Systematic Analysis of Efflux Pump-Mediated Antiseptic Resistance in Staphylococcus aureus Suggests a Need for Greater Antiseptic Stewardship

ABSTRACT Staphylococcus aureus-associated infections can be difficult to treat due to multidrug resistance. Thus, infection prevention is critical. Cationic antiseptics, such as chlorhexidine (CHX) and benzalkonium chloride (BKC), are liberally used in health care and community settings to prevent infection. However, increased administration of antiseptics has selected for S. aureus strains that show reduced susceptibilities to cationic antiseptics. This increased resistance has been associated with carriage of specific efflux pumps (QacA, QacC, and NorA). Since prior published studies focused on different strains and on strains carrying only a single efflux gene, the relative importance of these various systems to antiseptic resistance is difficult to ascertain. To overcome this, we engineered a collection of isogenic S. aureus strains that harbored norA, qacA, and qacC, individually or in combination. MIC assays showed that qacA was associated with increased resistance to CHX, cetrimide (CT), and BKC, qacC was associated with resistance to CT and BKC, and norA was necessary for basal-level resistance to the majority of tested antiseptics. When all three pumps were present in a single strain, an additive effect was observed in the MIC for CT. Transcriptional analysis revealed that expression of qacA and norA was significantly induced following exposure to BKC. Alarmingly, in a strain carrying qacA and norA, preexposure to BKC increased CHX tolerance. Overall, our results reveal increased antiseptic resistance in strains carrying multiple efflux pumps and indicate that preexposure to BKC, which is found in numerous daily-use products, can increase CHX tolerance. IMPORTANCE S. aureus remains a significant cause of disease within hospitals and communities. To reduce the burden of S. aureus infections, antiseptics are ubiquitously used in our daily lives. Furthermore, many antiseptic compounds are dual purpose and are found in household products. The increased abundance of antiseptic compounds has selected for S. aureus strains that carry efflux pumps that increase resistance to antiseptic compounds; however, the effect of carrying multiple pumps within S. aureus is unclear. We demonstrated that an isogenic strain carrying multiple efflux pumps had an additive resistance phenotype to cetrimide. Moreover, in a strain carrying qacA and norA, increased chlorhexidine tolerance was observed after the strain was preexposed to subinhibitory concentrations of a different common-use antiseptic. Taken together, our findings demonstrate cooperation between antiseptic resistance efflux pumps and suggest that their protective phenotype may be exacerbated by priming with subinhibitory concentrations of household antiseptics.

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Transcriptional Regulator TetR21 Controls the Expression of the Staphylococcus aureus LmrS Efflux Pump

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00649-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 3

Author(s):

Q. C. Truong-Bolduc ◽

Y. Wang ◽

C. Chen ◽

D. C. Hooper

Keyword(s):

Staphylococcus Aureus ◽

Parental Strain ◽

Efflux Pump ◽

Transcriptional Regulator ◽

Fold Increase ◽

Efflux Pumps ◽

Transcription Level ◽

Content Type

ABSTRACT TetR21 controls the expression of Tet38 and LmrS efflux pumps. A tetR21 mutant, QT21, exhibited a 4-fold increase in the transcription level of lmrS. Staphylococcus aureus lmrS overexpressor showed increases of 4-fold and 2-fold, respectively, in the MICs of chloramphenicol and erythromycin, while the MICs of lmrS mutant QT18 and lmrS-tetR21 mutant QT1821 remained similar to those of parental strain RN6390. TetR21 does not bind to the promoter of lmrS, suggesting indirect regulation of lmrS.

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Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus

Microbiology Spectrum ◽

10.1128/spectrum.00951-21 ◽

2021 ◽

Author(s):

Nisha Mahey ◽

Rushikesh Tambat ◽

Nishtha Chandal ◽

Dipesh Kumar Verma ◽

Krishan Gopal Thakur ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Nosocomial Infections ◽

Biofilm Formation ◽

Efflux Pump ◽

Efflux Pumps ◽

Drug Efflux ◽

Content Type ◽

Approved Drugs

Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections.

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Increased Usage of Antiseptics Is Associated with Reduced Susceptibility in Clinical Isolates of Staphylococcus aureus

mBio ◽

10.1128/mbio.00894-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 35

Author(s):

Katherine Hardy ◽

Katie Sunnucks ◽

Hannah Gil ◽

Sahida Shabir ◽

Eleftheria Trampari ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Efflux Pump ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Cross Resistance ◽

Single Strain ◽

Content Type ◽

Biocide Resistance ◽

The Face ◽

The Impact

ABSTRACT Hospital-acquired infection is a major cause of morbidity and mortality, and regimes to prevent infection are crucial in infection control. These include the decolonization of vulnerable patients with methicillin-resistant Staphylococcus aureus (MRSA) carriage using antiseptics, including chlorhexidine and octenidine. Concern has been raised, however, regarding the possible development of biocide resistance. In this study, we assembled a panel of S. aureus isolates, including isolates collected before the development of chlorhexidine and octenidine and isolates, from a major hospital trust in the United Kingdom during a period when the decolonization regimes were altered. We observed significant increases in the MIC and minimum bactericidal concentration (MBC) of chlorhexidine in isolates from periods of high usage of chlorhexidine. Isolates with increased MICs and MBCs of octenidine rapidly emerged after octenidine was introduced in the trust. There was no apparent cross-resistance between the two biocidal agents. A combination of variable-number tandem repeat (VNTR) analysis, PCR for qac genes, and whole-genome sequencing was used to type isolates and examine possible mechanisms of resistance. There was no expansion of a single strain associated with decreased biocide tolerance, and biocide susceptibility did not correlate with carriage of qac efflux pump genes. Mutations within the NorA or NorB efflux pumps, previously associated with chlorhexidine export, were identified, however, suggesting that this may be an important mechanism of biocide tolerance. We present evidence that isolates are evolving in the face of biocide challenge in patients and that changes in decolonization regimes are reflected in changes in susceptibility of isolates. IMPORTANCE Infection in hospitals remains a major cause of death and disease. One way in which we combat this is by decolonizing at-risk patients from carriage of bacteria which can cause disease such as MRSA. This is done with antiseptics, including chlorhexidine and octenidine. There is concern, however, that bacteria may be able to become resistant to these antiseptics. In this study, we looked at isolates of MRSA and found that there was a correlation between the use of antiseptics and increased resistance in the isolates. We also suggest that the mechanism by which these more tolerant isolates may become resistant to antiseptics is that of changing a transport pump that exports these agents. This information suggests that we need to study the impact of antiseptics on clinically important bacteria more closely.

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NorK, a novel norfloxacin efflux pump in Staphylococcus aureus

10.1101/850768 ◽

2019 ◽

Author(s):

Paul Briaud ◽

Jessica Baude ◽

Sylvère Bastien ◽

Laura Camus ◽

François Vandenesch ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Gene Family ◽

Efflux Pump ◽

Efflux Pumps ◽

Different Strains ◽

High Level

AbstractA novel efflux pump similar to Nor efflux pumps and designated as NorK was identified in Staphylococcus aureus. It contributes to norfloxacin resistance and presents a high level of expression in different strains. Its expression is not regulated by MgrA, unlike other genes of the nor gene family, suggesting that NorK could be important in the absence of expression of other nor genes.

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A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01252-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5858-5866 ◽

Cited By ~ 23

Author(s):

Somanon Bhattacharya ◽

Jack D. Sobel ◽

Theodore C. White

Keyword(s):

Candida Albicans ◽

Efflux Pump ◽

Pathogenic Fungus ◽

Resistance Mechanism ◽

Efflux Pumps ◽

Resistant Isolate ◽

Vaginal Infections ◽

Content Type ◽

Qrt Pcr ◽

Pump Activity

ABSTRACTCandida albicansis a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginalC. albicansisolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginalC. albicansisolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginalC. albicansisolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine β-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginalC. albicansisolates.

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Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01022-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 8

Author(s):

Raees A. Paul ◽

Shivaprakash M. Rudramurthy ◽

Manpreet Dhaliwal ◽

Pankaj Singh ◽

Anup K. Ghosh ◽

...

Keyword(s):

Aspergillus Flavus ◽

Efflux Pump ◽

Efflux Pumps ◽

Azole Resistance ◽

Wild Type ◽

Multidrug Efflux ◽

Environmental Isolates ◽

Content Type ◽

Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

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Transcriptional Profiling Analysis of the Global Regulator NorG, a GntR-Like Protein of Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.05847-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6207-6214 ◽

Cited By ~ 18

Author(s):

Q. C. Truong-Bolduc ◽

P. M. Dunman ◽

T. Eidem ◽

D. C. Hooper

Keyword(s):

Staphylococcus Aureus ◽

Target Genes ◽

Efflux Pump ◽

Transcriptional Profiling ◽

Fold Increase ◽

Regulatory Function ◽

Drug Transporter ◽

Content Type ◽

Global Regulators ◽

Inorganic Ion

The GntR-like protein NorG has been shown to affectStaphylococcus aureusgenes involved in resistance to quinolones and β-lactams, such as those encoding the NorB and AbcA transporters. To identify the target genes regulated by NorG, we carried out transcriptional-profiling assays usingS. aureusRN6390 and its isogenicnorG::catmutant. Our data showed that NorG positively affected the transcription of global regulatorsmgrA,arlS, andsarZ. The three putative drug efflux pump genes most positively affected by NorG were the NorB efflux pump (5.1-fold), the MmpL-like protein SACOL2566 (5.2-fold), and the BcrA-like drug transporter SACOL2525 (5.7-fold) genes. TheS. aureuspredicted MmpL protein showed 53% homology with the MmpL lipid transporter ofMycobacterium tuberculosis, and the putative SACOL2525 protein showed 87% homology with the bacitracin drug transporter BcrA ofStaphylococcus hominis. Two pump genes most negatively affected by NorG were the NorC (4-fold) and AbcA (6-fold) genes. Other categories of genes, such as those participating in amino acid, inorganic ion, or nucleotide transporters and metabolism, were also affected by NorG. Real-time reverse transcription (RT)-PCR assays formgrA,arlS,sarZ,norB,norC,abcA,mmpL, andbcrA-like were carried out to verify microarray data and showed the same level of up- or downregulation by NorG. ThenorGmutant showed a 2-fold increase in resistance to norfloxacin and rhodamine, both substrates of the NorC transporter, which is consistent with the resistance phenotype conferred by overexpression ofnorCon a plasmid. These data indicate that NorG has broad regulatory function inS. aureus.

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The Evolutionary Conservation of Escherichia coli Drug Efflux Pumps Supports Physiological Functions

Journal of Bacteriology ◽

10.1128/jb.00367-20 ◽

2020 ◽

Vol 202 (22) ◽

Cited By ~ 2

Author(s):

Tanisha Teelucksingh ◽

Laura K. Thompson ◽

Georgina Cox

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Efflux Pumps ◽

Evolutionary Conservation ◽

Drug Efflux ◽

Physiological Functions ◽

Content Type ◽

Bacterial Physiology ◽

E Coli ◽

Drug Efflux Pumps

ABSTRACT Bacteria harness an impressive repertoire of resistance mechanisms to evade the inhibitory action of antibiotics. One such mechanism involves efflux pump-mediated extrusion of drugs from the bacterial cell, which significantly contributes to multidrug resistance. Intriguingly, most drug efflux pumps are chromosomally encoded components of the intrinsic antibiotic resistome. In addition, in terms of xenobiotic detoxification, bacterial efflux systems often exhibit significant levels of functional redundancy. Efflux pumps are also considered to be highly conserved; however, the extent of conservation in many bacterial species has not been reported and the majority of genes that encode efflux pumps appear to be dispensable for growth. These observations, in combination with an increasing body of experimental evidence, imply alternative roles in bacterial physiology. Indeed, the ability of efflux pumps to facilitate antibiotic resistance could be a fortuitous by-product of ancient physiological functions. Using Escherichia coli as a model organism, we here evaluated the evolutionary conservation of drug efflux pumps and we provide phylogenetic analysis of the major efflux families. We show the E. coli drug efflux system has remained relatively stable and the majority (∼80%) of pumps are encoded in the core genome. This analysis further supports the importance of drug efflux pumps in E. coli physiology. In this review, we also provide an update on the roles of drug efflux pumps in the detoxification of endogenously synthesized substrates and pH homeostasis. Overall, gaining insight into drug efflux pump conservation, common evolutionary ancestors, and physiological functions could enable strategies to combat these intrinsic and ancient elements.

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Eugenol Reduces the Expression of Virulence-Related Exoproteins in Staphylococcus aureus

Applied and Environmental Microbiology ◽

10.1128/aem.00704-10 ◽

2010 ◽

Vol 76 (17) ◽

pp. 5846-5851 ◽

Cited By ~ 75

Author(s):

Jiazhang Qiu ◽

Haihua Feng ◽

Jing Lu ◽

Hua Xiang ◽

Dacheng Wang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Transcriptional Analysis ◽

Pcr Analysis ◽

Transcriptional Level ◽

Toxic Shock ◽

Release Assay ◽

Subinhibitory Concentrations ◽

Toxic Shock Syndrome Toxin ◽

Culture Supernatants ◽

Necrosis Factor

ABSTRACT Eugenol, an essential oil component in plants, has been demonstrated to possess activity against both Gram-positive and Gram-negative bacteria. This study examined the influence that subinhibitory concentrations of eugenol may have on the expression of the major exotoxins produced by Staphylococcus aureus. The results from a tumor necrosis factor (TNF) release assay and a hemolysin assay indicated that S. aureus cultured with graded subinhibitory concentrations of eugenol (16 to 128 μg/ml) dose dependently decreased the TNF-inducing and hemolytic activities of culture supernatants. Western blot analysis showed that eugenol significantly reduced the production of staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1 (the key exotoxins to induce TNF release), as well as the expression of α-hemolysin (the major hemolysin to cause hemolysis). In addition, this suppression was also evaluated at the transcriptional level via real-time reverse transcription (RT)-PCR analysis. The transcriptional analysis indicated that 128 μg/ml of eugenol remarkably repressed the transcription of the S. aureus sea, seb, tst, and hla genes. According to these results, eugenol has the potential to be rationally applied on food products as a novel food antimicrobial agent both to inhibit the growth of bacteria and to suppress the production of exotoxins by S. aureus.

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MroQ Is a Novel Abi-Domain Protein That Influences Virulence Gene Expression in Staphylococcus aureus via Modulation of Agr Activity

Infection and Immunity ◽

10.1128/iai.00002-19 ◽

2019 ◽

Vol 87 (5) ◽

Cited By ~ 3

Author(s):

Stephanie Marroquin ◽

Brittney Gimza ◽

Brooke Tomlinson ◽

Michelle Stein ◽

Andrew Frey ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Gene ◽

Transcriptional Analysis ◽

Data Sets ◽

Virulence Determinants ◽

Sequencing Data ◽

Content Type ◽

Virulence Gene Expression ◽

Function Mechanism ◽

Family Protein

ABSTRACT Numerous factors have, to date, been identified as playing a role in the regulation of Agr activity in Staphylococcus aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigated the product of SAUSA300_1984 (termed MroQ), a transmembrane Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a USA300 mroQ mutant, we observed a drastic reduction in proteolysis, hemolysis, and pigmentation that was fully complementable. This appears to result from diminished agr activity, as transcriptional analysis revealed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA sequencing data sets for both mroQ and agr mutants revealed a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr-controlling effect; however, this was not wielded at the level of AgrD processing. Virulence assessment demonstrated that both mroQ and agr mutants exhibited increased formation of renal abscesses but decreased skin abscess formation alongside diminished dermonecrosis. Collectively, we present the characterization of a novel agr effector in S. aureus which would appear to be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase.

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