Cryo-EM structure of antibacterial efflux transporter QacA from Staphylococcus aureus reveals a novel extracellular loop with allosteric role.

Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major facilitator superfamily antiporter, mediates proton-coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo-EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate-free QacA, complexed to two single-domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward-open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.

Efflux Pump (QacA, QacB, and QacC) and ß-Lactamase Inhibitors? An Evaluation of 1,8-Naphthyridines against Staphylococcus aureus Strains.

The bacterial species Staphylococcus aureus presents a variety of resistance mechanisms, among which the expression of ß-lactamases and efflux pumps stand out for providing a significant degree of resistance to clinically relevant antibiotics. The 1,8-naphthyridines are nitrogen heterocycles with a broad spectrum of biological activities and, as such, are promising research targets. However, the potential roles of these compounds on bacterial resistance management remain to be better investigated. Therefore, the present study evaluated the antibacterial activity of 1,8-naphthyridine sulfonamides, addressing their ability to act as inhibitors of ß-lactamases and efflux pump (QacA/B and QacC) against the strains SA-K4414 and SA-K4100 of S. aureus. All substances were prepared at an initial concentration of 1024 µg/mL, and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Subsequently, their effects on ß-lactamase- and efflux pump-mediated antibiotic resistance was evaluated from the reduction of the MIC of ethidium bromide (EtBr) and ß-lactam antibiotics, respectively. The 1,8-naphthyridines did not present direct antibacterial activity against the strains SA-K4414 and SA-K4100 of S. aureus. On the other hand, when associated with antibiotics against both strains, the compounds reduced the MIC of EtBr and ß-lactam antibiotics, suggesting that they may act by inhibiting ß-lactamases and efflux pumps such as QacC and QacA/B. However, further research is required to elucidate the molecular mechanisms underlying these observed effects.

Clinical and Molecular Characteristics of qacA- and qacB-Positive Methicillin-Resistant Staphylococcus aureus Causing Bloodstream Infections.

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-ß-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.

A Novel, Widespread qacA Allele Results in Reduced Chlorhexidine Susceptibility in Staphylococcus epidermidis.

Chlorhexidine gluconate (CHG) is a topical antiseptic widely used in health care settings. In Staphylococcus spp., the pump QacA effluxes CHG, while the closely related QacB cannot due to a single amino acid substitution. We characterized 1,050 cutaneous Staphylococcus isolates obtained from 173 pediatric oncology patients enrolled in a multicenter CHG bathing trial. CHG susceptibility testing revealed that 63 (6%) of these isolates had elevated CHG MICs (≥4 µg/ml). Screening of all 1,050 isolates for the qacA/B gene (the same qac gene with A or B allele) by restriction fragment length polymorphism (RFLP) yielded 56 isolates with a novel qacA/B RFLP pattern, qacA/B273 The CHG MIC was significantly higher for qacA/B273 -positive isolates (MIC50, 4 µg/ml; MIC range, 0.5 to 4 µg/ml) than for other qac groups: qacA-positive isolates (n = 559; MIC50, 1 µg/ml; MIC range, 0.5 to 4 µg/ml), qacB-positive isolates (n = 17; MIC50, 1 µg/ml; MIC range, 0.25 to 2 µg/ml), and qacA/B-negative isolates (n = 418, MIC50, 1 µg/ml; MIC range, 0.125 to 2 µg/ml) (P = 0.001). A high proportion of the qacA/B273 -positive isolates also displayed methicillin resistance (96.4%) compared to the other qac groups (24.9 to 61.7%) (P = 0.001). Whole-genome sequencing revealed that qacA/B273 -positive isolates encoded a variant of QacA with 2 amino acid substitutions. This new allele, named qacA4, was carried on the novel plasmid pAQZ1. The qacA4-carrying isolates belonged to the highly resistant Staphylococcus epidermidis sequence type 2 clone. By searching available sequence data sets, we identified 39 additional qacA4-carrying S. epidermidis strains from 5 countries. Curing an isolate of qacA4 resulted in a 4-fold decrease in the CHG MIC, confirming the role of qacA4 in the elevated CHG MIC. Our results highlight the importance of further studying qacA4 and its functional role in clinical staphylococci.

Decreased susceptibility to chlorhexidine and distribution of qacA/B genes among coagulase-negative Staphylococcus clinical samples.

BACKGROUND: Healthcare-associated infection (HAI) is a major public health problem. As a form of prevention and control, preparations of chlorhexidine are used extensively; however, the reduction of susceptibility to chlorhexidine has been reported. The aim of this study was to investigate the susceptibility to chlorhexidine and the distribution of the qacA/B genes in 211 clinical isolates of coagulase-negative Staphylococci (CoNS). METHODS: CoNS were identified by conventional biochemical tests. Antimicrobial susceptibility was tested by disk-diffusion. Minimum inhibitory concentration (MIC) of chlorhexidine was determined by agar dilution test; detection of the qacA/B and mecA genes were evaluated by PCR. RESULTS: The most frequently isolated species were S. epidermidis, S. hominis hominis, S. auricularis, and S. haemolyticus, respectively. The strains presented a multidrug resistance profile of 87%, including methicillin resistance. Reduced susceptibility to chlorhexidine was observed in 31%. The qacA/B genes were detected in samples resistant (32/32) and susceptible (17/32) to chlorhexidine. The vast majority (94%) of the samples with reduced susceptibility to chlorhexidine were multidrug resistant. CONCLUSIONS: Our results show that qacA/B genes are not restricted to strains expressing chlorhexidine resistance. Further studies are needed to understand how the expression of these genes occurs.

Impact of Health Care Exposure on Genotypic Antiseptic Tolerance in Staphylococcus aureus Infections in a Pediatric Population.

Staphylococcus aureus possessing either the smr gene or the qacA/B genes is associated with decreased susceptibility to chlorhexidine gluconate (CHG) and other antiseptics. Previous studies of antiseptic-tolerant staphylococci have focused largely on high-risk populations, and the exact role of health care exposure in the acquisition of these organisms is unclear. We sought to describe the risk factors and features of infection caused by antiseptic-tolerant S. aureus in a general pediatric population. Isolates were selected from an ongoing S. aureus surveillance study. Every third sequential isolate in the year 2014 was selected for inclusion. All isolates underwent PCR for the genes qacA/B and smr Medical records were reviewed. Five hundred six isolates were included in the study, with 377 (74.3%) being community acquired. One hundred (19.8%) isolates were smr positive and 79 (15.6%) qacA/B positive. In univariable analyses, the presence of either gene was associated with underlying medical conditions, nosocomial acquisition, recent hospitalization, central venous lines, and CHG exposure. In multivariable analyses, only differences between patients with chronic medical conditions (odds ratio [OR] = 1.72; 95% confidence interval [CI], 1.22 to 2.64) and nosocomial acquisition (OR = 2.48; 95% CI, 1.16 to 8.17) remained statistically significant. Among patients without risk factors, 27.9% had infection with an antiseptic-tolerant isolate. smr- or qacA/B-positive S. aureus isolates are common in children and are independently associated with nosocomial acquisition and underlying medical conditions. These findings imply a role for the health care environment in acquisition of these organisms. However, genotypic antiseptic tolerance was seen in >25% of healthy children with an S. aureus infection, indicating that these organism are prevalent in the community as well.

Characterization of Chlorhexidine Resistance Among Clinical Isolates of Coagulase-Negative Staphylococcus Species in a Tertiary Care Center, Chennai.

Background Coagulase-negative Staphylococci (CoNS) are potential pathogens and are often associated with healthcare-associated infections (HAIs). Chlorhexidine (CHX) is the most widely used antiseptic to reduce colonization and infection by all Staphylococci, including CoNS. Resistance to CHX among CoNS has been observed over the past few years, consequent to its widespread use. Phenotypic tolerance or reduced susceptibility to CHX is conferred by plasmid-mediated qac group of genes, mainly qacA/B and smr, which cause activation of efflux pumps over the bacterial cell wall. This study aims to characterize the phenotypic and genotypic resistance exhibited by CoNS species against CHX. Methods After ethical approval, 148 consecutive, non-repetitive isolates of clinically significant CoNS species of hospitalized patients, isolated from blood samples and exudative specimens, were included in the study. Speciation was performed by conventional biochemical identification and automated methods. Antimicrobial susceptibility testing was performed by disc diffusion technique and for vancomycin by minimum inhibitory concentration (MIC) determination, as per Clinical Laboratory Standards Institute (CLSI) M-100 2023 guidelines. Methicillin resistance was detected using a cefoxitin disc. MIC for CHX was performed by agar dilution method; reduced susceptibility was considered when MIC to CHX ≥4 µg/mL. The simplex polymerase chain reaction (PCR) was carried out with suitable controls to detect qacA/B and smr. Statistical analysis was conducted to determine the association of qacA/B and smr genes with MIC of CHX in the study isolates. Results Fifteen different species of CoNS were obtained from clinical samples. A high percentage of resistance was observed against various classes of antibiotics. Methicillin resistance was observed in 69.6% (103/148) of isolates. Of 148 CoNS, 52.7% (78/148) of isolates exhibited reduced susceptibility to CHX with an MIC ≥4 µg/mL. These isolates exhibited a higher percentage of methicillin resistance (75.6%, 59/78). By PCR, 34.5% (51/148) of isolates carried either or both genes. Gene qacA/B was solely detected in 27.02% (40/148) of isolates, of which 14 were CHX-tolerant and the remaining 26 were CHX-susceptible. Gene smr was solely detected in 4.1% (6/148) of isolates comprising three isolates each in CHX-tolerant and susceptible categories. There were 3.4% (5/148) of isolates that harbored both genes, of which only one isolate was CHX-susceptible, while the other four were CHX-tolerant. A proportion of isolates that were phenotypically tolerant to CHX did not carry either or both genes. A significant statistical association was found between reduced susceptibility to CHX and the presence of antiseptic resistance genes in the study isolates (p-value=0.033942). Conclusion To our knowledge, this is the first study from South India to investigate CHX resistance among CoNS using phenotypic and genotypic methods. The rise of antiseptic resistance among CoNS is an emerging threat to current infection control practices. The presence of qacA/B and smr genes, especially in CHX susceptible isolates, is concerning since these resistance genes are located on transferable plasmids, and the isolates can develop resistance eventually upon exposure to CHX.

Association of qacA/B and smr Carriage with Staphylococcus aureus Survival following Exposure to Antiseptics in an Ex Vivo Venous Catheter Disinfection Model.

Many health care centers have reported an association between Staphylococcus aureus isolates bearing efflux pump genes and an elevated MIC/minimal bactericidal concentration (MBC) to chlorhexidine gluconate (CHG) and other antiseptics. The significance of these organisms is uncertain, given that their MIC/MBC is typically far lower than the CHG concentration in most commercial preparations. We sought to evaluate the relationship between carriage of the efflux pump genes qacA/B and smr in S. aureus and the efficacy of CHG-based antisepsis in a venous catheter disinfection model. S. aureus isolates with and without smr and/or qacA/B were utilized. The CHG MICs were determined. Venous catheter hubs were inoculated and exposed to CHG, isopropanol, and CHG-isopropanol combinations. The microbiocidal effect was calculated as the percent reduction in CFU following exposure to the antiseptic relative to the control. The qacA/B- and smr-positive isolates had modest elevations in the CHG MIC90 compared to the qacA/B- and smr-negative isolates (0.125 mcg/ml vs. 0.06 mcg/ml, respectively). However, the CHG microbiocidal effect was significantly lower for qacA/B- and/or smr-positive strains than for susceptible isolates, even when the isolates were exposed to CHG concentrations up to 400 µg/mL (0.04%); this finding was most notable for isolates bearing both qacA/B and smr (89.3% versus 99.9% for the qacA/B- and smr-negative isolates; P = 0.04). Reductions in the median microbiocidal effect were also observed when these qacA/B- and smr-positive isolates were exposed to a solution of 400 µg/mL (0.04%) CHG and 70% isopropanol (89.5% versus 100% for the qacA/B- and smr-negative isolates; P = 0.002). qacA/B- and smr-positive S. aureus isolates have a survival advantage in the presence of CHG concentrations exceeding the MIC. These data suggest that traditional MIC/MBC testing may underestimate the ability of these organisms to resist the effects of CHG. IMPORTANCE Antiseptic agents, including chlorhexidine gluconate (CHG), are commonly utilized in the health care environment to reduce rates of health care-associated infections. A number of efflux pump genes, including smr and qacA/B, have been reported in Staphylococcus aureus isolates that are associated with higher MICs and minimum bactericidal concentrations (MBCs) to CHG. Several health care centers have reported an increase in the prevalence of these S. aureus strains following an escalation of CHG use in the hospital environment. The clinical significance of these organisms, however, is uncertain, given that the CHG MIC/MBC is far below the concentration in commercial preparations. We present the results of a novel surface disinfection assay utilizing venous catheter hubs. We found that qacA/B-positive and smr-positive S. aureus isolates resist killing by CHG at concentrations far exceeding the MIC/MBC in our model. These findings highlight that traditional MIC/MBC testing is insufficient to evaluate susceptibility to antimicrobials acting on medical devices.

Distribution of chlorhexidine resistance genes among Staphylococcus aureus clinical isolates: the challenge of antiseptic resistance.

Introduction: Chlorhexidine is an antiseptic agent which is extensively used to prevent nosocomial infections; however, this could result in reduction of its susceptibility. The aim of this work was to determine chlorhexidine susceptibility among Staphylococcus aureus isolates and to detect qacA/B and smr antiseptic resistance genes among these isolates. Furthermore, we aimed to identify possible risk factors for the reduction of chlorhexidine susceptibility among S. aureus isolates. Methods: Various clinical samples were collected from patients with evidence of S. aureus infection. Antimicrobial susceptibilities of identified S. aureus isolates were determined by disk diffusion method. Resistance to methicillin was identified by cefoxitin disk diffusion test besides mecA gene detection by PCR. Chlorhexidine minimum inhibitory concentration (MIC) values were measured by broth microdilution method while qacA/B and smr resistance genes were detected by multiplex PCR. Results: A total percentage of 25.9% of S. aureus isolates showed reduced susceptibility to chlorhexidine. Methicillin resistant S. aureus (MRSA) had a reported percentage of 39.5%, which was significantly higher than the 11.3% reported for methicillin susceptible S. aureus (MSSA), p<0.001. S. aureus isolates were found to harbor qacA/B and smr genes at 23.2% and 7.7% respectively. Risk factors for reduced susceptibility to chlorhexidine included; ICU setting (OR=2.02, 95%CI: 0.3-1.6), prolonged ICU stay (OR=1.7, 95%CI: 0.4-1.1), presence of central vascular catheter (OR=2.3, 95%CI: 0.2-1.9), mechanical ventilation (OR=1.88, 95%CI: 0.4-1.7) and acquisition of qacA/B (OR=15.7, 95%CI: 3.4-12.1) or smr gene (OR=15.7, 95%CI: 3.4-12.1). Conclusions: Our work highlighted the current challenge of antiseptic resistance in our locality. The frequencies of qacA/B and smr genes were significantly higher among MRSA than MSSA isolates. About two thirds of chlorhexidine tolerant isolates displayed an MDR profile. To maintain chlorhexidine efficiency, biocidal stewardship program and ongoing surveillance are essential.

Distribution of Biocide Resistance Genes and Association with Clonal Complex Genotypes in Staphylococcus aureus Isolated from School-Age Children in Guangzhou.

Purpose: Chlorhexidine and mupirocin are often prescribed to children in affected communities to prevent colonization and transmission of Staphylococcus aureus, but this has led to an increasing rate of biocide resistance. In this study, we aimed to determine the distribution of biocide resistance genes among S. aureus isolates from school-age children in Guangzhou, investigate chlorhexidine gluconate and mupirocin susceptibility and clonal complex (CC) genotypes in strains carrying biocide-resistance genes, and further explore the role of biofilms in this resistance. Patients and Methods: Antibiotic resistance and multilocus sequence genotyping were performed on 722 S. aureus isolates from previous study. The distribution of nine biocide genes (qacA/B, mupA, mepA, sepA, norA, lmrS, smr, mupB, qacG) was determined by PCR. Isolates carrying qacA/B or mupA genes were further tested for susceptibility to chlorhexidine gluconate (CHG) and mupirocin and biofilm formation abilities. Results: The most prevalent of the nine biocide resistance genes were mepA (95.57%), followed by norA (78.81%), lmrS (77.01%), and sepA (58.17%). The qacG gene was not detected. Distribution of sepA was significantly decreased in CC30 and CC45 genotypes, and presence of sepA was associated with resistance to antibiotics such as CLI, ERY, TCY, SXT, CIP, and LVX. In addition, 64 (94.1%, n=68) qacA/B+ isolates showed CHG resistance, 12 (100.0%, n=12) mupA+ isolates were mupirocin resistant, and 4 (80%, n=5) and 5 (100%, n=5) qacA/B+mupA+ isolates were CHG and mupirocin resistant, respectively. Of these 85 isolates, 98.8% (n=84) had different degrees of biofilm-forming abilities, which were positively associated with CHG and mupirocin resistance. Conclusion: The distribution of biocide resistance genes was associated with special CCs. The qacA/B and mupA genes are highly associated with resistance to CHG and mupirocin, and biofilm formation was found to contribute to this biocide resistance.

Distribution of mecA and qacA/B genes among coagulase negative staphylococci isolated from central venous catheters of intensive care unit patients.

PURPOSE: This study was designed to detect the prevalence of antibiotic and antiseptic resistance genes, mecA and qacA/B in coagulase negative Staphylococcus (CoNS) species isolated from intensive care unit patients with catheter related blood stream infections (CRBSI) or colonized central venous catheters (CVC). METHODS: Consecutive CoNS isolates from ICU patients with CRBSI or colonized central venous catheters were speciated and antibiotic susceptibilities were determined. The mecA and qacA/B genes were detected by polymerase chain reaction. RESULTS: Eighty-two CoNS isolates from ICU patients with CRBSI (n â€‹= â€‹8) or colonized CVC (n â€‹= â€‹74) were included. The mecA gene was detected in 62 CoNS isolates (76%). The commonest species isolated was S. haemolyticus (n â€‹= â€‹34; 41%) and 30 of these possessed mecA which was significantly higher compared to other CoNS species (p â€‹= â€‹0.036). The qacA/B gene was detected in 13 (16%) isolates. Eleven (13%) CoNS had both genes. A significant association was seen with the presence of mecA and resistance to cloxacillin (p â€‹< â€‹0.001) and erythromycin (p â€‹= â€‹0.046). Presence of qacA/B (p â€‹= â€‹0.007) or both mecA and qacA/B (p â€‹= â€‹0.014) was associated with a higher resistance to clindamycin. CONCLUSION: A considerably high prevalence of mecA and qacA/B genes as well as co-existence of both genes is noted among the CoNS isolated from ICU patients. This indicates the need of taking prompt actions in hospital acquired infection prevention including continuous surveillance.

Advancements in the Development of Non-Nitrogen-Based Amphiphilic Antiseptics to Overcome Pathogenic Bacterial Resistance.

The prevalence of quaternary ammonium compounds (QACs) as common disinfecting agents for the past century has led bacteria to develop resistance to such compounds. Given the alarming increase in resistant strains, new strategies are required to combat this rise in resistance. Recent efforts to probe and combat bacterial resistance have focused on studies of multiQACs. Relatively unexplored, however, have been changes to the primary atom bearing positive charge in these antiseptics. Here we review the current state of the field of both phosphonium and sulfonium amphiphilic antiseptics, both of which hold promise as novel means to address bacterial resistance.

Differentiation of qacA and qacB using high-resolution melt curve analysis, and both qacA and qacB but not qacC or norA types increase chlorhexidine minimal inhibitory concentrations in Staphylococcus aureus isolates.

BACKGROUND: Chlorhexidine is one of the most essential ingredients in infection control applications. Except qacA, the effects of other various efflux-medicated biocide genes (including qacB, qacC, qacEΔ1, qacH or norA) on biguanides resistance are still controversial. In addition, most of the studies have discussed the effect of qacA/B on clinical S. aureus isolates but not that qacA or qacB individually. METHODS: In total, 254 methicillin-resistant Staphylococcus aureus (MRSA), selected 30 methicillin-susceptible S. aureus (MSSA) clinical isolates from different patients during 2014-2015 and 15 S. aureus quality control strains (including Mu3 and Mu50) were included in the study. Various biocide genes, including qacA/B, qacC, qacH, qacEΔ1, and different types of norA, were determined through conventional PCR. S. aureus isolates with qacA/B (+) were analyzed using high-resolution melting curve (HRM) to differentiate qacA from qacB. The chlorhexidine MIC was determined using the agar dilution method. Univariate and multivariate statistics were analyzed to see which biocide resistant genes had effects on chlorhexidine MIC. RESULTS: Results of all HRM analyses (n = 22) were consistent with those of Sanger sequencing for differentiation of qacA from qacB. None of the isolates harbored qacH and only one MRSA harbored qacEΔ1. The harboring rate of qacA, qacB, and qacC among MRSA/MSSA isolates was 7.1% (n = 18)/0%, 38.2% (n = 97)/0%, and 7.5% (n = 19)/3.3% (n = 1), respectively. The most type of norA was norAI (n = 158), followed by norAIII (n = 87) and norAII (n = 9) among MRSA isolates. Based on the results of multivariate logistic regression analyses, only qacA and qacB would increase chlorhexidine MIC from ≤ 1 ug/ml to ≥ 2 ug/ml in MRSA isolates (P < 0.001) but not qacC or norA types (P=0.976 and 0.633 or 0.933, respectively). In addition, only qacA but not qacB was contributed to elevate chlorhexidine from ≤ 1 ug/ml to 4 ug/ml in MRSA isolates (P < 0.001 and 0.017, respectively). CONCLUSION: HRM analysis can be a great method to differentiate qacA from qacB. The biocide gene with the most effect on chlorhexidine MIC in S. aureus isolates was qacA, followed by qacB, but qacC and different types of norA did not have any effect on chlorhexidine susceptibility. Further investigation on the influence of qacB, qacC and types of norA on chlorhexidine susceptibility is necessary.

Decreased Prevalence of qacA-Positive Methicillin-Resistant Staphylococcus aureus in Hospitalized Patients in Tokyo, Japan.

Presence of methicillin-resistant Staphylococcus aureus (MRSA) strains carrying plasmid-borne multidrug efflux pump-encoding gene, qacA/B, is a serious issue for infection control in hospitals, because they can survive hand hygiene. The qacA/B genes are divided into five subtypes: qacA, qacBI, qacBII, qacBIII, and qacBIV. The aim of this study was to investigate the prevalence and risk factors of hospitalized patients infected by respective qacA/B-positive MRSA strains between 2010 and 2016 in Tokyo, Japan. Of the 600 total MRSA strains observed, the qacA/B-positive strains constituted 19.8% (199 isolates), of which 56.8% (113 isolates), 28.6% (57 isolates), and 14.6% (29 isolates) were classified as qacA, qacBIII, and qacBII-positive strains, respectively. The prevalence of qacA-positive MRSA strains significantly decreased from July 2010 to June 2011 (34.0%) to July 2015 to May 2016 (5.3%) (p < 0.05). When staphylococcal cassette chromosome (SCC)mec types of the respective qacA/B-positive strains were determined, 81.4% of the qacA-positive strains were classified into SCCmec type II, which has recently been decreasing in hospital-acquired MRSA in Japan. Risk factor analysis showed that there were no specific clinical departments associated with the presence of qacA-positive strains. Our findings suggest that change in the MRSA clonal lineages impact to decrease the prevalence of qacA-positive strains in Japanese hospitals.

Fast-acting bactericidal activity of olanexidine gluconate against qacA/B-positive methicillin-resistant Staphylococcus aureus.

The qacA/B gene is one of the major determinants of resistance to antiseptics in methicillin-resistant Staphylococcus aureus (MRSA). Here, we compared the fast-acting bactericidal activity of skin antiseptics, including olanexidine gluconate (OLG), a new biguanide antiseptic agent introduced in Japan, against clinical qacA/B-positive MRSA strains by determination of minimum bactericidal concentration and time-kill assay. Our findings provide, for the first time, data indicating that the fast-acting bactericidal activity of OLG against qacA/B-positive MRSA is higher than that of chlorhexidine gluconate, even though both are biguanide antiseptics.

A novel multidrug-resistant PVL-negative CC1-MRSA-IV clone emerging in Ireland and Germany likely originated in South-Eastern Europe.

This study investigated the recent emergence of multidrug-resistant Panton-Valentine leukocidin (PVL)-negative CC1-MRSA-IV in Ireland and Germany. Ten CC1-MSSA and 139 CC1-MRSA isolates recovered in Ireland between 2004 and 2017 were investigated. These were compared to 21 German CC1-MRSA, 10 Romanian CC1-MSSA, five Romanian CC1-MRSA and two UAE CC1-MRSA, which were selected from an extensive global database, based on similar DNA microarray profiles to the Irish isolates. All isolates subsequently underwent whole-genome sequencing, core-genome single nucleotide polymorphism (cgSNP) analysis and enhanced SCCmec subtyping. Two PVL-negative clades (A and B1) were identified among four main clades. Clade A included 20 German isolates, 119 Irish isolates, and all Romanian MRSA and MSSA isolates, the latter of which differed from clade A MRSA by 47-130 cgSNPs. Eighty-six Irish clade A isolates formed a tight subclade (A1) exhibiting 0-49 pairwise cgSNPs, 80 of which harboured a 46 kb conjugative plasmid carrying both ileS2, encoding high-level mupirocin resistance, and qacA, encoding chlorhexidine resistance. The resistance genes aadE, aphA3 and sat were detected in all clade A MRSA and the majority (8/10) of clade A MSSA isolates. None of the clade A isolates harboured any enterotoxin genes other than seh, which is universally present in CC1. Clade B1 included the remaining German isolate, 17 Irish isolates and the two UAE isolates, all of which corresponded to the Western Australia MRSA-1 (WA MRSA-1) clone based on genotypic characteristics. MRSA within clades A and B1 differed by 188 cgSNPs and clade-specific SCCmec characteristics were identified, indicating independent acquisition of the SCCmec element. This study demonstrated the existence of a European PVL-negative CC1-MRSA-IV clone that is distinctly different from the well-defined PVL-negative CC1-MRSA-IV clone, WA MRSA-1. Furthermore, cgSNP analysis revealed that this newly defined clone may have originated in South-Eastern Europe, before spreading to both Ireland and Germany.

Association between biofilm production, adhesion genes and drugs resistance in different SCCmec types of methicillin resistant Staphylococcus aureus strains isolated from several major hospitals of Iran.

OBJECTIVES: The ability of bacteria to produce biofilm and adhesion makes them more resistant to antibiotics. The current study aims to evaluate the biofilm formation by Staphylococcus aureus and to determine the prevalence of adhesion genes, also their correlation with drug resistance. MATERIALS AND METHODS: A total of 96 MRSA were collected from hospitals of Iran's western provinces during 2012 to 2013. The presence of ica A, B, C, D, clfA, cna, fnbA, mecA genes were determined by PCR technique. Biofilm formation was studied by microtiter plate assay, the clonal relations of the strains were examined by SCCmec and Spa typing. RESULTS: The results demonstrated that 96 % of isolates were biofilm producers. The distributions of biofilm formation between isolates were 4.2%, 54.2%, 35.4% as high, moderate and weak, respectivelly. The highest biofilm production was observed from blood culture isolates. All virulent genes icaA,B, C, D, clfA, cna, fnbA were observed in moderate and weak biofilm formation isolates. Among high biofilm formation isolates, icaB and cna genes were not seen. Statistical analysis showed that there was a significant correlation between ica, fnbA and the biofilm production, but there was not a significant correlation between the type of samples and drug resistance, spa type and SCCmec type with biofilm production (P>0.05). Frequency of All virulent genes in type III SCCmec was higher than other types. CONCLUSION: The majority of MRSA isolates were biofilm producers and blood isolates ranked as the great biofilm producer. In these isolates ica D and fnbA genes are correlated with biofilm production.

Development of a real-time assay to determine the frequency of qac genes in methicillin resistant Staphylococcus aureus.

PURPOSE: The emergence of antiseptic resistance and/or antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) may result in failure of decolonization treatments. Plasmid-encoded efflux pump genes qacA/B and qacC (smr) confer tolerance to chlorhexidine and quaternary ammonium compounds. The objective of this study was to develop and validate a multiplex real time-PCR assay for detection of antiseptic-resistance genes, apply the assay on 200 MRSA isolates and explore if carriage of these genes was associated with resistance to topical antibiotics. METHODOLOGY: A SYBR-Green based multiplex real time-PCR assay was developed to detect qacA/B, qacC, and mecA (internal control) simultaneously. The multiplex assay was compared against conventional single-plex PCR followed by agarose gel electrophoresis, using DNA from the first 73 MRSA isolates, followed by multiplex testing of the remaining 127 MRSA isolates. All 200 MRSA isolates were tested for susceptibility to mupirocin, retapamulin, neomycin, bacitracin and octenidine. The genetic diversity of the isolates was investigated by spa-typing. RESULTS: The concordance between multiplex and conventional PCR, in assignments of qacA/B and qacC status were 99%(72/73) and 100%(73/73) respectively. Among 200 MRSA isolates, 48(24%) and 44(23%) were found to harbour qacA/B and qacC genes, respectively. These isolates remained susceptible to many common decolonization agents, except mupirocin. The predominant spa-types were t020 and t1081 (41 and 32 isolates respectively). CONCLUSION: The real-time assay performed acceptably for the detection of qac genes. A high prevalence of antiseptic-resistance genes were detected in the MRSA isolates in our population and appeared to be associated with spa-type t1081.

Distribution of sasX, pvl, and qacA/B genes in epidemic methicillin-resistant Staphylococcus aureus strains isolated from East China.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen. Various virulence and antiseptic-resistant factors increase the pathogenicity of MRSA strains and allow for increased infection rates. PURPOSE: The purpose of this study was to investigate the prevalence and distribution of virulence-associated and antiseptic-resistant genes from epidemic MRSA strains isolated from East China. METHODS: A newly designed multiplex PCR assay was used to assess whether the virulence-associated genes sasX and pvl and the chlorhexidine tolerance gene qacA/B were present in 189 clinical isolates of MRSA. Multilocus sequence typing (MLST) and Staphylococcal protein A (spa) typing of these isolates were also performed. The frequency of these genes in isolates with epidemic sequence types (STs) was investigated. RESULTS: Twenty STs and 36 spa types with five epidemic clones (ST5-t311, ST59-t437, ST5-t002, ST239-t030, and ST239-t037) were identified. The prevalence of sasX, pvl, and qacA/B in all isolates was 5.8%, 10.1%, and 20.1%, respectively. The prevalences of these genes in isolates with ST5, ST59, ST239, and other ST genetic backgrounds were all significantly different (P<0.001). Isolates that had the highest frequency of sasX, pvl, or qacA/B were ST239 (33.3%), ST59 (28.9%), and ST5 (34.1%), respectively. The gene distribution pattern from all of the isolates showed that sasX-pvl-qacA/B+, sasX-pvl+qacA/B-, and sasX+pvl-qacA/B- were closely associated with epidemic clones ST5-t311, ST59-t437, and ST239-t037, respectively. CONCLUSION: There are significant differences in the prevalence of virulence-associated and antiseptic-resistant genes in epidemic MRSA strains. Using this information, more effective control and prevention strategies for nosocomial MRSA infections can be developed.

Distribution of biocide resistant genes and biocides susceptibility in multidrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii - A first report from the Kingdom of Saudi Arabia.

PURPOSES: The aim of this study was to determine the frequency of biocide resistant genes, qacA, qacE and cepA in multidrug resistant (MDR) bacteria: Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii and to correlate the presence or absence of resistant genes with biocides susceptibility. MATERIALS AND METHODS: The study included 44 MDR K. pneumoniae, P. aeruginosa and A. baumannii microorganisms. The bacteria were screened for the presence of biocide resistant genes by the polymerase chain reaction (PCR) method. The test organisms were isolated from various clinical specimens in the Qassim region, Saudi Arabia. The in vitro susceptibility tests of the three biocides (benzalkonium chloride, cetrimide and chlorhexidine gluconate) were studied against the test isolates by broth microdilution method following Clinical and Laboratory Standards Institute guidelines. RESULTS: With the distribution of biocide resistant genes in K. pneumoniae, all 9 isolates (100%) possessed cepA; 4 (44.4%) and 1 (11.1%) isolate contained qacA and qacE genes respectively. Among 24 isolates of A. baumannii tested, cepA, qacA and qacE genes were found in 54.2%, 16.7% and 33.3% of isolates respectively. Among 11 P. aeruginosa isolates, 63.6% contained cepA gene, 18.2% contained qacE genes, and none of the isolates harboured qacA gene. There was no significant correlation between presence or absence of biocide resistant genes and high MIC values of the test isolates (p≥0.2). CONCLUSION: Our observations imply that there was no significant correlation between presence or absence of biocide resistant genes and MICs observed in MDR K. pneumoniae, P. aeruginosa and A. baumannii. Further studies are required to find to confirm the trend of reduced susceptibility to biocides of problematic nosocomial pathogens.