Population Analysis Profile-area Under The Curve Research Articles

Genetic Evaluation of Heteroresistance among Carbapenem‐Susceptible Clinical Isolates of Enterobacterales

Carbapenems currently serve as the last line of defense when treating serious infections caused by multidrug‐resistant Enterobacterales species; however, heteroresistance of these species is thought to cause failure in the treatment with these broad‐spectrum antibiotics. This study was designed to determine the prevalence of carbapenem heteroresistance and associated genotypic modifications among phenotypically meropenem‐susceptible Escherichia coli and Klebsiella pneumoniae isolates. A total of 204 isolates of E. coli (n: 118) and K. pneumoniae (n: 86) from various clinical samples were included in this prospective experimental study. Identification and antimicrobial susceptibility testing of the isolates were performed by VITEK® (bioMérieux, France). Strains that were found susceptible to carbapenem group antibiotics (meropenem, imipenem, and ertapenem) with automated system were further investigated by disk diffusion method. The isolates with discrete colony growth within the clear inhibition zone among phenotypically meropenem‐susceptible strains were tested for heteroresistance with the “gold standard” population analysis profile‐area under the curve (PAP‐AUC) method. In addition, heteroresistant isolates were analyzed for the presence of carbapenemase genes with in‐house PCR method. The heteroresistance prevalence rate was 3.5% for E. coli and 18.1% for K. pneumoniae. The presence of heteroresistance in a total of 10 meropenem‐susceptible isolates (E. coli, n: 4; K. pneumoniae, n: 6) was confirmed by the PAP‐AUC method. The most frequently detected carbapenemase in heteroresistant isolates was OXA‐48 (6/10), followed by NDM‐1 (2/10). Meropenem is frequently preferred as initial empirical monotherapy in most of Gram‐negative infections in adult and pediatric patients. The presence of heteroresistance against meropenem is too important to ignore, and for this reason, it seems beneficial to prefer combined treatment regimens in clinical practice.

Resistance phenotype and genetic features of a heterogeneous vancomycin intermediate-resistant Staphylococcus aureus strain from an immunocompromised patient.

Strain C1 was successfully isolated from an immunosuppressed patient with persistent bacteremia, who had not previously been exposed to glycopeptide antibiotics. This strain was found to be a heterogeneous vancomycin intermediate-resistant Staphylococcus aureus (hVISA). It is noteworthy that, following a brief period of vancomycin treatment, strains C6, C8, and C9, which were obtained from blood and other body parts, exhibited a significant reduction in heterogeneity as determined by population analysis profile-area under the curve (PAP-AUC) detection. Genotyping analysis revealed that these bacterial strains belonged to the same SCCmecIVa-ST59-t437-agrI genotype and shared the same virulome and resistome. In this study, a comparative genomics analysis was conducted between strain C1 and strain N315 to identify potential hVISA-associated mutations. Ultimately, a total of 205 mutation sites in 19 candidate genes, likely associated with the hVISA phenotype, were identified.

Heterogeneous Vancomycin Intermediate Staphylococcus aureus Infections in Diabetic and Non-Diabetic Patients – A Hospital-Based Comparative Study

To study the infections caused by methicillin resistant Staphylococcus aureus (MRSA) with emphasis on heterogeneous vancomycin intermediate S. aureus (hVISA) in diabetic and non-diabetic patients and their comparison. S. aureus strains isolated from diabetic and non-diabetic patients admitted in four tertiary care hospitals in Coastal Karnataka, South India, were tested for methicillin resistance and included in the present study. Demographic and clinical data of the patients were collected using structured proforma. Antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method, and MLSB phenotypes were identified using the D-test. The minimum inhibitory concentration (MIC) of vancomycin was determined using agar dilution. MRSA isolates were tested for hVISA using vancomycin screen agar and population analysis profile - area under the curve (PAP-AUC) test. Statistical analysis of the results was done using the chi-square test. SPSS version 29.0 was used for this purpose. Out of 665 strains of S. aureus isolated, 220 (33.1%) were MRSA. Of these 220 MRSA strains, 122 (55.5%) and 98 (44.5%) were isolated from diabetic and non-diabetic patients, respectively. There was no significant difference in the antimicrobial resistance patterns of MRSA strains isolated from diabetic and non-diabetic patients. Foot infections and osteomyelitis caused by MRSA were significantly more among diabetic patients. Out of 220 strains of MRSA, 14 (6.4%) were hVISA. The rates of hVISA among MRSA isolated from diabetic and non-diabetic were 9.0% and 3.1%, respectively. This difference was statistically not significant. The rate of hVISA among all MRSA isolates was 6.4%. The risk of hVISA infection was three times more in diabetic patients. The results emphasize the importance of the detection of hVISA among MRSA isolates especially from diabetic patients.

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.

Detection of heterogeneous vancomycin-intermediate Staphylococcus aureus: A preliminary report from south India.

Background & objectives:Although there are reports of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) across the globe, there is a lack of reliable data on hVISA in India. The present study was undertaken to determine the rate of hVISA among the methicillin-resistant Staphylococcus aureus (MRSA) isolates, and to compare the brain heart infusion agar with vancomycin 4 μg/ml (BHIV4) method with population analysis profile-area under the curve (PAP-AUC) method for the detection of hVISA and to study the distribution of mobile genetic element that carries methicillin-resistance gene SCCmec (Staphylococcal cassette chromosome mec) types among these isolates.Methods:BHIV4 and PAP-AUC methods were employed to detect hVISA among 500 clinical isolates of MRSA. SCCmec typing of these isolates was performed by multiplex polymerase chain reaction. The clinical presentation, treatment with vancomycin and outcome was documented for patients with hVISA.Results:The rate of hVISA was 12.4 per cent by PAP-AUC method. Sensitivity, specificity, PPV, NPV and kappa agreement of BHIV4 with PAP-AUC was 58.06, 93.15, 54.55, 94.01 per cent and 0.498, respectively. The isolation of hVISA was significantly (P<0.01) higher in patients admitted to intensive care units and wards than in patients attending the outpatient departments. Only 38 per cent of the patients received vancomycin as therapy. Majority of the hVISA isolates carried SCCmec type V or IV.Interpretation & conclusions:The rate of hVISA isolation in our study was 12.4 per cent. The sensitivity of the BHIV4 screening test was low, and was in moderate agreement with PAP-AUC test. SCCmec type V was the predominant type seen in half of the isolates. More studies need to be done in different parts of the country on a large number of isolates to confirm our findings.

Phenotypic and Molecular Characterisation of Staphylococcus Aureus with Reduced Vancomycin Susceptibility Derivated in Vitro.

Vancomycin has been the primary agent used to treat serious Methicillin-resistant Staphylococcus aureus (MRSA) infection for many years. However, the rise of MRSA infection rates and the extensive use of vancomycin have led to the emergence of reduced vancomycin susceptibility. Therefore, four typical Staphylococcus aureus (S. aureus) strains from different clinical specimens were derivated by vancomycin in vitro to better clarify their phenotypic and molecular characteristics. Some experiments, such as stepwise selection of vancomycin-resistant strains, pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility test, population analysis profile-area under the curve (PAP-AUC), molecular typing, transmission electron microscopy, δ-hemolysin expression, autolysis assay, biofilm assay and quantitative real-time polymerase chain reaction (qPCR) for gene expression were carried out to compare the derivated bacteria with their parental strains. Results showed that the observed phenotypes of vancomycin-resistant strains such as hemolysin, autolysis and biofilm significantly reduced, which were associated with vancomycin resistance capability of the selected strain. The changes of phenotype and regulatory genes expression were inversely proportional to the vancomycin minimum inhibitory concentration (MICvan). Most heterogeneous vancomycin intermediate Staphylococcus aureus (hVISA) or VISA strains belonged to spa type t570 and agr group II. In summary, the clinical isolated vancomycin susceptible Staphylococcus aureus (VSSA), hVISA and VISA could be derivated into high vancomycin-resistant VISA in vitro, but it was difficult for them to develop into vancomycin resistant Staphylococcus aureus (VRSA). VISA and hVISA could gradually adapt to the environment with the vancomycin concentration that continuously elevates.

Screening for Intermediately Vancomycin-Susceptible and Vancomycin-Heteroresistant Staphylococcus aureus by Use of Vancomycin-Supplemented Brain Heart Infusion Agar Biplates: Defining Growth Interpretation Criteria Based on Gold Standard Confirmation.

BHI agars supplemented with vancomycin 4 (BHI-V4) and 3 (BHI-V3) mg/liter have been proposed for screening vancomycin intermediately susceptible Staphylococcus aureus (VISA) and heteroresistant (hVISA) phenotypes, respectively, but growth interpretation criteria have not been established. We reviewed the growth results (CFU) during population analysis profile-area under the curve (PAP-AUC) of consecutive methicillin-resistant Staphylococcus aureus (MRSA) blood isolates, which were saved intermittently between 1996 and 2012. CFU counts on BHI-V4 and BHI-V3 plates were stratified according to PAP-AUC interpretive criteria: <0.90 (susceptible [S-MRSA]), 0.90 to 1.3 (hVISA), and >1.3 (VISA). CFU cutoffs that best predict VISA and hVISA were determined with the use of receiver operating characteristic (ROC) curves. Mu3, Mu50, and methicillin-susceptible S. aureus (MSSA) controls were included. We also prospectively evaluated manufacturer-made BHI-V3/BHI-V4 biplates for screening of 2010-2012 isolates. The PAP-AUC of 616 clinical samples was consistent with S-MRSA, hVISA, and VISA in 550 (89.3%), 48 (7.8%), and 18 (2.9%) instances, respectively. For VISA screening on BHI-V4, a cutoff of 2 CFU/droplet provided 100% sensitivity and 97.7% specificity. To distinguish VISA from hVISA, a cutoff of 16 CFU provided 83.3% sensitivity and 94.7% specificity; the specificity was lowered to 89.5% with a 12-CFU cutoff. For detecting hVISA/VISA on BHI-V3, a 2-CFU/droplet cutoff provided 98.5% sensitivity and 93.8% specificity. These results suggest that 2-CFU/droplet cutoffs on BHI-V4 and BHI-V3 best approximate VISA and hVISA gold standard confirmation, respectively, with minimal overlap in samples with borderline PAP-AUC. Simultaneous screening for VISA/hVISA on manufacturer-made BHI-V4/BHI-V3 biplates is easy to standardize and may reduce the requirement for PAP-AUC confirmation.

An optimal method for generating stable vancomycin heteroresistance and intermediate susceptibility in methicillin-resistant Staphylococcus aureus blood isolates under in-vitro vancomycin pressure

Objectives: The relevance of vancomycin intermediately-susceptible (VISA) and hetero-resistant (hVISA) methicillin-resistant Staphylococcus aureus (MRSA) remains uncertain because of their low frequency. In vitro attempts to generate reduced susceptibility have been inconsistent. We describe a simple method for generating VISA/hVISA. Materials and methods: Twenty-four SCCmec type II and IV MRSA blood isolates plus USA100 and USA300 controls were cultured (107 CFU/ml) in BHI broth with 0, 2 and 3 mg/L vancomycin for 10 days followed by 10 days vancomycin-freepassage. We monitored vancomycin minimum inhibitory concentration (MIC; Etest) and population analysis profile-area under the curve (PAP-AUC) ratios of tested isolate-Mu3 during and after vancomycin pressure (VP). PAP-AUC ratios 1.3 were considered consistent with susceptible, hVISA and VISA, respectively. Results: VP at 2 mg/L (VP-2) increased MIC to 3 mg/L in 20 (83.3%) isolates and raised PAP-AUC ratio to hVISA (n=18; 75.0%) and VISA (six; 25.0%) ranges. VP-3 increased MIC to 3 and 4 mg/L in 19 (79.2%) and three (12.5%) isolates, respectively and raised PAP-AUC ratio to hVISA (n=3; 12.5%) and VISA (n=19; 79.2%) ranges. SCCmec IV isolates had lower pre-exposure PAP-AUC ratios (0.48±0.14 vs. 0.69±0.10; p<0.001) and lower MIC (1.6±0.2 vs. 1.8±0.2 mg/L; p=0.1) but MIC rise and VISA/ hVISA emergence was comparable. MIC and PAP-AUC ratio rises were stable in 20/22 isolates during drug-free passages. Conclusions: VISA/hVISA readily emerges among MRSA SCCmec type II and IV isolates under VP. VP at the MIC level generates hVISA while pressure at slightly higher level results in the VISA phenotype. J Microbiol Infect Dis 2012; 2(4): 129-134

An optimal method for generating stable vancomycin heteroresistance and intermediate susceptibility in methicillin-resistant Staphylococcus aureus blood isolates under in-vitro vancomycin pressure

Objectives: The relevance of vancomycin intermediately-susceptible (VISA) and hetero-resistant (hVISA) methicillin-resistant Staphylococcus aureus (MRSA) remains uncertain because of their low frequency. In vitro attempts to generate reduced susceptibility have been inconsistent. We describe a simple method for generating VISA/hVISA. Materials and methods: Twenty-four SCCmec type II and IV MRSA blood isolates plus USA100 and USA300 controls were cultured (107 CFU/ml) in BHI broth with 0, 2 and 3 mg/L vancomycin for 10 days followed by 10 days vancomycin-free-passage. We monitored vancomycin minimum inhibitory concentration (MIC; Etest) and population analysis profile-area under the curve (PAP-AUC) ratios of tested isolate/Mu3 during and after vancomycin pressure (VP). PAP-AUC ratios 1.3 were considered consistent with susceptible, hVISA and VISA, respectively. Results: VP at 2 mg/L (VP-2) increased MIC to 3 mg/L in 20 (83.3%) isolates and raised PAP-AUC ratio to hVISA (n=18; 75.0%) and VISA (six; 25.0%) ranges. VP-3 increased MIC to 3 and 4 mg/L in 19 (79.2%) and three (12.5%) isolates, respectively and raised PAP-AUC ratio to hVISA (n=3; 12.5%) and VISA (n=19; 79.2%) ranges. SCCmec IV isolates had lower pre-exposure PAP-AUC ratios (0.48&plusmn;0.14 vs. 0.69&plusmn;0.10; p<0.001) and lower MIC (1.6&plusmn;0.2 vs. 1.8&plusmn;0.2 mg/L; p=0.1) but MIC rise and hVISA/VISA emergence was comparable. MIC and PAP-AUC ratio rises were stable in 20/22 isolates during drug-free passages. Conclusions: VISA/hVISA readily emerges among MRSA SCCmec type II and IV isolates under VP. VP at the MIC level generates hVISA while pressure at slightly higher level results in the VISA phenotype.

The Prevalence of Vancomycin-Intermediate Staphylococcus aureus and Heterogeneous VISA Among Methicillin-Resistant Strains Isolated from Pediatric Population in a Turkish University Hospital.

There are limited data regarding the prevalence of vancomycin-intermediate Staphylococcus aureus (VISA)/heterogeneous VISA (hVISA) among pediatric population. Our objective was to determine the distribution of vancomycin and daptomycin minimum inhibitory concentrations (MICs) and explore the phenomenon of vancomycin MIC creep and the VISA/hVISA prevalence among the methicillin-resistant Staphylococcus aureus (MRSA) strains belonging to pediatric population by population analysis profile-area under the curve (PAP-AUC) and Etest macromethod. Vancomycin and daptomycin susceptibilities of 94 pediatric isolates of MRSA were tested by broth microdilution (BMD) and Etest methods. To determine the prevalence of VISA/hVISA, Etest macromethod and PAP-AUC was performed on all isolates. All isolates were susceptible to vancomycin and daptomycin by both BMD and Etest methods. Twenty-eight (29.8%) isolates had vancomycin MICs of 2 μg/ml by BMD. No increase in vancomycin MICs was observed over time. There were no VISA among 94 MRSA tested but 20 (21.3%) hVISA isolates were identified by PAP-AUC. Results of Etest macromethod were compared to PAP-AUC. Etest macromethod was 60.0% sensitive and 90.5% specific. The hVISA isolates represented 53.6% of isolates with vancomycin MICs of 2 μg/ml. Also, 75% of hVISA isolates had vancomycin MICs of 2 μg/ml. To our knowledge, this is the first study investigating the prevalence of VISA/hVISA among MRSA isolated from pediatric patients by PAP-AUC method. Based on our findings, MRSA isolates, which have vancomycin MIC of 2 μg/ml can be investigated for the presence of hVISA. In this study, daptomycin showed potent activity against all isolates and may represent a therapeutic option for MRSA infections.

Dissecting vancomycin-intermediate resistance in staphylococcus aureus using genome-wide association.

Vancomycin-intermediate Staphylococcus aureus (VISA) is currently defined as having minimal inhibitory concentration (MIC) of 4–8 µg/ml. VISA evolves through changes in multiple genetic loci with at least 16 candidate genes identified in clinical and in vitro-selected VISA strains. We report a whole-genome comparative analysis of 49 vancomycin-sensitive S. aureus and 26 VISA strains. Resistance to vancomycin was determined by broth microdilution, Etest, and population analysis profile-area under the curve (PAP-AUC). Genome-wide association studies (GWAS) of 55,977 single-nucleotide polymorphisms identified in one or more strains found one highly significant association (P = 8.78E-08) between a nonsynonymous mutation at codon 481 (H481) of the rpoB gene and increased vancomycin MIC. Additionally, we used a database of public S. aureus genome sequences to identify rare mutations in candidate genes associated with VISA. On the basis of these data, we proposed a preliminary model called ECM+RMCG for the VISA phenotype as a benchmark for future efforts. The model predicted VISA based on the presence of a rare mutation in a set of candidate genes (walKR, vraSR, graSR, and agrA) and/or three previously experimentally verified mutations (including the rpoB H481 locus) with an accuracy of 81% and a sensitivity of 73%. Further, the level of resistance measured by both Etest and PAP-AUC regressed positively with the number of mutations present in a strain. This study demonstrated 1) the power of GWAS for identifying common genetic variants associated with antibiotic resistance in bacteria and 2) that rare mutations in candidate gene, identified using large genomic data sets, can also be associated with resistance phenotypes.

Study of Decreased Susceptibility to Vancomycin in Methicillin-Resistant Staphylococcus aureus Strains isolated from a Romanian Multidisciplinary Emergency Hospital

The clinical relevance of Staphylococcus aureus strains with heterointermediate susceptibility to vancomycin (hVISA) is still controversial, however they could be responsible for treatment failures in patients treated with vancomycin. The lack of standardization and the complexity of testing methods are the main challenge in indentifying such strains. The aim of our study was to evaluate the frequency of hVISA strains in Targu-Mures Clinical Emergency Hospital. One hundred twenty-two, non-duplicate, methicillin-resistant S. aureus (MRSA) isolates susceptible to vancomycin using standard E-test (MIC≤2 mg/L) were screened for heteroresistance with Glycopeptide Resistance Detection test (E-test GRD). Population analysis profile-area under the curve (PAP/AUC) method was used for confirmation. Twenty-four strains (19.5%) were found positive with the screening method. Two of them (1.63%) were confirmed having hVISA phenotype and no strains with intermediate vancomycin susceptibility (VISA) were detected. In conclusion, the rate of MRSA strains with reduced vancomycin susceptibility was low. However, their monitoring may be useful, taking into consideration the wide usage of glycopeptides in the treatment of serious MRSA infections.

Vancomycin and daptomycin minimum inhibitory concentration distribution and occurrence of heteroresistance among methicillin-resistant Staphylococcus aureus blood isolates in Turkey

BackgroundThe aim of this study was to determine the distribution of vancomycin and daptomycin MICs among methicillin-resistant Staphylococcus aureus (MRSA) blood isolates, the prevalence of heterogeneous vancomycin-intermediate S. aureus (hVISA) and the relationship between hVISA and vancomycin MIC values.MethodsA total of 175 MRSA blood isolates were collected from seven university hospitals in Turkey. All isolates were tested for susceptibility to vancomycin and daptomycin by reference broth microdilution (BMD) and by standard Etest method. BMD test was performed according to CLSI guidelines and Etest was performed according to the instructions of the manufacturer. All isolates were screened for the presence of the hVISA by using macro Etest (MET) and population analysis profile-area under the curve (PAP-AUC) methods.ResultsThe vancomycin MIC50, MIC90 and MIC ranges were 1, 2, and 0.5-2 μg/ml, respectively, by both of BMD and Etest. The daptomycin MIC50, MIC90 and MIC ranges were 0.5, 1 and 0.125 -1 μg/ml by BMD and 0.25, 0.5 and 0.06-1 μg/ml by Etest, respectively. The vancomycin MIC for 40.6% (71/175) of the MRSA isolates tested was >1 μg/ml by BMD. No vancomycin and daptomycin resistance was found among MRSA isolates. Percent agreement of Etest MICs with BMD MICs within ±1 doubling dilution was 100% and 73.1% for vancomycin and daptomycin, respectively. The prevalence of hVISA among MRSA blood isolates was 13.7% (24/175) by PAP-AUC method. MET identified only 14 of the hVISA strains (sensitivity, 58.3%), and there were 12 strains identified as hVISA that were not subsequently confirmed by PAP-AUC (specificity, 92.1%).ConclusionsAgreement between BMD and Etest MICs is high both for vancomycin and daptomycin. Daptomycin was found to be highly active against MRSA isolates including hVISA. A considerable number of isolates are determined as hVISA among blood isolates. As it is impractical to use the reference method (PAP-AUC) for large numbers of isolates, laboratory methods for rapid and accurate identification of hVISA need to be developed.

Correlation of methicillin-resistant Staphylococcus aureus vancomycin minimal inhibitory concentration results by Etest and broth microdilution methods with population analysis profile: lack of Etest overestimation of the MIC

Methicillin-resistant Staphylococcus aureus (MRSA) vancomycin minimum inhibitory concentrations (V-MICs) are sometimes reported to be higher according to Etest versus broth microdilution (BMD). These observations are often interpreted as an Etest overestimation of the actual MIC. We measured V-MIC of 484 MRSA blood isolates using Etest, BMD, and a modified BMD (M-BMD) with incremental dilutions parallel to the Etest scale, correlated the results with population analysis profile-area under the curve (PAP-AUC). All MIC tests were done in parallel. The mean V-MIC was comparable (1.83 ± 0.44 [Etest], 1.88 ± 0.67 [BMD] and 1.75 ± 0.57 mg/L [M-BMD]; p = 0.9 [ANOVA]). The V-MICs/PAP-AUC correlation coefficient was 0.555 (Etest), 0.513 (BMD), and 0.586 (M-BMD). Etest MICs were equal (44.2 %), one dilution higher (21.9 %), two dilutions higher (2.5 %), one dilution lower (29.8 %), and two dilutions lower (1.6 %) than BMD MICs and were equal (61.5 %), one dilution higher (28.3 %), two dilutions higher (0.4 %), one dilution lower (9.5 %), and two dilutions lower (0.2 %) than M-BMD MICs. The mean PAP-AUC for Etest vs M-BMD among isolates with similar Etest/M-BMD MIC values was 0.25 ± 0.15 vs 0.35 ± 0.13 (p = 0.8), 0.46 ± 0.16 vs 0.50 ± 0.17 (p = 0.8), 0.64 ± 0.19 vs 0.67 ± 0.21 (p = 0.9), and 0.90 ± 0.31 vs 0.88 ± 0.25 (p = 1.0) for isolates with V-MIC of ≤ 1, 1.5, 2, and ≥ 3 mg/L respectively. These results suggest that Etest might not overestimate V-MIC in comparison to M-BMD or BMD; Etest and M-BMD tests depict comparable PAP-AUC and have a higher correlation with PAP-AUC than the conventional BMD, probably because of the more detailed results. Etest may be more suitable than conventional BMD for MIC outcome assessment because of the more detailed MICs.

The High Diversity of MRSA Clones Detected in a University Hospital in Istanbul

Background: To characterize the methicillin-resistant Staphylococcus aureus (MRSA) clones present in Istanbul, 102 MRSA isolates collected during a 5-year period at the Istanbul Medical Faculty Hospital were characterized using microarray analysis and phenotypic resistance profiles.Methods: Resistance to methicillin was detected with a cefoxitin disk diffusion assay and confirmed with a MRSA-agar and MRSA detection kit. Antimicrobial susceptibility testing was performed by a disk diffusion assay and interpreted according to the 2012 guidelines of the Antibiogram Committee of the French Society for Microbiology. Decreased susceptibility to glycopeptides was confirmed using the population analysis profile-area under the curve (PAP-AUC) method. The presence of the mecA gene was detected by polymerase chain reaction. Bacterial DNA was extracted according to the manufacturer's recommended protocol using commercial extraction kits. Strains were extensively characterized using the DNA microarray.Results: Isolates were grouped into six clonal complexes. The most frequently detected clone was the Vienna/Hungarian/Brazilian clone (ST239-MRSA-III), which accounted for 53.9% of the isolates. These isolates were resistant to multiple antibiotics, particularly penicillin, tetracycline, rifampicin, kanamycin, tobramycin, gentamicin, levofloxacin, erythromycin, lincomycin and fosfomycin. Furthermore, three isolates were detected by population analysis profile as heterogeneous vancomycin-intermediate S. aureus (hVISA). The UK-EMRSA-15 clone (ST22-MRSA-IV PVL negative) was detected in 9.8% of the isolates and was mainly susceptible to all anti-staphylococcal antibiotics. Seven isolates (6.9%) were positive for PVL genes and were assigned to the CC80-MRSA-IV clone (European CA-MRSA clone, three isolates), ST8-MRSA-IV clone (USA300 clone, two isolates, one ACME-positive) or ST22-MRSA-IV clone (“Regensburg EMRSA” clone, two isolates). All other clones were detected in one to six isolates and corresponded to well-known clones (e.g., Pediatric clone, Dublin EMRSA clone, WA MRSA-54/63, WA MRSA-1/57).Conclusions: This work highlighted both the high prevalence of ST239-MRSA-III clone and the large diversity of the other MRSA clones detected in a university hospital in Istanbul.

Utility of the Etest GRD for detecting Staphylococcus aureus with reduced susceptibility to glycopeptides in cystic fibrosis patients

Glycopeptide-intermediate S. aureus (GISA), particularly heterogeneous GISA (hGISA), remain difficult to detect in the routine practice of medical microbiology. Novel tools have been evaluated comparatively to the population analysis profile-area under the curve (PAP-AUC) reference method for detecting GISA/hGISA. Among them, the Etest GRD showed relatively high specificity (85.8-97%) and negative predictive value (97%) but lower sensibility (57-95%) and positive predictive value (30.8%). We investigated the utility of the Etest GRD for detecting GISA/hGISA among 180 strains isolated from 106 cystic fibrosis (CF) patients. Etest GRD was performed on all isolates, and those exhibiting a GISA/hGISA phenotype were further tested by PAP-AUC and other agar routine assays for GISA/hGISA detection. The Etest GRD allowed the detection of 15 GISA/hGISA strains, of which eight were confirmed by the reference method. Despite the 3.9% level of false positive results, the Etest GRD constitutes a useful routine tool for detecting GISA/hGISA overlooked by other routine assays, two strains being detected by the Etest GRD only. GISA/hGISA represented 7.7% of MRSA and 2.1% of MSSA, and were found in 4.7% of CF patients colonized/infected by S. aureus, which is the highest rate reported to date in this population.

Detection of Intermediately Vancomycin-Susceptible and Heterogeneous Staphylococcus aureus Isolates: Comparison of Etest and Agar Screening Methods

Detection of Staphylococcus aureus isolates with intermediate vancomycin susceptibility (VISA) and heteroresistance (hVISA) remains problematic. The population analysis profile/area under the curve (PAP/AUC) is the gold standard but is cumbersome. We compared the performance of two Etest screening methods (macromethod [MAC] and glycopeptide resistance detection [GRD]) plus brain heart infusion (BHI) agars supplemented with 3 (BHI-V3) or 4 (BHI-V4) mg/liter vancomycin in detecting hVISA and/or VISA phenotypes. Etest hVISA screenings were done in parallel for 485 saved methicillin-resistant S. aureus (MRSA) blood isolates according to the manufacturer's instructions. The PAP/AUC was measured for all isolates according to the modified method. PAP/AUC test isolate/Mu3 ratios of <0.9, 0.9 to 1.3, and >1.3 were considered positive for susceptible MRSA (S-MRSA), hVISA, and VISA, respectively. PAP/AUC revealed seven VISA and 33 hVISA phenotypes. MAC screening was positive for 30 (75.0%) hVISA/VISA and 49 (11.0%) S-MRSA isolates. GRD screening was positive for 28 (70.0%) hVISA/VISA and 63 (14.2%) S-MRSA isolates. Growth on BHI-V3 was noted in all hVISA/VISA and 24 (5.4%) S-MRSA isolates. Growth on BHI-V4 was noted in all VISA and four (12.1%) hVISA isolates. None of the S-MRSA isolates grew on BHI-V4 agar. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 75.0%, 89.0%, 38.0%, and 97.5% for MAC; 70.0%, 85.8%, 30.8%, and 97.0% for GRD; 100%, 94.6%, 62.5%, and 100% for BHI-V3; and 100, 99.2%, 63.6%, and 100% for BHI-V4 (for detecting VISA). These findings suggest that both Etest screening methods have excellent NPV, but positive results require confirmation. BHI-V3 and BHI-V4 agars provide more precise identification of hVISA and VISA, respectively; they may be reasonable alternatives to PAP/AUC.

The molecular characteristics of heteroresistant vancomycin-intermediate Staphylococcus aureus in China

Obiective To investigate the molecular characteristics of heteroresistant vancomycinintermediate Staphylococcus aureus(hVISA)in China and analyze the differences of the molecular characteristics between hVISA and VSSA(vancomycin-susceptible Staphylococcus aureus)isolates.Methods A total of 3 15 non-repetitive MBSA were collected from the national surveillance program in China in 2007.The isolates of hVISA were confirmed by modified population analysis profile-area under the curve(PAP-AUC).The genotypes of agr and SCCmec were determined by multiplex PCR,and spa typing was performed bv PCR and DNA sequencing.The pvl gene was detected bv PCR Results The prevalence of hVISA was 9.5%(30/315).Among 315 MRSA,SCCmec Ⅲ was the most popular type,which was found in 234 isolates(234/315,74.3%),followed by SCCmec Ⅱ,which was identified in 56 isolates (56/315,17.8%).The rate of SCCmec Ⅱ in hVISA(46.7%)was significantly hisher than in VSSA (14.7%,X2=18.93,P＜0.001).The most prevalent agr type among 315 MRSA was agr 1 accounting for 73.6%(232/315).The agr 2 accounted for 18.7%(59/315),and agr 3 and agr 4 were very rare in clinical isolates.It was different in agr types between the two groups.The rate of agr 2 in hVISA(53.4%)was higher than in VSSA(15.1%).X2 value was 26.08 and P value was less than 0.001 through X2 test.There was a statistical significance in the result.There were 4 spa types in hVISA isolates,including t002 (13 isolates),t037(9 isolates),t030(6 isolates),and 1548(2 isolates).The pvl positive MRSA isolates were very low,accounting for 1.6%(5/315).Conclusions The prevalence of hVISA was relatively higher in China.Compared to VSSA,the majority(53.4%)of the hVISA strains were agr 2,which was obviously different from VSSA.hVISA isolates were more diverse by spa typing, Key words: Staphylococcus aureus; Vancomycin resistance; Bacterial proteins; Transactivators

Lack of Heteroresistance among Staphylococcus aureus Isolates with Vancomycin MICs of 2 Micrograms per Milliliter by Automated Testing

Susceptibility testing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates from blood cultures collected at Emory University Hospital from March 2008 to July 2008 using the MicroScanWalkaway96 (software version 2.01; Siemens Healthcare Diagnostics, Inc., Deerfield, IL) identification/susceptibility system with the POS Combo29 broth microdilution panel and the PROMPT inoculation method revealed a modal vancomycin MIC of 2 μg/ml. All clinical MRSA blood isolates (collected between 26 March 2008 and 6 July 2008 with vancomycin MICs of 2 μg/ml were evaluated by additional MIC testing methods and for evidence of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) isolates by the population analysis profile-area-under-the-curve (PAP-AUC) method. Thirty isolates from unique patients were identified; repeat positive MRSA cultures from the same patient were excluded. Sixty-three percent of all MRSA isolates and 56% of all methicillin-susceptible Staphylococcus aureus isolates had vancomycin MICs of 2 μg/ml by MicroScanWalkaway96 during the study period. Isolates were stored at −70°C in BBL trypticase soy broth (Becton, Dickinson and Company, Sparks, MD) containing 20% glycerol and subcultured twice prior to testing. Vancomycin MICs were determined by standard Etest methods (bioMerieux, Durham, NC) using a 0.5 McFarland standard inoculum on Mueller-Hinton agar plates (Remel, Lenexa, KS). Reference broth microdilution vancomycin MICs were determined using standard CLSI methods (1, 2). Heteroresistance was evaluated by PAP-AUC measurement using the modified method of Wootton et al. (5), whereby 10−3 and 10−6 culture dilutions were spiral plated onto brain heart infusion agar containing increasing concentrations of vancomycin, and by the microdilution technique previously described by Pfeltz et al. (4). For both methods, the concentrations of vancomycin used were 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, and 8.0 μg/ml. Mu3 (the archetype hVISA), ATCC 29213 (vancomycin-susceptible Staphylococcus aureus), and Mu50 (vancomycin-intermediate Staphylococcus aureus) were included as controls (3). The results are depicted in Table ​Table1.1. All 30 isolates with initial vancomycin MICs of 2 μg/ml by the MicroScan method had lower vancomycin MICs by the reference broth microdilution method and Etest. The modal vancomycin MICs for all 30 isolates were 1 μg/ml by reference broth microdilution and 1.5 μg/ml by the Etest. The mean PAP-AUC ratio for the 30 MRSA clinical isolates to the hVISA strain Mu3 was 0.56, with a mode of 0.49, and no heteroresistance was detected among the 30 isolates by either method (a heteroresistant isolate was defined as one with an AUC ≥0.90 of that of Mu3). TABLE 1. Comparison of susceptibility testing methods: MRSA isolates with vancomycin MICs of 2μg/ml by MicroScan All isolates had lower vancomycin MICs by the Etest and reference broth microdilution methods than by the automated MicroScan method; however, differences between vancomycin MICs generated by automated testing and those generated by the CLSI reference broth microdilution method of (plus or minus) a single twofold dilution are within a widely accepted margin of error. Moreover, no definitive data demonstrating significant clinical implications for a single-dilution difference in vancomycin MIC within the range of 0.5 to 2 μg/ml exist. Although no hVISA isolates were identified in this collection, the emergence of MRSA with decreased vancomycin susceptibility presents major diagnostic and therapeutic challenges for clinicians and microbiologists. (This work was presented in part at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy/IDSA 46th Annual Meeting, Washington, DC, Oct 2008.)

Characterisation of laboratory-generated vancomycin intermediate resistant Staphylococcus aureus strains

Vancomycin has been the drug of choice for 30 years for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Emergence of decreased vancomycin susceptibility in MRSA strains presents a significant clinical problem with few therapeutic options. This study was performed to generate and characterise S. aureus strains with reduced susceptibility to vancomycin. Eighteen S. aureus strains were subjected to serial passaging on vancomycin to generate vancomycin intermediate resistant S. aureus (VISA) strains. Minimum inhibitory concentration (MIC) determination was performed for the parent and the passaged cultures with 13 different antibiotics. The strains were tested by the following five methods: simplified population analysis; CDC method; modified vancomycin agar screen; population analysis profile (PAP); and modified population analysis (PAP–area under the curve (AUC) ratio). Phenotypic changes such as doubling time, synergy with β-lactam antibiotics and effect on norA efflux pumps were also studied for these strains. The result indicated that 8 VISA mutants (vancomycin MICs, 8–16 μg/mL) were generated in vitro from the 18 S. aureus strains. The CDC and modified agar methods proved to be the most sensitive and specific methods for detection of VISA strains. The PAP for all the VISA strains ranged from 12 μg/mL to >16 μg/mL, with a PAP–AUC ratio of >1.3. All mutants showed increased doubling time compared with their parent isolate. Synergism of the vancomycin and β-lactam combinations was observed for all methicillin-resistant mutants. Upon acquisition of vancomycin resistance, a few mutants showed decreased oxacillin resistance. Two VISA strains were chosen for molecular characterisation of the mecA gene and one mutant showed genotypic changes with deletion of mecA. Loss of norA efflux pumps leading to fluoroquinolone sensitivity was also observed in four mutants.