Dodecanoic acid & palmitic acid disarms rifampicin resistance by putatively targeting mycobacterial efflux pump Rv1218c

Background & objectives: Drug-resistant tuberculosis (TB) jeopardizes the treatment process with poor outcomes. Efflux pumps (EPs) belonging to the ABC transporter family in Mycobacterium tuberculosis confer resistance to rifampicin (RMP) besides genetic mutations thus serving as a target for a potential adjunct therapeutic inhibitory molecule. Rv1218c is one such pump that was previously reported to be active in multidrug-resistant TB clinical isolates. Methods: In this study, the inhibition potential of Rv1218c-EP was tested on 8 molecules that were shortlisted by in silico methods. These molecules were subjected to the minimum inhibitory concentration (MIC) determination, checkerboard drug combination assay, ethidium bromide-DNA binding assay, and in vitro and ex vivo cytotoxicity assay. Results: Based on the outcome of the study, two molecules dodecanoic acid (DA) and palmitic acid (PA) were found to be potential enough to decrease the MIC of RMP by 8 to 1000 folds against multidrug-resistant clinical isolates and Rv1218c expressing recombinant Mycobacterium smegmatis. Interpretation & conclusions: These molecules were also found to reduce the time taken by RMP to kill these drug-resistant Mycobacteria to 48 h, unlike control isolates that survived more than 240 h of RMP exposure. The functional concentration of both molecules was non-toxic to the epithelial and blood mononuclear cells. With further comprehensive scientific validation, PA and DA could be recommended as adjunct therapeutic molecules with first-line anti-TB drugs to treat drug-resistant TB.

Research progress on efflux pump inhibitors of Mycobacterium tuberculosis in recent years / 药学学报

Currently, the resistance of first-line anti-tuberculosis drugs has made the prevention and treatment of tuberculosis increasingly difficult, posing a serious threat to global public health. Several studies have shown that efflux pumps are one of the important causes for bacteria to develop multi-drug resistance and extremely-drug resistance, and efflux pump inhibitors can inhibit the efflux of antibacterial drugs, thereby reducing bacterial drug resistance. Numerous natural products and synthetic compounds have been reported to possess efflux pump inhibitory activity, but they have not been applied in clinical settings because of their toxicity, pharmacokinetic properties, etc. Therefore, we summarized the efflux pump inhibitory activity, antimicrobial activity, and structure-activity relationships of reported efflux pump inhibitors against Mycobacterium tuberculosis in recent years, providing references for the development of new efflux pump inhibitors with better activity and lower toxicity.

Recent advances in the development of antibacterial potentiators / 中华检验医学杂志

Multidrug-resistant bacteria that can′t be treated with any common antibacterial drugs have become a global medical crisis. Therefore, there is an urgent need for new antibacterial potentiators to restore the sensitivity of bacteria to the antibacterial drug. This review elaborates on the novel antibacterial synergistic methods and their underlying mechanisms, clinical experimental data and efficacy, and the progress of drug research and development. This review aims to raise awareness about antibacterial potentiators among the public.

Antimicrobial resistance in Pseudomonas aeruginosa

ABSTRACT@#In recent years, antimicrobial resistance (AMR) has become a global public health concern. The growth of resistant bacteria is increasing dramatically, while the number of new antibiotics accessible is decreasing. This is especially true in the case of Pseudomonas aeruginosa, an important causative agent of healthcare-associated infections. The ability of P. aeruginosa to survive in different environments and on medical devices has made it more resistant to antibiotics. This causes bacteremia in hospitalized patients, ventilator-associated pneumonia, catheter-associated urinary tract infections and wound infections, particularly in patients with severe burns, bed ulcers and immunocompromised individuals. The rise in the AMR rate in both developed and developing countries may be attributed to a number of factors such as variations in the standard health care, large population, awareness about antibiotic resistance, inadequate training on rationale antibiotic usage and inadequate infection control facilities in many hospitals. The emergence of Extensive Drug Resistance (XDR) and Pan Drug Resistance (PDR) among organisms that cause various infections leads to increased treatment costs, morbidity and mortality, leaving no therapeutic options. This review highlights the different mechanisms of antibiotic resistance, including intrinsic and acquired resistance, which are frequently observed in P. aeruginosa.

Study on the susceptibility genes that mediate the resistance of Helicobacter pylori to clarithromycin and levofloxacin based on comprehensive antibiotic research database comparison / 中华消化杂志

Objective:Taking clinical strains of Helicobacter pylori ( H. pylori) with different antimicrobial resistance as the research object, to explore the new genes related to the resistance of H. pylori to clarithromycin (CLA) and levofloxacin (LVX) based on whole-genome sequencing. Methods:From September 1st, 2016 to August 31st, 2019, 1 749 patients with upper gastrointestinal symptoms and positive 13C urea breath test who visited the Department of Gastroenterology and Hepatology, the University of Hong Kong-Shenzhen Hospital were enrolled. After gastric mucosal biopsy, H. pylori was isolated and cultured from gastric mucosa. Ninety H. pylori strains were successfully preserved. According to the results of in vitro drug sensitivity test, a total of 40 strains including 10 strains with single-drug resistance to CLA (CLA group), 10 strains with single-drug resistance to LVX (LVX group), 10 strains with dual-resistance to CLA and LVX (dual resistance group) and 10 strains sensitive to CLA, LVX, amoxicillin, furazolidone, tetracycline and metronidazole (all sensitive group) were screened out. By whole-genome sequencing and comparison to the comprehensive antibiotic research database (CARD), single nucleotide variations (SNV) and indels were analyzed, genes related to H. pylori resistance to CLA and LVX were screened out and the differences of new genes among 4 groups were analyzed. Independent sample t test, one-way analysis of variance, least significant difference method and chi-square test were used for statistical analysis. Results:Among the 4 groups there were no statistically significant differences in the number of SNV (74 952.00±8 755.21, 77 128.10±3 191.35, 78 639.90±601.23 and 77 474.60±2 421.05) and Indels (2 582.20±265.45, 2 653.60±108.37, 2 667.10±43.82 and 2 641.10±80.25) (all P>0.05). Compared to CARD, a total of 223 drug resistance-related genes were detected, of which 19 genes related to CLA mono-resistance in CLA group, 24 genes related to LVX mono-resistance in LVX group, 16 genes related to CLA mono-resistance, 14 genes related to LVX mono-resistance, and 12 dual resistance-related genes in dual resistance group, and 11 genes related to CLA mono-resistance, 17 genes related to LVX mono-resistance, and 13 dual resistance-related genes in all sensitive group. Among the genes related to CLA mono-resistance, the detection rates of erythromycin esterase gene ( ere)B in CLA group, LVX group, dual resistance group and all sensitive group were 0/10, 0/10, 3/10, 0/10, respectively, and the difference was statistically significant( χ2=5.79, P=0.049). The detection rate of erythromycin ribosomal methylase gene ( erm) family in CLA group and dual resistance group was higher than that in LVX group and all sensitive group (45.0%, 9/20 vs. 10.0%, 2/20), and the difference was statistically significant ( χ2=6.14, P=0.013). The detection rates of free methionine-(R)-sulfoxide reductase gene ( msrC) in CLA group, LVX group, dual resistance group and all sensitive group were 10/10, 7/10, 6/10, 4/10, respectively, and the difference was statistically significant ( χ2=8.97, P=0.030). Among the genes related to LVX mono-resistance, the detection rate of quinolone resistance pentapeptide repeat protein gene ( qnr) family in LVX group and dual resistance group was higher than that in CLA group and all sensitive group (60.0%, 12/20 vs. 25.0%, 5/20), and the difference was statistically significant ( χ2=5.01, P=0.025). The detection rates of qnrB4 in CLA group, LVX group, dual resistance group and all sensitive group were 1/10, 3/10, 7/10, 1/10, respectively, and the difference was statistically significant ( χ2=10.17, P=0.010). The number of efflux transporter genes related to CLA mono-resistance in 4 groups were less than those of LVX mono-resistance and dual drug resistance (11 vs. 29 and 11 vs. 23), and the differences were statistically significant ( χ2=11.87, 5.80; P=0.001, 0.016). The detected numbers of LVX resistance-related efflux transport genes in CLA group, LVX group, dual resistance group and all sensitive group were 28, 40, 24 and 27, respectively, and the difference was statistically significant ( χ2=10.26, P=0.016). Conclusions:Erm family and msrC may be important genes that mediate the resistance of H. pylori to CLA, and qnr family is related to mediating the resistance of H. pylori to LVX. Efflux transport genes may play a synergistic role in the process of drug efflux, and are more likely to mediate H. pylori resistance to LVX.

Antibacterial activity and inhibition against Staphylococcus aureus NorA efflux pump by ferulic acid and its esterified derivatives

Objective: To evaluate the inhibitory activity of ferulic acid and four of its esterified derivatives (methyl, ethyl, propyl, and butyl) against resistance mechanisms in Staphylococcus aureus strains. Methods: Ferulic acid derivatives were obtained by esterification with methanol, ethanol, propanol, and butanol, and then characterized by hydrogen and carbon-13 nuclear magnetic resonance analysis. The minimum inhibitory concentrations (MIC) of ferulic acid and its esterified derivatives, ethidium bromide, and norfloxacin were obtained using the microdilution test, while the efflux pump inhibition test was conducted by examining reduction in the MICs of norfloxacin and ethidium bromide. Molecular docking was also carried out using the Schrodinger Suite 2015 molecular modeling software. A three-dimensional model of NorA efflux pump was generated using I-TASSER. The best scoring model was used as a receptor for ligand-receptor docking. Results: The methyl and butyl ester derivatives did not demonstrate significant antimicrobial activity. However, a significant synergic effect was evidenced when norfloxacin was combined with the ethyl and propyl esterified derivatives. The docking study demonstrated favorable energy of interaction between ferulate derivatives and NorA, and amino acid residues TYR57, TYR58, and LEU255 were present commonly in stabilizing all complexes. The PCA analysis corroborated the docking hypothesis that the lipophilic character and hydrogen bond interactions were the most relevant characteristics involved with NorA inhibitors. The pharmacokinetic parameters of ferulic acid derivatives showed good ADMET properties, demonstrating that they can be easily absorbed and have no effect or inhibit the cytochrome P450 enzyme complex, revealing their potential as drug candidates. Conclusions: This study provides strong evidence that the molecular basis for this activity is potentially due to the NorA efflux pump.

Perfil de tolerancia al triclosán y detección de los genes MexA, MexC, AcrB y oqxA relacionados con la expresión de bombas de expulsión en aislados clínicos del género Enterobacter aerogenes y Enterobacter cloacae / Triclosan tolerance profile and detection of MexA, MexC, AcrB and oqxA genes related to the expression of expulsion pumps in clinical isolates of Enterobacter aero- genes and Enterobacter cloacae / Perfil de tolerância ao triclosan e detecção dos genes mexA, mexC, acrB e oqxA relacionados à expressão de bombas de expulsão em isolados clínicos do gênero Enterobacter aerogenes e Enterobacter cloacae

Introducción. La resistencia a los antimicrobianos y la tolerancia a biocidas está dada por mecanismos comunes, generados por su uso en diferentes ambientes; mecanismos como la expresión de bombas de expulsión presentes en bacterias del género Enterobacter circulantes amenaza la eficacia de los antimicrobianos limitando las opciones de terapia antibiótica. Objetivos: Determinar el perfil de tolerancia al triclosán y detección de genes asociados a bombas de expulsión en aislados clínicos de Enterobacter aerogenes y Enterobacter cloacae. Materiales y Métodos: Se realizó un estudio observacional, descriptivo y de corte transversal, se determinaron perfiles de tolerancia al triclosán por microdilución, de susceptibilidad antimicrobiana, confirmación fenotípica de mecanismos de resistencia, por reacción en cadena de la polimerasa, se identificó la presencia de genes que codifican para bombas de expulsión. Resultados: El 17% correspondió a Enterobacter cloacae y el 6% Enterobacter aerogenes. El 93,7% de los aislados clínicos del género Enterobacter presentó el fenotipo de resistencia BLEE y AmpC. En el 81,3% de los aislamientos se obtuvo la presencia de al menos un gen relacionado con las expresión de bombas de expulsión, siendo frecuentes MexC y AcrB; no identificó presencia del gen oqxA. Conclusiones: La resistencia a diferentes grupos de antibióticos se identifica en especies de Enterobacter circulante, así la presencia de enzimas BLEE y AmpC, la presencia de genes relacionados con bombas de expulsión y la alta tolerancia al triclosán. Palabras clave: Triclosán, Resistencia, Bombas de expulsión, Genes, Biocida

Introduction. Antimicrobial resistance and tolerance to biocides is given by common mechanisms, generated by the use of antimicrobial and biocidal substances in different environments, these me- chanisms such as the expression of expulsion pumps present in bacteria of the Enterobacter genus circulating threatens the efficacy of antimicrobials by limiting antibiotic therapy options. Objective: to determine the triclosan tolerance profile and detection of genes associated with expul- sion pumps in clinical isolates of Enterobacter aerogenes and Enterobacter cloacae. Materials and Methods: An observational, descriptive and the cross-sectional study was performed, triclosan tolerance profiles were determined by microdilution, antimicrobial susceptibility, phenotypic confirmation of resistance mechanisms, by the presence of polymerase chain reaction, the presence of genes that code for expulsion pumps. Results: The 17% corresponded to Enterobacter cloacae and 6% Enterobacter aerogenes. 93.7% of the clinical isolates of the genus Enterobacter presented the ESBL and AmpC resistance phenotype. In 81.3% of the isolates, the presence of at least one gene related to the expression of ejection pumps was obtained, with MexC and AcrB being frequent; did not identify the presence of the oqxA gene. conclusions: The resistance to different groups of antibiotics is identified in circulating Enterobacter species, as well as the presence of ESBL and AmpC enzymes, the presence of genes related to ejection pumps, and high tolerance to triclosan.

Introdução.A resistência antimicrobiana e a tolerância a biocidas esta dada pelos mecanismos comuns gerados pelo uso em diferentes ambientes; mecanismos como a expressão de bombas de expulsão presentes em bactérias do gênero Enterobacter circulantes ameaza a eficácia das antimicrobiana limitando as opções de terapia antibiótica. Objetivos: Determinar o perfil de tolerância ao triclosan e detecção dos genes asociados a bombas de expulsão em isolados clínicos Enterobacter aerogenes e Enterobacter cloacae. Materiais e Métodos: Realizou-se um estudo observacional, descritivo e de corte transversal, deter- minaram-se perfiles de tolerância ao triclosan por microdiluição, de susceptibilidade antimicrobiana, confirmação de mecanismos de resistência fenotípica por reação em cadeia da polimerase, identifi- cou-se a presença de genes que codificam para bombas de expulsão. Resultados: 17% correspondeu ao Enterobacter cloacae e 6% ao Enterobacter aerogenes. 93,7% em isolados clínicos do gênero Enterobacter presentou o fenótipo de resistência BLEE e AmpC. No 81% dos isolamentos se obteve a presença de pelo menos um gen relacionado à expressão de bombas de expulsão, sindo frequentes mexC e acrB; não se identificou a presença do gen oqxA. Conclusões: A resistência de diferentes grupos de antibióticos se identificou em espécies de Entero- bacter circulante, assim a presença de enzimas BLEE e AmpC, a presença de genes relacionados com bombas de expulsão e a alta tolerância ao triclosan.

Plant-derived secondary metabolites as the main source of efflux pump inhibitors and methods for identification / 药物分析学报

The upsurge of multiple drug resistance (MDR) bacteria substantially diminishes the effectiveness of antibiotic arsenal and therefore intensifies the rate of therapeutic failure. The major factor in MDR is efflux pump-mediated resistance. A unique pump can make bacteria withstand a wide range of struc-turally diverse compounds. Therefore, their inhibition is a promising route to eliminate resistance phenomenon in bacteria. Phytochemicals are excellent alternatives as resistance-modifying agents. They can directly kill bacteria or interact with the crucial events of pathogenicity, thereby decreasing the ability of bacteria to develop resistance. Numerous botanicals display noteworthy efflux pumps inhibi-tory activities. Edible plants are of growing interest. Likewise, some plant families would be excellent sources of efflux pump inhibitors (EPIs) including Apocynaceae, Berberidaceae, Convolvulaceae, Cucur-bitaceae, Fabaceae, Lamiaceae, and Zingiberaceae. Easily applicable methods for screening plant-derived EPIs include checkerboard synergy test, berberine uptake assay and ethidium bromide test. In silico high-throughput virtual detection can be evaluated as a criterion of excluding compounds with efflux substrate-like characteristics, thereby improving the selection process and extending the identification of EPIs. To ascertain the efflux activity inhibition, real-time PCR and quantitative mass spectrometry can be applied. This review emphasizes on efflux pumps and their roles in transmitting bacterial resistance and an update plant-derived EPIs and strategies for identification.

Nordihydroguaiaretic acid reverses efflux pump system MexCD-OprJ mediated ceftazidime resistance of pseudomonas aeruginosa and its related mechanisms / 中草药

Objective: To evaluate the effect of nordihydroguaiaretic acid (NDGA) on ceftazidime resistance of Pseudomonas aeruginosa mediated by efflux pump system MexCD-OprJ and explore its mechanism. Methods: The bacterial solution with a concentration of 0.5 mcburney was diluted and inoculated in a 96-well plate, and NDGA and ceftazidime were added by the checkerboard dilution method. At the same time, the untreated control group, NDGA control group and ceftazidime control group were set; After being cultured for 24 h, the absorbance was measured by an enzyme micro-plate reader, the minimum inhibitory concentration (MIC) of each drug was recorded and the bacteriostatic rate and fractional inhibitory concentration (FIC) index were calculated. Bacteria were inoculated with the bacterial liquid coating method in the 96-well plates, and the bacterial colony number was counted after 24 h of culture. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the gene expressions of efflux pump membrane protein MexC, MexD, OprJ and nfxB. Results: Compared with ceftazidime or NDGA alone, combination of ceftazidime and NDGA significantly inhibited the growth of efflux pump system MexCD-OprJ-mediated ceftazidime-resistant P. aeruginosa (P < 0.05); The pharmacological effects of ceftazidime and NDGA showed synergistic or additive effects; After combined administration, the MIC values of ceftazidime and NDGA were significantly decreased, and the MIC value of some ceftazidime had no significant difference from that of ceftazidime-sensitive P. aeruginosa; Compared with ceftazidime alone, the gene expressions of efflux pump membrane proteins MexC, MexD and OprJ were significantly decreased after combined application of ceftazidime and NDGA (P < 0.05), while the expression of nfxB was significantly increased (P < 0.05). Conclusion: The mechanism of NDGA on ceftazidime resistance of P. aeruginosa mediated by efflux pump system MexCD-OprJ is related to its ability to down-regulate the gene expression of efflux pump membrane proteins MexC, MexD and OprJ, and up-regulate the gene expression of the negative regulatory gene nfxB of the above three proteins.

Effect of Tanreqing Injection and Antibiotics on Multi-drug Resistant Pseudomonas aeruginosa Efflux Pump / 中国实验方剂学杂志

Objective:To study the interaction between Tanreqing injection and commonly used antibiotics against multi-drug resistant Pseudomonas aeruginosa (MDR-PA) and the effect on bacterial efflux pump. Method:Antibiotic susceptibility test was performed with bacteria. Paper diffusion method (Kirby-Bauer, KB) combined with efflux pump inhibitor (50 mg·L-1) was used to measure the diameter of the inhibition zone, and real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect gene expression of efflux pump Positive efflux pump strain. KB method was used to observe the changes of Tanreqing (final concentration 3 g·L-1) and antibiotics on the diameter of the zone of inhibition. Strains were co-cultured with Tanreqing and antibiotic sub-inhibitory concentrations for Real-time PCR detection. KB method was used to observe the effect of Tanreqing on the diameter of bacteriostatic ring after the continuous use of efflux pump-positive bacteria. Result:Two MDR-PA efflux pump-positive strains were identified and screened. Tanreqing has synergistic antibacterial effect with aloxicillin, aztreonam, meropenem, ceftazidime, cefoperazone, and Shupushen. In inhibiting the expression levels of bacterial efflux pump genes, the four drugs were compared by the effect: cefoperazone>Tanreqing>ceftazidime>Shupushen. After Tanreqing continued to act on efflux pump-positive strains, it could have a better effect in combination with ceftazidime, cefoperazone, and Shupushen. Conclusion:Tanreqing, ceftazidime, cefoperazone, and Shupushen can reduce the drug resistance of bacteria by down-regulating the expressions of bacterial efflux pump genes, and reducing the clinical dose of antibiotics, and thus play a bacteriostatic effect.

Detection of efflux pump CmeABC in enrofloxacin resistant Campylobacter spp. strains isolated from broiler chickens (Gallus gallus domesticus) in the state of Rio de Janeiro, Brazil / Detecção da bomba de efluxo CmeABC em cepas de Campylobacter spp. resistentes à enrofloxacina isoladas de frangos de corte (Gallus gallus domesticus) no Estado do Rio de Janeiro, Brasil

Fowls are the main reservoirs of the highly important food-originating pathogen called Campylobacter spp. and broilers' meat and byproducts are the main vehicles of this microorganism. Increasing of Campylobacter spp. resistant strains to fluorquinolones, an antimicrobial class often employed in poultry farming and in human medicine has become a great concern to poultry breeders. In fact, several studies evaluated increasing bacterial resistance against these antimicrobial agents. The role of CmeABC efflux system has been underscored among the resistance mechanisms in Campylobacter spp. to fluorquinolones. This study investigated the occurrence of CmeABC efflux pump in 81 and 78 enrofloxacin resistant strains of Campylobacter jejuni and C. coli respectively, isolated from broilers collected from six abattoirs situated at São José do Vale do Rio Preto/RJ poultry center and from two commercial abattoirs situated at Metropolitan Region of Rio de Janeiro, from 2013 to 2016. The resistance to enrofloxacin was assessed by agar dilution to determine minimum inhibitory concentration (MIC). The CmeABC efflux system was investigated through the detection of genes genes cmeA, cmeB and cmeC by PCR. The activity of CmeABC efflux pump was investigated in 20 strains by using the efflux pump inhibitor Phenylalanine-Arginine ß-Naphthylamide (PAßN). The three genes cmeA, cmeB and cmeC were detected in 94.3% of the strains (C. jejuni = 80 and C. coli = 70), whereas the system was absent or incomplete in 5.7% of strains (C. jejuni = 1 and C. coli = 8). MIC varied between 0.5µg/ml and 64µg/ml, and 88.7% of strains were enrofloxacin resistant and 11.3% featuring intermediate resistance. The inhibition of the efflux pump by PAßN reduced the MIC to enrofloxacin up to eight times in fifteen strains (75%). These results indicate that this system is frequent and active in Campylobacter spp. Resistant strains in the presence of enrofloxacin.(AU)

As aves são os principais reservatórios de Campylobacter spp., importante patógeno de origem alimentar e a carne de frango e produtos derivados são os principais veículos desse microrganismo. O aumento de cepas de Campylobacter spp. resistentes às fluorquinolonas, uma classe antimicrobiana frequentemente empregada na avicultura e na medicina humana, tornou-se uma grande preocupação para os produtores de aves e vários estudos avaliaram o aumento da resistência bacteriana a esses antimicrobianos. O papel do sistema de efluxo CmeABC tem sido enfatizado entre os mecanismos de resistência em Campylobacter spp. à fluorquinolonas. O presente estudo investigou a ocorrência da bomba de efluxo CmeABC em 81 cepas de Campylobacter jejuni e 78 cepas de Campylobacter coli resistentes à enrofloxacina, isoladas de frangos de corte coletados em seis abatedouros situados no polo avícola de São José do Rio Preto/RJ e de dois abatedouros comerciais situados na Região Metropolitana do Rio de Janeiro, de 2013 a 2016. A resistência à enrofloxacina foi avaliada pelo método de diluição em ágar para determinar a concentração inibitória mínima (CIM). O sistema de efluxo CmeABC foi investigado através da detecção dos genes cmeA, cmeB e cmeC por PCR. A atividade da bomba de efluxo CmeABC foi investigada em 20 cepas utilizando o inibidor da bomba de efluxo Phenylalanine-Arginine ß-Naftilamida (PAßN). Os três genes cmeA, cmeB e cmeC foram detectados em 94,3% das cepas (C. jejuni = 80 e C. coli = 70), enquanto o sistema estava ausente ou incompleto em 5,7% das cepas (C. jejuni = 1 e C coli = 8). A CIM variou entre 0,5µg/ml e 64µg/ml e 88,7% das cepas foram resistentes à enrofloxacina, enquanto 11,3% apresentaram resistência intermediária. A inibição da bomba de efluxo pelo PAßN reduziu a CIM da enrofloxacina até oito vezes em quinze cepas (75%). Estes resultados indicam que este sistema é frequente e ativo em cepas resistentes de Campylobacter spp. na presença de enrofloxacina.(AU)

Distribution of adeG, adeB, adeE, adeY, abeM, and adeJ Efflux Pump Genes in Clinical Isolates of Acinetobacter Species from Korea

BACKGROUND: The aim of the present study was to determine the frequency of six efflux pump genes in Acinetobacter clinical isolates collected from South Korean hospitals. METHODS: In this study, we used a total of 339 Acinetobacter strains, comprising 279 Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex and 60 non-ACB complex strains. We performed specific PCR assays to detect adeG, adeB, adeE, adeY, abeM, and adeJ, transporter genes of the multidrug efflux pumps AdeFGH, AdeABC, AdeDE, AdeXYZ, AbeM, and AdeIJK, respectively. RESULTS: Frequencies of six efflux pump genes varied according to the species of Acinetobacter. Frequencies of adeE, abeM, and adeJ between A. baumannii group and A. nosocomialis group were found to be significantly different. Significant differences were found in the frequencies of adeB, adeE, adeY, and adeJ among the susceptible A. baumannii (SAB), multidrug-resistant A. baumannii (MDRAB), and extensively drug-resistant A. baumannii (XDRAB) groups within the 154 strains of A. baumannii. The frequencies of efflux pump genes in imipenem-susceptible and imipenem-nonsusceptible groups were significantly different for adeB, adeY, and adeJ. The frequencies of efflux pump genes in ciprofloxacin-susceptible and ciprofloxacin-nonsusceptible groups were significantly different for adeB and adeY. No significant difference was found in the frequency of efflux pump genes among groups sampled from different regions of Korea, across 86 strains of A. baumannii collected in 2012. CONCLUSIONS: The frequencies of six efflux pump genes obtained in this study demonstrate the fundamental epidemiological feature of efflux pump genes in Korean Acinetobacter clinical isolates.

Relationship between high expression of efflux pump AcrAB-TolC induced by quaternary ammonium compound and fluoroquinolones resistance in Escherichia coli / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of efflux pump AcrAB-TolC involved in the cross-resistance between quaternary ammonium compounds ( QAC) and fluoroquinolones ( FQ) in Escherich-ia coli. Methods Seventy-eight Escherichia coli strains were isolated from clinical samples and then tested for the sensitivity to benzalkonium bromide by ager dilution method. Six strains were randomly selected and induced with benzalkonium bromide. Changes in the sensitivity of these strains to benzalkonium bromide and ciprofloxacin were analyzed after induction. Expression of the efflux pump genes acrA, acrB and tolC at mRNA level in the induced strains and their parent strains were detected by quantitative real-time PCR. Re-sults Among the 78 strains, the minimum inhibitory concentrations ( MIC) ranged from 8 μg/ml to 64μg/ml and 47. 4％ of strains have higher MIC than the standard strain. Compared with the parent strains, the induced strains showed higher expression of acrA and tolC genes, and the differences between the two groups were statistically significant. The MIC values of ciprofloxacin to the six induced strains were 4-8 times higher than those to their parent strains. Conclusions It was speculated that the increased expression of acrA and tolC genes at transcription level in the induced strains were related to the cross-resistance between quaternary ammonium compounds and fluoroquinolone antibiotics.

Susceptilibity and molecular characterization of carbapenem resistant Klebsiella pneumoniae without producing carbapenemase / 临床检验杂志

Objective@#To analyze the susceptibility and distribution of β-lactamase encoding and efflux pump genes as well as the inhibitory effect of efflux pump inhibitors on the carbapenem resistance of the carbapenem resistant Klebsiella pneumoniae without producing carbapenemase (CRKPPC). @*Methods@#One hundred and eight strains of carbapenem non-susceptible Klebsiella pneumoniae were collected from our hospital during 2012-2014. The strains producing carbapenemase were screened by Mastdiscs combi Carba plus disc system. For CRKPPC strains, the susceptibilities to antimicrobial agents were determined by micro-dilution broth methods. PCR and DNA sequencing technology were used to analyze the prevalence of β-lactamase encoding extended-spectrum β-lactamase (ESBL) and plasmid mediated AmpC genes as well as efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The inhibitory experiments were implemented by using carbonylcyanide-m-chlorophenylhydrazone (CCCP) and Reserpine to observe the role of efflux pumps on the carbapenem resistance. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to analyze the expression of outer membrane proteins OmpK35 and OmpK36. @*Results@#Twenty-six strains out of the 108 carbapenem non-susceptible Klebsiella pneumoniae were identified to be CRKPPC strains which displayed quite high resistance to β-lactam and high resistance to amikacin, gentamicin, levofloxacin and ciprofloxacin. Very good sensitivities were observed to tigecycline, polymyxin, ceftazidime/avibatan and aztreonam/avibatan. The high prevalence of bla CTX-M and bla SHV were also displayed with the prevalent rates being 76.9% and 57.7%, respectively. The loss of outer membrane proteins OmpK35 and OmpK36 with varying degrees was observed, among which 57.7% strains lost ompK35 and 19.2% strains lost OmpK36. More than 80% strains contained efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The results of inhibitory experiments showed that CCCP displayed quite obviously inhibitory effects on the carbepenem resistance whereas no inhibitory effect was observed for Reserpine. @*Conclusion@#Tigecycline and polymyxin could be used as the basis of combined drug for CRKPPC. The prevalence of AmpC, ESBLs and loss of OmpK35 and OmpK36 with varying degrees among these strains were observed, but the over-expression of efflux pumps should be the main mechanism of the carbapenem resistance in the Klebsiella pneumoniae strains, and efflux inhibitors may have potential value in treating the infections caused by these bacteria.

Transcriptional response of AcrEF-TolC against fluoroquinolone and carbapenem in Escherichia coli of clinical origin

Introduction: Efflux pump systems constitute a major means of intrinsic resistance in Escherichia coli. AcrEF-TolC pump is known to exhibit higher expression level in quinolone resistant isolates. However, the transcriptional response of this pump is yet to be known when exposed to quinolone and other group of antibiotics. Objective: The present study analyses the transcriptional response of AcrEF-TolC in the presence of quinolones and carbapenems. Methodology: A total of 167 non-duplicate clinical isolates from Silchar medical college and Hospital, Silchar, India were included in this study. Of which 27 were devoid of any carbapenemase activity and among them 13 isolates showed overexpression of AcrE and AcrF gene. Transcriptional response of AcrE was directly proportional to increasing concentration of levofloxacin and ofloxacin. However, the response of AcrE and AcrF was inconsistent with carbapenems. Result: The study isolates showed susceptibility towards amikacin (68.4%), gentamicin (59.6%), cefepime (52.7%) and pipercillin/tazobactam (48.3%). The present investigation highlights that apart from qnr genes and mutational changes in gyr region, AcrEF-TolC plays a major role in fluoroquinolone resistance in this part of the world. Conclusion: Upregulation of AcrE in the presence of levofloxacin and ofloxacin warrants further investigation to establish their active role in efflux of this drug.

Overview of resistance mechanisms in Campylobacter / 中华预防医学杂志

Campylobacter is a major cause of food-borne gastroenteritis worldwide. While mortality is low when people was infected with Campylobacter, morbidity imparted by post-infectious sequelae such as Guillain-Barré syndrome and irritable bowel syndrome is significantly noteworthy. Although fluoroquinolones and macrolides were the first line drug for the treatment of Campylobacter infections, there is a tough challenge in clinical treatment with high antimicrobial resistant rate and multi antimicrobial resistance arise. Based on the latest literature acquired in this work, we have chosen five classes of antibiotics always used in clinical, and discussed antibiotic resistance mechanisms and transmission of Campylobacter, in order to provide proper therapy both in the veterinary and human populations, and support basis data for the development of new drugs.

Research progress of biofilm formation in Candida albicans / 中华临床感染病杂志

Biofilm formation is associated with invasive fungal infection, which is considered to be an important virulence factor.Candida albicans has become one of the important bacterial flora in hospital infection,and it is the most common clinical fungi strain, mainly isolated from respiratory tract.The formation of biofilm can promote the adhesion,proliferation and proliferation of Candida albicans, resulting in a significant increase in the minimum inhibitory concentration and drug resistance.This article reviews the current research advances of the mechanism of Candida albicans biofilm formation,structural characteristics and its prevention and treatment,to provide a reference for the development of clinical prevention and control strategies of Candida albicans biofilm formation.

Expression and purification of multidrug resistance related membrane protein MmpL6 of Mycobacterium tuberculosis / 安徽医科大学学报

Objective To construct the prokaryotic expression vector carrying Mycobacterium tuberculosis gene mmpL6 (rv1557), overexpress and purify the recombinant MmpL6 (Rv1557) protein in Escherichia coli (E. coli).Methods The DNA fragment of MmpL6 was amplified by polymerase chain reaction using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template. The target gene was cloned into pET21b vector to construct the pET21b-MmpL6 expression plasmid, and then transformed into E. coli for protein expression. Recombinant protein expression was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and detected by SDS-PAGE combined with Western blot. The overexpressed MmpL6 protein was purified by Ni-affinity chromatography and gel filtration chromatography. Results The recombinant pET21b-MmpL6 plasmid was successfully constructed and the highest expression level was obtained at 25 ℃ in Rosetta strain induced by IPTG. Conclusion The successful expression and purification of MmpL6 in E. coli lays the foundation for the further structure and function studies of the protein, and also provides clues to the design of anti-tuberculosis drugs targeting efflux pump proteins.

Relationship between expression and antimicrobial resistance of RND efflux pump of Acinetobacter baumannii / 中国感染控制杂志

Objective To detect the distribution of resistance-nodulation (RND)efflux pump system of Acineto-bacter baumannii (AB),and explore the relationship between its’expression and antimicrobial resistance.Methods Fifty-nine strains of multidrug-resistant AB isolated from clinical specimens in The First Affiliated Hospital of Nan-chang University were identified and performed antimicrobial susceptibility analysis,distribution of RND efflux sys-tem of AB was detected by polymerase chain reaction(PCR),expression of efflux pump genes in different drug-re-sistant phenotypes of AB was compared,relationship between the expression level and drug resistance was analyzed, amplified products of RND efflux system were sequenced.Results Resistance rates of AB to ampicillin/sulbactam, imipenem,gentamicin,ciprofloxacin,and levofloxacin were 93.2%,94.9%,88.1%,96.6%,and 52.5% respec-tively.PCR detection results of efflux pump and integron genes of 59 AB strains revealed that the carrying rates of adeR,adeS,adeB,adeJ,and adeG genes were 81.4%,91.5%,93.2%,100.0%,and 61.0% respectively.The expression of efflux pump genes in different strains was different,expression levels of ade B and adeJ genes among gentamicin,imipenem,ampicillin/sulbactam resistant AB group and non-resistant AB group were significantly dif- ferent (all P＜0.05).There was no mutation or insertion sequence in the base sequences of regulatory genes ade R and ade S of adeABC efflux pump.Conclusion RND efflux pump system is universally present in AB,the expres-sion upregulation of ade B and ade J genes in RND efflux pump system is related with antimicrobial resistance of bacteria to gentamycin,imipenem,and ampicillin-sulbactam.

Detection and analysis of different interactions between resistance mechanisms and carbapenems in clinical isolates of Klebsiella pneumoniae

Abstract Carbapenems are considered last-line agents for the treatment of serious infections caused by Klebsiella pneumoniae, and this microorganism may exhibit resistance to β-lactam antibiotics due to different mechanisms of resistance. We evaluated 27 isolates of K. pneumoniae resistant to carbapenems recovered from inpatients at the University Hospital of Santa Maria-RS from July 2013 to August 2014. We carried out antimicrobial susceptibility, carbapenemase detection, testing for the presence of efflux pump by broth microdilution and loss of porin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Genetic similarity was evaluated by ERIC-PCR. High levels of resistance were verified by the minimum inhibitory concentration for the antimicrobials tested. The blaKPC gene was present in 89% of the clinical isolates. Blue-Carba and combined disk with AFB tests showed 100% concordance, while the combined disk test with EDTA showed a high number of false-positives (48%) compared with the gold-standard genotypic test. Four isolates showed a phenotypic resistance profile consistent with the overexpression of the efflux pump, and all clinical isolates had lost one or both porins. The ERIC-PCR dendrogram demonstrated the presence of nine clusters. The main mechanism of resistance to carbapenems found in the assessed isolates was the presence of the blaKPC gene.