeiro between 2016 and 2018. The detection of MRSA and trimethoprim-sulfamethoxazole resistant isolates was performed using the disk diffusion test, while the minimum inhibitory concentrations (MICs) were evaluated for 5 antimicrobials using the broth microdilution method. The MICs for ceftaroline and vancomycin of the MRSA isolates were determined using the E test. The presence of hVISA isolates was evaluated for isolates with vancomycin MICs of 1 and 2 μg/mL by screening on BHI agar added with vancomycin. The population profile was divided by the area under the curve (ie, PAP/AUC test). SCC mec was evaluated by PCR and the clonal profile by PFGE method. Among 123 S. aureus isolates from BSI, 31% were MRSA. MIC50 and MIC90 were daptomycin 2 and 2 μg/mL; linezolid, 1 and 1 μg/mL; oxacillin 1 and 256 μg mL; teicoplanin, 0.5 and 0.5 μg/mL and vancomycin 1 and 1 μg/ml. MIC values for ceftaroline and vancomycin were 0.75 and 2 μg/mL. The frequency of isolates not susceptible to daptomycin was 75%. The clonal lineages and SCCmec types found were USA100/ST5-II (50%), USA800/ST5-IV (22%), USA300/ST8-IV (15.8%), USA1100/ST30-IV (5.3%), BEC/ST239-III (5.3%), and 1 isolate carrying SCCmecV/ST1. We found 1 VISA isolate, and the PAP/AUC analysis detected 3 hVISA isolates that were associated with the USA100 and USA300 lineages. Overall, 85% of patients had a vascular catheter. More advanced age was associated with MRSA infection as was higher mortality. Patients with end-stage renal disease were more affected by MSSA infection. Daptomycin nonsusceptibility and VISA and hVISA phenotypes associated with prevalent clonal lineages were described. In addition, MRSA infections presented higher mortality, which emphasizes the importance of epidemiological studies.Funding: NoneDisclosures: None

Etest Methods for Screening Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA) strains.

The importance of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) as a preceding stage for the development of vancomycin-resistant S. aureus is growing. We analyzed the prevalence of hVISA among bacteremia with methicillin-resistant S. aureus (MRSA) using two Etests and evaluated the clinical characteristics and outcomes. Ninety-eight MRSA isolates from blood were collected at two University hospitals in Korea. Macrodilution Etest and glycopeptide resistance detection Etests were used for detection of hVISA. Staphylococcal cassette chromosome mec (SCCmec) typing was performed by multiplex PCR. Clinical data were collected retrospectively from patient medical records. About 30% of MRSA strains were identified as hVISA. Diabetes mellitus was associated (P = 0.047) with hVISA infections. The hVISA isolates were associated with high teicoplanin MIC and multidrug resistance (P = 0.001). SCCmec type II accounted for the majority (79.3%) of hVISA strains. The prevalence of hVISA strains was increased and can lead to the development of multidrug-resistant strains. Patients with diabetes were found to have a greater risk for infection with hVISA strains. As the impact of hVISA on clinical outcome is not yet clear, large-scale studies about clinical outcomes and optimal detection methods of hVISA are needed. In conclusion, hVISA strains have a high prevalence in bloodstream MRSA infections. Awareness of the increase in hVISA strains should motivate laboratories to establish a system to detect and monitor hVISA.

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.

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.