Efflux Pump Genes Research Articles

Genomic Characterization of Multidrug-Resistant Extended Spectrum β-Lactamase-Producing Klebsiella pneumoniae from Clinical Samples of a Tertiary Hospital in South Kivu Province, Eastern Democratic Republic of Congo.

Multidrug-resistant (MDR) and extended spectrum β-lactamase (ESBL)-producing extra-intestinal K. pneumoniae are associated with increased morbidity and mortality. This study aimed to characterize the resistance and virulence profiles of extra-intestinal MDR ESBL-producing K. pneumoniae associated with infections at a tertiary hospital in South-Kivu province, DRC. Whole-genome sequencing (WGS) was carried out on 37 K. pneumoniae isolates displaying MDR and ESBL-producing phenotype. The assembled genomes were analysed for phylogeny, virulence factors and antimicrobial resistance genes (ARG) determinants. These isolates were compared to sub-Saharan counterparts. K. pneumoniae isolates displayed a high genetic variability with up to 16 sequence types (ST). AMR was widespread against β-lactamases (including third and fourth-generation cephalosporins, but not carbapenems), aminoglycosides, ciprofloxacin, tetracycline, erythromycin, nitrofurantoin, and cotrimoxazole. The blaCTX-M-15 gene was the most common β-lactamase gene among K. pneumoniae isolates. No carbapenemase gene was found. ARG for aminoglycosides, quinolones, phenicols, tetracyclines, sulfonamides, nitrofurantoin were widely distributed among the isolates. Nine isolates had the colistin-resistant R256G substitution in the pmrB efflux pump gene without displaying reduced susceptibility to colistin. Despite carrying virulence genes, none had hypervirulence genes. Our results highlight the genetic diversity of MDR ESBL-producing K. pneumoniae isolates and underscore the importance of monitoring simultaneously the evolution of phenotypic and genotypic AMR in Bukavu and DRC, while calling for caution in administering colistin and carbapenem to patients.

Molecular characteristics and genetic analysis of the genes related to carbapenemase, efflux pump, and outer membrane proteins in carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a life-threatening pathogen that has not been fully investigated on a molecular basis. Therefore, the molecular mechanisms of carbapenem resistance in CRKP collected from medical institutions in Hyogo Prefecture has been analyzed. Antimicrobial susceptibilities and the presence of carbapenemase along with epidemiological analyzes using multilocus sequence typing (MLST) have been investigated. The relative expression of efflux pump genes and mutations of ompK35 and ompK36, encoding the outer membrane porin, were also assessed for their relationship with carbapenem resistance. Most of the collected 22 CRKP isolates were non-susceptible to imipenem (68.2%), meropenem (90.9%), and ertapenem (81.8%), but all 22 strains were susceptible to colistin. Twelve strains (54.5%) were detected for carbapenemase genes such as blaIMP-6. Sequence type 37 was detected by MLST in 10 strains (45.5%). Non-carbapenemase-producing strains had high resistance rates for three carbapenems, and the main cause of resistance was ompK35 mutation. In conclusion, the main cause of resistance was imipenemase metallo-β-lactamase (IMP-6) production in carbapenemase-producing strains, and ompK35 mutation in non-carbapenemase-producing strains. Susceptibility to carbapenem did not differ in CRKP regardless of carbapenemase production, except for imipenem susceptibility. This result contributes to a more insightful understanding of the mechanisms of CRKP in Japan.

Association of Antibacterial Susceptibility Profile with the Prevalence of Genes Encoding Efflux Proteins in the Bangladeshi Clinical Isolates of Staphylococcus aureus

Expelling antibiotic molecules out of the cell wall through multiple efflux pumps is one of the potential mechanisms of developing resistance against a wide number of antibiotics in Staphylococcus aureus. The aim of this study was to investigate the association between the antibiotic susceptibility profile and the prevalence of different efflux pump genes i.e., norA, norB, norC, mepA, sepA, mdeA, qacA/B, and smr in the clinical isolates of S. aureus. Sixty clinical isolates were collected from a tertiary level hospital in Bangladesh. The disc diffusion method using ten antibiotics of different classes was used to discern the susceptibility profile. polymerase chain reaction (PCR) was employed to observe the resistance patterns and to detect the presence of plasmid and chromosomal encoded genes. Among the clinical isolates, 60% (36 out of 60) of the samples were Methicillin-resistant Staphylococcus aureus (MRSA), whereas 55% (33 out of 60) of the bacterial samples were found to be multi-drug resistant. The bacteria showed higher resistance to vancomycin (73.33%), followed by ciprofloxacin (60%), cefixime (53.33%), azithromycin (43.33%), and amoxicillin (31.67%). The prevalence of the chromosomally-encoded efflux genes norA (91.67%), norB (90%), norC (93.33%), mepA (93.33%), sepA (98.33%), and mdeA (93.33%) were extremely high with a minor portion of them carrying the plasmid-encoded genes qacA/B (20%) and smr (8.33%). Several genetic combinations of efflux pump genes were revealed, among which norA + norB + norC + mepA + sepA + mdeA was the most widely distributed combination among MRSA and MSSA bacteria that conferred resistance against ciprofloxacin and probably vancomycin. Based on the present study, it is evident that the presence of multiple efflux genes potentiated the drug extrusion activity and may play a pivotal role in the development of multidrug resistance in S. aureus.

4-Allyl-2-methoxyphenol modulates the expression of genes involved in efflux pump, biofilm formation and sterol biosynthesis in azole resistant Aspergillus fumigatus.

Antifungal therapy for aspergillosis is becoming problematic because of the toxicity of currently available drugs, biofilm formation on host surface, and increasing prevalence of azole resistance in Aspergillus fumigatus. Plants are rich source of bioactive molecules and antimicrobial activity of aromatic bioactive compounds draws attention because of its promising biological properties. The present study elucidated the antibiofilm activity of 4-allyl-2-methoxyphenol (eugenol) against azole-resistant environmental A.fumigatus isolates. Soil samples were collected from agricultural fields across India; azole-resistant A. fumigatus (ARAF) were isolated followed by their molecular identification. Antibiofilm activity of eugenol was calculated via tetrazolium based-MTT assay. The expression of the multidrug efflux pumps genes MDR1, MDR4, transporters of the MFS gene, erg11A gene encoding 14α demethylase, and transcription regulatory genes, MedA, SomA and SrbA, involved in biofilm formation of A.fumigatus were calculated by quantitative real time PCR. Out of 89 A.fumigatus isolates, 10 were identified as azole resistant. Eugenol exhibited antibiofilm activity against ARAF isolates, ranging from 312 to 500 µg/mL. Confocal laser scanning microscopy analysis revealed absence of extracellular matrix of ARAF biofilm after eugenol treatment. The gene expression indicated significantly low expression of efflux pumps genes MDR1, MDR4, erg11A and MedA in eugenol treated ARAF isolates when compared with untreated isolates. Our results demonstrate that eugenol effects the expression of efflux pump and biofilm associated genes as well as inhibits biofilm formation in azole resistant isolates of A. fumigatus.

Emergence and Inter- and Intrahost Evolution of Pandrug-Resistant Klebsiella pneumoniae Coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2.

Klebsiella pneumoniae is capable of acquiring various exogenous genetic elements and subsequently conferring high antimicrobial resistance. Recently, a plasmid-mediated RND family multidrug efflux pump gene cluster, tmexCD1-toprJ1, was discovered in K. pneumoniae. In this study, we analyzed tigecycline-resistant K. pneumoniae isolates from patients from surveillance from 2017 to 2021. In addition to phenotype detection, including growth curves, plasmid transferability and stability, hypermucoviscosity, biofilm formation, and serum survival, by whole-genome sequencing, we analyzed the phylogenetic relationships of the isolates harboring tmexCD1-toprJ1 and discovered the composition of plasmids carrying tmexCD1-toprJ1. In total, we discovered that 12 tigecycline-resistant isolates from 5 patients possessed tmexCD1-toprJ1, designated sequence type 22 (ST22) and ST3691. An ST11 isolate harbored a partial tmexD1, and a complete toprJ1 (tmexC1 was lost) was tigecycline sensitive. All the ST22 tigecycline-resistant isolates coharbored tmexCD1-toprJ1, blaNDM-1, and blaKPC-2. tmexCD1-toprJ1 was encoded by a novel IncU plasmid in ST22 and an IncFIB/HI1B plasmid in ST3691, which presented differences in mobility and stability. Interestingly, isolates from the same patients presented heteroresistance to tigecycline, not only among isolates from different specimens but also those from the same sample, which might be attributed to the differential expression of tmexCD1-toprJ1 due to the dynamic genetic heterogeneity caused by relocating tmexCD1-toprJ1 close to the replication origin of plasmid. Here, we reported the emergence of K. pneumoniae isolates coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2. The results highlight the impact of in vivo genetic heterogeneity of tmexCD1-toprJ1-carrying elements on the in vivo variation of tigecycline resistance, which might have notable influences on antimicrobial treatment. IMPORTANCE Pandrug-resistant (PDR) Klebsiella pneumoniae poses a great challenge to public health, and tigecycline is an essential choice for antimicrobial treatment. In this study, we reported the emergence of PDR K. pneumoniae coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2, which belongs to ST22 and ST3691. By whole-genome analysis, we reconstructed the evolutionary map of the ST22 ancestor to become the PDR superbug by acquiring multiple genetic elements encoding tmexCD1-toprJ1 or blaNDM-1. Importantly, the genetic contexts of tmexCD1-toprJ1 among the ST22 isolates are different and present with various mobilities and stabilities. Furthermore, we also discovered the heterogeneity of tigecycline resistance during long-term infection of ST22, which might be attributed to the differential expression of tmexCD1-toprJ1 due to the dynamic genetic heterogeneity caused by relocating tmexCD1-toprJ1 close to the replication origin of plasmid. This study tracks the inter- and intrahost microevolution of the superbug PDR K. pneumoniae and highlights the importance of timely monitoring of the variation of pathogens during antimicrobial treatment.

Efflux pump effects on Mycobacterium tuberculosis drug resistance

Resistance and tolerance to antituberculosis drugs have become serious problems in disease treatment. This multi-phase study investigated the contributions of efflux pumps to Mycobacterium tuberculosis drug resistance. In the first phase, the minimum inhibitory concentration (MIC) levels of antibiotics were determined. In the second phase, MIC levels were determined in the presence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP), verapamil, reserpine and thioridazine. In the third phase, MIC levels were reduced in 6 M. tuberculosis isolates in the presence of efflux pump inhibitors to determine the expression of putative efflux pump genes by reverse transcriptase-polymerase chain reaction (RT-PCR). MIC levels of fluoroquinolones decreased in 6 (6.52%) isolates, MIC of rifampicin in 4 (4.34%), and MIC of streptomycin in 3 (3.26%) in the presence of efflux pump inhibitors reserpine, CCCP and verapamil. The efflux pump inhibitors CCCP, verapamil, and reserpine changed MICs 2- to 16-fold. Overexpression of all 15 efflux pump genes was observed in 6 isolates with a reduction in MIC values in the presence of efflux pump inhibitors. The overexpression of efflux-related genes in resistant isolates suggests that efflux pumps are associated with resistance development.

Functional Expression of Recombinant Candida auris Proteins in Saccharomyces cerevisiae Enables Azole Susceptibility Evaluation and Drug Discovery

Candida auris infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in C. auris are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes CDR1 and MDR1. We report the establishment of a novel platform for molecular analysis and drug screening based on acquired azole-resistance mechanisms found in C. auris. Constitutive functional overexpression of wild-type C. auris Erg11, Erg11 with amino acid substitutions Y132F or K143R and the recombinant efflux pumps Cdr1 and Mdr1 has been achieved in Saccharomyces cerevisiae. Phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. Strains overexpressing the Cdr1 protein were pan-azole resistant. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Type II binding spectra showed tight azole binding to the affinity-purified recombinant CauErg11 protein. The Nile Red assay confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 exhibited ATPase activity that was inhibited by Oligomycin. The S. cerevisiae overexpression platform enables evaluation of the interaction of existing and novel azole drugs with their primary target CauErg11 and their susceptibility to drug efflux.

Emergence of high-level colistin resistance mediated by multiple determinants, including mcr-1.1, mcr-8.2 and crrB mutations, combined with tigecycline resistance in an ST656 Klebsiella pneumoniae.

Colistin and tigecycline are usually regarded as the last resort for multidrug-resistant Klebsiella pneumoniae infection treatment. Emergence of colistin and tigecycline resistance poses a global healthcare challenge and is associated with high mortality due to limited therapeutic options. Here, we report the ST656 extensively drug-resistant K. pneumoniae strain KP15-652, which was isolated from a patient's urine in China. Antimicrobial susceptibility testing showed it to be resistant to tigecycline, amikacin, levofloxacin, ciprofloxacin, and high-level colistin resistance (> 2048 mg/L). Whole-genome sequencing revealed that it harbors one chromosome and seven plasmids, including four plasmids carrying multiple acquired resistance genes. Transformation/conjugation tests and plasmid curing assays confirmed that mcr-1.1, mcr-8.2 and crrB mutations are responsible for the high-level colistin resistance and that a series of efflux pump genes, such as tmexCD1-toprJ1, tet(A) and tet(M), contribute to tigecycline resistance. mcr-1.1 and tet(M) are located on an IncX1 plasmid, which has conjugation transfer potential. mcr-8.2 and tet(A) are located on a multireplicon IncR/IncN plasmid but unable to be transferred via conjugation. Moreover, another conjugable and fusion plasmid carries the tmexCD1-toprJ1 gene cluster, which may have arisen due to IS26-mediated replicative transposition based on 8-bp target-site duplications. Importantly, a complex class 1 integron carrying various resistance genes was detected on this fusion plasmid. In conclusion, it is possible that the high-level of colistin resistance is caused by the accumulated effect of several factors on the chromosome and mcr-carrying plasmids, combined with many other resistances, including tigecycline. Effective surveillance should be performed to prevent further dissemination.

Gene-Gene Interactions Reduce Aminoglycoside Susceptibility of Pseudomonas aeruginosa through Efflux Pump-Dependent and -Independent Mechanisms.

Pseudomonas aeruginosa causes a wide range of acute and chronic infections. Aminoglycosides are a cornerstone of treatment, but isolates are often resistant. The purpose of this research was to better understand the genetic basis of aminoglycoside resistance in P. aeruginosa. Bioinformatic approaches identified mutations in resistance-associated genes in the clinical isolates of P. aeruginosa. The common mutations were then engineered into the genome of P. aeruginosa reference strain PAO1. Mutations in the elongation factor gene fusA1 caused the biggest reduction in aminoglycoside susceptibility, with mutations in the two-component regulator gene amgS and the efflux pump regulator gene mexZ having less impact. This susceptibility was further reduced by combinations of mutations. Mutations in fusA1, amgS and mexZ all increased the expression of the mexXY efflux pump that is strongly associated with aminoglycoside resistance. Furthermore, the fusA1 amgS mexZ triple mutant had the highest efflux pump gene expression. Engineering fusA1 and amgS mutants lacking this efflux pump showed that fusA1 and amgS also reduce aminoglycoside susceptibility through additional mechanisms. The fusA1 and amgS mutations reduced bacterial growth, showing that these mutations have a fitness cost. Our findings demonstrate the complex interplay between mutations, efflux pump expression and other mechanisms for reducing the susceptibility of P. aeruginosa to aminoglycosides.

Migration, Transformation, and Toxicity of Quaternary Ammonium Antimicrobial Agents in the Environment

Quaternary ammonium compounds (QACs) are one type of widely used cationic biocide, and their usage amount is growing rapidly due to the flu and COVID-19 pandemic. Many QACs were released into the environment in or after the course of their use, and thus they were widely detected in water, sediment, soil, and other environmental media. QACs have stronger surface activity and non-specific biotoxicity, which poses a potential threat to the ecosystem. In this study, the environmental fate and potential toxicity of QACs were documented in terms of their migration and transformation process, biological toxicity effects, and the main mechanisms of bacterial resistance to QACs. Aerobic biodegradation was the main natural way of eliminating QACs in the environment, and the reaction was mainly initiated by the hydroxylation of C atoms at different positions of QACs and finally mineralized to CO2and H2O through decarboxylation, demethylation, and β-oxidation reaction. Toxicological studies showed that QACs at environmental concentrations could not pose acute toxicity to the selected biotas but threatened the growth and reproduction of aquatic organisms like Daphnia magna. Their toxicity effects depended on their molecular structure, the tested species, and the exposed durations. Additionally, our team first investigated the toxicity effects and mechanisms of QACs toward Microcystis aeruginosa, which showed that QACs depressed the algae growth through the denaturation of photosynthetic organelles, suppression of electron transport, and then induction of cell membrane damage. In the environment, the concentrations of QACs were always lower than their bactericidal concentrations, and their degradation could induce the formation of a concentration gradient, which facilitated microbes resistant to QACs. The known resistance mechanisms of bacteria to QACs mainly included the change in cell membrane structure and composition, formation of biofilm, overexpression of the efflux pump gene, and acquisition of resistance genes. Due to the similar targets and mechanisms, QACs could also induce the occurrence of antibiotic resistance, mainly through co-resistance and cross-resistance. Based on the existing data, future research should emphasize the toxicity effect and the potential QACs resistance mechanism of microorganisms in real environmental conditions.

Deterministic Effect of pH on Shaping Soil Resistome Revealed by Metagenomic Analysis.

Soil is recognized as the major reservoir of antibiotic resistance genes (ARGs), harboring the most diverse naturally evolved ARGs on the planet. Multidrug resistance genes are a class of ARGs, and their high prevalence in natural soil ecosystems has recently raised concerns. Since most of these genes express proton motive force (PMF) driven efflux pumps, studying whether soil pH is a determinant for the selection of multidrug efflux pump genes and thus shaping the soil resistome are of great interest. In this study, we collected 108 soils with pH values ranging from 4.37 to 9.69 from multiple ecosystems and profiled the composition of ARGs for metagenomes and metagenome-assembled genomes. We observed the multidrug efflux pump genes enriched in the acidic soil resistome, and their abundances have significant soil pH dependence. This reflects the benefits of high soil proton activity on the multidrug efflux pump genes, especially for the PMF-driven inner membrane transferase. In addition, we preliminary indicate the putative microbial participants in pH shaping the soil resistome by applying ecological analyzing tools such as stepwise regression and random forest model fitting. The decisive influence of proton activity on shaping the resistome is more impactful than any other examined factors, and as the consequence, we revisited the influence of edaphic factors on the soil resistome; i.e., the deterministic selection of resistance mechanisms by edaphic factors could lead to the bottom-up shaping of the ARG composition. Such natural developing mechanisms of the resistome are herein suggested to be considered in assessing human-driven ARG transmissions.

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.

Molecular typing methods & resistance mechanisms of MDR Klebsiella pneumoniae.

The emergence and transmission of carbapenem-resistant Klebsiella pneumoniae (CRKP) have been recognized as a major public health concern. Here, we investigated the molecular epidemiology and its correlation with the mechanisms of resistance in CRKP isolates by compiling studies on the molecular epidemiology of CRKP strains worldwide. CRKP is increasing worldwide, with poorly characterized epidemiology in many parts of the world. Biofilm formation, high efflux pump gene expression, elevated rates of resistance, and the presence of different virulence factors in various clones of K. pneumoniae strains are important health concerns in clinical settings. A wide range of techniques has been implemented to study the global epidemiology of CRKP, such as conjugation assays, 16S-23S rDNA, string tests, capsular genotyping, multilocus sequence typing, whole-genome sequencing-based surveys, sequence-based PCR, and pulsed-field gel electrophoresis. There is an urgent need to conduct global epidemiological studies on multidrug-resistant infections of K. pneumoniae across all healthcare institutions worldwide to develop infection prevention and control strategies. In this review, we discuss different typing methods and resistance mechanisms to explore the epidemiology of K. pneumoniae pertaining to human infections.

Antibiotic Resistance of Hemophilus influenzae Isolated from Children in Southwest China.

Hemophilus influenzae (Hi) is one of the major pediatric bacterial pneumonia pathogens that heavily threatens children's lives and global health. With widespread usage as first-line treatment, the prevalence of β-lactam-resistant strains is increasing sharply. In order to treat Hi more effectively, a systematic study on the antibiotic resistance profiles, β-lactamase-negative ampicillin-resistant (BLNAR) strains isolation rate, and potential BLNAR resistance mechanism in our region is needed. This study analyzed antimicrobial susceptibility of Hi, and clinical data of Hi-infected patients retrospectively. BLNAR and β-lactamase-positive ampicillin-clavulanate resistant strains (BLPACR) were confirmed by the Kirby-Bauer method and β-lactamase test. ftsI gene in BLNAR was sequenced to find out whether resistance was induced by penicillin-binding protein mutation. Ampicillin susceptibility test with or without efflux pump inhibitors were done to assess efflux pump contribution in BLNAR. RT-PCR was performed to evaluate the efflux pump genes' transcription levels. A total of 2,561 Hi strains were isolated in our hospital from January 2016 to December 2019. Male to female ratio was 1.52:1. Median age was 10 months. Infant (< 3 years old) infection accounted for 83.72%. Hi resistance rates to sulfamethoxazole-trimethoprim, ampicillin, cefathiamidine, cefaclor, cefuroxime, cephalothin, amoxicillin-clavulanate, tetracycline, chloramphenicol, ofloxacin, cefotaxime, and rifampin were 84.28%, 78.01%, 49.80%, 41.98%, 36.58%, 33.64%, 4.55%, 4.1%, 3.37%, 1.77%, 0.99%, and 0.12%, respectively, while 1.33% were BLNAR. BLNARs were classified into four groups by mutation patterns in ftsI gene and most strains were divided to Group Ⅲ/Ⅲ-like. EmrB, ydeA and norM transcription levels in some ampicillin-resistant strains were higher than their sensitive counterparts. Ampicillin is not sufficiently effective as a first-line Hi infection treatment. However, ampicillin-clavulanate and cefotaxime may be a better choice. Efflux pumps, emrB, ydeA and norM play roles in the high resistance to ampicillin.

Molecular Detection of Some Efflux Pumps Genes in Escherichia Coli Isolated from Patients with Urinary Tract Infections in Baqubah City, Iraq

Molecular Detection of Some Efflux Pumps Genes in Escherichia Coli Isolated from Patients with Urinary Tract Infections in Baqubah City, Iraq

Analysis of Efflux Pump System and Other Drug Resistance Related Gene Mutations in Tigecycline-Resistant Acinetobacter baumannii.

The isolation of tigecycline-resistant Acinetobacter baumannii in recent years has brought great difficulties to clinical prevention and treatment. To explore the effect of efflux pump system and other resistance related gene mutations on tigecycline resistance in Acinetobacter baumannii. Fluorescence quantitative PCR was used to detect the expression levels of major efflux pump genes (adeB, adeJ, and adeG) in extensive drug-resistant Acinetobacter baumannii. The minimum inhibitory concentration (MIC) of tigecycline was detected by the broth microdilution testing and efflux pump inhibition experiment to assess the role of efflux pump in tigecycline resistance of Acinetobacter baumannii. Efflux pump regulatory genes (adeR and adeS) and tigecycline resistance related genes (rpsJ, trm, and plsC) were amplified by PCR and sequenced. By sequence alignment, tigecycline sensitive and tigecycline-insensitive Acinetobacter baumannii were compared with standard strains to analyze the presence of mutations in these genes. The relative expression of adeB in the tigecycline-insensitive Acinetobacter baumannii was significantly higher than that in the tigecycline sensitive Acinetobacter baumannii (114.70 (89.53-157.43) vs 86.12 (27.23-129.34), P = 0.025). When efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added, the percentage of tigecycline-insensitive Acinetobacter baumannii with tigecycline MIC decreased was significantly higher than that of tigecycline-sensitive Acinetobacter baumannii (10/13 (76.9%) vs 26/59 (44.1%)), P = 0.032); the relative expression of adeB in the MIC decreased group was significantly higher than that in the MIC unchanged group (110.29 (63.62-147.15) vs 50.06 (26.10-122.59), P = 0.02); The relative expression levels of efflux pumps adeG and adeJ did not increase significantly, and there was no significant difference between these groups. One adeR point mutation (Gly232Ala) and eight adeS point mutations (Ala97Thr, Leu105Phe, Leu172Pro, Arg195Gln, Gln203Leu, Tyr303Phe, Lys315Asn, Gly319Ser) were newly detected. Consistent mutations in trm and plsC genes were detected in both tigecycline-insensitive and tigecycline-sensitive Acinetobacter baumannii, but no mutation in rpsJ gene was detected in them. Tigecycline-insensitive Acinetobacter baumannii efflux pump adeABC overexpression was an important mechanism for tigecycline resistance, and the mutations of efflux pump regulator genes (adeR and adeS) are responsible for adeABC overexpression. The effect of trm, plsC, and rpsJ gene mutations on the development of tigecycline resistance in Acinetobacter baumannii remains controversial.

دور مضخة الدفق في مقاومة البكتيريا للمضادات الحيوية

The multi-drug resistant efflux pump is a glycoprotein pump whose function is to push foreign substances. The efflux pump is found in humans, animals. It also has wide-ranging properties in bacteria and fungi. They are found in all species of bacteria, and efflux pump genes can be found in bacterial chromosomes or mobile genetic elements, such as plasmids. The most sensitive function that leads to a global problem is its resistance to antibiotics in bacterial cells, which increases the ability to bacteria from becoming strong virulence factors that most or all antibiotics cannot kill. It also has other important functions. It is related to the defense mechanism of the horse by pushing all harmful substances. This pump is divided into five families. The division depends on the source of energy, symmetry, and the general structure. The major facilitator (MFS) superfamily; the multidrug and toxic compound extrusion (MATE) family and the adenosine-triphosphate (ATP)-binding cassette (ABC); superfamily the resistance-nodulation-cell division (RND) superfamily and the small multidrug resistance (SMR). The ABC family uses ATP as a source of energy for export, while other flow pumps use the driving force of the proton. These hosts participate in many cellular processes such as intercellular communication, formation of cell membranes, virulence factors of bacterial cell, extrusion of toxic substances, disinfectants, toxic metabolic by-products, and antibiotics. It is also one of the important formations in bacteria that give bacteria the ability to expel antibiotics through various and diverse genes that are located either on the chromosome or plasmid of different pump families. Evidence indicates that the efflux pump has physiological functions in bacteria, and its overexpression has a role in inducing multi-drug resistance and biofilm formation. Hence, the regulation of efflux pump action is an orderly, strict action in response to physiological and environmental signals.

Evaluation of Antibiotic Resistance and adeABC, adeR, adeS Efflux Pump Genes among Foodborne and Clinical Acinetobacter spp. in Türkiye.

The adeABC efflux pump has a crucial role in the resistance of Acinetobacter baumannii strains to antimicrobial agents; it is encoded by adeABC, adeR, adeS genes. We evaluated antibiotic resistance, efflux pump genes, clonal relationships, and analyzed a probable correlation that can exist between antibiotic resistance and the aforementioned genes. We conducted this study on 27 food-originated and 50 human clinical Acinetobacter spp. in Southern Türkiye. MALDI-TOF system and disc diffusion/agar dilution (colistin) methods were used for the identification and antibiotic susceptibility. The efflux pump genes and genetic relatedness of the two groups were investigated by (PCR) and (PFGE) methods. Foodborne A. dijkshoorniae strain was multidrug- resistant (MDR), and none of them resistant to colistin. Most of the clinical isolates (92%) were Extensive-Drug Resistant (XDR); highest resistant to ceftazidime, piperacillin-tazobactam, and imipenem (47, 94%), and were lowest to colistin (7, 14%), respectively. adeABC, and adeR, adeS genes were (23, 85.2%), (9, 33.3%), (27, 100%) and (10, 37.3%), (18, 66.7%) in foodborne strains respectively. These rates were (43, 86%), (48, 96%), (50, 100%), and (34, 68%), (48, 96.7%) in clinical strains respectively. A positive correlation existed between adeA gene positivity and piperacillintazobactam, ceftazidime, gentamycin, imipenem (P=0.048), amikacin (P=0.007) and trimethoprimsulfamethoxazole (P=0.029) resistance in clinical strains. A positive correlation of trimethoprimsulfamethoxazole resistance and adeS gene positivity was seen in foodborne strains (P=0.018). Multiple-efflux pump genes rise in parallel to multidrug-resistance in clinical isolates, while susceptible to diverse antibiotics; food may be a potential provenance for the dissemination of adeABC, adeR and adeS genes.

Deciphering the microbial community tolerance mechanism and alteration of antibiotic resistance genes during chloramphenicol wastewater treatment

Excessive use of chloramphenicol (CAP) and its residues has emerged as a potential threat to human health and ecological safety. It is challenging to reveal the key microbial species in the CAP biodegradation process and interactions of the microbial community with the antibiotic resistance genes (ARGs). The present study sought to investigate the interactions of microbial community with ARGs during the sequential batch reactor (SBR) treatment of CAP wastewater to explore the potential antibiotic resistance mechanisms. The treatment showed excellent performance with 95.5% CAP removal efficiency based on the core genera of Candidatus Competibacter and Hydrogenophaga with strong antibiotic resistance and adaptability to the increasing CAP concentration in the influent. Meanwhile, Candidatus Competibacter and Hydrogenophaga were significantly associated with the primary CAP resistance genes of cml_e3 and cml_e8, which developed resistance against CAP pressure through efflux pump mechanism. Furthermore, the two CAP efflux pumps genes (floR and cmlA) were stimulated at low CAP level (0.01–0.1 g L−1) while suppressed at high CAP level (0.2 g L−1). This study emphasizes that SBR is an eco-friendly strategy to remove CAP by enriching the core genera of Candidatus Competibacter and Hydrogenophaga through the efflux pump mechanism to against the CAP pressure.

A comprehensive description of the TolC effect on the antimicrobial susceptibility profile in Enterobacter bugandensis.

Enterobacter bugandensis is an emerging human pathogen in which multidrug resistant strains have been continuously isolated from various environments. Thus, this organism possesses the potential to pose challenges in human healthcare. However, the mechanisms, especially the efflux pumps, responsible for the multidrug resistance in E. bugandensis remain to be well elucidated. The Enterobacter strain CMCC(B) 45301 was specifically identified using whole genome sequencing. The specific CMCC(B) 45301 homologues of the TolC dependent efflux-pump genes characterized in Escherichia coli were identified. The tolC deletion mutant in CMCC(B) 45301 was constructed and subjected to susceptibility tests using 26 different antimicrobial agents, along with the wild type strain. The synergistic effects combining the Bacillus crude extract (BCE) and several other TolC-affected compounds against CMCC(B) 45301 were assayed. We reclassified the Enterobacter CMCC(B) 45301 strain from species cloacae to bugandensis, on the basis of its whole genome sequence. We found that the CMCC(B) 45301 TolC, AcrAB, AcrD, AcrEF, MdtABC, EmrAB, and MacAB exhibit high similarity with their respective homologues in E. coli and Enterobacter cloacae. Our results for the susceptibility tests revealed that lacking tolC causes 4- to 256-fold decrease in the minimal inhibitory concentrations of piperacillin, gentamicin, kanamycin, tetracycline, norfloxacin, ciprofloxacin, chloramphenicol, and erythromycin against CMCC(B) 45301. In addition, the inhibition zones formed by cefuroxime, cefoperazone, amikacin, streptomycin, minocycline, doxycycline, levofloxacin, florfenicol, trimethoprim-sulfamethoxazole, azithromycin, lincomycin, and clindamycin for the tolC mutant were larger or more obvious than that for the parent. Our data suggested the important role played by TolC in CMCC(B) 45301 susceptibility to common antibiotic families covering ß-lactam, aminoglycoside, tetracycline, fluoroquinolone, phenicol, folate pathway antagonist, macrolide, and lincosamide. Deletion for tolC also increased the susceptibility of CMCC(B) 45301 to berberine hydrochloride and BCE, two natural product-based agents. Finally, we found that erythromycin, norfloxacin, and ciprofloxacin can potentiate the antibacterial activity of BCE against CMCC(B) 45301. The present study elaborated the comprehensive TolC effect on the antimicrobial susceptibility profile in E. bugandensis, which might contribute to the development of more therapeutic options against this nosocomial pathogen.