Impact of Molecular Epidemiology and Reduced Susceptibility to Glycopeptides and Daptomycin on Outcomes of Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia

Background Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia was associated with high mortality, but the risk factors associated with mortality remain controversial. Methods A retrospective cohort study was designed. All patients with MRSA bacteremia admitted were screened and collected for their clinical presentations and laboratory characteristics. Minimum inhibitory concentration (MIC) and staphylococcal cassette chromosome mec (SCCmec) type of bacterial isolates were determined. Risk factors for mortality were analyzed. Results Most MRSA isolates from the 189 enrolled patients showed reduced susceptibility to antibiotics, including MIC of vancomycin ≥ 1.5 mg/L (79.9%), teicoplanin ≥ 2 mg/L (86.2%), daptomycin ≥ 0.38 mg/L (73.0%) and linezolid ≥ 1.5 mg/L (64.0%). MRSA with vancomycin MIC ≥ 1.5 mg/L and inappropriate initial therapy were the two most important risk factors for mortality (both P < 0.05; odds ratio = 7.88 and 6.78). Hospital-associated MRSA (HA-MRSA), carrying SCCmec type I, II, or III, was associated with reduced susceptibility to vancomycin, teicoplanin or daptomycin and also with higher attributable mortality (all P < 0.05). Creeping vancomycin MIC was linked to higher MIC of teicoplanin and daptomycin (both P < 0.001), but not linezolid (P = 0.759). Conclusions Giving empirical broad-spectrum antibiotics for at least 5 days to treat catheter-related infections, pneumonia, soft tissue infection and other infections was the most important risk factor for acquiring subsequent HA-MRSA infection. Choice of effective anti-MRSA agents for treating MRSA bacteremia should be based on MIC of vancomycin, teicoplanin and daptomycin. Initiation of an effective anti-MRSA agent without elevated MIC in 2 days is crucial for reducing mortality.


Introduction
Antimicrobial susceptibility testing and vancomycin-heteroresistant S. aureus screening The MICs of vancomycin, daptomycin, teicoplanin, and linezolid were determined by the Etest (AB Biodisk, Solna, Sweden) [14]. The interpretation was based on Clinical and Laboratory Standards Institute (CLSI) standards [15]. Breakpoints for vancomycin, teicoplanin, daptomycin and linezolid were summarized as shown in supplemental information (S1 Table). Screening for vancomycin-heteroresistant S. aureus (hVISA) was performed by the vancomycinteicoplanin Etest macromethod, according to the manufacturer's instructions (AB Biodisk). ATCC29213 was used as a control in the MIC and hVISA screening tests. An isolate was considered hVISA when growing in the presence of teicoplanin 12 μg alone or 8 μg for both vancomycin and teicoplanin [15].

Staphylococcal cassette chromosome mec typing
The staphylococcal cassette chromosome mec (SCCmec) types of all 189 isolates were determined by a multiplex polymerase chain reaction (PCR) described previously [16], and another multiplex PCR strategy was used if this method was not successful in identifying a type [17]. The identification of SCCmec type V T was verified with a method described by Boyle-Vavra et al [18].

Clinical data collection and definitions
Clinical data were collected retrospectively. Mortality was defined as bacteremia-attributable death, i.e., death before resolution of symptoms and signs of bacteremia and at least a blood culture positive for MRSA [21]. Considering "time at risk" for bacteria to become resistant to antimicrobial agents under antimicrobial selective pressure, prior exposure to these agents was defined as at least 5 days of therapy during the 14 days before the isolation of MRSA [21,22]. Appropriate initial antimicrobial therapy was defined as giving patients with at least one susceptible antimicrobial agent, except single aminoglycoside or rifampicin treatment, within 2 days after the onset of bacteremia [12]. Multidrug resistance (MDR) was defined as resistance to at least 3 antibiotic classes [21]. Culture detecting time was the interval (days) from culture sampling to reporting [21]. Community-associated MRSA (CA-MRSA) was defined as an isolate possessing the SCCmec type IV or V genes, and hospital-associated MRSA (HA-MRSA) was defined as an isolate carrying SCCmec type I, II, or III genes [11]. Community-acquired infection (CAI) was defined as the isolation of MRSA from bloodstream within 48 h of admission, while hospital-acquired infection (HAI) as beyond that time [23]. Catheter-related infection was defined by the evidence of infected intravascular catheter which was considered to be the portal of entry if a catheter-tip culture was positive for S. aureus or if inflammation was present around the catheter insertion site.

Multi-stages risk factor analysis
An analytical model with multiple stages was proposed from the appearance of MRSA infection to mortality for patients with mortality [21]. The most important main risk factor (MRF1) associated with mortality due to MRSA infection was the first one to be analyzed by multivariate logistic analysis. Then main risk factor (MRF2) correlated with MRF1 was the second one to be analyzed by the same method. In this way, MRF1, MRF2, MRF3, MRF4, etc. would be identified as the most important independent risk factor in each stage backward from death to infection [21].

Statistical analysis
Data were recorded and entered into a database. Analyses were performed using SPSS software, v. 17.0 (SPSS Inc., Chicago, IL, USA). The Student's t-test, the Chi-square test, Fisher's exact test or ANOVA was used when appropriate to compare proportions. Variables with a P value < 0.2 in the univariate analysis were added in a stepwise manner and selected to determine the final model for multivariable analysis. All statistical analyses were two-sided, and significance was set at P < 0.05.

Bacterial isolates and patients
A total of 501 isolates among patients with S. aureus bacteraemia treated in Chang Gung Memorial Hospital from January 2010 to December 2011 were collected. A total of 252 MRSA isolates was collected and the resistance rate to oxacillin among S. aureus was 50.3%. After review, 63 patients were excluded including 4 patients < 18 years old, 32 with polymicrobial infection, 25 non-first, repetitive episodes, and 2 with insufficient medical records. A total of 189 patients with symptoms and isolation of MRSA from bloodstream met the inclusion criteria.

Reduced susceptibility in MRSA with various SCCmec types
We found correlations of SCCmec types with MICs of antimicrobial agents (Table 1). In comparison with CA-MRSA (SCCmec type IV, V and V T ), most HA-MRSA (SCCmec type I, II, III, IIIa and IIIb) showed higher incidence of vancomycin MIC 1.5 mg/L, teicoplanin MIC 2 mg/L, daptomycin MIC 0.38 mg/L, and linezolid < 1.5 mg/L ( Table 1).

Genotyping
The predominant ST in isolates of HA-MRSA is ST239 (66.3%) and that in CA-MRSA is ST59 (53.2%) as shown in supplemental information (S1 Fig and S2 Table).

Multi-stages risk factor analysis
Many factors correlated with mortality, including clinical severity (Pitt bacteraemia score, white blood cell count, C-reactive protein level), specific source (catheter-related infection), infection by specific SCCmec type (IIIA), HA-MRSA, or hVISA, infection by pathogen with higher vancomycin MIC ( 2 mg/L or 1.5 mg/L), teicoplanin MIC ( 6 mg/L, 4 mg/L, 3mg/L, 2mg/L), and daptomycin MIC ( 0.38 mg/L) and inappropriate initial therapy were associated with mortality (Table 3). After multivariate analysis, only Pitt bacteraemia score, Creactive protein level, white blood cell count, catheter-related infection as infection source, infection by pathogen with vancomycin MIC 2 mg/L, infection by pathogen with vancomycin MIC 1.5 mg/L and inappropriate therapy were independent risk factors for 30-day mortality. Among these independent risk factors, infection by pathogen with vancomycin MIC 1.5 mg/L was the major risk factor (MRF1) for mortality with the highest odds ratio (adjusted odds ratio [AOR], 7.88; P = 0.010) ( Table 3).

Backward root analysis for mortality
After the above multi-stages risk factor analysis, we were able to summarize a development stages for mortality due to MRSA bacteremia (Table 4). A prior infection, such as catheterrelated infections, pneumonia, or soft tissue infection, was stage 1. Broad-spectrum antibiotic use for treating one of these prior infections was stage 2. From stage 3 to stage 5, HA-MRSA with higher vancomycin MIC, higher teicoplanin MIC, and higher daptomycin MIC (Table 1) was selected from broad-spectrum antibiotic pressure from stage 2. After analysis by log-rank test, higher incidence of mortality correlated with MRSA with higher vancomycin MIC (P = 0.003), higher teicoplanin MIC (P = 0.005), and higher daptomycin MIC (P = 0.012) (Fig 2). Therefore, infection by HA-MRSA was associated with high mortality (P = 0.010 by logrank test).

Discussion
At least three important findings in MRSA bacteremia were reported in this study. Firstly, in contrast to CA-MRSA (53.2% ST59), HA-MRSA (66.3% ST239) was found associated with increased vancomycin MIC, teicoplanin MIC, and daptomycin MIC. HA-MRSA tended to simultaneously express reduced susceptibility to most anti-MRSA agents, except linezolid. Secondly, although patients infected by MRSA with vancomycin MIC 1.5 mg/L or teicoplanin MIC > 1.5 mg/L have been proved to have a higher mortality rate [3,6], we further found in this study that bacteremic patients infected by MRSA with daptomycin MIC 0.38 mg/L also showed a higher mortality. Thirdly, by backward root analysis, there were multiple stages from the source of infection to mortality due to MRSA bacteremia. MRSA with vancomycin MIC 1.5 mg/L and inappropriate initial therapy were the two most important risk factors for mortality. Risk factors, including HA-MRSA, MRSA with vancomycin MIC 1.5 mg/L, teicoplanin MIC 1.5 mg/L, and daptomycin MIC 0.38 mg/L, were associated with each other and the combined effect is a higher risk of mortality in the bacteremic patients. Similar to previous reports, we found in this study that catheter-related infections, pneumonia, and soft tissue infections usually preceded MRSA bacteremia [6]. Broad-spectrum antibiotics were prescribed for the empirical treatment of such infections. This therefore generated a high antibiotic selective pressure for subsequent breakthrough infection caused by drug-resistant organisms, such as MRSA with a higher antimicrobial MIC. Likely in previous studies, use Factors Influencing the Outcome of MRSA Bacteremia of broad-spectrum antibiotics such as fluoroquinolones has been found in association with increased rates of MRSA acquisition [24].
We found that HA-MRSA with higher MIC of anti-MRSA agents were selected by the prior use of broad-spectrum antibiotics to cause subsequent bacteremia. The association of genotypes of MRSA with increased vancomycin MIC was examined in previous studies [8,9]. A significant association between SCCmec II/III and elevated vancomycin MIC was reported [8].
Higher vancomycin MICs were also linked to specific clonal complexes (CCs) and HA-MRSA [10,11]. CC8 was associated with elevated vancomycin MIC, and in contrast, low vancomycin MIC with CC22, CC88, and CC188 [9]. SCCmec is a mobile element that was spread through horizontal gene transfer. We observed that HA-MRSA with increased MIC of anti-MRSA drugs (except linezolid) was also associated with higher mortality in this study, suggesting that other intrinsic microbial factors were involved in the pathogenesis of MRSA bacteremia with a high mortality. In fact, in a previous study, in comparison to patients with CA-MRSA infections, the higher risk for treatment failure among patients with HA-MRSA infections suggested that HA-MRSA could possess an intrinsic strain-specific virulence factor [10].
Before 2006, MRSA isolates were considered susceptible to vancomycin when MIC was 4 mg/mL; however, this breakpoint was decreased to 2 mg/L due to poor therapeutic results in patients with infections caused by MRSA with MIC > 2 mg/L [25]. Thereafter, elevated vancomycin MIC ( 1.5 mg/L in MRSA and > 1.5 mg/L in methicillin-susceptible S. aureus [MSSA]) has been proved an independent risk factor for 30-day mortality of patients with S. aureus bacteremia, regardless of resistance to methicillin or the treatment administered [4,26]. Given the results, and others, not only vancomycin MIC but also teicoplanin MIC and daptomycin MIC could be considered surrogate markers for pathogen-specific factors responsible for worse outcomes or increased virulence secondary to antibiotic resistance [27]. Although vancomycin remains to be the first-line therapy for severe MRSA infections [28], there are now sufficient data demonstrating the efficacy of daptomycin and linezolid for both MSSA and MRSA infections [29,30]. For the treatment of MRSA bacteremia, only daptomycin and vancomycin have been approved by Food and Drug Administration (FDA), but therapy with linezolid, an alternative antibiotic, showed outcomes non-inferior to vancomycin treatment in patients [30]. Infection caused by MRSA with higher vancomycin MIC, which showed thicker cell wall, has been reported associated with higher risk of daptomycin treatment failure. This is a phenomenon that can be explained by the inability of daptomycin, molecular weight of which is 4.8 times larger than linezolid, to diffuse to its active site through a thickened cell wall [29]. Indeed, our results showed a positive correlation between vancomycin MIC and daptomycin MIC in MRSA. On the other hand, our results revealed that the linezolid MIC did not increase along with the creeping vancomycin MIC in MRSA. Therefore, therapy with linezolid may be better than that with daptomycin for infection caused by MRSA with higher vancomycin MIC.
In this study, inappropriate initial therapy was found the second most important risk factor for mortality. This was also described in previous studies that identified delay in the initiation of appropriate antimicrobial therapy as an integral determinant of poor clinical outcomes for severe diseases, such as MRSA bacteremia [12,13]. Based on this, in case of persistent MRSA bacteremia following initial treatment, the 7-day threshold to seeking alternative combination antibiotic therapy is recommended to be shortened to 3-4 days [30].
In summary, MIC of vancomycin, teicoplanin and daptomycin must be checked in 2 days for choosing an effective initial antibiotic therapy for patients with MRSA bacteremia. If a patient shows a poor response to vancomycin and only vancomycin MIC is known, the study suggests that therapy with linezolid might lead to a better outcome for MRSA isolates with vancomycin MIC 1.5 mg/L. Such isolates usually showed a reduced susceptibility to daptomycin.
Giving an effective anti-MRSA agent without an MIC indicating reduced susceptibility in 2 days is crucial for reducing mortality of MRSA bacteremia.  Table. Sequence types by multilocus sequence typing identified among isolates with different staphylococcal cassette chromosome mec types with or without Panton-Valentine leucocidins.