oqxA Genes Research Articles

Effect of Mentha longifolia essential oil on oqxA efflux pump gene expression and biofilm formation in ciprofloxacin-resistant Klebsiella pneumoniae strains.

Today, medicinal plants and their derivatives are considered to reduce the prevalence of antibiotic resistance. The aim of this study was to investigate the effect of Mentha longifolia essential oil on oqxA efflux pump gene expression and biofilm formation in ciprofloxacin-resistant Klebsiella pneumoniae strains. A total of 50 clinical strains of K. pneumoniae resistant to ciprofloxacin were studied. The minimum inhibitory concentration (MIC) of M. longifolia essential oil and its synergistic effect with ciprofloxacin were determined using the microbroth dilution method and the fractional inhibitory concentration (FIC) method. Minimum biofilm inhibition concentration (MBIC) of M. longifolia essential oil was detected. The effect of essential oils on the expression level of the oqxA gene was detected by Real-time PCR. M. longifolia essential oil showed inhibitory activity against ciprofloxacin-resistant strains of K. pneumoniae. When M. longifolia essential oil was combined with ciprofloxacin, the MIC was reduced 2-4 times. In 28% of the strains, M. longifolia with ciprofloxacin showed a synergistic effect. M. longifolia essential oil reduces the strength of biofilm formation and alters the biofilm phenotype. A significant decrease in oqxA gene expression was observed in all isolates after treatment with M. longifolia essential oil. Based on the results of this study, it was observed that supplementing M. longifolia essential oil can help reduce ciprofloxacin resistance and inhibit biofilm formation in fluoroquinolone-resistant K. pneumoniae strains.

Molecular insights into novel environmental strains of Klebsiella quasipneumoniae harboring different antimicrobial-resistance genes.

The emergence of bacterial pathogens in environmental hosts represents a major risk to public health. This study aimed at characterizing seven novel environmental strains of K. quasipneumoniae using a genomic approach which was misidentified by phenotypic methods in a previous batch of 27 species thought to be K. pneumoniae. Whole-genome sequencing was performed using the Illumina platform, and the generated raw reads were de novo assembled. Comparative genomic, resistome, virulome, mobilome, and phylogeny were then investigated using dierent bioinformatics tools. Six strains were identified as K. quasipneumoniae subsp similipneumoniae and one as K. quasipneumoniae subsp. quasipneumoniae. All isolates were resistant to ampicillin, cephalexin, and amoxicillin-clavulanic acid and harbored the fosA, bla OKP types, oqxB, and oqxA genes. One isolate additionally harbored a gene cassettes consisting of bla SHV-1, bla OXA-1, aac(6')-Ib-cr, catB genes. The aminoglycoside-modifying enzyme gene aph(3")-Ia was bracketed by two insertion elements. Plasmid analyses showed that IncFIBK was the most prevalent plasmid, circulating in six isolates, while one isolate exhibited seven different plasmids. The isolates have virulence genes responsible for capsule formation, lipopolysaccharide, iron uptake aerobactin (iutA), salmochelins (iroE, iroN), enterobactin siderophore, adherence, and biofilm formation (mrkA, mrkB, mrkC, mrkD, mrkF, and mrkH). Our study highlights the ecology and transmission of K. quasipneumoniae (which have the ability to disseminate to other environmental sources including animals) outside the clinical setting and the contribution of water, vegetables, and table surfaces as potential reservoirs of farm-to-fork transmission of disease via local markets in Khartoum, Sudan.

Relationship of OqxAB efflux pump to antibiotic resistance, mainly fluoroquinolones in Klebsiella pneumoniae, isolated from hospitalized patients.

This research was designed to study the prevalence of OqxAB efflux pump genes and also to investigate the relationship between efflux pump and resistance to antibiotics, especially to fluoroquinolones, evaluate the expression levels of OqxAB genes, and molecular typing of Klebsiella pneumoniae isolated from hospitalized patients in Hamadan hospitals, west of Iran. In a cross-sectional study, 100 clinical strains of K. pneumoniae were isolated from hospitalized patients in three major teaching hospitals from January to June 2021. The antibiotic susceptibility of isolates was evaluated by the disk-diffusion agar method. The frequency of genes encoding oqxA and oqxB of efflux pump genes was investigated by PCR, and the expression of the oqxA efflux pump gene was investigated by the Real-time PCR method. The genetic relationship of K. pneumoniae isolates was analyzed by the Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR technique. According to our results, the multi-drug resistance phenotype (MDR) in 65% and high prevalence resistance to ciprofloxacin in 89% of K. pneumoniae isolates was detected. The higher prevalence of oqxA (95%) and oqxB (98%) was also detected. There was a significant relationship between ciprofloxacin resistance and the oqxB gene as well as between ceftriaxone and chloramphenicol resistance and the oqxA gene. The expression of the oqxA gene was higher in ciprofloxacin-resistant isolates. The results of this study suggest a potential reservoir for the spread of OqxAB genes among hospital-acquired bacteria. Infection control strategies should be used prudently to reduce the spread of resistant strains of K. pneumoniae in hospitals.

The Molecular Epidemiology of Resistance to Antibiotics among Klebsiella pneumoniae Isolates in Azerbaijan, Iran.

Introduction Klebsiella pneumoniae (K. pneumoniae) is one of the leading causes of hospital-acquired and community-acquired infections in the world. This study was conducted to investigate the molecular epidemiology of drug resistance in clinical isolates of K. pneumoniae in Azerbaijan, Iran. Materials and Methods A total of 100 nonduplicated isolates were obtained from the different wards of Azerbaijan state hospitals, Iran, from 2019 to 2020. Antibiotic susceptibility testing was done. The DNA was extracted, and the PCR for evaluation of the resistance genes was carried out. Results The highest antibiotic resistance was shown to ampicillin (96%), and the highest susceptibility was shown to tigecycline (9%), and 85% of isolates were multidrug resistant. The most frequent ESBL gene in the tested isolates was blaSHV-1 in 58%, followed by blaCTXM-15 (55%) and blaSHV-11(42%). The qepA, oqxB, and oqxA genes were found to be 95%, 87.5%, and 70%, respectively. We detected tetB in 42%, tetA in 32%, tetD in 21%, and tetC in 16%. Seventy isolates were resistant to co-trimoxazole, and the rate of resistance genes was sul1 in 71%, followed by sul2 (43%), dfr (29%), and sul3 (7%). The most common aminoglycoside resistance genes were ant3Ia, aac6Ib, aph3Ib, and APHs in 44%, 32%, 32%, and 31.4%, respectively. The most frequent resistance gene to fosfomycin was fosA (40%) and fosX (40%) followed by fosC (20%). Conclusion The results of this study indicate the high frequency of drug resistance among K. pneumoniae isolated from hospitals of Azerbaijan state. The present study shows the presence of high levels of drug-resistant genes in various antibiotics, which are usually used in the treatment of infections due to K. pneumoniae.

Emergence of NDM-5-Producing Carbapenem-Resistant Klebsiella pneumoniae and SIM-Producing Hypervirulent Klebsiella pneumoniae Isolated from Aseptic Body Fluid in a Large Tertiary Hospital, 2017-2018: Genetic Traits of blaNDM-Like and blaSIM-Like Genes as Determined by NGS.

PurposeTo characterize the clinical, resistance, and virulence features of carbapenem-resistant Klebsiella pneumonaie (CRKP) and hypervirulent Klebsiella pneumoniae (hvKP) and also provide an effective selection of drug in CRKP and hvKP treatment.Materials and MethodsTwelve strains were collected and investigated these isolates for their antimicrobial susceptibility and molecular features. Resistance mechanisms, virulence-associated genes, multilocus sequence typing (MLST), and serotypes were detected by PCR and sequencing. Next general sequencing (NGS) was carried out to determine the features of carbapenem resistance and virulence. The synergistic activity of tigecycline–imipenem (TGC+IPM), tigecycline–meropenem (TGC+MEM), and tigecycline–aztreonam (TGC+ATM) combinations were performed by microdilution checkerboard method.ResultsEleven CRKP and one hvKP strains were collected. All strains showed highly sensitive rates to tigecycline (TGC) and amikacin (AMK). NDM (33.3%, 4/12) was the main resistance mechanism and MLST assigned 3 of them to ST11. CTX-M-producing (n = 1) and KPC-2-producing (n = 1) isolates belonged to ST147 and ST11, respectively. The MICs of ATM and quinolones in NDM-1 CRKP and NDM-5 CRKP strains were different. The serotype of the majority strains was KL22KL137 (58.3%, 7/12), hvKP stain belonged to K64. CRKP strains harbored plasmid-mediated quinolone resistance genes (oqxA, oqxB, qnrS, qnrB), β-lactams (blaCTX-M-3), aminoglycosides, type I and type III fimbriae genes, siderophore genes, and transporter and pumps. SIM-producing ST1764 K64 showed typical features of hvKP, showing hypermucoviscosity phenotype. The virulence genes, including rmpA2, alls and aerobactin genes, linked to hvKP, were found in ST1764 hvKP. hvKP was sensitive to quinolone; also, oqxA gene was detected. All TGC combinations showed highly synergistic effects and TGC+IPM was more effective treatment.ConclusionWe first identified the NDM-5-producing ST690 CRKP and SIM-producing ST1764 hvKP strains in Shanxi province. Tigecycline-carbapenem combinations were available treatments for CRKP.

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

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.

Antibiotic Resistance Characterization of Environmental E. coli Isolated from River Mula-Mutha, Pune District, India

In the current study, ceftazidime- and ciprofloxacin-resistant—or dual drug-resistant (DDR)—E. coli were isolated from river Mula-Mutha, which flows through rural Pune district and Pune city. The DDR E. coli were further examined for antibiotic resistance to six additional antibiotics. The study also included detection of genes responsible for ceftazidime and ciprofloxacin resistance and vectors for horizontal gene transfer. Twenty-eight percent of the identified DDR E. coli were resistant to more than six antibiotics, with 12% being resistant to all eight antibiotics tested. Quinolone resistance was determined through the detection of qnrA, qnrB, qnrS and oqxA genes, whereas cephalosporin resistance was confirmed through detection of TEM, CTX-M-15, CTX-M-27 and SHV genes. Out of 219 DDR E. coli, 8.2% were qnrS positive and 0.4% were qnrB positive. Percentage of isolates positive for the TEM, CTX-M-15 and CTX-M-27 genes were 32%, 46% and 0.9%, respectively. None of the DDR E. coli tested carried the qnrA, SHV and oqxA genes. Percentage of DDR E. coli carrying Class 1 and 2 integrons (mobile genetic elements) were 47% and 8%, respectively. The results showed that antibiotic resistance genes (ARGs) and integrons were present in the E. coli isolated from the river at points adjoining and downstream of Pune city.

Quinolone Resistance Detection by PCR-RFLP and Multiplex-PCR among Extended- Spectrum β- Lactamase Producing Enterobacteriaceae

Quinolone resistance limits the therapeutic potential for the Extended-Spectrum β- lactamase (ESBL) producing Enterobacteriaceae. The aim of this study was to investigate the most common mechanisms of quinolones resistance in ESBL- producing Enterobacteriaceae using both phenotypic and genotypic methods. Out of 1766 clinical isolates collected between October 2012 and September 2013, 219 Enterobacteriaceae clinical isolates were ESBL producers, nalidixic acid and ciprofloxacin resistant were identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDITOF/ MS) and the Minimal Inhibitory Concentration (MIC) values of ciprofloxacin and nalidixic acid wasdetected before and after addition of phenylalanine-arginine β-naphthylamide (PaβN) efflux pump inhibitor. Thirty three isolates were selected for screening of the plasmid-mediated fluoroquinolone resistance (PMQR) genes; (qnr, aac(6’)-Ib-cr and qepA) and the efflux pump genes (oqxAB genes) by multiplex PCR. Whereas GyrA and ParC genes mutations were detected by PCR-RFLP assay. The PaβN changed the MIC values of 28 isolates. The GyrA gene mutation was detected in 24/33 (72.7%), while the par C gene mutation was detected in 3/33 (9.1%). Qnr-genes were detected in 13/33 (39.4%), aac(6')-Ib gene was detected in 24/33 (72.7%). qepA gene was detected in only one Klebsiella pneumoniae isolate. Finally the oqxA gene was detected in 16/33 (48.5%) of the studied isolates. The present study indicated that the PaβN was an effective phenotypic screening method for quinolones resistance efflux pump; moreover PCR-RFLP offered simple and rapid method for detection of ciprofloxacin-resistance that could be useful for clinical diagnosis and epidemiological studies.

Contribution of OqxAB efflux pumps to quinolone resistance in extended-spectrum- -lactamase-producing Klebsiella pneumoniae

The aims of this study were to analyse the presence of oqxA and oqxB genes in a collection of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae strains, to determine their chromosomal and/or plasmidic locations and to analyse expression levels in relation to susceptibility or resistance to quinolones. A collection of 114 non-repetitive isolates of ESBL-producing K. pneumoniae was used. K. pneumoniae ATCC 27799 and K. pneumoniae ATCC 700603 were also included. Detection of oqxA and oqxB genes was performed by PCR. Testing for chromosomal and/or plasmidic location was carried out using plasmid DNA and subsequent hybridization. oqxA gene expression was analysed using real-time RT-PCR. Transfer of the plasmid-encoded OqxAB was evaluated. The prevalence of both oqxA and oqxB detected in K. pneumoniae was high: 76% and 75%, respectively. Hybridization assays showed that oqxA (16%) and oqxB (13%) were simultaneously present in locations on the chromosome and on large plasmids. The plasmids were transferable by transformation into K. pneumoniae. RT-PCR assays showed higher expression (4-fold) in strains with reduced susceptibility to quinolones than in susceptible strains. Interestingly, K. pneumoniae ATCC 700603 showed an 18-fold higher expression than K. pneumoniae ATCC 27799. These differences were in accordance with quinolone susceptibility. The prevalence of the OqxAB efflux pump (both chromosomal and plasmid encoded) in ESBL-producing K. pneumoniae is high in Spain and represents a potential reservoir for the spread of these genes. High expression of this pump contributes to reduced susceptibility to quinolones in clinical isolates of ESBL-producing K. pneumoniae.

Development of Quinoxaline 1, 4-Dioxides Resistance in Escherichia coli and Molecular Change under Resistance Selection

Quinoxaline 1, 4-dioxides (QdNOs) has been used in animals as antimicrobial agents and growth promoters for decades. However, the resistance to QdNOs in pathogenic bacteria raises worldwide concern but it is barely known. To explore the molecular mechanism involved in development of QdNOs resistance in Escherichia coli, 6 strains selected by QdNOs in vitro and 21 strains isolated from QdNOs-used swine farm were subjected to MIC determination and PCR amplification of oqxA gene. A conjugative transfer was carried out to evaluate the transfer risk of QdNOs resistant determinant. Furthermore, the transcriptional profile of a QdNOs-resistant E. coli (79O4-2) selected in vitro with its parent strain 79–161 was assayed with a prokaryotic suppression subtractive hybridization (SSH) PCR cDNA subtraction. The result showed that more than 95% (20/21) clinical isolates were oqxA positive, while all the 6 induced QdNOs-resistant strains carried no oqxA gene and exhibited low frequency of conjugation. 44 fragments were identified by SSH PCR subtraction in the QdNOs-resistant strain 79O4-2. 18 cDNAs were involved in biosynthesis of Fe-S cluster (narH), protein (rpoA, trmD, truA, glyS, ileS, rplFCX, rpsH, fusA), lipoate (lipA), lipid A (lpxC), trehalose (otsA), CTP(pyrG) and others molecular. The 11 cDNAs were related to metabolism or degradation of glycolysis (gpmA and pgi) and proteins (clpX, clpA, pepN and fkpB). The atpADG and ubiB genes were associated with ATP biosynthesis and electron transport chain. The pathway of the functional genes revealed that E. coli may adapt the stress generated by QdNOs or develop specific QdNOs-resistance by activation of antioxidative agents biosynthesis (lipoate and trehalose), protein biosynthesis, glycolysis and oxidative phosphorylation. This study initially reveals the possible molecular mechanism involved in the development of QdNOs-resistance in E. coli, providing with novel insights in prediction and assessment of the emergency and horizontal transfer of QdNOs-resistance in E. coli.

The Prevalence of the OqxAB Multidrug Efflux Pump amongst Olaquindox-ResistantEscherichia coliin Pigs

The quinoxaline olaquindox has been used extensively as a growth promoter for pigs. Recently, we isolated a plasmid (pOLA52) conferring resistance to olaquindox from swine manure. On this plasmid, the oqxA and oqxB genes encode an RND-family multidrug efflux pump, OqxAB. It facilitates resistance to olaquindox as well as resistance to other antimicrobials like chloramphenicol. In this study, 10 of the 556 (1.8%) previously isolated Escherichia coli strains were shown to have an MIC >or= 64 microg/ml olaquindox. In nine of the ten strains, the oqxA gene was detected. Sequencing of an internal fragment of oqxA from the oqxA-positive strains showed no variation, indicating highly conserved oqxA genes. All of the oqxA-positive strains contain plasmids with replicons similar to that of pOLA52. It was verified by Southern hybridization that the oqxAB operon was situated on plasmids in most, if not all, resistant strains. Furthermore, horizontal transfer of olaquindox resistance from three olaquindox-resistant isolates was achieved using an olaquindox-sensitive E. coli as recipient.