Clinical impact of and microbiological risk factors for qacA/B positivity in ICU-acquired ST5-methicillin-resistant SCCmec type II Staphylococcus aureus bacteremia

Concern about resistance to chlorhexidine has increased due to the wide use of the latter. The impact of the qacA/B and smr chlorhexidine tolerance genes on the outcome of methicillin-resistant Staphylococcus aureus (MRSA) infections is unclear. We evaluated the prevalence and clinical impact of, and microbiological risk factors for, qacA/B tolerance in MRSA bacteremia. MRSA bacteremia that occurred more than two days after intensive care unit admission between January 2009 and December 2018 was identified from a prospective cohort of S. aureus bacteremia in a tertiary-care hospital from South Korea. A total of 183 MRSA blood isolates was identified, and the major genotype found was ST5-MRSA-II (87.4%). The prevalences of qacA/B and smr were 67.2% and 3.8%, respectively. qacA/B-positive isolates were predominantly ST5-MRSA-II (96.7% [119/123]), the dominant hospital clone. In a homogenous ST5-MRSA-II background, qacA/B positivity was independently associated with septic shock (aOR, 4.85), gentamicin resistance (aOR, 74.43), and non-t002 spa type (aOR, 74.12). qacA/B positivity was found to have decreased significantly in ST5-MRSA-II in association with a decline in qacA/B-positive t2460, despite the increasing use of chlorhexidine since 2010 (P < 0.001 for trend). Continuous surveillance of the qac genes, and molecular characterization of their plasmids, are needed to understand their role in MRSA epidemiology.

www.nature.com/scientificreports/ bathing is estimated to reduce MRSA colonization and bacteremia in the ICU by 41% and 36%, respectively 5 . While there is benefit from chlorhexidine bathing and nasal mupirocin application, concern persists about the potential emergence of increased resistance due to antiseptic exposure. The qacA and qacB genes encode multidrug efflux pumps associated with chlorhexidine tolerance 6,7 . Several studies have shown an increase in the proportion of these genes after the widespread use of chlorhexidine in hospitals [8][9][10] . In addition, the presence of the qacA or qacB (qacA/B) gene has been linked to resistance to gentamicin, clindamycin, and penicillin, as well as to a high vancomycin minimal inhibitory concentration (MIC) [11][12][13] . Previous studies have shown an association between qacA/B positivity and specific MRSA genotypes, including ST5-and ST239-MRSA 6,7,14 . ST5-MRSA is a major hospital MRSA clone in South Korea, while ST72-MRSA is a community-associated MRSA 2 . Because ST5-MRSA is associated with higher mortality and more accessory gene regulator (agr) dysfunctional than ST72-MRSA 15,16 , clinical and microbiological risk factors for qacA/B positivity might be affected by the characteristics of specific genotypes. Thus, this study aimed to evaluate the prevalence and clinical impact of, and microbiological risk factors for, qacA/B positivity in MRSA bacteremia in a homogenous genotype background after the implementation of chlorhexidine use in a tertiary hospital from South Korea.

Methods
Study population and design. A retrospective analysis of a prospective observational cohort of patients with S. aureus bacteremia was conducted at the Asan Medical Center, a 2700-bed tertiary care hospital with 118 adult ICU beds, in Seoul, South Korea. All consecutive adult patients (≥ 16 years old) who had MRSA bacteremia that developed more than two days after ICU admission between January 2009 and December 2018 were identified, and only patients whose MRSA isolates were collected and available for laboratory tests were included in the study 17 . Only the initial episode of MRSA bacteremia in any given patient was included. Recurrence of S. aureus bacteremia more than three months from the first bacteremia episode was regarded as a new episode and included in the analysis.
To evaluate the clinical impact of qacA/B positivity in a single genotypic background, multilocus sequence typing (MLST) of the MRSA isolates was performed. The clinical, microbiological, and genotypic characteristics of qacA/B-positive MRSA isolates, and risk factors for qacA/B positivity, were analyzed. Treatment outcome of bacteremia was evaluated based on 14-day, 30-day, and 12-week all-cause mortality. This study was approved by the Asan Medical Center Institutional Review Board (IRB number: 2008-0274). The requirement for written informed consent was waived by the Asan Medical Center Institutional Review Board, given the observational nature of the study. All methods were performed in accordance with the relevant guidelines and regulations.

Study definitions.
The prognosis for the underlying disease was classified according to the system of McCabe and Jackson as rapidly fatal (when death was expected within a few months), ultimately fatal (when death was expected within 4 years), and nonfatal (when life expectancy was > 4 years) 18 . Charlson's comorbidity index was used to measure comorbid burden 19 . The severity of bacteremia was measured by the Acute Physiology and Chronic Health Evaluation II (APACHE II) score and the Pitt bacteremia score 20  Microbiological data and determination of genotypes. All S. aureus isolates were identified by standard methods. Antimicrobial susceptibilities were determined with a MicroScan (Dade Behring, West Sacramento, CA, USA) and the standard criteria of the Clinical and Laboratory Standards Institute (CLSI) 23 . Methicillin resistance was confirmed by detection of the mecA gene by PCR. Vancomycin minimal inhibitory concentrations (MICs) were determined by the vancomycin E-test (AB Biodisk, Piscataway, NJ, USA) on Mueller-Hinton agar. Heterogeneous vancomycin-intermediate S. aureus (hVISA) was detected by population analysis profiling, as previously described 24 .
Chlorhexidine MIC was determined by the broth microdilution test described by CLSI 30 . Chlorhexidine digluconate 20% aqueous solution (Sigma-Aldrich, St. Louis, MO) was used as the starting material. The bacterial inocula (5 × 10 5 colony-forming unit/mL) were seeded to wells containing chlorhexidine from 0.125 to 128 mg/L and incubated at 35 ℃ for 18 h. S. aureus ATCC 29213 was used as a quality control strain in susceptibility testing. Mupirocin resistance (low and high level) was detected by the disk diffusion method 31 , and high-level mupirocin resistance was confirmed by PCR detection of the mupA gene. The qacA/ qacB, smr, and mupA genes were detected by PCR using primers described elsewhere 32  www.nature.com/scientificreports/ Statistical analysis. We analyzed categorical variables using the chi-square or Fisher's exact test, and continuous variables using Student's t-test or the Mann-Whitney U test. A univariate analysis of the risk factors for qacA/B-positive MRSA was performed. All variables with P values < 0.1 in the univariate analysis were included in a multiple logistic regression model to identify independent risk factors. These variables were examined for correlations before inclusion in the multivariate analysis. Categorical variables were analyzed by the chi-square test, and continuous variables over time were analyzed by linear regression for trend. All tests of significance were two-tailed, and P < 0.05 was considered statistically significant. All statistical analyses were performed with SPSS, version 24.0 K for Windows (SPSS Inc, Chicago, IL, USA) and MedCalc software (Mariakerke, Belgium).

Results
Prevalence of qacA/B in MRSA blood isolates. During the study period, 238 episodes of ICU-acquired MRSA bacteremia were identified. When the 183 episodes with available MRSA isolates were analyzed, the two major genotypes were ST5-MRSA-II-agr group II (87.4%), the hospital MRSA clone, and ST72-MRSA-IV-agr group I (7.1%), the community-associated MRSA clone in South Korea. Major spa types in ST5-MRSA-II were t2460 (59.4%), t9353 (14.4%), and t002 (  Microbiological and genotypic characteristics of the qacA/B-positive ST5-MRSA-II isolates. The most common spa type of ST5-MRSA-II was t2460 (59.4%), followed by t9353 (14.4%) and t002 (8.1%). As shown in Table 2 The proportion of qacA/B-positive isolates decreased from 88.1 to 50% over time (P < 0.001 for trend) (Fig. 1a), mainly due to the lowered qacA/B-positive rate in t2460 (P < 0.001 for trend) (Fig. 1b).  Tables 1 and 2 were included in a logistic regression model to identify independent risk factors for qacA/B-positive MRSA. Because the t9353 spa type and fusidic acid resistance were highly correlated with gentamicin resistance, we retained gentamicin resistance only. Also, we included length of ICU stay before bacteremia rather than length of hospital stay before bacteremia because the former is a more clinically meaningful variable in ICU-acquired MRSA bacteremia. Thus, included in the multivariate regression modeling were length of ICU stay before bacteremia (per 10 days), recent surgery, septic shock, gentamicin resistance, non-t002 spa type, and agr dysfunction. The independent risk factors associated with qacA/B positivity were found to be septic shock (adjusted odds ratio [

Discussion
In this study we found that the chlorhexidine tolerance genes qacA/B and smr were mainly present in the specific hospital MRSA clone, ST5-MRSA-II, after the implementation of chlorhexidine use in a tertiary hospital from South Korea. The qacA/B gene was independently associated with septic shock, gentamicin resistance, and non-t002 spa type (especially spa type t9353) in a homogenous ST5-MRSA-II background. This implies that the qacA/B coexists well with the t9353 spa type but not the t002 spa type. During the study period, qacA/B positivity decreased significantly in ST5-MRSA-II mainly due to a reduction in qacA/B-positive t2460 isolates despite the increasing use of chlorhexidine in this hospital since 2010.
Previous clinical and microbiological studies of qacA/B and smr had been performed on strains with heterogeneous genotypic and clinical backgrounds, including both community and healthcare S. aureus strains 6,13,33,34 . Therefore, the proportion of qacA/B-positive isolates in the present study, which included only ICU-acquired ST5-MRSA-II bacteremia, was higher (74.4%) than in previous studies. Interestingly, the proportion of www.nature.com/scientificreports/ smr-positive isolates was very low (3.8%), in contrast with previous studies 13,33,34 . In another study conducted in South Korea 6 , the proportion of smr-positive MRSA was also low (2.8%), and qacA/B positivity was closely associated with ST5-MRSA, the dominant and generally agr dysfunctional hospital clone 16 . ST72-MRSA, a community-associated MRSA clone in South Korea that is usually agr functional 16 , was also significantly associated with qacA/B negativity in that study. Therefore, factors that were found to be independently associated with qacA/B positivity in that study, such as agr dysfunction, nosocomial infection, and previous antibiotic use, could have been reflections of the characteristics of the specific MLST, ST5-MRSA, because all MRSA genotypes were www.nature.com/scientificreports/ included. The clonal association of qacA/B with hospital MRSA clones such as ST239, ST5, and ST241 have been found in Asia 14,35 . Therefore, the clonal association between qacA/B and ST5-MRSA-II isolates in present study could be influenced by the dominant regional hospital MRSA clone in South Korea.
In contrast to other studies that showed the increased proportion of qacA/B positivity after the widespread use of chlorhexidine in hospitals [8][9][10] , the present study revealed that qacA/B positivity in ST5-MRSA-II decreased over time. This finding could be influenced by the decreased proportion of qacA/B-positive rate in t2460, which was main spa type in ST5-MRSA-II.
Several studies have shown that healthcare exposure and underlying medical condition are independently associated with qacA/B positivity in S. aureus isolates 6,13,34 . In the present study, we found that septic shock, gentamicin resistance, and fusidic acid resistance were also closely associated with qacA/B positivity in nosocomial ST5-MRSA-II blood isolates in a homogenous MRSA genotypic background. The qacA/B genes are usually carried by large plasmids that encode efflux pumps, and some of these plasmids contain other antibiotic resistance genes 11,12,36 . This fact should influence the likelihood of co-resistance to other antimicrobials such as gentamicin and fusidic acid. In addition, the presence of virulence genes on certain qac plasmids could affect the severity of www.nature.com/scientificreports/  www.nature.com/scientificreports/ the illness of patients with qacA/B-positive MRSA bacteremia 37 . Further detailed analysis of our qac plasmids would clarify these associations. Interestingly, the non-t002 spa type was significantly associated with qacA/B positivity in ST5-MRSA-II isolates. t2460 and t9353 are the major spa types of MRSA in South Korea 6,38 . While a previous study showed that t2460 was associated with the qacA/B genes in MRSA strains as a whole 6 , we found that only t9353 was strongly associated with qacA/B positivity in a homogenous ST5-MRSA-II background. As shown in Fig. 1b, there has been no outbreak of qacA/B-positive t9353, and the proportion of qacA/B-positivity in t2460 strains has decreased over time (P < 0.001 for trend). Potential hypotheses for such findings are that t9353 spa type might maintain the qacA/B gene efficiently, and t2460 may have a tendency to lose a plasmid carrying qacA/B. This study has several limitations. First, it was conducted in a single tertiary-care hospital and included exclusively MRSA bacteremia occurring in ICUs. Hence, further studies are needed to confirm our results in other hospital settings. Second, because almost all the patients had a recent history of antibiotic use, it was difficult to determine whether antibiotic use exerts selective pressure for chlorhexidine tolerance. Third, despite the proportion of available MRSA blood isolates tested was high (183/238, 77%), these results may not reflect the true prevalence of the genotypes found. Therefore, caution is needed in generalizing the results to other situations.
In conclusion, the qacA/B and smr antiseptic tolerance genes were mainly encountered in ST5-MRSA-II, the dominant hospital MRSA clone, after the implementation of chlorhexidine use in a tertiary hospital from South Korea since 2010. Septic shock, gentamicin resistance, and non-t002 spa type were independent predictors of the presence of qacA/B in ST5-MRSA-II blood isolates. Continuous surveillance for the qac genes and molecular characterization of the corresponding plasmids are needed to understand the role of these genes in MRSA epidemiology.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files. The genotypic data have been deposited to the figshare database at https:// doi. org/ 10. 6084/ m9. figsh are. 19114 976. v2.