Resistance Efflux Pumps Research Articles

Early transcriptional changes of heavy metal resistance and multiple efflux genes in Xanthomonas campestris pv. campestris under copper and heavy metal ion stress

BackgroundCopper-induced gene expression in Xanthomonas campestris pv. campestris (Xcc) is typically evaluated using targeted approaches involving qPCR. The global response to copper stress in Xcc and resistance to metal induced damage is not well understood. However, homologs of heavy metal efflux genes from the related Stenotrophomonas genus are found in Xanthomonas which suggests that metal related efflux may also be present.Methods and ResultsGene expression in Xcc strain BrA1 exposed to 0.8 mM CuSO4.5H2O for 15 minutes was captured using RNA-seq analysis. Changes in expression was noted for genes related to general stress responses and oxidoreductases, biofilm formation, protein folding chaperones, heat-shock proteins, membrane lipid profile, multiple drug and efflux (MDR) transporters, and DNA repair were documented. At this timepoint only the cohL (copper homeostasis/tolerance) gene was upregulated as well as a chromosomal czcCBA efflux operon. An additional screen up to 4 hrs using qPCR was conducted using a wider range of heavy metals. Target genes included a cop-containing heavy metal resistance island and putative metal efflux genes. Several efflux pumps, including a copper resistance associated homolog from S. maltophilia, were upregulated under toxic copper stress. However, these pumps were also upregulated in response to other toxic heavy metals. Additionally, the temporal expression of the coh and cop operons was also observed, demonstrating co-expression of tolerance responses and later activation of part of the cop operon.ConclusionsOverall, initial transcriptional responses focused on combating oxidative stress, mitigating protein damage and potentially increasing resistance to heavy metals and other biocides. A putative copper responsive efflux gene and others which might play a role in broader heavy metal resistance were also identified. Furthermore, the expression patterns of the cop operon in conjunction with other copper responsive genes allowed for a better understanding of the fate of copper ions in Xanthomonas. This work provides useful evidence for further evaluating MDR and other efflux pumps in metal-specific homeostasis and tolerance phenotypes in the Xanthomonas genus. Furthermore, non-canonical copper tolerance and resistance efflux pumps were potentially identified. These findings have implications for interpreting MIC differences among strains with homologous copLAB resistance genes, understanding survival under copper stress, and resistance in disease management.

Comparative analysis of antimicrobial resistance phenotype and genotype of Riemerella anatipestifer

Riemerella anatipestifer is one of the important bacterial pathogens that threaten the waterfowl farming industry. In this study, 157 suspected R. anatipestifer strains were isolated from diseased ducks and geese from seven regions of China during 2019–2020, and identified using multiple polymerase chain reaction (PCR). Antimicrobial susceptibility tests and whole-genome sequence (WGS) analysis were then performed for comparative analysis of antimicrobial resistance phenotypes and genotypes. The results showed that these strains were susceptible to florfenicol, ceftriaxone, spectinomycin, sulfafurazole and cefepime, but resistant to kanamycin, amikacin, gentamicin, and streptomycin, exhibiting multiple antimicrobial resistance phenotypes. WGS analysis revealed a wide distribution of genotypes among the 157 strains with no apparent regional pattern. Through next-generation sequencing analysis of antimicrobial resistance genes, a total of 88 resistance genes were identified. Of them, 19 tetracycline resistance genes were most commonly found, followed by 15 efflux pump resistance genes, 11 glycopeptide resistance genes and seven macrolide resistance genes. The 157 R. anatipestifer strains contained 42–55 resistance genes each, with the strains carrying 47 different resistance genes being the most abundant. By comparing the antimicrobial resistance phenotype and genotype, it was observed that a high correlation between them for most antimicrobial resistance properties was detected, except for a difference in aminoglycoside resistance phenotype and genotype. In conclusion, 157 R. anatipestifer strains exhibited severe multiple antimicrobial resistance phenotypes and genotypes, emphasizing the need for improved antimicrobial usage guidelines. The wide distribution and diverse types of resistance genes among these strains provide a foundation for studying novel mechanisms of antimicrobial resistance.

Effect of Selenium Nanoparticles on the Expression of OqxBGene in Clinical Isolates of Klebsiella pneumoniae

Background: OqxB is an efflux pump that has emerged as a factor contributing to antibiotic resistance in Klebsiella pneumoniae. The objective of this study was to investigate the occurrence of AcrAB efflux pump resistance genes in clinical samples of K. pneumoniae and to evaluate the influence of selenium nanoparticles (Se-NPs), loaded with ampicillin, on the expression of the OqxB gene associated with efflux pumps. Methods: A total of 500 clinical samples were collected from hospitalized patients, and 60 strains of K. pneumoniae were isolated using standard microbiological methods. These strains were then analyzed using phenotypic and polymerase chain reaction (PCR) techniques to detect the frequency of KPSM, MRK, OqxA, and OqxB genes through multiplex PCR. The impact of Se-NPs loaded with ampicillin on the expression of the OqxB resistance gene was investigated using a real-time PCR technique. Results: Based on the results of this study, it was found that the KPSM gene is not present in any of the investigated K. pneumoniae isolates. However, the MRK, OqxA, and OqxB genes were detected in 57, 55, and 54 isolates, respectively. The minimum inhibitory concentration (MIC) values for Se-NP and Se-NPs with ampicillin were reported to be 1500 μg/mL and 375 μg/mL, respectively. Notably, the SeNPs with ampicillin could significantly down-regulate the expression of the OqxB gene. These findings demonstrated the potential of Se-NPs as a promising strategy for reducing antibiotic resistance in K. pneumoniae infections. Conclusion: The findings emphasize the notable prevalence of drug resistance genes, specifically those associated with efflux pump production, in clinical samples of K. pneumoniae. Remarkably, the utilization of Se nanoparticles loaded with ampicillin demonstrated its efficacy in suppressing the expression of the OqxB gene and enhancing bacterial susceptibility to ampicillin. The results further imply that Se-NPs could serve as a promising avenue for the development of innovative antibacterial agents, aimed at combating antibiotic resistance in K. pneumoniae infections.

Synthesis, spectroscopic characterization, and antibacterial activity of chalcone (2E)-1-(3'-aminophenyl)-3-(4-dimethylaminophenyl)-prop-2-en-1-one against multiresistant Staphylococcus aureus carrier of efflux pump mechanisms and β-lactamase.

The bacterium Staphylococcus aureus has stood out for presenting a high adaptability, acquiring resistance to multiple drugs. The search for natural or synthetic compounds with antibacterial properties capable of reversing the resistance of S. aureus is the main challenge to be overcome today. Natural products such as chalcones are substances present in the secondary metabolism of plants, presenting important biological activities such as antitumor, antidiabetic, and antimicrobial activity. In this context, the aim of this work was to synthesize the chalcone (2E)-1-(3'-aminophenyl)-3-(4-dimethylaminophenyl)-prop-2-en-1-one with nomenclature CMADMA, confirm its structure by nuclear magnetic resonance (NMR), and evaluate its antibacterial properties. The synthesis methodology used was that of Claisen-Schmidt, and spectroscopic characterization was performed by NMR. For microbiological assays, the broth microdilution methodology was adopted in order to analyze the antibacterial potential of chalcones and to analyze their ability to act as a possible inhibitor of β-lactamase and efflux pump resistance mechanisms, present in S. aureus strain K4100. The results obtained show that CMADMA does not show direct antibacterial activity, expressing a MIC of ≥1024 μg/mL, or on the enzymatic mechanism of β-lactamase; however, when associated with ethidium bromide in efflux pump inhibition assays, CMADMA showed promising activity by reducing the MIC of the bromide from 64 to 32 μg/mL. We conclude that the chalcone synthesized in this study is a promising substance to combat bacterial resistance, possibly acting in the inhibition of the QacC efflux pump present in S. aureus strain K4100, as evidenced by the reduction in the MIC of ethidium bromide.

Drug resistance and physiological roles of RND multidrug efflux pumps in Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa.

Drug efflux pumps transport antimicrobial agents out of bacteria, thereby reducing the intracellular antimicrobial concentration, which is associated with intrinsic and acquired bacterial resistance to these antimicrobials. As genome analysis has advanced, many drug efflux pump genes have been detected in the genomes of bacterial species. In addition to drug resistance, these pumps are involved in various essential physiological functions, such as bacterial adaptation to hostile environments, toxin and metabolite efflux, biofilm formation and quorum sensing. In Gram-negative bacteria, efflux pumps in the resistance–nodulation–division (RND) superfamily play a clinically important role. In this review, we focus on Gram-negative bacteria, including Salmonella enterica , Escherichia coli and Pseudomonas aeruginosa , and discuss the role of RND efflux pumps in drug resistance and physiological functions.

Plant Secondary Metabolites on Efflux-Mediated Antibiotic Resistant Stenotrophomonas Maltophilia: Potential of Herbal-Derived Efflux Pump Inhibitors.

During the process of adapting to metal contamination, plants produce secondary metabolites that have the potential to modulate multidrug-resistant (MDR) phenotypes; this is achieved by inhibiting the activity of efflux pumps to reduce the minimum inhibitory concentrations (MICs) of antimicrobial substrates. Our study evaluated the effect of secondary metabolites of belowground parts of Pteris vittata L. and Fallopia japonica, two metal-tolerant plants from northern Vietnam, on six antibiotic-resistant Stenotrophomonas maltophilia strains possessing efflux pump resistance mechanisms that were isolated from soil and clinical samples. The chemical composition of aqueous and dichloromethane (DCM) fractions extracted from P. vittata and F. japonica was determined using UHPLC-DAD-ESI/QTOF analysis. The antibacterial and efflux pump inhibitory activities of the four fractions were evaluated for the six strains (K279a, 0366, BurA1, BurE1, PierC1, and 502) using a microdilution assay at fraction concentrations of 62.5, 125, and 250 μg/mL. The DCM fraction of F. japonica exhibited remarkable antibacterial activity against strain 0366, with a MIC of 31.25 μg/mL. Furthermore, this fraction also significantly decreased gentamicin MIC: four-fold and eight-fold reductions for BurA1 and BurE1 strains, respectively (when tested at 250 μg/mL), and two-fold and eight-fold reductions for K279a and BurE1 strains, respectively (when tested at 125 μg/mL). Pure emodin, the main component identified in the DCM fraction of F. japonica, and sennidine A&B only reduced by half the MIC of gentamicin (when tested at 30 μg/mL). Our results suggest that the DCM fraction components of F. japonica underground parts may be potential candidates for new bacterial efflux pump inhibitors (EPIs).

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.

Susceptibility Tests and Predictions of Transporter Profile in Serratia Species.

Disinfectants and biosecurity are critically important to control microbial diseases. Resistance to disinfectants compromises sectors such as agriculture and healthcare systems. Currently, efflux pumps are the most common mechanism of antimicrobial resistance. This study aimed to identify the efflux transporters responsible for disinfectant resistance in a multidrug-resistant isolate Serratia sp. HRI compared to a susceptible Serratia sp. type strain. An efflux system profile was generated using the Transporter Automatic Annotation Pipeline (TransAAP) for both isolates. Thereafter, the efflux pump inhibitors, reserpine (RSP) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were used to reveal the role of efflux pumps in susceptibility to three disinfectants (Didecyldimethylammonium chloride, HyperCide®, and benzalkonium chloride). Interestingly, the resistant isolate had fewer efflux systems in total compared to the type strain and fewer efflux systems classified as resistance efflux pumps. After the addition of RSP, a significant reduction in resistance capabilities against all three antimicrobials was observed for both isolates. However, CCCP supplementation produced mixed results with some outcomes suggesting the involvement of the Eagle effect. This study provides evidence that efflux pumps are responsible for the disinfectant resistance phenotype of the Serratia species due to the increased susceptibility when efflux pump inhibitors are added.

In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties.

Lactiplantibacillus pentosus CF2-10 N, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. High throughput sequencing and annotation of genome sequences underline the potential of L. pentosus CF2-10 N as excellent probiotic candidate of vegetable origin. In a previous study we could show the probiotic potential of CF2-10 N in vitro, while in this study in silico analysis of its genome revealed new insights into its safety and functionality. Our findings highlight the microorganism's ecological flexibility and adaptability to a broad range of environmental niches, food matrices and the gastrointestinal tract. These features are shared by both phylogenetically very close L. pentosus strains (CF2-10 N and MP-10) isolated from the same ecological niche with respect to their genome size (≅ 3.6 Mbp), the presence of plasmids (4-5) and several other properties. Nonetheless, additional and unique features are reported in the present study for L. pentosus CF2-10 N. Notably, the safety of L. pentosus CF2-10 N was shown by the absence of virulence determinants and the determination of acquired antibiotic resistance genes, i.e., resistome, which is mostly represented by efflux-pump resistance genes responsible for the intrinsic resistance. On the other hand, defense mechanisms of L. pentosus CF2-10 N include eight prophage regions and a CRISPR/cas system (CRISPR-I and CRISPR-II) as acquired immune system against mobile elements. Finally, the probiotic potential of this strain was further demonstrated by the presence of genes coding for proteins involved in adhesion, exopolysaccharide biosynthesis, tolerance to low pH and bile salts, immunomodulation, and vitamin and enzyme production. Taken together these results, we propose the use of L. pentosus CF2-10 N as a potential and promising probiotic candidate able to colonize several niches and adapt to different lifestyles. The strain can provide attractive functional and probiotic features necessary for its application as starter culture and probiotic.

Emergence of the resistance-nodulation-division efflux pump tmexCD3-toprJ3 gene confers resistance to tigecycline in Pseudomonas juntendi and Proteus terrae isolated from a pig farm in China.

Tigecycline is regarded as a last-resort antimicrobial agent against multidrug resistance (MDR). However, resistance-nodulation-division efflux pump resistance genes, such as tmexCD3-toprJ3, that confer resistance to tigecycline have emerged. This study aimed to characterise the tmexCD3-toprJ3 gene cluster in Pseudomonas juntendi and Proteus terrae isolated from a pig farm. Samples were obtained from a Chinese piggery and included 92 pig faecal samples, 56 wastewater samples, 23 drinking water samples, 28 sow vagina samples and nine sow uterus swabs. The presence of tmexCD3-toprJ3 was detected using a polymerase chain reaction assay, and the antimicrobial susceptibility profile of the tmexCD3-toprJ3-positive isolates was determined using the broth dilution method. Genomic locations were identified using whole-genome sequencing and bioinformatics. We identified two tmexCD3-toprJ3-positive isolates, and both isolates were highly resistant to antibiotics such as tigecycline. In addition, we identified several mobile elements (ISPa7, ISCfr1, ISVsa3) and insertion sequences (TnAs2, TnAs3) in tmexCD3-toprJ3-positive isolates that could increase the potential for the spread of MDR. To the best of our knowledge, this study is the first to report the detection of tmexCD3-toprJ3 in P. juntendi and P. terrae isolated from a pig farm.

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.

Synthesis, antibiotic modifying activity, ADMET study and molecular docking of chalcone (E)-3-(2,4-dichlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one in strains of Staphylococcus aureus carrying MepA efflux pumps.

The Staphylococcus aureus bacteria is a Gram-positive, immobile, non-spore bacterium, with catalase and positive coagulase, among other characteristics. It is responsible for important infections caused in the population and for hospital infections. Because of that many strategies are being developed to combat the resistance of microorganisms to drugs, in recent times, chalcones have been studied for this purpose. Chalcones are found in parts of plants and can be found, for example, in the roots, leaves, bark, among others, but are mainly found as petal pigments, they are a class of compounds considered an exceptional model due to chemical simplicity and a wide variety of biological activities. This study aimed to evaluate the ability of chalcone (E)-3-(2,4-dichlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one to reverse the efflux pump resistance, present in the bacteria S. aureus 1199B and S. aureus K2068. The synthetic chalcone (E)-3-(2,4-dichlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one was able to synergistically modulate the antibiotic Ciprofloxacino and Ethidium Bromide against the bacterial strain S. aureus K2068, and with the antibiotic Norfloxacino against the strain 1199B. Thus, it is suggested that this chalcone may be acting by inhibiting the efflux pump mechanism of these bactéria. The theoretical physicochemical and pharmacokinetic properties of chalcone showed that the chalocne did not present a severe risk of toxicity, such as genetic mutation or cardiotoxicity. Molecular docking showed that the chalcone could act as a competitive inhibitor of the MepA efflux pump, as at hinders the binding of other substrates, such as EtBr.

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

ABSTRACTStaphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations.IMPORTANCE 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. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Molecular detection of gyrA and mexA genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the types of bacteria that arises resistance toward fluoroquinolos antibiotics remarkably in recent years. Fifty P. aeruginosa isolates were isolated from one hundred clinical samples, investigated the antibiogram activity toward eight different groups of antibiotics. Screening about gyrA gene was done by conventional PCR further more qualitative gene expression of mexA gene was done by using Real-time PCR in 22 MDR isolates, furthermore Relative gene expression analysis of gyrA and mexA was done. The rate of P. aeruginosa isolates was (41.6%) from total clinical samples, the antibiogram test showed high resistance toward Ceftazidime, Ciprofloxacin, Levofloxacin and Gentamicin (100%), while the sensitivity was observed towards colistin (100%). Screening of gyrA that was achieved by PCR technique showed 22 positive isolates. Furthermore, the 22 isolates appeared high expression level of the efflux pump resistance gene mexA and gyrA gene compared with housekeeping gene rspL gene within fold change ranging (0.18-36 and 1-28.84 respectively) with a mean of 18.46 ct and 18.59 (respectively). All P. aeruginosa isolates were MDR with high level of efflux pump expression of mexA gene as well as gyrA gene.

Upregulation of abeM, amvA, and qacEΔ1 efflux pump genes associated with resistance of Acinetobacter baumannii strains to disinfectants.

Background and aimsAcinetobacter baumannii is among the most concerning cause of nosocomial infections due to its high level of antibiotic resistance and high mortality. The aim of this study was to determine the role of efflux pumps in resistance of A. baumannii strains to three disinfectants, including MICROZED ID‐MAX, NANOSIL D2, and OPIDEX OPA.MethodsTwenty‐eight environmental and clinical isolates of A. baumannii were collected from selected hospitals of central Iran. The minimum inhibitory concentrations of the disinfectants were determined and real time reverse transcriptase‐PCR was performed to investigate the expression level of qacEΔ1, amvA, abeM, and adeB efflux pump genes.ResultsConsidering both clinical and environmental isolates, there was a significant difference in the mean expression level of qacEΔ1 gene between susceptible and resistant strains to MICROZED ID‐MAX disinfectant, of amvA and abeM genes between susceptible and resistant strains to NANOSIL D2 disinfectant and of abeM gene in susceptible and resistant strains to OPIDEX OPA disinfectant (all P ˂ .05). The expression levels of abeM and amvA genes were higher in the environmental isolates that were resistant to NANOSIL D2 disinfectant compared to those that were susceptible (P ˂ .05).ConclusionsThis study provided evidence for the role of abeM and amvA genes in the resistance of environmental isolates to disinfectants, particularly hydrogen peroxide derivatives.

MICROARRAY-BASED ANALYSIS OF STAPHYLOCOCCUS AUREUS ISOLATES FROM NON-CLINICAL SOURCE

This study was aimed to genotyped virulence, toxin and resistance genes of 30 S. aureus isolates from pigs using DNA microarray. The 30 isolates analyzed in this study all belonged to clonal complexes (CC) CC1 (3; 10%), CC5 (3; 10%), CC15 (16; 53.33%) and CC152 (8; 26.66%), respectively. Of antibiotic resistance-associated genes reported; Twenty out of 30 isolates (66.66%) carried the beta-lactamase operon (blaZ/I/R) and the tetracycline resistance gene (tetK), respectively. Also, the macrolide resistance gene (ermA/msrA) were detected in ten isolates (33.33%), the multidrug efflux pump resistance gene (sdrM) was detected in seven isolates (23.33%) and one isolates (3.33%) harboured the chloramphenicol resistance gene (cat). None of the isolates harboured genes conferring methicillin resistance. In terms of genes encoding enterotoxins; seb was detected among all isolates of CC15 while the enterotoxin gene cluster egc and a distinct variant of the enterotoxin A (sea-N315) were detected among isolates of CC5 and CC152, respectively. Two CC152 isolate was positive for genes encoding the Panton-Valentine leukocidin (lukF-PV+lukS-PV). In addition, all the isolates harboured the genes encoding for intracellular adhesion proteins (icaA/C/D) and the biofilm-associated genes (clfA and clfB) except for CC1 isolates. The capsular genes cap5 and cap8 were detected in isolates of CC5 and CC15 while only cap5 was present in CC152 isolates. The study provides detailed genotyping data on the population structure, presence of toxins and antibiotic resistance markers of S. aureus isolates and indicates the importance of the microarray technique in analyzing multiple gene contents of bacteria.

Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

BackgroundThis study was aimed to characterize the genetic diversity and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii.MethodsAntimicrobial susceptibility testing of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by the broth microdilution method. Moreover, the MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of the RND efflux pumps (adeRS, adeL, adeN and baeSR) were subjected to sequencing. The relative expression of adeB, adeG and adeJ genes was determined by quantitative real-time PCR (qRT-PCR).ResultsOverall, the majority of isolates (94%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of the isolates against multiple antibiotics mainly tigecycline. However, we found no efflux activity against imipenem. Several amino acid substitutions were detected in the products of regulatory genes; except in AdeN. Of note, G186V mutation in AdeS was found to be associated with overexpression of its efflux pump. No insertion sequences were detected.ConclusionsOur findings outlined the role of RND efflux pumps in resistance of A. baumannii to multiple antibiotics particularly tigecycline, and pointed out the importance of a variety of single mutations in the corresponding regulatory systems. Further studies are required to decipher the precise role of RND efflux pumps in multidrug-resistant clinical isolates of A. baumannii.

Detection of multidrug resistant environmental isolates of acinetobacter and Stenotrophomonas maltophilia: a possible threat for community acquired infections?

Acinetobacter spp. and Stenotrophomonas maltophilia are bacteria commonly associated with infections at the clinical settings. Reports of infections caused by environmental isolates are rare. Therefore, this study focused on determination of the antibiotic resistance patterns, antibiotic resistance genes, efflux pumps and virulence signatures of Acinetobacter spp. and S. maltophilia recovered from river water, plant rhizosphere and river sediment samples. The isolates were identified and confirmed using biochemical tests and PCR. The antimicrobial resistance profiles of the isolates were determined using Kirby Bauer disk diffusion assay and presence of antibiotic resistance and virulence genes were detected using PCR. S. maltophilia was more frequent in plant rhizosphere and sediment samples than the water samples. Acinetobacter spp. were mostly resistant to trimethoprim-sulfamethoxazole (96% of isolates), followed by polymyxin b (86%), cefixime (54%), colistin (42%), ampicillin (35%) and meropenem (19%). The S. maltophilia isolates displayed total resistance (100%) to trimethoprim- sulfamethoxazole, meropenem, imipenem, ampicillin and cefixime, while 80% of the isolates were resistant to ceftazidime. Acinetobacter spp. contained different antibiotic resistance genes such as sul1 (24% of isolates), sul2 (29%), blaOXA 23/51 (21%) and blaTEM (29%), while S. maltophilia harbored sul1 (8%) and blaTEM (20%). Additionally, efflux pump genes were present in all S. maltophilia isolates. The presence of multidrug resistant Acinetobacter spp. and Stenotrophomonas maltophilia in surface water raises concerns for community-acquired infections as this water is directly been used by the community for various purposes. Therefore, there is the need to institute measures aimed at reducing the risks of these infections and the resulting burden this may have on the health care system within the study area.

Molecular Detection and Quantification Gene Expression of Efflux Pump Antibiotic Resistance Genes in Extensive Drug Resistance Pseudomonas Aeruginosa Isolated from Clinical Infection Patients in Al Diwaniyah City of Iraq

The study was included investigated 16 extensive drug resistance pseudomonas aeruginosa isolateswhich previously isolated from sputum, burns, urine, and ear swab clinical patients samples. The reversetranscription Real Time PCR (RT-qPCR) was performed for molecular detection and quantification of geneexpression levels for efflux pump antibiotic resistance gene (mexA, mexB, mexC, mexE, and mexY) andchromosomal lactamase (AmpC) gene, as well as outer membrane prion protein (OprD) gene in extensivedrug pseudomonas aeruginosa isolates. The relative expression analysis was done by used the 2-??CT (Livak)method, this method was used for normalization of expression levels (mRNA transcript levels) in targetgenes with suitable housekeeping gene. In this study 30S ribosomal protein S12 (rpsL) housekeeping genewas used in normalization of antibiotic resistance genes expression levels in extensive drug pseudomonasaeruginosa isolates. The efflux pump resistance gene expression were by RT-qPCR technique. The relativegene expression analysis were showed highest frequency of overexpression at (100%, 100%, 87.5%,81.25%, 56.25%) for (mexB, mexE, mexA,mexC, and mexY) with fold change ranged (2.16- 184.82) foldchange compared to wild type PA control isolate. The relative gene expression analysis of chromosomallactamase AmpC gene was showed highest frequency of overexpression (100%) in XDR isolates withexpression levels ranged (6.77- 73.1) fold change compared to wild type PA control isolate. Whereas, therelative gene expression analysis of OprD gene was showed (68.75%) frequency of down expression inXDR Pseudomonas aeruginosa isolates with expression levels ranged (0.01-0.16) fold change compared towild type PA control isolate. In conclusion, The XDR Pseudomonas aeruginosa isolates can ability to overexpression production of inducible chromosomal lactamase AmpC gene, efflux pumps protein genes, andlow expression outer membrane prion protein (OprD) gene as important antimicrobial mechanism againstCephalosporins and Carbapenems antibiotic classes.

Lactobacillus spp. attenuate antibiotic-induced immune and microbiota dysregulation in honey bees

Widespread antibiotic usage in apiculture contributes substantially to the global dissemination of antimicrobial resistance and has the potential to negatively influence bacterial symbionts of honey bees (Apis mellifera). Here, we show that routine antibiotic administration with oxytetracycline selectively increased tetB (efflux pump resistance gene) abundance in the gut microbiota of adult workers while concurrently depleting several key symbionts known to regulate immune function and nutrient metabolism such as Frischella perrera and Lactobacillus Firm-5 strains. These microbial changes were functionally characterized by decreased capped brood counts (marker of hive nutritional status and productivity) and reduced antimicrobial capacity of adult hemolymph (indicator of immune competence). Importantly, combination therapy with three immunostimulatory Lactobacillus strains could mitigate antibiotic-associated microbiota dysbiosis and immune deficits in adult workers, as well as maximize the intended benefit of oxytetracycline by suppressing larval pathogen loads to near-undetectable levels. We conclude that microbial-based therapeutics may offer a simple but effective solution to reduce honey bee disease burden, environmental xenobiotic exposure, and spread of antimicrobial resistance.