RNA-seq analysis reveals resistome genes and signalling pathway associated with vancomycin-intermediate Staphylococcus aureus

Context: Vancomycin-intermediate Staphylococcus aureus remains one of the most prevalent multidrug-resistant pathogens causing healthcare infections that are difficult to treat. Aims: This study uses a comprehensive computational analysis to systematically investigate various gene expression profiles of resistant and sensitive S. aureus strains on exposure to antibiotics. Settings and Design: The transcriptional changes leading to the development of multiple antibiotic resistance were examined by an integrative analysis of nine differential expression experiments under selected conditions of vancomycin-intermediate and -sensitive strains for four different antibiotics using publicly available RNA-Seq datasets. Materials and Methods: For each antibiotic, three experimental conditions for expression analysis were selected to identify those genes that are particularly involved in the development of resistance. The results were further scrutinised to generate a resistome that can be analysed for their role in the development or adaptation to antibiotic resistance. Results: The 99 genes in the resistome are then compiled to create a multiple drug resistome of 25 known and novel genes identified to play a part in antibiotic resistance. The inclusion of agr genes and associated virulence factors in the identified resistome supports the role of agr quorum sensing system in multiple drug resistance. In addition, enrichment analysis also identified the kyoto encyclopedia of genes and genomes (KEGG) pathways – quorum sensing and two-component system pathways – in the resistome gene set. Conclusion: Further studies on understanding the role of the identified molecular targets such as SAA6008_00181, SAA6008_01127, agrA, agrC and coa in adapting to the pressure of antibiotics at sub-inhibitory concentrations can help in learning the molecular mechanisms causing resistance to the pathogens as well as finding other potential therapeutics.

The study of reduced susceptibility of methicillin resistant Staphylococcus aureus to various antibiotics with special reference to glycopeptides in a tertiary care hospital in central India

Background: Staphylococcus aureus has emerged over the past several decades as a leading cause of hospital-associated and community acquired infections. Methicillin resistant S. aureus (MRSA), which are often resistant to several classes of antibiotics, is the most common cause of nosocomial infections and pose a great threat to the world. Vancomycin is regarded as the first-line drug for treatment of MRSA but resistance to this drug is being reported now a day.Methods: It was carried out for a period between January 2014 to June 2017 in the microbiology diagnostic laboratory. MRSA detection was performed by cefoxitin disk diffusion method. Screening for the vancomycin intermediate and the vancomycin resistant S. aureus (VISA and VRSA respectively) was carried out by using vancomycin screen. MIC (minimum inhibitory concentration) of vancomycin was tested by agar dilution method and E strip on all MRSA isolates.Results: A total of 287 S. aureus clinical isolates were included in the study. All MRSA were inoculated on vancomycin screen agar. Visible growth was present in 8 isolates. Five (3.73%) MRSA isolates with MIC of 4 were termed VISA (vancomycin intermediate S. aureus) by agar dilution method. Six isolates had the MIC of 4 and were termed as VISA.Conclusions: As disc diffusion method is not recommended by CLSI for S. aureus, vancomycin screen agar and MIC determination by either of the methods viz. agar dilution or E test can be used.

Epidemiological and clinical infection features of heterogeneous vancomycin-intermediate Staphylococcus aureus / 中国感染与化疗杂志

Objective To investigate the prevalence and clinical characteristics of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) in the First Affiliated Hospital of Chongqing Medical University. Methods Clinical isolates of S. aureus were collected from the hospital during the period from 2012 to 2015 and were tested for susceptibility to vancomycin using agar dilution method. The results were interpreted according to CLSI 2016 breakpoints. VISA and hVISA strains were screened out by population analysis profile-area under the curve (PAP-AUC). E-test was carried out to determine the MIC of VISA. The clinical data of the patients infected with S. aureus were reviewed retrospectively. Results A total of 105 patients were included in this analysis. And 105 strains of S. aureus were isolated from these patients, including methicillin-resistant S. aureus (MRSA) strains (58.1％, 61/105). PAP-AUC identified 19 (18.1％) hVISA strains and 10 (9.5％) VISA strains. Overall, 52 of the 105 patients were nosocomial infections and 53 community infections. The prevalence of MRSA was 69.2％ (36/52) in nosocomial infections, higher than that in community infections (47.2％, 25/53) (P＜0.05). The prevalence of hVISA in community infections (20.8％, 11/53) did not show significant difference from that in nosocomial infections (15.4％, 8/52) (P＞0.05). The clinical outcome (P＞0.05) and length of hospital stay (P＞0.05) did not show significant difference between hVISA and non-hVISA infections, or between VISA and non-VISA infections. Conclusions The prevalence of hVISA is high in this hospital, which does not show difference between S. aureus nosocomial infection and community infection, or between MRSA and MSSA. The length of hospital stay of hVISA infection is not significantly longer than that of nonhVISA infection. The clinical outcome of hVISA infection does not show difference from that of non-hVISA infection. Larger sample size is required to better understand the prevalence and clinical features of hVISA.

Molecular Typing and Resistance Profiles of Vancomycin-Intermediate Staphylococcus aureus in Korea: Results from a National Surveillance Study, 2007-2013 / 대한임상미생물학회지

BACKGROUND: To investigate the national molecular epidemiology and resistance profiles of vancomycin-intermediate Staphylococcus aureus (VISA), we analyzed the characteristics of methicillin-resistant Staphylococcus aureus (MRSA) collected from clinical samples at tertiary or general hospitals participating in a nationwide surveillance program for VISA and vancomycin-resistant Staphylococcus aureus (VRSA) in Korea during an 12-week period in each year from 2007 to 2013. METHODS: VISA was defined by agar dilution, broth dilution and E-test methods with vancomycin minimum inhibitory concentrations of >2 μg/mL. All VISA isolates were characterized by multilocus sequence typing, staphylococcal cassette chromosome mec typing, spa typing, accessory gene regulator typing, Diversilab analysis, and antibiogram analysis. RESULTS: Of 109,345 MRSA isolates, 87,354 were screened and 426 isolates were identified as positive on brain heart infusion agar containing 4 μg/mL vancomycin (BHI-V4). Of 426 isolates, 76 isolates were identified as VISA. No VRSA isolates were detected among the isolates. Overall, a total of 6 genotypes were identified among VISA strains and the predominant clones were ST5-II-t2460, ST72-IV-t324, and ST239-III-t037 (44.7%, 15.8%, and 10.5%, respectively). Of note, ST72-IV-t324 clones are known to be a typical community-associated MRSA. ST239-III-t037 strains were more resistant to trimethoprim-sulfamethoxazole than any other type of strain. ST72-IV-t324 strains were susceptible to all of the antimicrobial agents tested except erythromycin and daptomycin. All of the VISA isolates were susceptible to linezolid and quinupristin-dalfopristin. CONCLUSION: Although VRSA is still rare, continuous monitoring of VRSA occurrence is needed, as well as VISA prevalence, epidemic clonal shift, and antimicrobial resistance.

Prevalence and Clinical Impact of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Isolated From Hospitalized Patients

BACKGROUND: We estimated the prevalence and clinical impact of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA). The concordance between macromethod and glycopeptide resistance detection (GRD) E tests was determined. In addition, predictors of clinical outcomes in hospitalized patients with S. aureus bacteremia (SAB) or pneumonia (SAP) were evaluated. METHODS: We obtained 229 consecutive S. aureus isolates from all hospitalized patients at two university hospitals located in Busan and Yangsan, Korea. Standard, macromethod, and GRD E tests were performed. Additionally, we reviewed the medical records of all patients. Among the 229 patients, predictors of clinical outcomes were analyzed for 107 patients with SAB and 39 with SAP. RESULTS: Among the 229 isolates, 34.5% of S. aureus isolates and 50.7% of methicillin-resistant S. aureus isolates exhibited the hVISA phenotype based on the macromethod E test. hVISA was nearly associated with treatment failure in patients with SAB (P=0.054) and was significantly associated with treatment failure in patients with SAP (P=0.014). However, hVISA was not associated with 30-day mortality in patients with SAB or SAP. The concordance between the macromethod and GRD E tests was 84.2%. CONCLUSIONS: hVISA is quite common in the southeastern part of Korea. hVISA is associated with treatment failure in patients with SAP.

Comparative proteomics profiling reveals down-regulation of Staphylococcus aureus virulence in achieving intermediate vancomycin resistance

Aims: VraSR and GraSR were shown to be important in conferring intermediate vancomycin resistance in VISA. Nevertheless, the exact mechanism modulated by these systems leading to the development of VISA remains unclear. We employed a proteomic approach to determine the VraS and GraR regulons and subsequently derive the possible vancomycin resistance regulatory pathway(s) in the Mu50 lineage of Staphylococcus aureus. Methodology and results: Staphylococcus aureus strains Mu50Ω, Mu50Ω-vraSm and Mu50Ω-vraSm-graRm are isogenic strains with ascending levels of vancomycin resistance. Total proteins were extracted from the 3 strains and trypsin digested prior to protein isolation and identification by LC-ESI MS/MS and PLGS 2.4. Expression profiles of resulting proteins were analyzed using Progenesis LC/MS software. Differential expression profiles revealed 3 regulons, each controlled by VraS (Mu50Ω-vraSm vs Mu50Ω), GraR (Mu50Ω-vraSm-graRm vs Mu50Ω-vraSm) and VraS-GraR (Mu50Ω-vraSm-graRm vs Mu50Ω), respectively. The regulon down-regulated by VraS in Mu50Ω-vraSm were proteins associated with virulence (MgrA, Rot, and SarA), while GraR up-regulated resistance-associated proteins (TpiA, ArcB and IsaA) in Mu50Ω-vraSm-graRm. The VraS-GraR regulon mediated both up-regulation of resistance-associated proteins (ArgF, ArcB, VraR and SerS) and down-regulation of virulence-associated protein GapB. Conclusion, significance and impact of study: Down-regulation of virulence- in concert with up-regulation of resistance-associated proteins appears to be integral for development of intermediate-vancomycin resistance in the Mu50 lineage of S. aureus.

Epidemiology of heterogeneous vancomycin-intermediate Staphylococcus aureus from sterile body fluid specimens / 国际检验医学杂志

Objective To investigate the prevalence and molecular characteristic of heterogeneous‐ vancomycin intermediate Staphylococcus aureus(hVISA) among methicillin‐resistant Staphylococcus aureus (MRSA)strains isolated from sterile body fluid specimens from 2009 to 2013 in Huangzhou District People′s Hospital in Huanggang City .Methods The minimum inhibitory con‐centrations (MIC) of antibiotics was determined by agar dilution method .The hVISA strains were detected by population analysis profile/area under the curve method (PAP/AUC) .The staphylococcal cassette chromosome mec (SCCmec) ,multilocus‐sequence typing (MLST) ,accessory gene regulator (agr) and Staphylococcus aureus protein A(spa) typing of hVISA strains were detected using PCR method .Results 32 hVISA strains were detected among 285 MRSA strains ,the prevalence rate of hVISA was 11 .2% , and the detection rates of hVISA from 2009 to 2013 were 0 .0% ,6 .4% ,9 .0% ,14 .3% and 18 .8% ,respectively ,showed an increas‐ing trend .The main hVISA epidemic clone was ST239‐SCCmecIII‐ t030‐agrI type(28strains ,accounting for 87 .5% ) .Conclusion The detection rate of hVISA showed an increasing trend in the past 5 years ,should be paid attention to strictly control the utiliza‐tion of glycopeptide drugs .

Vancomycin Resistance of Staphylococcus aureus in Korean Primary Hospitals

According to a United States study, 13 cases of vancomycin-resistant Staphylococcus aureus (VRSA) have been reported to date. In 2001, a survey conducted in Korea revealed that 0.5% of methicillin-resistant S. aureus (MRSA) isolates have a vancomycin minimum inhibitory concentration (MIC) of 4 microg/ml, and are thus referred to as vancomycin intermediate S. aureus (VISA). However there are no reports of VISA found in primary hospitals. We evaluated the MIC of vancomycin in MRSA samples obtained from primary hospitals to determine whether VISA was present in primary hospitals. The population analysis was performed to determine whether hetero-VISA was present in primary hospitals. As a result, twenty of the 103 isolates were S. aureus which were all MRSA and the vancomycin MIC was similar to that seen in tertiary hospitals. Population analysis confirmed that three strains were hetero-VISA, by showing that one strain grew in 8 microg/ml vancomycin and that two strains grew in 4 microg/ml vancomycin. In conclusion, hetero-VISA was detected in Korean primary hospitals, which may develop into VISA, however a larger sample size will be needed to confirm these results.

The incidence and risk factors for heterogeneous vancomycin intermediate Staphylococcus aureus / 中华内科杂志

Objectives To investigate the prevalence of heterogeneous vancomycin intermediate Staphylococcus aureus(hVISA) and the sensitivity of hVISA to novel antibiotics,and to explore the risk factors and infection attributable mortality associated with hVISA infection.Methods A total of 456 methicillin resistant Staphylococcus aureus (MRSA) isolates were isolated in Zhongshan Hospital from January,2008 to November,2010.All MRSA isolates were investigated for hVISA by two agar screening methods BHIA5T (brain-heart infusion containing teicoplanin 5 mg/L)or BHIA6V (brain-heart infusion containing vancomycin 6 mg/L),as well as macroEtest method(MET).Possible hVISA isolates were tested by modified population analysis profile-area under the curve (PAP-AUC).The minimal inhibitory concentrations(MICs) of vancomycin,teicoplanin and linezolid were determined by microbroth dilution as recommended by Clinical Laboratory Standards Institute(CLSI).The contribution difference between hVISA and vancomycin susceptible Staphylococcus aureus (VSSA) in different MIC range was compared.A retrospective case-control study of the patients with hVISA infection or VSSA infection was carried out and statistical analysis was performed using t test,Mann-Whitney test,x2 test and Fisher exact test.Results A total of 105 isolates of hVISA were screened by BHIA5T and BHIA6V (23.0％) with other 23 isolates by MET(5.0％) and 21 by PAP-AUC(4.6％).All isolates were 100％ sensitive to vancomycin,teicoplanin and linezolid.The vancomycin MIC [(1.76 ±-0.16) mg/L] in hVISA group was significantly higher than that in VSSA group[(1.09 ± 0.07)mg/L,P ＜ 0.01],which was a potential risk factor for hVISA infection.The retrospective study showed chronic obstructive pulmonary disease (COPD) was also a risk factor for hVISA infection of the lower respiratory tract.No significant difference in infection attributable mortality was showed between the hVISA group and the VSSA group.Conclusions The overall prevalence of hVISA in Zhongshan Hospital is estimated as 4.6％,while the prevalence of hVISA isolated from blood is as high as 12.5％.All isolates are 100％ sensitive to vancomycin and linezolid.COPD is a risk factor for hVISA infection of the lower respiratory tract.

Be alert to the alterations in the biological characteristics in heterogeneous vancomycin-intermediate Staphylococcus aureus.

The development of reduced vancomycin susceptibility in Staphylococcus aureus in many cases appears to be associated with characteristic changes. These changes may have pitfall of identifying S. aureus by automated testing methods like Vitek 32. In this study, we retested 24 heterogeneous vancomycin-intermediate Staphylococcus haemolyticus (h-VISH) collected in 2008-2010 at the Department of Clinical Microbiology by conventional biochemical tests and polymerase chain reaction (PCR). The heterogeneous vancomycin-intermediate S. aureus (hVISA) reversion test and electron microscopic examination were also used. Six isolates of 24 h-VISH possessed nuc, coa, and 16S rRNA genes, and could be reversed into S. aureus. It suggested that biochemical and morphological changes in hVISA and vancomycin-intermediate S. aureus (VISA) should be considered, and the detection of S. aureus, especially reduced vancomycin susceptibility isolates, requires more attention and different techniques.

An evaluation of 2.0 McFarland Etest method for detection of heterogeneous vancomycin-intermediate Staphylococcus aureus

Background: Staphylococcus aureus with reduced susceptibility to vancomycin or heterogeneous vancomycinintermediate S. aureus (hVISA) have become increasingly reported from various parts of the world. hVISA cannot be detected by routine test for minimal inhibitory concentration (MIC) for vancomycin. The gold standard method for detection, population analysis profiles (PAP) method, is complicated, time-consuming, expensive, and needs well-trained microbiologists. Objective: Evaluate of 2.0 McFarland Etest method, in comparison with the PAP method, for detection of hVISA in clinical specimens. Methods: All methicillin-resistant S. aureus strains from clinical specimens isolated from consecutive patients attended at King Chulalongkorn Memorial Hospital and Siriraj Hospital, Bangkok between 2006 and 2007 were studied. 1 hundred nineteen specimens were obtained. The PAP method detected six hVISA strains 5 from blood and from cultures) from four patients at King Chulalongkorn Memorial Hospital, accounting for a prevalence of 6.35%. The MIC determined by agar dilution method was in the range of 2-3 μg/mL. Results: 2.0 McFarland Etest method detected no false positive and five false negatives (42%), and gave a sensitivity and a specificity of 16.7% and 100%, respectively. The one-point population analysis screening method detected two false positives and 1 false negative, and gave a sensitivity of 83.3% and a specificity and 98.2%. Conclusion: The 2.0 McFarland Etest method had a very good specificity but a poor sensitivity for detecting hVISA. It may be used as an alternative method to confirm detection of hVISA.

Two Cases of Vancomycin-intermediate Staphylococcus aureus Isolated from Joint Tissue or Wound / 대한진단검사의학회지

Since its first isolation in 1997, vancomycin-intermediate Staphylococcus aureus (VISA) has been a clinical concern because it may lead to treatment failure. Up to the present, there were two reports of clinical VISA cases in Korea. We now report two additional cases of VISA with the minimum inhibitory concentration (MIC) of 4 microgram/mL. The first patient was a 59 yr-old man who had undergone total hip replacement arthroplasty in 1999 due to avascular necrosis of femur heads. He had recurrent episodes of infected hip caused by methicillin-resistant Staphylococcus aureus (MRSA) and was treated with vancomycin. He underwent replacement operation of prosthesis. Cultures of joint fluid and joint tissue grew S. aureus. Vancomycin MIC as determined by a broth microdilution method was 4 microgram/mL for the both isolates. The patient was treated with high enough doses of vancomycin to maintain serum trough concentrations at 20-25 microgram/mL for 52 days and was discharged. The second patient was a 57 yr-old man with diabetes. He lost consciousness from drinking. After recovery of consciousness, he was diagnosed with aspiration pneumonia. MRSA and Acinetobacter baumannii were cultured from sputum and the patient was treated with vancomycin and meropenem. During hospitalization, bed sores developed in his ankle and back. A wound culture from the sore grew S. aureus with vancomycin MIC of 4 microgram/mL. Since infection was localized, systemic antibiotics did not seem necessary, and the patient was transferred to another hospital for isolation and management.