Role Of Efflux Pumps Research Articles

The tigecycline resistance mechanisms in Gram-negative bacilli.

Tigecycline, hailed as a pivotal agent in combating multidrug-resistant bacterial infections, confronts obstacles posed by the emergence of resistance mechanisms in Gram-negative bacilli. This study explores the complex mechanisms of tigecycline resistance in Gram-negative bacilli, with a particular focus on the role of efflux pumps and drug modification in resistance. By summarizing these mechanisms, our objective is to provide a comprehensive understanding of tigecycline resistance in Gram-negative bacilli, thereby illuminating the evolving landscape of antimicrobial resistance. This review contributes to the elucidation of current existing tigecycline resistance mechanisms and provides insights into the development of effective strategies to manage the control of antimicrobial resistance in the clinical setting, as well as potential new targets for the treatment of tigecycline-resistant bacterial infections.

Identification of the Major Facilitator Superfamily Efflux Pump KpsrMFS in Klebsiella pneumoniae That Is Down-Regulated in the Presence of Multi-Stress Factors.

Efflux pumps play important roles in bacterial detoxification and some of them are stress-response elements that are up-regulated when the host is treated with antibiotics. However, efflux pumps that are down-regulated by stimulations are rarely discovered. Herein, we analyzed multiple transcriptome data and discovered a special (Major Facilitator Superfamily) MFS efflux pump, KpsrMFS, from Klebsiella pneumoniae, which was down-regulated when treated with antibiotics or extra carbon sources. Interestingly, overexpression of kpsrmfs resulted in halted cell growth in normal conditions, while the viable cells were rarely affected. The function of KpsrMFS was further analyzed and this efflux pump was determined to be a proton-driven transporter that can reduce the intracellular tetracycline concentration. In normal conditions, the expression of kpsrmfs was at a low level, while artificial overexpression of it led to increased endogenous reactive oxygen species (ROS) production. Moreover, by comparing the functions of adjacent genes of kpsrmfs, we further discovered another four genes that can confer similar phenotypes, indicating a special regulon that regulates cell growth. Our work provides new insights into the roles of efflux pumps and suggests a possible regulon that may regulate cell growth and endogenous ROS levels.

Hypoxia increases Neisseria gonorrhoeae resistance to resazomycins: Role of Efflux Pumps and Oxidative Stress

Neisseria gonorrhoeae (Ng) has developed resistance to all antibiotics previously and currently recommended for treatment of gonorrhea, underscoring the need for novel antimicrobials against this pathogen. Resazomycins are highly effective antimicrobials against Ng in vitro. However, when these compounds were tested in a mouse model of gonorrhea, only one resazomycin, resorufin pentyl ether (RPE), exhibited any therapeutic effect. One reason for this difference between in vitro and in vivo therapeutic efficacy is increased resistance to resazomycins at low levels of oxygen (2%). We hypothesized this difference in susceptibility was due to altered activity of multi-drug efflux pumps. To test this, we screened a selection of Ngmutants that do not express or overexpress either the MtrCDE or NorM efflux pumps for Rz susceptibility. Overexpression of MtrCDE resulted in increased resistance to Rz at both 2% and ~20% oxygen suggesting resazurin may be a substrate of this efflux pump. Loss of expression of either MtrCDE or NorM had no effect on the increased resistance of Ng to resazurin at low oxygen. We next sought to determine whether the increased susceptibility of Rz at 20% oxygen is due to oxidative stress. To test this, we measured the susceptibility of Ng to Rz in the presence and absence of the antioxidants, cysteine HCl and glutathione, at 20% oxygen. In the presence of cysteine HCl or glutathione, multiple Ng strains had a higher Rz MIC at 20% oxygen. Here, we have shown oxygen concentration affects Ngsusceptibility to Rz due to increased oxidative stress.

The Role of Efflux Pumps in Antibiotic Resistance of Gram Negative Rods

Antibiotic resistance is an important public health problem today, causing increased morbidity and mortality. Resistance to antibiotics in bacteria can develop by various mechanisms such as a change in the target site of the drug, a change in the outer membrane permeability, enzymatic defusing of the drug and efflux of the antimicrobial compound. Some bacteria have the potential to develop resistance to more than one drug by using several mechanisms together. One of the important resistance mechanisms of bacteria is active efflux pumps (EPs). EPs are pump proteins found in all cell types, located in the cell membrane. They are responsible for the excretion of various intracellular and extracellular substances (antibiotics, etc.) out of the cell. There is much research on various antimicrobials that cause antibiotic resistance in Gram negative rods, but studies on EPs are relatively few. Due to the concern that antibiotics will be insufficient in the treatment of diseases, a good understanding of EPs and the discovery of new EP inhibitors will shed light on the future of humanity. In this review, the structure of bacterial EPs in Gram negative bacteria, the role of EPs in multidrug resistance, the importance of EP inhibitors in the fight against antibiotic resistance and the phenotypic and genotypic detection methods of EPs are discussed.

Efflux Pumps and Different Genetic Contexts of tet(X4) Contribute to High Tigecycline Resistance in Escherichia fergusonii from Pigs.

Tigecycline is a last-resort antibiotic for the treatment of infections caused by multidrug-resistant bacteria. The emergence of plasmid-mediated tigecycline resistance genes is posing a serious threat to food safety and human health and has attracted worldwide attention. In this study, we characterized six tigecycline-resistant Escherichia fergusonii strains from porcine nasal swab samples collected from 50 swine farms in China. All the E. fergusonii isolates were highly resistant to tigecycline with minimal inhibitory concentration (MIC) values of 16-32 mg/L, and all contained the tet(X4) gene. In addition, 13-19 multiple resistance genes were identified in these isolates, revealed by whole-genome sequencing analysis. The tet(X4) gene was identified as being located in two different genetic structures, hp-abh-tet(X4)-ISCR2 in five isolates and hp-abh-tet(X4)-ΔISCR2-ISEc57-IS26 in one isolate. The role of efflux pumps in tigecycline resistance was evaluated by using inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The MIC values of tigecycline showed a 2- to 4-fold reduction in the presence of CCCP, indicating the involvement of active efflux pumps in tigecycline resistance in E. fergusonii. The tet(X4) gene was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquisition of tigcycline resistances in the transconjugants. Whole-genome multilocus sequence typing (wgMLST) and phylogenetic analysis showed a close relationship of five isolates originating from different pig farms, suggesting the transmission of tet(X4)-positive E. fergusonii between farms. In conclusion, our findings suggest that E. fergusonii strains in pigs are reservoirs of a transferable tet(X4) gene and provide insights into the tigecycline resistance mechanism as well as the diversity and complexity of the genetic context of tet(X4) in E. fergusonii.

The RND Efflux Pump Gene Expression in the Biofilm Formation of Acinetobacter baumannii.

Multidrug resistant (MDR) Acinetobacter baumannii is a critical opportunistic pathogen in healthcare-associated infections (HAI). This is attributed to several factors, including its ability to develop biofilms that can enhance antimicrobial resistance (AMR) in addition to creating an environment for horizontal transfer of antibiotic resistance genes. The role of the efflux pump in biofilm formation is important for studies on alternative treatments for biofilms. One of the significant efflux pump families is the RND efflux pump family, which is common in Gram negative bacteria. The aim is to study the role of the RND efflux pump in biofilm formation by A. baumannii. The biofilm formation potential of thirty-four MDR A. baumannii isolates was evaluated by crystal violet assays. The effect of efflux pump inhibition and activation was studied using the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the RND efflux pump substrate levofloxacin (at sub-MIC), respectively. The isolates were genotypically grouped by enterobacterial repetitive intergenic consensus (ERIC) typing and the expression of adeABC, adeFGH, and adeIJK efflux pump genes was measured by qPCR. Overall, 88.2% (30/34) of isolates were biofilm producers (the phenotype was variable including strong and weak producers). Efflux pump inhibition by CCCP reduced the biofilm formation significantly (p < 0.05) in 17.6% (6/34) of some isolates, whereas sub-MICs of the substrate levofloxacin increased biofilm formation in 20.5% (7/34) of other isolates. Overexpression of the three RND efflux pump genes was detected in five out of eleven selected isolates for qPCR with remarkable overexpression in the adeJ gene. No correlation was detected between the biofilm phenotype pattern and the RND efflux pump gene expression in biofilm cells relative to planktonic cells. In conclusion, the role of the RND efflux pumps AdeABC, AdeFGH, and AdeIJK in biofilm formation does not appear to be pivotal and the expression differs according to the genetic background of each strain. Thus, these pumps may not be a promising target for biofilm inhibition.

Role of Efflux Pumps in Reduced Susceptibility to Tigecycline among Clinical Isolates of Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) is a very well-known emerging pathogen and has become a major burden on healthcare system especially in intensive care units (ICUs). Tigecycline is the last resort drug for treatment of multidrug-resistant A. baumannii infections. However, non-susceptibility to this drug is a rising problem. Resistance to tigecycline is mediated by Resistance-nodulation-cell division (RND) efflux pumps. This study was done to detect efflux pump genes (adeABC) and regulator genes (adeS,adeR) responsible for tigecycline resistance among the clinical isolates of A. baumannii. A total of 150 OXA-51 confirmed clinical isolates were subjected for tigecycline susceptibility test by broth microdilution (BMD) method. All isolates irrespective of their MIC were subjected to conventional PCR for detection of efflux genes (adeABC) and regulator genes (adeRS). Prevalence of tigecycline resistance was found to be 14 (9.33%) by the reference broth microdilution method (BMD). Overall prevalence of efflux genes was highest for adeB (69%) and lowest for adeR (29%). Combination of genes especially three, four or five were found more prevalent among resistant isolates with higher minimum inhibitory concentration (MIC). Combination of efflux genes confer higher MIC and can be a major contributor for resistance to tigecycline.

The Role of Efflux Pumps in the Antibiotic Resistance of Campylobacter spp. Isolated From Domestic Animals and Poultry

Background: Recently, the rate of antibiotic resistance of Campylobacter has been reported to be increasing and the mechanism of this resistance has been reported to be related to the activity of efflux pumps. The purpose of this study was to isolate Campylobacter strains from domestic animals such as poultry and cows and evaluate the role of efflux pumps in antibiotic resistance property of them. Methods: A total of 300 fecal samples were collected from poultry and cows and subjected to isolation of Campylobacter by preT-KB method. The isolates were identified and confirmed by phenotypic and genotypic methods and their antibiotic susceptibility was evaluated using the disk diffusion method. Efflux pump activity in the isolates was assessed by EtBr-agar cartwheel method and the presence of efflux pump cmeABC was evaluated in all isolates. Finally, the correlation between efflux pump activity and antibiotic resistance was evaluated in the isolates using inhibition of efflux pump activity of Phe-Arg β-naphthylamide. Results: Of all samples, 10 (3.3%) Campylobacter strains were isolated. Seven (70%) and three (30%) strains were isolated from poultry and cows, respectively. Of all isolates, 9 belonged to Campylobacter jejuni and 1 belonged to Campylobacter coli. The isolates were resistant to three antibiotics, namely Ciprofloxacin, Ceftriaxone, and Cefotaxime. Efflux pump activity was observed in all isolates; however, cmeABC genes were not present in all of them. In addition, resistance to Erythromycin and Ciprofloxacin was associated with efflux pump activity. Conclusions: All Campylobacter isolates in the current study showed antibiotic resistance and the activity of efflux pumps could induce antibiotic resistance and decrease the antibacterial activity of many drug families in Campylobacter. In addition, the activity of efflux pumps can be considered a mechanism of antibiotic resistance and elimination of this activity might increase the effectiveness of antibiotics.

Rv1258c acts as a drug efflux pump and growth controlling factor in Mycobacterium tuberculosis

The possible role of efflux pump as a survival mechanism in Mycobacterium tuberculosis (M. tb) is gaining an increasing attention. Previously, Rv1258c (Tap) and its certain mutations confer the clinically relevant drug resistance. In this study, we found new mutations of Rv1258c in G195C, T297P and I328T. Effect of modulating T297P and I328T on the drug resistance by knockout and complement in M. tb H37Rv showed that M. tb ΔRv1258c showed a slightly lower MIC for rifampin, ethambutol, ofloxacin, amikacin, capreomycin and streptomycin than M. tb H37Rv WT and the complement. Rv1258c T297P and Rv1258c I328T showed an increased drug resistance to ethambutol and capreomycin than the complement of Rv1258c WT. Most importantly, M. tb ΔRv1258c exhibited a slow growth in the normal culture medium. TMT-based quantitative proteomics analysis of M. tb ΔRv1258c and WT showed that the knockout of Rv1258c greatly down-regulated the expression of the ribosome system and one of the special five type VII secretion systems, ESX-3, which impaired the bacterial growth. These results indicate that the newly found T297P and I328T mutations of Rv1258c contributed to an increased resistance to ethambutol and capreomycin, and Rv1258c as growth controlling factor influencing the growth of M. tb.

The Future of Bacteriophage Therapy Will Promote Antimicrobial Susceptibility.

ABSTRACTRising antimicrobial resistance severely limits efforts to treat infections and is a cause for critical concern. Renewed interest in bacteriophage therapy has advanced understanding of the breadth of species capable of targeting bacterial antimicrobial resistance mechanisms, but many questions concerning ideal application remain unanswered. The following minireview examines bacterial resistance mechanisms, the current state of bacteriophage therapy, and how bacteriophage therapy can augment strategies to combat resistance with a focus on the clinically relevant bacterium Pseudomonas aeruginosa, as well as the role of efflux pumps in antimicrobial resistance. Methods to prevent antimicrobial efflux using efflux pump inhibitors and phage steering, a type of bacteriophage therapy, are also covered. The evolutionary context underlying antimicrobial resistance and the need to include theory in the ongoing development of bacteriophage therapy are also discussed.

The impact and fate of clarithromycin in anaerobic digestion of waste activated sludge for biogas production

Clarithromycin retained in waste activated sludge (WAS) inevitably enters the anaerobic digestion system. So far, the complex impacts and fate of clarithromycin in continuous operated WAS anaerobic digestion system are still unclear. In this study, two semi-continuous long-term reactors were set up to investigate the effect of clarithromycin on biogas production and antibiotic resistance genes (ARGs) during WAS anaerobic digestion, and a batch test was carried out to explore the potential metabolic mechanism. Experimental results showed that clarithromycin at lower concentrations (i.e., 0.1 and 1.0 mg/L) did not affect biogas production, whereas the decrease in biogas production was observed when the concentration of clarithromycin was further increased to 10 mg/L. Correspondingly, the relative abundance of functional bacteria in WAS anaerobic digestion (i.e., Anaerolineaceae and Microtrichales) was reduced with long-term clarithromycin exposure. The investigation of ARGs suggested that the effect of methylation belonging to the target site modification played a critical role for the anaerobic microorganisms in the expression of antibiotic resistance, and ermF, played dominated ARGs, presented the most remarkable proliferation. In comparison, the role of efflux pump was weakened with a significant decrease of two detected efflux genes. During WAS anaerobic digestion, clarithromycin could be partially degraded into metabolites with lower antimicrobial activity including oleandomycin and 5-O-desosaminyl-6-O-methylerythronolide and other metabolites without antimicrobial activity.

In-depth analysis of multi- and extensively drug-resistant mycobacterium tuberculosis isolates in the capital of iran

Aims and objectives: The emergence and spread of multidrug resistant (MDR) Mycobacterium tuberculosis (M. tuberculosis) strains is a critical global health problem. In order to shorten the course of treatment and its effectiveness, it is essential to gain an in-depth insight into the genetic diversity, drug resistance mechanisms and host-pathogen interactions of drug resistant M. tuberculosis. Methods: Between 2014 and 2018, 606 MTBC strains were isolated from 13,892 suspected pulmonary tuberculosis (TB) patients in Tehran, Iran, including 16 MDR-TB cases. A combination of phenotypic (conventional drug susceptibility testing, MIC, Verapamil effectiveness) and genotypic methods (whole-genome sequencing and qPCR) was employed for the identification of additional drug resistances, the role of efflux pumps and strain-to-strain genetic distances as a marker for recent transmission events. Finally, the consequences of mutations in genes encoding the beta subunit of RNA polymerase (rpoB) for host-pathogen interactions was investigated by means of THP-1-derived macrophages infection and Cytokines and Chemokines Real-Time RT2 Profiler PCR Array. Results: MDR and extensively drug-resistant (XDR) TB cases were almost exclusively infected by lineage 2/Beijing strains (14/16, P &lt; 0.001). Out of the 16 MDR/pre-XDR/XDR isolates, 6 (37.5%) and 3 (18.8%) isolates demonstrated a significant decrease in rifampicin (RIF) MIC and isoniazid (INH) MIC due to the Verapamil exposure (64 μg/mL), respectively. More importantly, it was found that the administration of Verapamil profoundly decreased the MIC of Bedaquiline (BDQ) among most of the studied clinical isolates. Moreover, the efflux pump genes of Rv2938, Rv2936, Rv1145, Rv1146, Rv933, Rv1250, Rv876, Rv2333, Rv2459, Rv849, and Rv1819 were overexpressed in the presence of anti-TB drugs, showing the contribution of these efflux pumps to the overall resistance phenotype. Finally, the macrophages immune responses induced in response to infection with drug-resistant M. tuberculosis (RMR strain) may not be substantially different from drug susceptible M. tuberculosis (H37Rv strain), but the severity of the responses was different in some cases specially IL-6, IL-8, CNTF, IL-13, TNFSF10 and CD40LG in response to RMR and H37Rv strains. In addition, RMR strain showing significantly higher intracellular bacillary growth compared to H37Rv strain. Conclusions: The crucial finding is that almost all of our MDR/pre-XDR/XDR strains are of the Beijing lineage (14/16) and more than half of them are probably from recent transmission (8/14). Our results clearly showed that efflux systems, besides spontaneous mutations, play a role in the development of INH/RIF resistance. In addition, Verapamil was very successful at the phenotypic level specially in response to BDQ. Finally, the results initially demonstrate that the M. tuberculosis carrying the rpoB-S450L can modulate macrophage responses to mediate bacterial survival.

The Role of Efflux Pumps in the Transition from Low-Level to Clinical Antibiotic Resistance.

Antibiotic resistance is on the rise and has become one of the biggest public health challenges of our time. Bacteria are able to adapt to the selective pressure exerted by antibiotics in numerous ways, including the (over)expression of efflux pumps, which represents an ancient bacterial defense mechanism. Several studies show that overexpression of efflux pumps rarely provides clinical resistance but contributes to a low-level resistance, which allows the bacteria to persist at the infection site. Furthermore, recent studies show that efflux pumps, apart from pumping out toxic substances, are also linked to persister formation and increased spontaneous mutation rates, both of which could aid persistence at the infection site. Surviving at the infection site provides the low-level-resistant population an opportunity to evolve by acquiring secondary mutations in antibiotic target genes, resulting in clinical resistance to the treating antibiotic. Thus, this emphasizes the importance and challenge for clinicians to be able to monitor overexpression of efflux pumps before low-level resistance develops to clinical resistance. One possible treatment option could be an efflux pump-targeted approach using efflux pump inhibitors.

Alterations in AdeS and AdeR regulatory proteins in 1-(1-naphthylmethyl)-piperazine responsive colistin resistance of Acinetobacter baumannii

Colistin resistant Acinetobacter baumannii strains are of great concern worldwide. However, the role of efflux pumps in colistin resistance needs to be elucidated. We investigated the changes in colistin MICs of 29 colistin resistant A. baumannii isolates in response to resistance-nodulation-division (RND)-type efflux pump inhibitor (EPI) and the alterations in AdeR and AdeS two-component regulatory proteins previously associated with the overproduction of AdeAB. The EPI, 1-(1-naphthylmethyl)-piperazine (NMP), led to significant reductions in colistin MICs. At least one of the following amino acid substitutions was found in AdeS proteins from 18 of the isolates: L172P, A94V, V27I, V32I, G186V, and G164A. Besides, A136V and V120I alterations were identified in AdeR from five isolates. Therefore, EPI-responsive colistin resistance in our isolates is most likely due to the action of an RND-type efflux system. The underlying mechanism of resistance might be the result of certain AdeRS alterations, leading to AdeAB overexpression.

Tetracycline resistance mediated by tet efflux pumps in clinical isolates of Acinetobacter baumannii.

ABSTRACT Acinetobacter baumannii is one of the most frequent nosocomial pathogen capable of acquiring resistance to different antimicrobials. The aim of this study was to investigate the activity of tetracycline, doxycycline and minocycline, the prevalence of tet(A) and tet(B) determinants, and the role of efflux pump in tetracycline resistance among the A. baumannii clinical isolates. Susceptibility of 98 A. baumannii isolates to tetracyclines was evaluated by disk diffusion method. The presence of active efflux pump was investigated by determination of the minimum inhibitory concentration (MIC) of tetracycline using the carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Polymerase chain reaction (PCR) was performed to investigate the presence of tet(A) and tet(B) determinants in tetracycline-resistant isolates. The rate of resistance to tetracycline, doxycycline and minocycline was 47.95%, 0%, and 30.61%, respectively. Among the 47 tetracycline-resistant isolates, 29.79% were originated from burned patients and showed MIC ranging from 128-256 μg/mL with both MIC 50 and MIC90 values of 256 μg/mL, while 70.21% were from ventilator-associated pneumonia (VAP) patients and had MIC values ranging from 32-1024 μg/mL, with MIC50 and MIC90 of 512 μg/mL and 1024 μg/mL, respectively. The tet(B) gene was found in 61.7% of tetracycline-resistant isolates, while none of the isolates carried the tet(A) gene. CCCP led to 2-128-fold reduction in tetracycline MIC of the tested isolates. The results showed that doxycycline and minocycline are promising agents for the treatment of A. baumannii infections. This study has also revealed the role of efflux activity in the resistance to tetracycline of A. baumannii isolates. The emergence of resistance to these agents is likely due to the spread of clones presenting with a higher prevalence of resistance determinants.

Are Drug Efflux Genes Present among &amp;lt;i&amp;gt;Mycobacterium tuberculosis&amp;lt;/i&amp;gt; Isolates from Patients in Lagos, Nigeria?

A major challenge in the treatment of Tuberculosis (TB) is emergence of Multi-Drug Resistant Mycobacterium tuberculosis (MDRTB) strains. Efflux genes have been established to be among factors for drug resistance in Mycobacterium tuberculosis (M. tuberculosis) pulmonary infections by conferring bacterial ability to pump-out drugs from intracellular compartment, making it impossible for drugs to attain intracellular concentration lethal to the organism. There is paucity of data on the role of efflux pump in MDRTB in Nigerian strains of M. tuberculosis. Hence, the aim of this study was to detect the carriage, distribution and frequency of efflux pump genes among MDRTB and non-MDRTB isolates from participants with pulmonary tuberculosis in Lagos, Nigeria. This study was carried out on M. tuberculosis isolated from 1020 participants suspected of pulmonary tuberculosis in Lagos State, Nigeria. A total of 78 M. tuberculosis isolates were obtained from the participants suspected of pulmonary tuberculosis. Forty Eight isolates were confirmed as MDRTB and 30 non-MDRTB. Efflux pump genes were investigated in the isolates using the conventional polymerase chain reaction. Statistical analysis was carried out using the Statistical Package for Social Science (SPSS version 20) to compare the efflux pump gene results between MDRTB and non- MDRTB isolates. Different efflux genes types and frequency were detected in MDRTB and non-MDRTB isolates. Carriage of 2 or more alleles of efflux gene types Rv2486c (efpA), Rv2459c (jefA), Rv1877, Rv1002c, Rv0342, Rv2686c and drrC associated with MDR were detected. Additionally, the frequency of efflux genes alleles in MDRTB was significantly different from those in non- MDRTB isolates.

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa

The Role of Efflux Pumps and Environmental pH in Bacterial Multidrug Resistance.

One of the most studied bacterial resistance mechanisms is the resistance related to multidrug efflux pumps. In our study the pump activity of the Escherichia coli K-12 AG100 strain expressing the AcrAB-TolC pump system was investigated at pH 7 and pH 5 in the presence of the efflux pump inhibitor (EPI) promethazine (PMZ). The EPI activity was assessed by real-time fluorimetry. The influence of PMZ treatment on the relative expression of the pump genes acrA, acrB and their regulators marA, marB, marR, the stress genes soxS, rob, as well as the bacterial growth control genes ftsI, and sdiA were determined by RT-qPCR. The EPI activity of PMZ was more effective at neutral pH. The PMZ treatment induced a significant stress response in the bacterium at acidic pH by the up-regulation of genes. The genetic system that regulates the activity of the main efflux pump is pH-dependent.

Mutation rate and efflux response of bacteria exposed to a novel antimicrobial iodo-thiocyanate complex

ObjectivesAntimicrobials, at sub-lethal concentrations, can act as selectors and promoters of resistance by increasing mutation rates. We measured the rate of Escherichia coli mutation from levofloxacin (LVX) sensitivity to resistance when it was grown under the near-lethal challenge of the novel biocidal iodo-thiocyanate complex (ITC). Another relevant factor affecting the emergence of antimicrobial resistance is the role of efflux pumps. Consequently, we evaluated whether ITC could potentially be a substrate for efflux pumps, and thus that efflux-mediated resistance could arise towards ITC. MethodsThe mutation rate was measured by fluctuation analysis, when multiple parallel E. coli cultures were grown in the absence and presence of ITC. Then the mutational events, which occurred independently in each culture, were scored by plating the fraction of the culture in LVX-selective solid media and compared with the total cell number. To detect if ITC is a substrate for efflux pumps, minimum inhibitory concentrations (MICs) were determined against Pseudomonas aeruginosa in the absence and presence of the efflux pump inhibitor (EPI). ResultsWe have found that the E. coli exposed to the near-lethal level of ITC had a slight, but not significant, increase in mutation rate compared with unexposed cultures. Furthermore, the presence of EPI decreased the MIC of ITC by a modest 2-fold, showing that ITC was not a target for efflux pumps. ConclusionsITC usage most likely will not promote resistance development via increased mutation rates, and efflux-mediated resistance emergence to it is less likely than for some other antimicrobials.

EFFLUX MEDIATED MULTIDRUG RESISTANT PSEUDOMONAS AERUGINOSA ISOLATED FROM DIFFERENT ENVIRONMENTAL SOURCES

Pseudomonas aeruginosa is an important opportunistic pathogen and one of the leading causes of multi-drug resistant nosocomial infections. This study was therefore carried out to determine the resistance nature, and the role of efflux pump in multidrug resistance of Pseudomonas aeruginosa isolated from different environmental sources using the efflux pump inhibitor, Carbonyl Cyanide 3-Chlorophenylhydrazone (CCCP). A total of 220 environmental samples were collected and processed following standard techniques. Susceptibility to antibiotics was performed using disc diffusion methods as described by the Clinical and Laboratory Standards Institute. Activity of the efflux pump system was carried out using the efflux pump inhibitor, CCCP. Results obtained identified 100 (45.5%) Pseudomonas aeruginosa and 72 (32.7%) other strains of Pseudomonas spp. The susceptibility testing revealed that all the identified strains of Pseudomonas aeruginosa that were subjected to susceptibility test were significantly resistant to ampicillin and cefotaxime, But the resistance profile of isolates to tetracycline, chloramphenicol, ceftriaxone, cefuroxime and perfloxacin were 93%, 72.1%, 79.1%, 58.1% and 51.2% respectively. However, imipenem was the most sensitive (100%), followed by cefepime (65%) and gentamicin (44%). Carbonyl Cyanide 3-Chlorophenylhydrazone decreased the minimum inhibitory concentration (MIC) of the isolates by 2 folds. Results obtained have shown the ubiquitous presence of multi-drug resistant P. aeruginosa from the environmental samples examined. Furthermore, it indicated the role of efflux pump in antibiotics resistance in P. aeruginosa isolates which indicate that P. aeruginosa strains from environmental sources could resist antibiotics by the efflux mechanism. &#x0D; &#x0D;