scholarly journals Antibiotic Resistant, Virulence-associated Genes, Biofilm and Efflux Pump Gene Expression and Molecular Typing of Klebsiella Pneumoniae Strains Recovered from Clinical Samples

2020 ◽  
Author(s):  
Amir Mirzaie ◽  
Reza Ranjbar
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Molecular Typing ◽  
Efflux Pump ◽  
Clinical Samples ◽  
Public Health Issue ◽  
Pcr Analysis ◽  
Antibiotic Resistant ◽  
Microbiological Methods ◽  
High Level

Abstract BackgroundMultidrug-resistant (MDR) Klebsiella pneumoniae strains are one of the most important life-threatening nosocomial pathogens. In the current study, antibiotic resistant, virulence-associated genes, gene expression of efflux pumps and biofilm genes as well as molecular typing of K. pneumoniae strains were investigated. A total of 505 clinical specimens were collected from hospitalized patients and K. pneumoniae strains were isolated by standard microbiological methods. Antibiotic resistant profile, prevalence of virulence associated genes, biofilm and efflux pump genes were investigated. The gene expression analysis of biofilm and efflux pump genes were analused quantitative Real Time PCR. Moreover, molecular typing of K. pneumoniae strains using Repetitive element sequence-based PCR (rep-PCR) technique was also carried out. ResultsOne hundred K. pneumoniae strains out of 500 clinical samples were isolated and the highest prevalence of resistance was observed against ciprofloxacin (75%), Trimethoprim-sulfamethoxazole (73%) and Nitrofurantoin (38%). Virulence associated genes including entB, traT and rmpA were found in 80%, 62% and 48%, respectively. Gene prevalence for biofilm association gene including mrkA, fimH and mrkD were 42% for all genes. The AcrAB, TolC and mdtK efflux pump genes were observed in 41%, 33% and 26%, respectively. In addition, most MDR strains formed biofilm, as well as, AcrAB efflux pump and mrkA biofilm gene expression was up-regulated in MDR K. pneumoniae strains and a significant statistically association was also observed between MDR strains and high expression of efflux pump and biofilm genes. In addition, the K. pneumoniae strains differentiated into 11 different genetic clusters by rep-PCR analysis. ConclusionsHigh prevalence of resistance, presence of diver’s virulence factors and high level of efflux pump and biofilm gene expression in diverse clones of K. pneumoniae strains pose an important public health issue.

scholarly journals Antibiotic resistance, virulence-associated genes analysis and molecular typing of Klebsiella pneumoniae strains recovered from clinical samples

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Amir Mirzaie ◽  
Reza Ranjbar
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Molecular Typing ◽  
Efflux Pump ◽  
Opportunistic Pathogen ◽  
Relevant Information ◽  
Multidrug Resistant ◽  
Clinical Samples ◽  
Microbiological Methods ◽  
High Level

AbstractKlebsiella pneumoniae is a multidrug-resistant (MDR) opportunistic pathogen that causes nosocomial infections. Virulence analysis and molecular typing as powerful approaches can provide relevant information on K. pneumoniae infection. In the current study, antibiotic resistance, virulence-associated genes analysis, as well as molecular typing of K. pneumoniae strains were investigated. Out of 505 clinical samples collected from hospitalized patients, 100 K. pneumoniae strains were isolated by standard microbiological methods and subjected to the phenotypic and genotyping analysis. The highest prevalence of resistance was observed against ciprofloxacin (75%), trimethoprim–sulfamethoxazole (73%) and nitrofurantoin (68%). Virulence associated genes including entB, traT, ybts, magA, iucC, htrA and rmpA were found in 80%, 62%, 75%, 5%, 30%, 72% and 48%, of the isolates, respectively. The prevalence of biofilm-associated genes including mrkA, fimH, and mrkD were equally 88% for all tested isolates. Moreover, the efflux pump genes including AcrAB, TolC and mdtK were observed in 41 (41%), 33 (33%) and 26 (26%) of the strains respectively. A significant statistical association was observed between MDR strains and high expression of efflux pump and biofilm genes. The K. pneumoniae strains were differentiated into 11 different genetic patterns using the repetitive element sequence-based PCR (rep-PCR) technique. High prevalence of resistance, presence of various virulence factors, high level of efflux pump, and biofilm gene expression in diverse clones of K. pneumoniae strains pose an important health issue in clinical settings.

scholarly journals The impact of Grammosciadium platycarpum Boiss. & Hausskn. extract on oqxA efflux pump gene expression in antibiotic resistant clinical isolates of Klebsiella pneumoniae using real time PCR

2020 ◽  
Vol 19 (75) ◽  
pp. 291-304
Author(s):  
Faezeh Mohammadpour Bishak ◽  
Fatemeh Ashrafi ◽  
Soheila Moradi Bidhendi ◽  
Amir Mirzaie ◽  
Hassan Noorbazargan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Efflux Pump ◽  
Clinical Isolates ◽  
Antibiotic Resistant ◽  
The Impact

scholarly journals Molecular Epidemiology and Characterization of Carbapenem-Resistant Klebsiella pneumoniae Isolated from Urine at a Teaching Hospital in Taiwan

2021 ◽  
Vol 9 (2) ◽  
pp. 271
Author(s):  
Yuarn-Jang Lee ◽  
Chih-Hung Huang ◽  
Noor Andryan Ilsan ◽  
I-Hui Lee ◽  
Tzu-Wen Huang
Keyword(s):  
Klebsiella Pneumoniae ◽  
Teaching Hospital ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Pcr Analysis ◽  
Carbapenem Resistant ◽  
High Prevalence ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests ◽  
Tract Infections

Urinary tract infections (UTIs) are common in clinics and hospitals and are associated with a high economic burden. Enterobacterium Klebsiella pneumoniae is a prevalent agent causing UTIs. A high prevalence of carbapenem-resistant K. pneumoniae (CRKP) has emerged recently and is continuing to increase. Seventeen urinary CRKP isolates collected at a teaching hospital in Taiwan from December 2016 to September 2017 were analyzed to elucidate their drug resistance mechanisms. Two-thirds of the isolates were obtained from outpatients. Antimicrobial susceptibility tests demonstrated multidrug resistance in all the isolates. Multilocus sequence typing analysis showed high diversity among the isolates. PCR analysis demonstrated the presence of carbapenemases in three isolates. All isolates carried at least one other extended-spectrum β-lactamase, including TEM, DHA, and CTX-M. Fifteen isolates contained mutations in one of the outer membrane porins that were assessed. The expression levels of the acrB and/or oqxB efflux pump genes, as determined by qRT-PCR, were upregulated in 11 isolates. Six isolates might have utilized other efflux pumps or antimicrobial resistance mechanisms. These analyses demonstrated a highly diverse population and the presence of complex resistance mechanisms in urinary isolates of K. pneumoniae.

scholarly journals Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Saeed Sharifi ◽  
Bita Bakhshi ◽  
Shahin Najar-peerayeh
Keyword(s):  
Gene Expression ◽  
Point Mutation ◽  
Campylobacter Jejuni ◽  
Significant Contribution ◽  
Efflux Pump ◽  
Campylobacter Coli ◽  
Rapd Pcr ◽  
Resistant Strains ◽  
High Level ◽  
Level Resistance

Abstract Background Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. Methods A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. Results Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. Conclusion Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

scholarly journals Efflux-Related Resistance to Norfloxacin, Dyes, and Biocides in Bloodstream Isolates of Staphylococcus aureus

2007 ◽  
Vol 51 (9) ◽  
pp. 3235-3239 ◽  
Author(s):  
Carmen E. DeMarco ◽  
Laurel A. Cushing ◽  
Emmanuel Frempong-Manso ◽  
Susan M. Seo ◽  
Tinevimbo A. A. Jaravaza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Efflux Pump Inhibitor ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Environmental Surfaces ◽  
Genes Encoding ◽  
High Level

ABSTRACT Efflux is an important resistance mechanism in Staphylococcus aureus, but its frequency in patients with bacteremia is unknown. Nonreplicate bloodstream isolates were collected over an 8-month period, and MICs of four common efflux pump substrates, with and without the broad-spectrum efflux pump inhibitor reserpine, were determined (n = 232). A reserpine-associated fourfold decrease in MIC was considered indicative of efflux. Strains exhibiting efflux of at least two of the four substrates were identified (“effluxing strains” [n = 114]). For these strains, MICs with or without reserpine for an array of typical substrates and the expression of mepA, mdeA, norA, norB, norC, and qacA/B were determined using quantitative real-time reverse transcription-PCR (qRT-PCR). A fourfold or greater increase in gene expression was considered significant. The most commonly effluxed substrates were ethidium bromide and chlorhexidine (100 and 96% of effluxing strains, respectively). qRT-PCR identified strains overexpressing mepA (5 [4.4%]), mdeA (13 [11.4%]), norA (26 [22.8%]), norB (29 [25.4%]), and norC (19 [16.7%]); 23 strains overexpressed two or more genes. Mutations probably associated with increased gene expression included a MepR-inactivating substitution and norA promoter region insertions or deletions. Mutations possibly associated with increased expression of the other analyzed genes were also observed. Effluxing strains comprised 49% of all strains studied (114/232 strains), with nearly half of these overexpressing genes encoding MepA, MdeA, and/or NorABC (54/114 strains). Reduced susceptibility to biocides may contribute to persistence on environmental surfaces, and efflux of drugs such as fluoroquinolones may predispose strains to high-level target-based resistance.

scholarly journals Investigation of Antibiotic Resistance and Biofilm Formation in Clinical Isolates of Klebsiella pneumoniae

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Kiana Karimi ◽  
Omid Zarei ◽  
Parinaz Sedighi ◽  
Mohammad Taheri ◽  
Amin Doosti-Irani ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Resistance Pattern ◽  
Chi Square ◽  
Disk Diffusion Method ◽  
High Level ◽  
Tract Infections

Aim. Klebsiella pneumoniae (K. pneumoniae) is an encapsulated Gram-negative bacterium that can lead to 14–20% of nosocomial infections. The ability of biofilm formation in this bacterium decreases the host immune response and antibiotic efficacy. This may impose a huge impact on patients and healthcare settings. This study aimed to evaluate the antibiotic resistance pattern and biofilm formation in K. pneumoniae strains isolated from two major Hamadan hospitals, west of Iran. Methods. A total of 83 K. pneumoniae strains were isolated from clinical samples of patients in different wards of Hamadan hospitals from September 2018 to March 2019. Determination of antimicrobial susceptibility was performed using the disk diffusion method. Biofilm formation was evaluated by the crystal violet method. Data were analyzed by the SPSS software and chi-square test. Results. The results showed that clinical samples included 18 urinary tract samples (22%), 6 wound samples (7%), 6 blood samples (7%), 17 tracheal tube aspiration samples (20%), 32 throat cultures (38%), 2 sputum samples (2.5%), and 2 abscess drain cultures (2.5%). High-level resistance to cefotaxime was detected in 92%, and all of isolates were susceptible to colistin. Biofilm formation was seen in 62 (75%) isolates. Strong biofilm formation was observed in 17 (20%) strains. A significant correlation was seen between biofilm formation and antibiotic resistance ( P value <0.05). Conclusion. Our findings emphasize the need for proper diagnosis, control, and treatment of infections caused by K. pneumoniae especially in respiratory tract infections due to the strong biofilm formation and high antibiotic resistance in these strains.

scholarly journals Outer Membrane Profiles of Clonally Related Klebsiella pneumoniae Isolates from Clinical Samples and Activities of Cephalosporins and Carbapenems

1998 ◽  
Vol 42 (7) ◽  
pp. 1636-1640 ◽  
Author(s):  
Carmen Ardanuy ◽  
Josefina Liñares ◽  
María Angeles Domínguez ◽  
Santiago Hernández-Allés ◽  
Vicente J. Benedí ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Parental Strain ◽  
Electrophoretic Analysis ◽  
Clinical Samples ◽  
Nosocomial Outbreak ◽  
Chromosomal Dna ◽  
Clonal Type ◽  
Extended Spectrum ◽  
Gene Coding ◽  
High Level

ABSTRACT Fifteen isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBLs) isolated during a nosocomial outbreak were studied. The strains belonged to the same clonal type, as shown by pulsed-field gel electrophoretic analysis of chromosomal DNA. All the isolates were resistant to extended-spectrum cephalosporins, aztreonam, gentamicin, and fluoroquinolones and were susceptible to carbapenems, tobramycin, netilmicin, and amikacin. None of the isolates expressed the OmpK36 porin. Eight isolates, for which the MICs of cefoxitin were ≥64 μg/ml, showed a diminished level or no expression of a 35-kDa porin. The MICs of meropenem, cefotaxime, and cefpirome were three to eight times higher for porin-deficient isolates than for isolates expressing the 35-kDa porin, but the MICs of imipenem increased two times for porin-deficient isolates compared to those for isolates expressing the porin. This MIC increase reverted to a level similar to that for the parental strain when porin-deficient isolates were transformed with the gene coding for theK. pneumoniae porin OmpK36. It is concluded that the high level of resistance to cefoxitin and the increase in the MICs of meropenem, cefotaxime, and cefpirome for the ESBL-producingK. pneumoniae isolates studied are associated with porin deficiency.

scholarly journals Molecular Typing of Klebsiella pneumoniae Clinical Isolates by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Parinaz Sedighi ◽  
Omid Zarei ◽  
Kiana Karimi ◽  
Mohammad Taheri ◽  
Pezhman Karami ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Polymerase Chain Reaction ◽  
Klebsiella Pneumoniae ◽  
Molecular Typing ◽  
Genetic Relatedness ◽  
Clinical Isolates ◽  
Enterobacterial Repetitive Intergenic Consensus ◽  
Chain Reaction ◽  
Microbiological Methods ◽  
Polymerase Chain

Aim. Klebsiella pneumoniae is one of the most important causes of nosocomial infections, including pneumonia, sepsis, and urinary tract infection. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique is a quick, reliable, and cost-effective method for molecular typing of Enterobacteriaceae family members. This study aimed to detect genetic relatedness among K. pneumoniae isolates from hospitals in Hamadan city, using ERIC-PCR technique. Materials and Methods. A total of 72 K. pneumoniae isolates were collected from patients admitted to Besat and Sina hospitals. After detection and confirmation of K. pneumonia isolates by chemical and conventional microbiological methods, DNAs were extracted after 24 hours of incubation at 37°C, using the boiling method. ERIC-PCR technique was carried out, and the ERIC patterns were analyzed by online data analysis service (inslico.ehu.es). ERIC profiles were compared using Dice method and clustered by UPGMA (unweighted pair group method with arithmetic mean) program. Also, the samples were evaluated by PCR method for the detection of aerobactin gene within their genome. Finding. The genetic relatedness among K. pneumoniae isolates was studied, and results established the genetic diversity of the clinical isolates by detecting 25 different ERIC types, including 14 common types and 11 unique types. Also, none of the isolates had aerobactin gene. Discussion. The results of this study showed high genetic diversity among K. pneumoniae strains, indicating the polyclonal distribution of K. pneumoniae isolates in Hamadan hospitals. This diversity causes problems for the treatment of infections due to the circulation of diverse K. pneumoniae clones, which possibly have different antimicrobial susceptibility patterns.

scholarly journals Evaluation of Antibiotic Sensitivity Pattern and Plasmid Isolation of Klebsiella Pneumoniae Isolates from Clinical Specimens in Sylhet City, Bangladesh

2017 ◽  
Vol 9 (1) ◽  
pp. 79-86
Author(s):  
P. Bhattacharjee ◽  
M. Z. Alam ◽  
S. M. A. Sayem
Keyword(s):  
Klebsiella Pneumoniae ◽  
Antimicrobial Agents ◽  
Bacterial Pathogen ◽  
Antibiotic Sensitivity ◽  
High Rate ◽  
Multi Drug Resistance ◽  
Clinical Samples ◽  
Antimicrobial Sensitivity ◽  
Microbiological Methods ◽  
Sensitivity Pattern

Klebsiella pneumoniae is recognized as an emerging opportunistic bacterial pathogen of clinical relevance for its association with both community acquired and nosocomial infections. Moreover, increased resistance to common antimicrobial agents complicates and limits available therapeutic options. The aim of this study was to investigate an antibiotic sensitivity pattern of K. pneumoniae isolated from clinical samples against commonly used antibiotics and to focus on the associated therapeutic challenges. Presumptive K. pneumoniae isolates were collected from a variety of sources like urine sample, pus, wound swab etc. and further analyzed through standard microbiological methods. Following the identification of 23 bacterium isolates from the collected specimens, an antimicrobial sensitivity test was carried out. The results of this test indicated that the organisms possess diversity of susceptibility profiles towards the drugs tested. A high rate of resistance to the first line antibiotics like Amoxicillin (100%), Vancomycin (90.90%), Erythromycin (83.33%), and Cotrimoxazole (83.33%), designates a critical therapeutic challenge. However, inspite of a moderate sensitivity to the Gentamicin (43.5%), increased number of intermediate sensitivity to the drugs tested clearly point towards the multi-drug resistance of the K. pneumoniae isolates. Plasmid profiling experiment revealed that the size of the isolated plasmids varied in length, ranging from 800-900bp.

scholarly journals Expression of the qepA1 gene is induced under antibiotic exposure

2021 ◽  
Author(s):  
Gerrit Brandis ◽  
Jonas Gockel ◽  
Linnéa Garoff ◽  
Lionel Guy ◽  
Diarmaid Hughes
Keyword(s):  
Gene Expression ◽  
Efflux Pump ◽  
Class I ◽  
Upstream Region ◽  
Antibiotic Exposure ◽  
Negative Effects ◽  
Integrase Gene ◽  
Bacterial Fitness ◽  
High Level

Abstract Background The qepA1 gene encodes an efflux pump that reduces susceptibility to ciprofloxacin. Little is known about the regulation of qepA1 expression. Objectives To assess the potential role of ciprofloxacin and other antibiotics in the regulation of qepA1 gene expression. To identify the promoter that drives qepA1 expression and other factors involved in expression regulation. To assess whether the identified features are universal among qepA alleles. Methods A translational qepA1-yfp fusion under the control of the qepA1 upstream region was cloned into the Escherichia coli chromosome. Expression of the fusion protein was measured in the presence of various antibiotics. Deletions within the upstream region were introduced to identify regions involved in gene expression and regulation. The qepA1 coding sequence and upstream region were compared with all available qepA sequences. Results Cellular stress caused by the presence of various antibiotics can induce qepA1 expression. The qepA1 gene is fused to a class I integron and gene expression is driven by the Pc promoter within the integrase gene. A segment within the integron belonging to a truncated dfrB4 gene is essential for the regulation of qepA1 expression. This genetic context is universal among all sequenced qepA alleles. Conclusions The fusion of the qepA1 gene to a class I integron has created a novel regulatory unit that enables qepA1 expression to be under the control of antibiotic exposure. This setup mitigates potential negative effects of QepA1 production on bacterial fitness by restricting high-level expression to environmental conditions in which QepA1 is beneficial.

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