Clinical Implications of Vancomycin Heteroresistant and Intermediately Susceptible Staphylococcus aureus

Staphylococcus aureus (S. aureus) has proven to be a major pathogen with the emergence of methicillin‐resistant S. aureus (MRSA) infections and recently with heteroresistant vancomycin‐intermediate S. aureus (hVISA) and vancomycin‐intermediate S. aureus (VISA) infections. Although vancomycin is traditionally a first‐line and relatively effective antibiotic, its continued use is under question because reports of heteroresistance in S. aureus isolates are increasing. Both hVISA and VISA infections are associated with complicated clinical courses and treatment failures. The prevalence, mechanism of resistance, clinical significance, and laboratory detection of hVISA and VISA infections are not conclusive, making it difficult to apply research findings to clinical situations. We provide an evidence‐based review of S. aureus isolates expressing heterogenic and reduced susceptibility to vancomycin.

estimates vary widely because of nonstandardized detection methodologies or the absence of routine hVISA screening, variation in interpretation, geographic location, clinical setting, and differing patient populations. [12][13][14][15][16][17][18][19] Reported rates of hVISA throughout the world range from 0-73.7%. 18 One retrospective study evaluated MRSA strains with heterogenic intermediate resistance to vancomycin over a 22-year period in three Detroit hospitals. The prevalence of these organisms increased from 2.2% (1986-1993) and 7.6% (1992-2002) to 8.3% between 2003 and 2007. 16 Only 14 of the 1498 (0.93%) MRSA isolates were identified as VISA. There was no apparent pattern of increasing prevalence over the three time periods for VISA isolates. An increase in hVISA was also described in a similar retrospective study from Turkey of 1.6% in 1998 to 36% in 2001. 20 Because clonality was not evaluated in either study, the increase in prevalence may have reflected clonal spread rather than true prevalence. Prevalence also may have been underestimated because the isolates were stored for prolonged periods in glycopeptide-free media, which may result in a loss of resistance. 21 Two surveillance studies conducted in 2009 and 2011 in over 40 U.S. medical centers determined rates of antimicrobial resistance among S. aureus isolates collected from patients with infections. 22,23 The rates of hVISA among MRSA isolates in 2011 were higher than in 2009 (1.2% vs 0.4%, p=0.003). 22 Of note, strains of VISA were not detected. 22,23 Although the current prevalence of VISA is low, these organisms may become more common in the future. Data suggest that heteroresistance is a precursor to VISA; therefore, the suspected increase in prevalence of hVISA may predict more VISA infections. 5 Increased use of vancomycin provides selection pressure for further emergence of VISA. Based on available data, hVISA appears to be on the rise, yet VISA still remains a rare occurrence. Additional studies are needed to determine appropriate surveillance methods because retrospective studies are complicated by the ability of hVISA to revert back to vancomycin-susceptible S. aureus (VSSA) and VISA to revert back to hVISA.

hVISA and VISA Laboratory Detection and Interpretation
Further discussion of hVISA and VISA require that clinical and microbiologic definitions are addressed. In 2006, the Clinical and Laboratory Standards Institute (CLSI) lowered vancomycin minimum inhibitory concentration (MIC) breakpoints for S. aureus. 24 The CLSI breakpoints by broth microdilution (BMD) currently define vancomycin susceptibility as an MIC of 2lg/ml or lower, vancomycin-intermediate susceptibility as an MIC of 4-8 lg/ml, and vancomycin resistance as an MIC of 16 lg/ml or higher (Table 1). 25 Vancomycin MIC breakpoints were lowered in an effort to increase the detection of potentially heterogeneous-intermediate isolates because of reported associations between vancomycin treatment failure and S. aureus isolates with MICs of 4 lg/ml or higher. 7,8,25 Heteroresistance refers to the presence of less susceptible subsets within a larger population of fully antimicrobial-susceptible microorganisms. 5 When tested using routine methods, hVISA isolates are susceptible to vancomycin (MIC of 2 lg/ml or lower) but contain subpopulations that express reduced vancomycin susceptibility (MIC of 4 lg/ml or higher). 11 Detection of hVISA is a great challenge in clinical microbiology laboratories because reliable and practical methods are not currently available for routine use. Heteroresistant subpopulations are present in low frequencies (1 9 10 6 or higher) and can grow in higher vancomycin concentrations than the MIC predicts. Such small populations may not be detected by the inocula (5 9 10 5 CFU/ml) used in standard CLSI microbiology methods. As a result, hVISA isolates are likely undetected in clinical laboratories that use traditional MIC testing methodology. 5 Population analysis profiling with area under the curve (PAP-AUC) is the current reference standard method for confirming hVISA and is the most reliable and reproducible test. However PAP-AUC is labor intensive, time consuming (3-5 days), and costly for use in clinical microbiology laboratories. 17,19,26 Consequently, several screening methods have been developed, such as glycopeptide resistance detection, macromethod Etest, and brain heart infusion screen agar plates ( Table 2). [27][28][29] However, none of these tests have the same degree of sensitivity and specificity as the PAP-AUC test, with issues of reproducibility and variability, in reporting results. 19 Until a suitable hVISA detection method becomes available for use in clinical microbiology laboratories, routine testing is not currently recommended. 2 Currently, clinical screening for hVISA isolates in high-risk patients is favored, particularly in patients who do not respond to vancomycin. Further research is warranted to develop a detection method that is practical, cost-effective, and reliable for routine use in clinical settings. Nonautomated MIC methods for the detection of VISA are recommended by the Centers for Disease Prevention and Control (CDC). 30 Acceptable nonautomated MIC methods for detecting VISA include BMD per CLSI, agar dilution, and Etest (0.5 McFarland). 30 Although automated methods and vancomycin screen agar plates can be useful in the detection of VISA isolates with a vancomycin MIC of 8 lg/ml, sensitivity levels have not been determined for S. aureus with vancomycin MICs of 4 lg/ml. 30 In these situations, a second method, such as BMD per CLSI criteria, should be used to confirm VISA isolates. 30 Current susceptibility testing methods do not consistently distinguish between MICs of 1 and 2 lg/ml. 2, 31 Therefore laboratory results should indicate the methodology used because vancomycin MIC results will differ between methods and may alter treatment decisions. 11 In comparison with the CLSI BMD method, automated detection methods, particularly Phoenix system and Vitek, tend to underestimate the MIC, whereas the Etest and MicroScan (prompt method) may overestimate the MIC. 31 Precision of these methods is clinically important because higher vancomycin MICs (1.5 lg/ml or higher) are associated with poorer outcomes (e.g., increased mortality, recurrence, delayed response, treatment failure, prolonged hospitalization), particularly in high-inoculum infections and with a higher proportion of hVISA presence. 25,32 Alternative therapies should be considered for patients receiving vancomycin therapy who are persistently bacteremic (7 days or longer) or who have no clinical improvement despite source control with an MIC of 1.5 lg/ml or higher by Etest. 2,31,32 Resistance Mechanisms of hVISA and VISA Evidence suggests that hVISA and VISA arise during continued or suboptimal exposure to vancomycin. 7,33 The proposed mechanism is selective pressure by vancomycin resulting in the development of rare vancomycin-resistant clones that progress to hVISA and, with continued-exposure, to a uniform population of VISA clones. 5,9 These isolates have significant differences in cell physiology including morphologic changes and genetic alterations. Strains of hVISA  17,19,27 Results ready to read following 24 hrs of incubation Unreliable specificity and sensitivity Uses standard bacterial inoculum MET or high-inoculum method 11,29 100% reproducibility Testing performed on nonstandard media while utilizing a standard McFarland suspension Easily performed Results of MET are cut-off points, not true MICs BHI screen agar plates 7,17,28 Easily performed Poor reproducibility Many variations; some studies screened with a different agar, inoculum size, or used suspensions with higher bacterial concentration BHI = brain heart infusion; GRD = glycopeptide resistance detection; MET = macromethod Etest; MIC = minimum inhibitory concentration; PAP = population analysis profiling. and VISA are characterized by thicker cell walls that correlate with increased vancomycin MICs. 34 Cell wall thickening impairs intracellular penetration of vancomycin rendering it ineffective. 5,34 In addition, hVISA and VISA are associated with slower growth rates than fully susceptible strains, which may contribute to persistent and recurrent infections. 35 Other mechanisms of resistance include alterations in transcriptional and metabolic genes and loss-offunction mutations that disturb critical cell wall biosynthesis. 11 The accessory gene regulator (agr) operon directs many critical virulence pathways, particularly the production of exotoxins. 11 In hVISA and VISA strains, agr function is reduced, favoring the development of vancomycin resistance and potentially promoting biofilm production that ultimately enhances the survival of hVISA and VISA. 33,36,37 Risk Factors and Outcomes Associated with hVISA and VISA Heteroresistance has been reported in MRSA isolates with MICs as low as 0.5 lg/ml and in cases where vancomycin was minimally effective. 6,16 Several studies have noted an increase in vancomycin treatment failures and mortality with vancomycin-susceptible MRSA strains, particularly those with MICs of 1.5 or 2 lg/ml. 25,32,[38][39][40] A 2013 meta-analysis of 20 studies evaluated high versus low vancomycin MICs (1.5 or higher vs lower than 1.5 lg/ml, respectively) on clinical outcomes in adults with MRSA infections. 40 An increased risk of failure was observed in the high MIC group compared with the low MIC group (relative risk [RR] 1.40; 95% confidence interval [CI] 1.15-1.71). There was also a greater risk of overall mortality (RR 1.45; 95% CI 1.08-1.87) in the high MIC group. Although the investigators attempted to exclude hVISA isolates, hVISA presence was not tested in every study, which may have contributed to vancomycin treatment responses. Although most of the isolates were from blood, clinical heterogeneity cannot be excluded. Another study evaluated 559 MRSA isolates and found an increased incidence of hVISA when the vancomycin MIC shifted from 1-2 lg/ml. 41 The incidence of hVI-SA was nearly 40% in isolates with an MIC of 2 lg/ml, supporting the results of other studies that suggest the proportion of hVISA isolates is directly related to increases in vancomycin MIC. 6,15,23,41 Increases in vancomycin MICs are hospital specific and perhaps caused by clonal outbreaks. However, this highlights the trends of vancomycin tolerance that may be caused by overuse of vancomycin, subtherapeutic vancomycin concentrations, high bacterial load, or slow vancomycin bactericidal activity. 3,42 Both hVISA and VISA have been identified in hospital and community strains of MRSA and in MSSA. 16 The findings of studies that evaluated clinical predictors and outcomes of hVISA infections are inconsistent. This may be attributed to the considerable heterogeneity of these studies including differences in study design, clinical definitions, selection of isolates (initial isolate, final isolate, or random selection), patient populations, and testing methodologies. Commonly reported associations with hVISA infections include vancomycin treatment failure and highinoculum MRSA infections (e.g., bacteremia, infective endocarditis, osteomyelitis, deep abscesses, and prosthetic device infections). 6,7,14,33,43,44 Other potential predictors of hVISA and VISA infections are prior MRSA infection or colonization (previous 3 mo), previous vancomycin exposure (prior 6 mo), initial low serum vancomycin trough levels (lower than 10 lg/ml), persistent bacteremia (7 days or longer), and the presence of indwelling devices (Table 3). 7,8,12,14,[44][45][46] Patients with hVISA infections tend to experience prolonged clinical courses, suboptimal response to vancomycin therapy, and prolonged hospital stays. 6-8, 14, 33, 42, 44 One retrospective case-control study compared the clinical features and outcomes of hVISA bacteremia of 27 subjects and MRSA bacteremia of 223 subjects. 14 Compared with MRSA bacteremia, patients with hVISA infections had significantly more days of bacteremia (median duration 12 vs 2 days, respectively; p=0.005) and significantly higher rates of endocarditis (18.5% vs 3.6%, respectively; p=0.007) and osteomyelitis (25.9% vs 7.2%, respectively; p=0.006). 14 Of note, patients in the hVISA group had significantly more prosthetic/implant devices (e.g., artificial heart valves, pacemakers, or orthopedic implants) and surgical site infections (in the previous month) at baseline that may have attributed to poorer outcomes. In a small case series, glycopeptide treatment failure (defined as a positive S. aureus blood culture after 7 days or longer of glycopeptide therapy or a sterile site culture positive for S. aureus after 21 days or longer of glycopeptide therapy) occurred in 19 of 25 patients (76%) with hVISA infections (bacteremia, endocarditis, osteomyelitis, or septic arthritis). 8 A retrospective, multicenter, matched cohort study compared the outcomes of hVISA versus vancomycin-susceptible-MRSA (VS-MRSA) bloodstream infections (BSIs) and found similar results. 6 Study investigators concluded that rates of vancomycin treatment failure were 11 times higher for a patient with hVISA BSI (50/61 [82%]) than VS-MRSA BSI (20/61 [32.8%]; p<0.001). Patients with hVISA BSI were also more likely than patients with VS-MRSA BSI to have persistent bacteremia (59% vs 21.3%, respectively; p<0.001), infection recurrence at 60 days (25.5% vs 1.9%, respectively; p<0.001), and longer hospital length of stay (median in days 24 vs 16, respectively; p=0.022). Differences in 30-day MRSA infection-related mortality and all-cause 30-day mortality were not observed between the hVISA BSI group and VS-MRSA BSI group (21.3% vs 9.8%; p=0.081 and 24.6% vs 11.5%; p=0.076, respectively). Similarly, no other studies have been powered to detect a significant difference in mortality between hVISA and non-hVISA infections. A systematic review and meta-analysis evaluated 30-day mortality from eight comparative hVISA studies. 18 After combining the data, 30-day mortality between hVISA and VSSA infections was similar (odds ratio 1.18, 95% CI 0.81-1.74). 18 These findings may be limited by the variability in definitions used and the predominantly retrospective designs of the original studies. Although the lack of association between hVISA and mortality can be partly explained by strain characteristics (e.g., decreased virulence) and host immune responses, sufficiently sized studies are needed to accurately determine if such an association exists. 47 Infections caused by VISA may also lead to recurrent infections, prolonged fevers and bacteremia, vancomycin treatment failure, and increased hospital stay. 7,12,33,44 In a singlecenter retrospective study, 6 patients with VISA had a significantly longer duration of bacteremia compared with 22 patients with hVISA (12.1 AE 13.1 vs 3.3 AE 3.9 days, respectively; p=0.001). 43 Significant differences in mortality between VISA and hVISA were not observed. However, rates of attributable mortality between hVISA and VSSA (215 patients) were similar (9.1% vs 8.4%, respectively), whereas those between VISA and VSSA (33.3% vs 8.4%) were not. 43 Although this study had several limitations, including a small sample size and bias through selective inclusion of isolates, the findings suggest that VISA may have more severe clinical implications and impact on patient outcomes. To date, no other published study has evaluated the outcomes of VISA infections, possibly because of the rarity of VISA infections.

Treatment Options for hVISA/VISA Infections
Although reports of vancomycin failure have emerged, no data demonstrate superior outcomes with alternative antimicrobials. Alternative antimicrobial agents with activity against hVISA/VISA include daptomycin, linezolid, ceftaroline, trimethoprim/sulfamethoxazole, tigecycline, quinupristin/dalfopristin, and the combination of vancomycin or daptomycin with a b-lactam. 12 Daptomycin Daptomycin is a potential treatment option for hVISA and VISA infections, and although it does have activity against MRSA, previous vancomycin exposure can result in some degree of cross-resistance to daptomycin. 48,49 Several studies have noted an in vitro association between increasing vancomycin MICs and increasing daptomycin nonsusceptibility. [48][49][50] The highest rate of daptomycin nonsusceptibility was reported in a study evaluating 47 Australian hVISA and VISA isolates never exposed to daptomycin. 50 The investigators noted daptomycin nonsusceptibly in 15% of hVISA and 38% of VISA strains. 50 Because bactericidal activity with daptomycin is concentration dependent, higher doses may be necessary to treat hVISA and VISA infections with elevated daptomycin MICs, highinoculum infections (e.g., endocarditis), and infection sites characterized by poor antimicrobial penetration. 51 High-dose daptomycin may prevent the selection or development of isolates with reduced susceptibility to daptomycin and subsequent treatment failure. 51 An in vitro study observed a more rapid reduction of bacterial burden of hVISA and VISA in simulated endocardial vegetations with highdose daptomycin (10 mg/kg/day for 8 days) and dose deescalation (10 mg/kg/day for 4 days followed by 6 mg/kg/day for 4 days) regimens compared with that of the standard (6 mg/kg/ day for 8 days) and dose escalation (6 mg/kg/ day for 4 days followed by 10 mg/kg/day for 4 days) regimens. 51 With respect to hVISA, the dose deescalation regimen had a significantly increased killing effect on the hVISA strain compared with the dose escalation regimen (p<0.024). 51 The investigators concluded that these daptomycin dosing approaches may lead to a faster cure of bacteremia in vivo and prevent the emergence of daptomycin nonsusceptibility. 51 However, no in vivo studies evaluating deescalation dosing and the appropriate duration of high-dose daptomycin have been published. The role of high-dose daptomycin alone in patients with hVISA or VISA infections is unclear. Until more evidence is available, caution is required when considering daptomycin in patients who may be at risk for hVISA or VISA infections (e.g., high-bacterial load infections, vancomycin failure). The determination of daptomycin susceptibility in these patients may also guide therapeutic decision making.

Linezolid
The role of linezolid for the treatment of invasive hVISA and VISA infections is also in question. Successful use of linezolid alone or in combination with other antimicrobial agents has been described in several case reports of vancomycin-heteroresistant and intermediate MRSA endocarditis and bacteremias after vancomycin failure and in some cases after daptomycin failure. 8,[52][53][54][55] In one case report, a 60-year-old man with an automatic implantable cardioverterdefibrillator presented with bacteremia and endocarditis initially caused by MRSA that later developed into hVISA, then daptomycin-nonsusceptible VISA after exposure to vancomycin and daptomycin. 55 The patient initially received 6 weeks of vancomycin (trough concentrations between 15 and 21 lg/ml), followed by~25 days of daptomycin (6 mg/kg every 48 hrs; renal dose adjusted). During therapy with daptomycin the defibrillator generator and leads were removed; however, the patient was persistently bacteremic and febrile. Blood cultures cleared after therapy was switched to linezolid and trimethoprim/sulfamethoxazole. The patient received at least 28 days of the combination and 6 weeks of linezolid monotherapy in total since his last positive blood culture. One year posttreatment the patient had no infection recurrence. After failing vancomycin and daptomycin therapy, this patient's VISA infection was successfully treated with linezolid. Although other case reports have shown similar outcomes with the use of linezolid, in vitro studies have not shown the same efficacy. 56 Evidence to recommend the use of linezolid for hVISA and VISA is insufficient. Further study is needed to evaluate linezolid alone or in combination for hVISA and VISA infections.

Ceftaroline
Ceftaroline has potent in vitro bactericidal activity against MRSA including hVISA, VISA, and daptomycin-nonsusceptible (DNS) MRSA strains. 57 The use of ceftaroline in the treatment of invasive infections (e.g., endocarditis, bacteremia, osteomyelitis) caused by hVISA, VISA, and DNS MRSA is supported by data from in vivo animal studies and human case reports. [58][59][60][61] In a 2012 case series report, a patient with DNS VISA bacteremia and endocarditis was successfully treated with 6 weeks of ceftaroline. The patient initially received and failed vancomycin therapy. 62 Blood cultures cleared within 48 hours of switching to daptomycin (6 mg/kg/day). However, subsequent blood cultures were positive and revealed DNS VISA. Daptomycin was discontinued, and ceftaroline (600 mg intravenously every 8 hrs) was initiated. While on ceftaroline, blood cultures cleared within 48 hours and remained sterile. In vitro pharmacokinetic/pharmacodynamic studies reported enhanced ceftaroline activity against hVISA, VISA, and DNS MRSA as vancomycin and daptomycin susceptibilities decreased, which have been referred to as the "seesaw effect." [58][59][60] Although further study is needed, ceftaroline appears to be a safe and effective alternative in the treatment of invasive hVISA, VISA, and DNS MRSA infections given its bactericidal activity, favorable safety profile, and emerging data.

Combination Therapy
The combination of vancomycin or daptomycin and a b-lactam antimicrobial has also been studied for the treatment of hVISA and VISA infections. b-Lactams that have been evaluated for synergistic activity with vancomycin or daptomycin include ceftaroline, cefazolin, and piperacillin-tazobactam. [63][64][65][66] In vitro and clinical case report data evaluating the combination of high-dose daptomycin (10 mg/kg/day) and trimethoprim/sulfamethoxazole also appear promising for the treatment of hVISA, VISA, and DNS MRSA infections. 67,68 In vitro studies have demonstrated improved kill rates with these antimicrobial combinations. [63][64][65] Investigators hypothesize that b-lactam exposure may influence vancomycin and cell wall interactions to improve vancomycin activity, although further investigation is warranted. 63 In summary, preliminary experimental studies show possible prospects for the treatment of hVISA and VISA infections. However, it is not yet clear which treatment options correlate with optimal clinical outcomes for patients with confirmed hVISA or VISA infections.
Infection Control: Preventing the Dissemination of hVISA/VISA As with MRSA, hVISA and VISA can colonize humans and the environment despite eradication efforts. The CDC has made several recommendations in an attempt to prevent the emergence of vancomycin nonsusceptible infections. 42 Infections with confirmed VISA should be reported to infection-control personnel, the patient's primary caregiver, medical ward staff, local and state departments of health, and the CDC. Patients and their caregivers should be educated regarding wound care, physical hygiene, and signs of infection. 69 Contact isolation in both the inpatient and outpatient setting may also limit further emergence. Adherence to recommended infection prevention and control guidelines, appropriate antibiotic prescribing through antimicrobial stewardship programs, and active surveillance in a cohesive health care system are essential to prevent further emergence of hVISA and VISA colonization and infection.

Conclusions
The evolution of S. aureus to MRSA and now to hVISA and VISA is an important and ongoing public health concern. Vancomycin is the drug of choice for invasive MRSA infections; however, its use is under question. Overuse, suboptimal concentrations, or inappropriate use of vancomycin is speculated to be a major contributor in the emergence of hVISA and VISA. Most alarming are the poor outcomes that have been associated with hVISA and VISA infections and the limited antimicrobials available to treat these infections. Proper detection methods are necessary for accurate surveillance, guidance on therapeutic decision making, and a full understanding of the implications of hVISA/VISA infections. Until then, patients who are at risk for hVISA/ VISA infections and failing vancomycin therapy may warrant further confirmatory testing for hVISA/VISA. Based on currently available data, clinicians should, with vigilance, continue to use vancomycin per the Infectious Diseases Society of America guidelines. 2,3 Alternative therapies should be considered in patients with risk factors for hVISA/VISA who are not responding clinically to vancomycin despite source control and a vancomycin MIC of 2 lg/ml or lower. In patients infected with VISA (vancomycin MIC 4-8 lg/ml), an alternative antimicrobial should be considered. Caution is advised when deciding to use daptomycin in patients with hVISA/VISA infections because of the potential for crossresistance. To prevent further resistance, appropriate use of antimicrobials and implementation of infection-control guidelines are imperative.