The increasing use of chlorhexidine for methicillin-resistant Staphylococcus aureus (MRSA) decolonization has raised concerns about the emergence of resistance to these agents. However, the clinical significance of MRSA positive for the qacA and qacB chlorhexidine tolerance genes has not been established.
KEYWORDS: Staphylococcus aureus; chlorhexidine; qacA, qacB
ABSTRACT
The increasing use of chlorhexidine for methicillin-resistant Staphylococcus aureus (MRSA) decolonization has raised concerns about the emergence of resistance to these agents. However, the clinical significance of MRSA positive for the qacA and qacB chlorhexidine tolerance genes has not been established. We investigated the clinical features and predictive factors of MRSA bloodstream infection (BSI) isolates, caused by qacA- and qacB-positive MRSA, from 2010 to 2016 at a tertiary hospital in South Korea. A total of 246 MRSA BSI isolates were included; 71 (28.9%) isolates carried qacA/B. The annual frequency of qacA- and qacB-positive MRSA bacteremia did not change significantly over the study period. Patients infected with qacA- and qacB-positive MRSA had common risk factors for health care-associated infections, including prior antibiotic use, central venous catheterization in situ, intensive care unit-acquired bacteremia, and nosocomial infection. The qacA- and qacB-positive isolates were also associated with an increasing chlorhexidine MIC and resistance to non-β-lactam antibiotics. The qacA- and qacB-positive isolates were more likely to belong to sequence type 5 (ST5), which is a common health care-associated MRSA strain in South Korea. In multivariable analyses, qacA- and qacB-positive MRSA isolates were found to be associated with agr dysfunction (adjusted odds ratio [aOR], 6.45; 95% confidence interval [CI], 2.59 to 16.10), ST5 MRSA strain (aOR, 4.96; 95% CI, 1.85 to 13.26), nosocomial infection (aOR, 4.88; 95% CI, 2.20 to 10.83), and antibiotic use within the previous 3 months (aOR, 2.59; 95% CI, 1.20 to 5.59). These findings suggest that the microbiological features of qacA and qacB carriage provide a selective advantage for specific MRSA strains in hospital environments.
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) has long been a major cause of health care-associated infections (HAIs), and it is associated with increased morbidity, mortality, and excess hospital costs (1–3). Chlorhexidine gluconate (CHG) is widely applied to minimize MRSA transmission between hospitalized patients as a part of infection control programs, being used in central venous catheter dressings and CHG-based body washes (4, 5). However, the widespread use of these topical antimicrobials has raised concerns regarding the emergence of MRSA strains resistant or tolerant to CHG (6). In S. aureus, resistance to or tolerance of chlorhexidine is associated with the qacA and qacB genes, which encode multidrug efflux pumps (7, 8). Moreover, some plasmids harboring the qacA and qacB genes may cotransmit with other antibiotic resistance genes as a result of selective pressure in certain S. aureus strains (9).
The clinical implications of qacA- and qacB-positive MRSA have not been established. A large clinical trial of CHG-based decolonization for the control of MRSA revealed the prevalence of qacA and qacB genes to be low and the impact of chlorhexidine tolerance on decolonization failure to be minimal (5, 10). However, some studies found that qacA and qacB carriage in MRSA was not uncommon in hospital environments, and the prevalence of certain MRSA clones, such as CC22 or ST239 (where CC stands for clonal complex and ST stands for sequence type), increased after the implementation of a chlorhexidine-based decolonization (11, 12). Another study reported that qacA and qacB genes in combination with low-level mupirocin resistance was related to failure of MRSA decolonization (13). Although a recent study reported that health care exposure, specifically nosocomial S. aureus acquisition and underlying medical conditions, was associated with chlorhexidine tolerance genes, factors predictive of the presence of qacA and qacB genes have not been well described (14). In the present study, we investigated the molecular epidemiology of qacA- and qacB-positive MRSA in bloodstream infection (BSI) isolates, and we describe the clinical significance of these isolates.
(These data were presented in part at IDWeek 2017 in San Diego, CA [15].)
RESULTS
Frequency of chlorhexidine tolerance genes in MRSA BSI isolates.
Of 246 MRSA BSI isolates, 77 (31.3%) possessed one or both chlorhexidine tolerance genes. Seventy-one isolates (28.9%) were positive for qacA and qacB, and seven (2.8%) were positive for smr, including one isolate (0.4%) that was positive for all three genes. The annual frequencies of qacA- and qacB-positive isolates of the overall and nosocomial MRSA BSIs did not change significantly during the 7-year study period (P = 0.808 and P = 0.578, respectively) (Fig. 1). Most BSIs caused by qacA- and qacB-positive MRSA were nosocomial (78.9%) or community-onset health care-associated (CO-HCA) (18.3%) infections. Two cases (2.8%) were community acquired.
FIG 1.
Annual frequency of qacA- and qacB-positive isolates in overall and nosocomial MRSA bloodstream infection isolates that were available for microbiological tests.
Clinical and microbiological characteristics of patients infected with qacA- and qacB-positive MRSA BSI isolates.
The clinical characteristics of 246 patients with MRSA BSIs are shown in Table 1. The most common cause of infection was central venous catheter infection (38.2%), followed by unknown primary bacteremia (22.4%) and soft-tissue, bone, and joint infection (19.5%). There were no significant differences in sex, underlying diseases, Charlson comorbidity score, or hospitalization within the previous year between the qacA- and qacB-positive and qacA- and qacB-negative MRSA isolates. However, qacA- and qacB-positive isolates were more likely to be associated with older age (72 versus 69 years; P = 0.043), antibiotic use within the past 3 months (69.0% versus 41.1%; P < 0.001), central venous catheter in situ (69.0% versus 31.4%; P < 0.001), and intensive care unit (ICU)-acquired bacteremia (28.2% versus 8.0%; P < 0.001). Patients with qacA- and qacB-positive isolates also had more central venous catheter-related infections (63.4% versus 28.0%; P < 0.001) but fewer soft-tissue, bone, and joint infections (7.0% versus 24.6%; P = 0.001) than did patients with qacA- and qacB-negative isolates. However, there were no significant differences in the incidence of septic shock on bacteremia presentation, the length of hospital stay after bacteremia, and mortality between the two groups.
TABLE 1.
Comparison of clinical characteristics of patients with MRSA bacteremia based on qacA and qacB status
| Variablea | Value(s) by qacA and qacB status |
P value | |
|---|---|---|---|
| Positive (n = 71) | Negative (n = 175) | ||
| Age, median (IQR) yr | 72 (61.5–79.5) | 69 (57–75) | 0.043 |
| Male gender | 45 (63.4) | 106 (60.6) | 0.773 |
| Mode of acquisition | |||
| Community onset | 15 (21.1) | 109 (62.3) | <0.001 |
| Community acquired | 2 (2.8) | 42 (24.0) | <0.001 |
| Healthcare associated | 13 (18.3) | 67 (38.3) | 0.003 |
| Nosocomial | 56 (78.9) | 66 (37.7) | <0.001 |
| Comorbidity | |||
| Diabetes mellitus | 25 (35.2) | 77 (44.0) | 0.253 |
| Solid cancer | 10 (14.1) | 27 (15.4) | 0.847 |
| Hematologic malignancy | 1 (1.4) | 7 (4.0) | 0.444 |
| Liver cirrhosis | 6 (8.5) | 23 (13.1) | 0.385 |
| End-stage renal disease | 8 (11.3) | 20 (11.4) | 0.999 |
| Chronic pulmonary disease | 6 (8.5) | 10 (5.7) | 0.408 |
| Charlson comorbidity score, median (IQR) | 4 (3 – 5) | 4 (3 – 6) | 0.554 |
| Polymicrobial BSI | 8 (11.3) | 15 (8.6) | 0.481 |
| Hospitalization in the prior 1 yr | 30 (42.3) | 86 (49.1) | 0.398 |
| Surgery in the prior 3 months | 20 (28.2) | 29 (16.6) | 0.052 |
| Antibiotic use within the previous 3 months | 49 (69.0) | 72 (41.1) | <0.001 |
| Central venous catheter in place | 49 (69.0) | 55 (31.4) | <0.001 |
| Chlorhexidine-impregnated catheterb | 24/46 (52.2) | 25/54 (46.3) | 0.688 |
| ICU-acquired bacteremia | 20 (28.2) | 14 (8.0) | <0.001 |
| Primary site of infection | |||
| Central venous catheter-related infection | 45 (63.4) | 49 (28.0) | <0.001 |
| Pneumonia | 3 (4.2) | 14 (8.0) | 0.409 |
| Surgical wound infection | 4 (5.6) | 5 (2.9) | 0.285 |
| Infective endocarditis | 0 | 8 (4.6) | 0.109 |
| Soft-tissue, bone, and joint infection | 5 (7.0) | 43 (24.6) | 0.001 |
| Unknown | 15 (21.1) | 40 (22.9) | 0.866 |
| Others | 2 (2.8) | 15 (8.6) | 0.164 |
| Septic shock on bacteremia presentation | 16 (22.5) | 36 (20.6) | 0.733 |
| Median length of hospital stay after bacteremia, days (IQR) | 24 (9–45.5) | 20 (10–34.5) | 0.265 |
| 30-Day mortality | 19 (26.8) | 54 (30.9) | 0.543 |
| In-hospital mortality | 25 (35.2) | 56 (32.0) | 0.655 |
Data are no. (%) of patients unless otherwise indicated.
Among 104 patients with central venous catheters, four were excluded because of a lack of information on the catheter type.
The microbiological characteristics of the MRSA BSI isolates based on qacA and qacB status are shown in Table 2. The qacA- and qacB-positive MRSA isolates were significantly associated with higher chlorhexidine MICs (median, 4 versus 2 μg/ml; range, 2 to 8 versus 1 to 32 μg/ml) (P < 0.001) and agr dysfunction (84.5% versus 25.7%; P < 0.001), and they were more often resistant to several non-β-lactam antibiotics, including mupirocin, clindamycin, ciprofloxacin, tetracycline, erythromycin, fusidic acid, and gentamicin. In addition, there was a significant association between agr dysfunction and higher chlorhexidine MICs in overall MRSA isolates (median, 4 versus 2 μg/ml; range, 1 to 16 versus 1 to 32 μg/ml) (P < 0.001). However, this association was not evident when the MRSA isolates were stratified by qacA and qacB status (see Fig. S1 in the supplemental material).
TABLE 2.
Microbiological characteristics of MRSA bloodstream infection isolates based on qacA and qacB status
| Variablea | Value(s) by qacA and qacB status |
P value | |
|---|---|---|---|
| Positive (n = 71) | Negative (n = 175) | ||
| Chlorhexidine MIC (BMD) | <0.001 | ||
| ≤2 mg/liter | 4 (5.6) | 120 (68.6) | |
| 4 mg/liter | 64 (90.1) | 44 (25.1) | |
| ≥8 mg/liter | 3 (4.2) | 11 (6.3) | |
| Vancomycin MIC of ≥2 mg/liter (BMD) | 18 (25.4) | 26 (14.9) | 0.066 |
| Carriage of smr | 1 (1.4) | 6 (3.4) | 0.677 |
| Dysfunctional agr | 60 (84.5) | 45 (25.7) | <0.001 |
| Resistance to | |||
| Mupirocin | 9 (12.7) | 7 (4.0) | 0.020 |
| Clindamycin | 70 (98.6) | 113 (64.6) | <0.001 |
| Ciprofloxacin | 70 (98.6) | 70 (40.0) | <0.001 |
| Tetracycline | 68 (95.8) | 52 (29.7) | <0.001 |
| Erythromycin | 70 (98.6) | 113 (64.6) | <0.001 |
| Fusidic acid | 63 (88.7) | 29 (16.6) | <0.001 |
| Gentamicin | 67 (94.4) | 47 (26.9) | <0.001 |
| Rifampin | 3 (4.2) | 2 (1.1) | 0.146 |
| Trimethoprim/sulfamethoxazole | 5 (7.0) | 5 (2.9) | 0.158 |
| Multidrug resistanceb | 70 (98.6) | 72 (41.1) | <0.001 |
Data are no. (%) of patients unless otherwise indicated.
Isolates were considered multidrug resistant if they were resistant to three or more different classes of non-β-lactam antimicrobials.
Genotypic characteristics of qacA- and qacB-positive MRSA BSIs.
Table 3 shows the genotypic characteristics of MRSA BSIs based on qacA and qacB status. The qacA- and qacB-positive isolates were more likely to belong to the staphylococcal cassette chromosome mec II (SCCmec II) or SCCmec III groups, which are common health care-associated MRSA strains in South Korea, than the SCCmec IV group (97.1% versus 2.8%; P < 0.001). ST5 (88.7%) was the most common sequence type in the qacA- and qacB-positive MRSA isolates. The dominant spa types in the qacA- and qacB-positive isolates were t2460 (71.8%) and t9353 (11.3%).
TABLE 3.
Genotypic characteristics of MRSA bloodstream infection isolates based on qacA and qacB status
| Variablea | Value(s) by qacA and qacB status |
P value | |
|---|---|---|---|
| Positive (n = 71) | Negative (n = 175) | ||
| SCCmec type | <0.001 | ||
| II | 65 (91.5) | 59 (33.7) | |
| III | 4 (5.6) | 4(2.3) | |
| IV | 2 (2.8) | 112 (64.0) | |
| MLST | <0.001 | ||
| ST5 | 63 (88.7) | 49 (28.0) | |
| ST72 | 1 (1.4) | 104 (59.4) | |
| Otherb | 7 (9.9) | 22 (12.6) | |
| spa type | <0.001 | ||
| t2460 | 51 (71.8) | 15 (8.5) | |
| t664 | 0 | 56 (32.0) | |
| t324 | 0 | 29 (16.6) | |
| t002 | 1 (1.4) | 26 (14.9) | |
| Otherc | 19 (26.8) | 49 (28.0) | |
Data are no. (%) of patients unless otherwise indicated.
The other MLSTs were ST239 (n = 5), ST518 (n = 1), and nontypeable (n = 1) in the qac-positive group and ST188 (n = 4), ST89 (n = 3), ST8 (n = 2), ST239 (n = 2), ST2084 (n = 1), and nontypeable (n = 10) in the qac-negative group.
The other spa types were t9353 (n = 8), t037 (n = 3), t111 (n = 1), t264 (n = 1), t2029 (n = 1), t2139 (n = 1), t2703 (n = 1), and nontypeable (n = 2) in the qac-positive group and t148 (n = 5), t189 (n = 3), t375 (n = 3), t1368 (n = 3), t901 (n = 2), t4359 (n = 2), t9353 (n = 2), t008 (n = 1), t037 (n = 1), t045 (n = 1), t126 (n = 1), t242 (n = 1), t535 (n = 1), t601 (n = 1), t1767 (n = 1), t2431 (n = 1), t2461 (n = 1), t2882 (n = 1), t4331 (n = 1), t4705 (n = 1), t5071 (n = 1), t5229 (n = 1), t5440 (n = 1), t5716 (n = 1), t12703 (n = 1), and nontypeable (n = 8) in the qac-negative group.
Factors associated with the presence of qacA and qacB genes.
When the clinical and microbiological factors that were significantly associated with the presence of qacA and qacB genes in univariate analyses were included in a multiple logistic regression analysis, qacA- and qacB-positive MRSA isolates were independently associated with agr dysfunction (adjusted odds ratio [aOR], 6.45; 95% confidence interval [CI], 2.59 to 16.10), ST5 MRSA strain (aOR, 4.96; 95% CI, 1.85 to 13.26), nosocomial infection (aOR, 4.88; 95% CI, 2.20 to 10.83), and antibiotic use within the previous 3 months (aOR, 2.59; 95% CI, 1.20 to 5.59) (Table 4).
TABLE 4.
Univariate and multivariable analyses of risk factors associated with the presence of qacA and qacB genes in MRSA bloodstream infection isolates
| Variable | Univariate analysis |
Multivariable analysis |
||
|---|---|---|---|---|
| OR (95% CI) | P value | aOR (95% CI) | P value | |
| Increased age per 10 yr | 1.17 (0.86–1.60) | 0.320 | ||
| Surgery in the prior 3 mo | 1.51 (0.54–4.25) | 0.432 | ||
| Central venous catheter in place | 1.17 (0.45–3.02) | 0.746 | ||
| ICU-acquired bacteremia | 1.34 (0.48–3.74) | 0.579 | ||
| Soft-tissue, bone, and joint infection | 0.95 (0.22–4.09) | 0.949 | ||
| Vancomycin MIC of ≥2 μg/ml | 1.13 (0.39–3.30) | 0.823 | ||
| Nosocomial infection | 3.88 (1.43–10.54) | 0.008 | 4.88 (2.20–10.83) | <0.001 |
| Antibiotic use within the previous 3 mo | 2.43 (1.06–5.56) | 0.036 | 2.59 (1.20–5.59) | 0.016 |
| Dysfunctional agr | 6.41 (2.48–16.57) | <0.001 | 6.45 (2.59–16.10) | <0.001 |
| MLST ST5 | 4.64 (1.70–12.67) | 0.003 | 4.96 (1.85–13.26) | 0.001 |
Because of the high proportion of ST5 in qacA- and qacB-positive MRSA isolates, we further assessed the relationship between the specific ST of MRSA and two other important risk factors for the presence of qacA and qacB genes: agr dysfunction and nosocomial infection (Tables S1 and S2). When the study group was classified into ST5 and non-ST5 MRSA, qacA and qacB carriage was significantly associated with agr dysfunction in ST5 (93.7% versus 57.1%: P < 0.001) but not in non-ST5 MRSA (12.5% versus 13.5%; P = 0.999). There was also a significant association between the presence of qacA and qacB genes and agr dysfunction in both nosocomial (83.9% versus 27.3%; P < 0.001) and community-onset MRSA infections (86.7% versus 24.8%; P < 0.001).
DISCUSSION
Although numerous studies have investigated the prevalence and clinical implications of the presence of qacA and qacB in S. aureus, few have addressed those issues in the context of bloodstream MRSA isolates (11, 16). Of about 250 samples of MRSA BSI isolates, we found that qacA- and qacB-positive MRSA isolates were not uncommon (28.9%) and were related to the ST5 strain, which is the most common health care-associated MRSA clone in South Korea (17). Although this study was conducted over a relatively short period, the frequency of qacA- and qacB-positive MRSA did not change significantly over 7 years. A study of MRSA isolates collected from 11 Asian countries between 1998 and 1999 showed that, of the isolates collected from South Korea, 32.4% (34/105) carried the qacA and qacB genes and 0% the smr genes (18). These findings imply that genotypic antiseptic tolerance is widely distributed in South Korea, despite the lack of a nationwide longitudinal study reporting the prevalence of qacA and qacB genes in S. aureus.
The clonal association of qacA and qacB in MRSA has been controversial. Two UK studies reported an increasing prevalence of CC22 and ST239 clones during the implementation of the CHG-bathing protocol (11, 19), and some Asian studies have revealed a high frequency of qacA and qacB among the ST239, ST5, and ST241 clones (12, 20). Meanwhile, U.S. studies did not demonstrate this clonal predominance (10, 21). Our previous study, performed at a surgical ICU, revealed that most qacA- and qacB-positive MRSA isolates were identified as ST5-SCCmec II (69.2%) and ST239-SCCmce III (23.1%), which are common healthcare-associated MRSA strains in South Korea (22). This clonal association between the qacA and qacB genes and the ST5 clone was also demonstrated in this study. However, no association between qacA and qacB and ST239 was evident, because the number of ST239 MRSA BSIs was too low to be analyzed statistically.
In the present study, patients infected with qacA- and qacB-positive MRSA had common risk factors for health care-associated infections, including prior antibiotic use, central venous catheter in situ, ICU-acquired bacteremia, and nosocomial infection. These findings are consistent with previous studies revealing an association between the presence of qacA and qacB and health care exposure and provide evidence that the widespread use of CHG in hospital settings selects for genotypic chlorhexidine-tolerant strains that are able to survive in the presence of this antiseptic (14, 19, 23). In addition, as several previous studies have reported, we also found an association between chlorhexidine tolerance genes and resistance to non-β-lactam antibiotics and a higher chlorhexidine MIC (11, 14, 23). Although prior antibiotic use was independently associated with qacA- and qacB-positive MRSA in the present study, we could not determine whether there was antibiotic pressure on the selection of these microorganisms or whether this finding was simply a reflection of the coexistence of antimicrobial resistance genes on mobile genetic elements (24).
We also found that the presence of qacA and qacB genes in MRSA BSIs was independently associated with agr dysfunction, consistent with our previous study (22). Loss of agr function in S. aureus has been reported to have certain advantages in hospital environments, in that the strain may be associated with attenuated vancomycin activity, vancomycin heteroresistance, persistent bacteremia, and increased biofilm production (25, 26). Since the concentration of chlorhexidine used in practice (10,000 to 40,000 μg/ml) is much higher than the higher MICs (4 to 16 μg/ml) of some isolates in vitro, chlorhexidine tolerance in S. aureus is regarded as an insignificant clinical problem (11). However, if MRSA strains with agr dysfunction form a biofilm, chlorhexidine tolerance could matter, because bacteria within biofilms can be more resistant or tolerant to this antiseptic than their planktonic counterparts, as reported by Bonez et al. (27, 28). In the present study, we could not draw a conclusion about whether MRSA with agr dysfunction was independently associated with higher chlorhexidine MICs, and we could not determine the causal relationship between qacA and qacB genes and agr dysfunction in MRSA. However, our data appear to provide evidence supporting the selection of qacA- and qacB-positive MRSA strains in hospital environments under chlorhexidine exposure.
Our study had some limitations. First, considering the high proportion of ST5 MRSA isolates in this study, it is possible that the predictive factors of qacA and qacB genes are merely reflecting the characteristics of the ST5 phenotype itself. Second, since it was conducted on only MRSA isolates in a single institution, our findings should not be generalized to methicillin-susceptible strains or to other hospital settings. Third, we did not perform microbiological tests on MRSA isolates from paired surveillance cultures, so we could not evaluate whether patients colonized with qacA- and qacB-positive MRSA strains were more vulnerable to BSIs, especially to central venous BSIs, than those with qacA- and qacB-negative strains under the pressure of chlorhexidine. Fourth, caution should be exercised in interpreting the results of chlorhexidine MICs, because there was no internationally agreed methodology for the detection of reduced chlorhexidine susceptibility until now (11). Fifth, we did not measure RNA expression of qacA and qacB, and the presence of these genes does not guarantee phenotypic chlorhexidine tolerance or resistance (24).
In conclusion, the present study demonstrated that qacA- and qacB-positive isolates were not uncommon among MRSA BSI isolates collected over a 7-year period. The qacA- and qacB-positive MRSA isolates were independently associated with agr dysfunction, nosocomial infection, antibiotic use within the previous 3 months, and the ST5 MRSA strain. Our data support the hypothesis that the microbiological features of qacA- and qacB-positive MRSA isolates, such as increased resistance to non-β-lactam antibiotics, higher MIC of chlorhexidine, and agr dysfunction, provide these strains with an advantage when exposed to chlorhexidine in hospital settings (12, 16, 22).
MATERIALS AND METHODS
Study setting, patients, and MRSA isolates.
This study was conducted at Gyeongsang National University Hospital (GNUH), an 890-bed community-based tertiary hospital in Jinju, South Korea, between March 2010 and December 2016. Because an increase in the incidence of MRSA infection in ICUs was noted, a CHG bathing protocol was introduced at a surgical ICU beginning in December 2012 and at a medical ICU beginning in January 2015. Chlorhexidine-impregnated catheters have been used as one type of central venous catheter since 2009. The detailed infection control programs at our institution have been described previously (22).
In total, 365 patients with MRSA bacteremia were identified at GNUH during the 7-year study period. Of these, 246 (67.4%) nonduplicate BSI isolates were available for laboratory tests and were therefore included in this study. Clinical data were reviewed for the patients whose MRSA BSI isolates were available for the tests. We compared the clinical and microbiological characteristics of MRSA isolates between qacA- and qacB-positive and qacA- and qacB-negative MRSA bacteremia.
Clinical data and definitions.
The following data were retrospectively reviewed for this study: age, sex, mode of acquisition of infection, comorbidity, Charlson comorbidity score, hospitalization within the previous year, antibiotic use within the previous 3 months, central venous catheter in place during the bacteremia, ICU-acquired bacteremia, primary site of bacteremia, septic shock on bacteremia presentation, length of hospital stay after bacteremia, 30-day mortality, and in-hospital mortality. The modes of acquisition of infection were classified as follows (29). Community-onset (CO) infection was defined as an infection diagnosed within 48 h of hospitalization that did not satisfy the criteria for nosocomial infection. CO-HCA infection was considered if any of the following criteria were present: hospitalization for >48 h in the previous 12 months; hemodialysis, intravenous medication, or home wound care in the previous 3 months; or residence in a nursing home or long-term-care facility. Nosocomial infections were those in which the patient developed signs/symptoms of infection ≥72 h after hospital admission. ICU-acquired bacteremia was defined as a bacteremia infection ≥72 h after ICU admission.
Microbiological methods.
All S. aureus isolates were identified by standard methods. Antimicrobial susceptibility was identified using a Vitek-2 system (bioMérieux, Marcy l'Etoile, France), and the MICs of vancomycin were determined using the broth microdilution (BMD) method according to Clinical and Laboratory Standards Institute guidelines (30). The MICs of chlorhexidine were determined using a broth microdilution method and a 20% (wt/vol) CHG solution (Sigma-Aldrich, St. Louis, MO, USA); the final concentrations of the antiseptic ranged from 0.5 to 32 mg/liter (22). We used δ-hemolysin activity to determine agr functionality, as described elsewhere (31).
Detection of the qacA and qacB genes and smr gene was carried out by PCR using previously published primers (18). Three S. aureus strains (TS77, TPS162, and L20) were used as positive controls for qacA, qacB, and smr (Riken BRC, Ibaraki, Japan), respectively. Staphylococcal protein A (spa) typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing were conducted according to previously published methods (32–34).
Statistical analysis.
The χ2 test for trend was used to compare the frequency of qacA- and qacB-positive MRSA BSI isolates by year during the study period. Continuous and categorical variables were compared using the Mann-Whitney U test and Fisher’s exact test, respectively. When multivariable logistic regression was performed, the risk factors of qacA and qacB carriage included in the model were selected according to the following steps. First, the risk factors were chosen based on the P value being less than 0.15 in univariate analysis. Second, to identify the multicollinearity problem, the correlation between the risk factors was confirmed using Pearson or Spearman correlation analysis. Finally, a model with a stepwise method was selected for combinations of risk factors included in the multivariable analysis. In this case, to prevent overfitting problems due to insufficient qacA- and qacB-positive cases, the number of risk factors to be included in the final model was limited to fewer than six. Multivariable logistic regression and model selection were performed by SAS, version 9.4 (SAS Institute, Cary, NC, USA), software. All other analyses were performed using IBM SPSS software (ver. 20.0; IBM Corporation, Armonk, NY, USA). All tests were two tailed, and a P value of <0.05 was considered statistically significant. The GNUH Institutional Review Board approved this study.
Supplementary Material
ACKNOWLEDGMENTS
The pathogen resources for this study were provided by the Gyeongsang National University Hospital Branch of the National Culture Collection for Pathogens (GNUH-NCCP). This work was supported by the Biomedical Research Institute Fund from the Gyeongsang National University Hospital (GNUHBRIF-2017-0001). We have no conflicts of interest to declare.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02157-18.
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