Efflux Pump-mediated Resistance Research Articles

Sub-MIC antibiotics increased the fitness cost of CRISPR-Cas in Acinetobacter baumannii.

The escalating prevalence of bacterial resistance, particularly multidrug-resistant bacteria like Acinetobacter baumannii, has become a significant global public health concern. The CRISPR-Cas system, a crucial defense mechanism in bacteria against foreign genetic elements, provides a competitive advantage. Type I-Fb and Type I-Fa are two subtypes of CRISPR-Cas systems that were found in A. baumannii, and the I-Fb CRISPR-Cas system regulates antibiotic resistance in A. baumannii. However, it is noteworthy that a majority of clinical isolates of A. baumannii lack or have incomplete CRISPR-Cas systems and most of them are multidrug-resistant. In light of this, our study aimed to examine the impact of antibiotic pressure on the fitness cost of the I-Fb CRISPR-Cas system in A. baumannii. In the study, we conducted in vitro competition experiments to investigate the influence of sub-minimum inhibitory concentration (sub-MIC) on the CRISPR-Cas systems' fitness cost in A. baumannii. We found that the fitness cost of the CRISPR-Cas system was increased under sub-MIC conditions. The expression of CRISPR-Cas-related genes was decreased, while the conjugation frequency was increased in AB43 under sub-MIC conditions. Through metabolomic analysis, we identified that sub-MIC conditions primarily affected energy metabolism pathways. In particular, we observed increased carbon metabolism, nitrogen metabolism, and intracellular ATP. Notably, the CRISPR-Cas system demonstrated resistance to the efflux pump-mediated resistance. Furthermore, the expression of efflux pump-related genes was increased under sub-MIC conditions. Our findings suggest that the I-Fb CRISPR-Cas system confers a significant competitive advantage in A. baumanni. However, under sub-MIC conditions, its function and the ability to inhibit the energy required for efflux pumps are reduced, resulting in an increased fitness cost and loss of competitive advantage.

A novel major facilitator superfamily-type tripartite efflux system CprABC mediates resistance to polymyxins in Chryseobacterium sp. PL22-22A.

Polymyxin B (PMB) and polymyxin E (colistin, CST) are polymyxin antibiotics, which are considered last-line therapeutic options against multidrug-resistant Gram-negative bacteria in serious infections. However, there is increasing risk of resistance to antimicrobial drugs. Effective efflux pump inhibitors (EPIs) should be developed to help combat efflux pump-mediated antibiotic resistance. Chryseobacterium sp. PL22-22A was isolated from aquaculture sewage under selection with 8 mg/L PMB, and then its genome was sequenced using Oxford Nanopore and BGISEQ-500 platforms. Cpr (Chryseobacterium Polymyxins Resistance) genes encoding a major facilitator superfamily-type tripartite efflux system, were found in the genome. These genes, and the gene encoding a truncation mutant of CprB from which sequence called CprBc was deleted, were amplified and expressed/co-expressed in Escherichia coli DH5α. Minimum inhibitory concentrations (MICs) of polymyxins toward the various E. coli heterologous expression strains were tested in the presence of 2-128 mg/L PMB or CST. The pumping activity of CprABC was assessed via structural modeling using Discovery Studio 2.0 software. Moreover, the influence on MICs of baicalin, a novel MFS EPI, was determined, and the effect was analyzed based on homology modeling. Multidrug-resistant bacterial strain Chryseobacterium sp. PL22-22A was isolated in this work; it has notable resistance to polymyxin, with MICs for PMB and CST of 96 and 128 mg/L, respectively. A novel MFS-type tripartite efflux system, named CprABC, was identified in the genome of Chryseobacterium sp. PL22-22A. Heterologous expression and EPI assays indicated that the CprABC system is responsible for the polymyxin resistance of Chryseobacterium sp. PL22-22A. Structural modeling suggested that this efflux system provides a continuous conduit that runs from the CprB funnel through the CprC porin domain to pump polymyxins out of the cell. A specific C-terminal α-helix, CprBc, has an activation function on polymyxin excretion by CprB. The flavonoid compound baicalin was found to affect the allostery of CprB and/or obstruct the substrate conduit, and thus to inhibit extracellular polymyxin transport by CprABC. Novel MFS-type tripartite efflux system CprABC in Chryseobacterium sp. PL22-22A mediates resistance to polymyxins, and baicalin is a promising EPI.

Inhibiting Efflux Pumps and Resistance Mechanisms: A Mini Review

Efflux pumps play a significant role in the development and spread of antibiotic resistance among bacteria. These specialized membrane proteins actively pump out various antibiotics and other toxic compounds, allowing bacteria to survive and thrive in the presence of these substances. Understanding the mechanisms by which efflux pumps contribute to antibiotic resistance is crucial for devising effective strategies to combat this global health threat. This article delves into the intricacies of efflux pump-mediated resistance, exploring the types and functions of efflux pumps, their role in antibiotic resistance, and the strategies employed to inhibit their activity. Additionally, it examines the challenges and future prospects in developing efflux pump inhibitors, highlighting case studies that showcase both successes and failures in this field. By shedding light on the inhibitory potential of efflux pumps, this article aims to contribute to the broader efforts in combating antibiotic resistance.

Efflux Pumps In Antimicrobial Resistance: Mechanism, Regulation And Therapeutic Implications

Efflux pumps play a crucial role in antimicrobial resistance, enabling bacteria to extrude a wide range of antibiotics and other antimicrobial compounds, thereby reducing their intracellular concentration and rendering them ineffective. Understanding the mechanisms underlying efflux pump-mediated resistance is essential for the development of effective strategies to combat this growing threat. This review paper provides an overview of the various efflux pump systems, their regulation mechanisms, and their impact on antimicrobial resistance. Additionally, we discuss the potential therapeutic interventions that target efflux pumps to restore the efficacy of antimicrobial agents.

Efflux Pump (QacA, QacB, and QacC) and β-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 β-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 β-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 β-lactamase- and efflux pump-mediated antibiotic resistance was evaluated from the reduction of the MIC of ethidium bromide (EtBr) and β-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 β-lactam antibiotics, suggesting that they may act by inhibiting β-lactamases and efflux pumps such as QacC and QacA/B. However, further research is required to elucidate the molecular mechanisms underlying these observed effects.

Riparin-B as a Potential Inhibitor of AdeABC Efflux System from Acinetobacter baumannii.

Acinetobacter baumannii is an important opportunistic pathogen that causes serious health-related infections, especially in intensive care units. The present study aimed to investigate the antimicrobial activity of Riparin-B (Rip-B) alone and in association with norfloxacin against multidrug-resistant clinical isolates of A. baumannii. For this, the minimum inhibitory concentrations were determined by the microdilution method. For the evaluation of resistance-modulating activity, MIC values for antibiotics were determined in the presence or absence of subinhibitory concentrations of Rip-B or chlorpromazine (CPZ). The AdeABC-AdeRS efflux system genes from these isolates were detected by PCR. Docking studies were also carried out to evaluate the interaction of Riparin-B and the AdeABC-AdeRS efflux system. The study was conducted from 2017 to 2019. The results showed that Rip-B showed weak intrinsic activity against the strains tested. On the other hand, Rip-B was able to modulate norfloxacin's response against A. baumannii strains that express efflux pump-mediated resistance. Docking studies provided projections of the interaction between Rip-B and EtBr with the AdeB protein, suggesting that Rip-B acts by competitive inhibition with the drug. Results found by in vitro and in silico assays suggest that Rip-B, in combination with norfloxacin, has the potential to treat infections caused by multidrug-resistant A. baumanni with efflux pump resistance.

Role of Efflux Pump-Mediated Antibiotic Resistance in Quorum Sensing-Regulated Biofilm Formation by Salmonella Typhimurium.

This study was designed to assess the influence of efflux pump activity on the biofilm formation in Salmonella Typhimurium. Salmonella enterica subsp. enterica serovar Typhimurium ATCC 19585 (STWT) and clinically isolated S. Typhimurium CCARM 8009 (STCI) were treated with ceftriaxone (CEF), chloramphenicol (CHL), ciprofloxacin (CIP), erythromycin (ERY), norfloxacin (NOR), and tetracycline (TET) in autoinducer-containing media in the absence and presence of phenylalanine-arginine β-naphthylamide (PAβN) to compare efflux pump activity with biofilm-forming ability. The susceptibilities of STWT and STCI were increased in the presence of PAβN. ERY+PAβN showed the highest decrease in the minimum inhibitory concentration (MIC) of ERY from 256 to 2 μg/mL against STWT and STCI. The antimicrobial activity of NOR against planktonic cells was significantly increased in the presence of PAβN, showing the lowest numbers of STWT (3.2 log CFU/cm2), and the TET+PAβN effectively inhibited the growth of STCI (5.2 log CFU/cm2). The lowest biofilm-forming abilities were observed at NOR+PAβN against STWT (biofilm-forming index, BFI < 0.41) and CEF+PAβN against STCI (BFI = 0.32). The bacteria swimming motility and relative fitness varied depending on the antibiotic and PAβN treatments. The motility diameters of STWT were significantly decreased by NOR+PAβN (6 mm) and TET+PAβN (15 mm), while the lowest motility of STCI was observed at CIP+PAβN (8 mm). The significant decrease in the relative fitness levels of STWT and STCI was observed at CIP+PAβN and NOR+PAβN. The PAβN as an efflux pump inhibitor (EPI) can improve the antimicrobial and anti-biofilm efficacy of antibiotics against S. Typhimurium. This study provides useful information for understanding the role of efflux pump activity in quorum sensing-regulated biofilm formation and also emphasizes the necessity of the discovery of novel EPIs for controlling biofilm formation by antibiotic-resistant pathogens.

Emergence of tigecycline-resistant Klebsiella pneumoniae ST11 clone in patients without exposure to tigecycline.

Tigecycline is a unique class of semi-synthetic glycylcyclines developed to treat infections caused by multidrug-resistant Klebsiella pneumoniae. In the past decades, eight tigecycline-resistant Acinetobacter baumannii isolates have been identified in Tehran and no Klebsiella pneumoniae has been reported. To elucidate the mechanism of K. pneumoniae efflux pump-mediated resistance, the expression of efflux pump genes (oqxA, oqxB, acrA, acrB, tolC) and regulators (acrR, ramA, marA, soxS, rarA, rob) was investigated by real-time RT-PCR. Multilocus sequence typing (MLST) of tigecycline-resistant strains was also performed. Compared to the tigecycline sensitive strain K32 (negative control), all resistant strains showed higher expression levels of efflux genes and regulatory factors. Three tigecycline-resistant strains (K53, K67, K79) showed higher levels of rarA expression (38.1-fold, 41-fold and 24-fold, respectively) and oqxB pump gene (48.2-fold, 60-fold and 58-fold, respectively). The increased expression of acrB was associated with the expression of ramA. However, to the best of our knowledge, studies on the mechanisms of resistance of K. pneumoniae strains to tigecycline are limited, especially in developing countries such as Iran. In the present study, we found that both AcrAB-TolC and OqxAB efflux pumps may play an important role in tigecycline resistance in K. pneumoniae isolates. Finally, the emergence of ST11 molecular type of resistant isolates should be monitored in hospitals to identify factors leading to tigecycline resistance.

Overexpression of the adeB Efflux Pump Gene in Tigecycline-Resistant Acinetobacter baumannii Clinical Isolates and Its Inhibition by (+)Usnic Acid as an Adjuvant.

Acinetobacter species are among the most life-threatening Gram-negative bacilli, causing hospital-acquired infections, and they are associated with high morbidity and mortality. They show multidrug resistance that acts via various mechanisms. In Acinetobacter baumannii, efflux pump-mediated resistance to many antimicrobial compounds, including tigecycline, has been widely reported. Natural compounds have been used for their various pharmacological properties, including anti-efflux pump activity. The present study aimed to evaluate the efflux pump-mediated resistance mechanism of Acinetobacter baumannii and the effect of (+)Usnic acid as an efflux pump inhibitor with tigecycline. For detecting the efflux pump activity of tigecycline-resistant Acinetobacter baumannii isolates, microbroth dilution method and real-time quantitative reverse transcription–polymerase chain reaction was used. (+)Usnic acid was added to tigecycline and tested by the checkerboard method to evaluate its efficacy as an efflux pump inhibitor. qRT-PCR analysis was carried out to show the downregulation of the efflux pump in the isolates. Out of 42 tigecycline-resistant Acinetobacter baumannii isolates, 19 showed efflux pump activity. All 19 strains expressed the adeB gene. (+)Usnic acid as an adjuvant showed better efficacy in lowering the minimum inhibitory concentration compared with the conventional efflux pump inhibitor, carbonyl cyanide phenylhydrazone.

Multi-drug resistant (MDR), extended spectrum beta-lactamase (ESBL) producing and carbapenem resistant Escherichia coli in rescued Sloth bears (Melursus ursinus), India.

The study reports the multi-drug resistant (MDR), extended spectrum beta-lactamase (ESBL) producing and carbapenem resistant Escherichia coli (CRE) isolated from rescued sloth bear (Melursus ursinus), India. Non-duplicate faecal samples from 21 adult rescued sloth bears were collected at once during 2015-2016 and processed for isolation of E. coli and antibacterial susceptibility pattern. From 21 samples, 45 E. coli were isolated and on phenotypic screening, 23 were MDR, 17 were ESBL producers, and five were carbapenem-resistant (CR). Three E. coli isolates (6.67%, 3/45) showed no resistance, however 42 isolates (93.33%, 42/45) exhibited resistant to at least one antibiotics. The MDR isolates carried beta-lactamase, chloramphenicol, aminoglycosides, tetracycline, fluroquinolone, and sulphadimidine resistance genes. All the phenotypic ESBL producing isolates harbored blaCTX-M genes. On genotypic screening, three CRE (60.0%, 3/5) were positive for blaNDM carbapenemase gene and efflux pump-mediated carbapenem resistance was detected in two CRE isolates (40.0%, 2/5) which were negative for carbapenemase genes. The CRE isolates (n = 5) also co-harbored AMR genes like blaTEM-1, blaAmpC, qnrA, qnrB, qnrS, tetA, tetB and sulI. Virulence screening of the resistant isolates detected the presence of Stx1(n = 1), Stx2 (n = 3), eaeA (n = 4) and hlyA (n = 3) genes. Plasmid incompatibility (Inc) typing revealed that two isolates harboured blaNDM-5 gene on Incl1 and one isolate on IncF plasmid. Apart from the NDM gene, the plasmids also carried tetracycline, beta-lactamase and quinolone resistance genes. The plasmid multilocus sequence typing (pMLST) of the E. coli Incl1 plasmid showed the Sequence Type (ST) 297. This appears to be the first report of MDR, ESBL producing and blaNDM-5 genes on Incl1 and IncF plasmids from rescued sloth bear.

The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance

The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

Carbapenem resistant Escherichia coli and Pseudomonas aeruginosa in captive blackbucks (Antilope cervicapra) and leopards (Panthera pardus) from India

The study aimed to investigate the occurrence of carbapenem resistant E. coli and P. aeruginosa in apparently healthy, captive blackbucks and leopards of India. Faecal samples of blackbucks (n = 7) and leopards (n = 7) were processed to isolate carbapenem resistant E. coli (CRE) and P. aeruginosa (CRP). Forty (leopards n = 26; blackbuck n = 14) E. coli and two P. aeruginosa (blackbuck n = 2) samples were isolated from the faecal samples (n = 14). Eleven carbapenem resistant isolates were recovered, of which 10 were CRE and one was CRP. The minimum inhibitory concentration (MIC) was determined for meropenem for carbapenem resistant isolates and was between 8 and 64 μg/mL. All the CRE and CRP were phenotypically multidrug resistant, and six CRE were extended-spectrum beta-lactamases (ESBL) producers. On genotypic screening, seven CRE and one CRP were positive for the blaNDM carbapenemase gene. Efflux pump-mediated carbapenem resistance was noticed in four CRE isolates (36.4%, 4/11). Of the six ESBL producing CRE, four isolates carried blaCTX-M-1 genes. The CRE isolates also harbored blaTEM-1, blaAmpC, qnrA, qnrB, qnrS, tetA, tetB and sul1 resistance genes. On Shiga toxin virulence screening, Stx1, Stx2 genes were detected in two and one isolates, respectively. Plasmid typing of CRE revealed that the blaNDM genes were carried on an Incl1 plasmid. The plasmid multilocus sequence typing (pMLST) of the isolates showed the Sequence Type (ST) 297. The occurrence of carbapenem resistance bacteria in captive wildlife should be a major public health priority.

Impact of marbofloxacin administration on the emergence of marbofloxacin-resistant E. coli in faecal flora of goats and elucidation of molecular basis of resistance

ObjectivesThe level of resistance immediately prior to slaughter in food-producing animals is of great public health significance because of likely transmission of resistant bacteria via the food chain. MethodsMarbofloxacin was administered to goats at the dose of 2 mg/kg body weight by intramuscular route for 5 days. Faecal Escherichia coli population was monitored and examined for bacteriological procedures. DNA sequencing of gyrA and parC genes was performed to identify mutations at quinolone-resistance determining region, and interaction between marbofloxacin and GyrA was studied by in silico docking. E. coli isolates were screened for plasmid-mediated quinolone resistance genes qnrA, qnrB, qnrS, aac(6′)Ib-cr, qepA, oqxA and oqxB. Efflux pump-mediated resistance was evaluated by ethidium bromide assay, reduction in minimum inhibitory concentration (MIC) values in the presence of efflux pump inhibitors and relative expression of AcrAB-TolC efflux pump. ResultsDuring the treatment period, emergence of marbofloxacin-resistant E. coli strains was observed in gut flora. Quinolone resistance determining regions (QRDRs) in gyrA identified amino acid codon mutations Ser83Leu and Asp87Asn, and Ser80Ile in parC. Docking analysis implied that marbofloxacin could not form strong complexes with mutated DNA-gyrase. A high prevalnce of PMQR genes, especially qnrS, was observed along with overexpression of AcrAB-TolC efflux pump. ConclusionsThe study highlighted the high prevalence of transferable mechanisms of quinolone resistance and over expression of efflux pumps in marbofloxacin-resistant E. coli isolates apart from classic QRDR mutations. The present study recommends to consider the period of dominance of resistant commensals, being excreted by animals during the antimicrobial treatments, while formulating the withdrawal period for drugs, especially in food-producing animals.

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

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.IMPORTANCES. 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.

Detection of Overexpression of Efflux Pump Expression in Fluoroquinolone-Resistant Pseudomonas aeruginosa Isolates.

Context:Fluoroquinolones are the most effective antibiotics against Pseudomonas aeruginosa; many strains, however, have shown resistance due to mutations in DNA gyrase, topoisomerase IV, or in the efflux pumps. Little is known about P. aeruginosa efflux pump resistance mechanisms in the Kingdom of Bahrain.Aim:The aim was to study efflux pump-mediated fluoroquinolone resistance among P. aeruginosa isolates using phenotypic (E-test and agar dilution) and genotypic (real-time-polymerase chain reaction [RT-PCR]) methods.Materials and Methods:Fifty ciprofloxacin-resistant P. aeruginosa isolates were included in this study. Genus and species of P. aeruginosa were confirmed by conventional PCR. The minimum inhibitory concentration (MIC) of ciprofloxacin with and without carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was determined by E-test and agar dilution test. The overexpression of genes MexB, MexD, MexF, and MexY was measured by RT-PCR.Results:All isolates were confirmed as P. aeruginosa. Among the fifty isolates, four showed reduction in ciprofloxacin MIC after addition of CCCP. These four isolates showed upregulation of expression of at least one of the four genes by RT-PCR. The mean gene expression of MexB, MexD, MexF, and MexY increased by 1.6, 4.65, 3.4, and 3.68-fold, respectively.Conclusion:The results demonstrate the presence and type of efflux pump overexpression, mandating for large multicentric studies.

Genome-facilitated discovery of RND efflux pump-mediated resistance to cephalosporins in Vibrio spp. isolated from the mummichog fish gut.

This study examined the role of resistance-nodulation-cell division (RND) efflux pumps in resistance to first-generation and third-generation cephalosporins, and the potential contribution to increased virulence in two Vibrio isolates from the gut microbiota of a forage-feeder fish. Phenotypic MIC testing was performed in the presence and absence of an RND efflux pump inhibitor, phenylalanine-arginine-beta-napthylamide (PAβN). Genomes of the two Vibrio spp. were compared to characterise RND efflux pump gene homologs. The study identified 13 and 12 RND operons, respectively, in Vibrio spp. T21 and T9, with Vibrio sp. T21 containing an additional RND operon compared with other V. parahaemolyticus strains. Both the inner-membrane protein (IMP) and the membrane facilitator protein (MFP) sequences of this operon were homologous to VexD and VexC, respectively, which is an RND operon in Vibrio cholerae. More generally, the other RND proteins in these strains showed homology to RND efflux pumps characterised in Escherichia coli and Vibrio cholerae. Decreased resistance to cefoperazone and cephradine was observed in Vibrio sp. T21, and to cefoperazone and cefsulodin in Vibrio sp. T9 in the presence of PaβN. The RND pumps may also mediate transport of kanamycin. By analysing the genomes of two Vibrio spp. isolated from the mummichog fish gut, RND efflux pump-mediated resistance to first-generation and third-generation cephalosporins was discovered in these strains. This work highlights the need for further research into this unique Vibrio spp. operon and, more generally, RND efflux pumps in Vibrio spp., as Vibrio spp. often cause seafood-borne illness.

Low nutrient levels as drinking water conditions can reduce the fitness cost of efflux pump-mediated ciprofloxacin resistance in Pseudomonas aeruginosa

The long-term persistence of antibiotic resistance in the environment, especially in drinking water, is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, usually confers a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persisted in low-nutrient environments (TOC < 10 mg/L) such as drinking and source water in which antibiotic selective pressure might be very low or even absent. Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacin-resistant mutants revealed that the fitness cost of ciprofloxacin resistance significantly decreased (p < 0.05) under low-nutrient (0.5 mg/L total organic carbon (TOC)) relative to high-nutrient (500 mg/L TOC) conditions. Mechanisms underlying this fitness cost were analyzed. The mexD gene expression in resistant bacteria (cip_3 strain) was significantly lower (p < 0.05) in low-nutrient conditions, with 10 mg/L TOC ((8.01 ± 0.82)-fold), than in high-nutrient conditions, with 500 mg/L TOC ((48.89 ± 4.16)-fold). Moreover, rpoS gene expression in resistant bacteria ((1.36 ± 0.13)-fold) was significantly lower (p < 0.05) than that in the wild-type strain ((2.78 ± 0.29)-fold) under low-nutrient conditions (10 mg/L TOC), suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller (p < 0.05) in low-nutrient conditions (5 and 0.5 mg/L TOC). These results suggest that nutrient levels are a key factor in determining the persistence of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics.

Essential Oil, Extracts, and Sesquiterpenes Obtained From the Heartwood of Pilgerodendron uviferum Act as Potential Inhibitors of the Staphylococcus aureus NorA Multidrug Efflux Pump.

Staphylococcus aureus is a serious human pathogen that is highly adaptive to environmental conditions and rapidly develops antibiotic resistance. The use of efflux pumps to reduce antibiotic concentrations at the intracellular level is one of the main mechanisms by which bacteria develop antibiotic resistance. The management of efflux pumps, specifically NorA, which is expressed by S. aureus strains, is a valuable strategy for restoring susceptibility in strains resistant to antibacterial agents. In recent years, many studies have focused on searching for natural substances that can reverse efflux pump-mediated resistance in S. aureus. Extracts and compounds obtained from plants can be efficient efflux pump inhibitors (EPIs) and represent a potentially patient-friendly strategy for controlling S. aureus. In the present study, we evaluated the ability of essential oils, petroleum ether extracts, dichloromethane extract (DCME) and six compounds isolated from the heartwood of Pilgerodendron uviferum (Cupressaceae) and two synthetic derivatives to inhibit efflux in NorA pumps in the following three S. aureus strains: K2378, which overexpressed the norA gene (norA++), K1902 (norA-deleted, ΔnorA) and the parental strain, NCTC 8325-4. Efflux activity was evaluated using a fluorometric method that measured the accumulation of the universal efflux pump substrate ethidium bromide (EtBr). Only DCME and the compounds 15-copaenol and epi-cubenol inhibited EtBr efflux by K2378. Even the lowest concentration of 15-copaenol exhibited a stronger inhibitory effect than carbonyl cyanide m-chlorophenyl hydrazone on EtBr efflux by K2378. 15-copaenal only showed inhibition of EtBr efflux in K2378 cells at 125 μg/mL, but not superior to the control inhibitor and 15-copaenyl acetate exerted no intrinsic EPI activity against K2378. Fractional inhibitory concentration index (FICI) values obtained in the checkerboard assays, indicated that all combinations between DCME, epi-cubenol and 15-copaenol, and tested antibiotics showed a synergistic effect in wild type, norA++ and ΔnorA strains. Moreover, those were not toxic for the HeLa cell line at concentrations in which the synergistic effect and inhibitory activity of efflux pumps was determined. Other extracts and compounds obtained from P. uviferum did not display EtBr efflux-inhibiting activity against the evaluated S. aureus strains.

Efflux pump-mediated resistance to antifungal compounds can be prevented by conjugation with triphenylphosphonium cation

Antifungal resistance due to upregulation of efflux pumps is prevalent in clinical Candida isolates. Potential efflux pump substrates (PEPSs), which are active against strains deficient in efflux pumps but inactive against wild-type strains, are usually missed in routine antifungal screening. Here we present a method for identification of PEPSs, and show that conjugation with mitochondria-targeting triphenylphosphonium cation (TPP+) can enhance or restore the compounds’ antifungal activity. The screening method involves co-culturing a wild-type C. albicans strain and a Cdr efflux pump-deficient strain, labelled with different fluorescent proteins. We identify several PEPSs from a library of natural terpenes, and restore their antifungal activity against wild-type and azole-resistant C. albicans by conjugation with TPP+. The most active conjugate (IS-2-Pi-TPP) kills C. albicans cells, prevents biofilm formation and eliminates preformed biofilms, without inducing significant resistance. The antifungal activity is accompanied by mitochondrial dysfunction and increased levels of intracellular reactive oxygen species. In addition, IS-2-Pi-TPP is effective against C. albicans in a mouse model of skin infection.

Antimicrobial Resistance, Biofilm Formation, and Phylogenetic Grouping of Uropathogenic Escherichia coli Isolates in Egypt: The Role of Efflux Pump-Mediated Resistance

Background: Urinary tract infections are the most commonly encountered infections in clinics and outpatient settings and are mainly caused by Uropathogenic Escherichia coli (UPEC). Multidrug-resistant bacteria (MDR) have become a major public health threat, worldwide. Objectives: This study aimed to investigate the prevalence of the MDR phenotype, efflux pump-mediated resistance, and the ability for in vitro biofilm formation among UPEC clinical isolates from Egypt. Methods: Uropathogenic E. coli isolates were collected from two Egyptian governorates, identified, and classified to their corresponding phylogenetic group by the polymerase chain reaction (PCR). Antimicrobial susceptibility testing was done using the Kirby-Bauer disk diffusion method. AcrAB-TolC efflux pump major genes were detected by PCR; efflux pump-mediated resistance was determined by the efflux pump inhibitor microplate-based assay. The ability for in-vitro biofilm formation was also tested. Results: The phylogenetic analysis of the UPEC isolates revealed that most of the isolates belonged to groups B2 and D. The MDR phenotype was detected in 90.8% of UPEC isolates; efflux pump-mediated resistance was detected in all MDR isolates. The acrA, acrB, and tolC were detected in 74.84% of MDR isolates. The ability for in-vitro biofilm formation was recorded in 76.5% of the UPEC isolates. Conclusions: The MDR phenotype and the ability for in-vitro biofilm formation were predominant among UPEC in Egypt. The high prevalence of MDR efflux pumps necessitates the application of new treatment strategies to inhibit this phenomenon.