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

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.

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

10.21203/rs.3.rs-96879/v1 ◽

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.

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Association of Efflux Pump and OMPs with Antibiotic Susceptibility Among ESBL-Producing Klebsiella Pneumoniae Clinical Strains in Malaysia

10.21203/rs.3.rs-375267/v1 ◽

2021 ◽

Author(s):

Golnaz Mobasseri ◽

Thong Kwai Lin ◽

Cindy Shuan Ju Teh

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Antibiotic Susceptibility ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Multidrug Resistant ◽

Efflux Pump Inhibitor ◽

Pump System ◽

Extended Spectrum Beta Lactamases ◽

Amoxicillin Clavulanate

Abstract Multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) poses a serious public health threat. K. pneumoniae strains that produce extended-spectrum beta-lactamases (ESBL) are becoming increasingly reported in nosocomial and community-acquired infections. Besides resistance genes, integrons, and plasmids, altered membrane permeability caused by porin loss and energy-dependent efflux have also contributed to antibiotic resistance in K. pneumoniae. The objective of this study was to determine the correlation between the reduction of antibiotic susceptibility and overexpression of efflux pump as well as the lack of outer membrane proteins (OMPs) among clinical ESBLs resistant K. pneumoniae. The expression levels of ramA, acrA, ompK35 and ompK36 in 12 MDR K. pneumoniae strains with varying MICs levels were analyzed using quantitative real time-Polymerase Chain Reaction (qRT-PCR). The role of efflux pump on antibiotic resistance was also studied by using minimum inhibitory concentration (MICs) method with//without efflux pump inhibitor. The result indicated that the strains with highest resistance to cefotaxime showed the lowest level of ompK35 and ompK36 genes expression while the strains with lowest MIC level of resistance to cefotaxime showed the highest level of expression of acrA and ramA. Our finding also revealed the effect of efflux pump inhibitor phenyl-arginine-b-naphthylamide (PAβN) on the MIC levels of ceftazidime, amoxicillin-clavulanate and cefotaxime which were significantly reduced around 1–7 folds MIC levels. These results suggest that Efflux pump system and deficiently of OMPs contributing role in antibiotic susceptibility which should be taken seriously to prevent the treatment failure due to antimicrobial resistance.

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

International Journal of Microbiology ◽

10.1155/2021/5573388 ◽

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.

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Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates

Biochemical Journal ◽

10.1042/bj20030963 ◽

2003 ◽

Vol 376 (3) ◽

pp. 801-805 ◽

Cited By ~ 75

Author(s):

Monique MALLÉA ◽

Abdallah MAHAMOUD ◽

Jacqueline CHEVALIER ◽

Sandrine ALIBERT-FRANCO ◽

Pierre BROUANT ◽

...

Keyword(s):

Antibiotic Resistance ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Microbial Pathogens ◽

Gram Negative Bacteria ◽

Gram Negative ◽

High Level ◽

Antibiotic Efflux

Over the last decade, MDR (multidrug resistance) has increased worldwide in microbial pathogens by efflux mechanisms, leading to treatment failures in human infections. Several Gram-negative bacteria efflux pumps have been described. These proteinaceous channels are capable of expelling structurally different drugs across the envelope and conferring antibiotic resistance in various bacterial pathogens. Combating antibiotic resistance is an urgency and the blocking of efflux pumps is an attractive response to the emergence of MDR phenotypes in infectious bacteria. In the present study, various alkylaminoquinolines were tested as potential inhibitors of drug transporters. We showed that alkylaminoquinolines are capable of restoring susceptibilities to structurally unrelated antibiotics in clinical isolates of MDR Gram-negative bacteria. Antibiotic efflux studies indicated that 7-nitro-8-methyl-4-[2´-(piperidino)ethyl]aminoquinoline acts as an inhibitor of the AcrAB–TolC efflux pump and restores a high level of intracellular drug concentration. Inhibitory activity of this alkylaminoquinoline is observed on clinical isolates showing different resistance phenotypes.

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Virulence Factors, Antibiotic Resistance patterns, and Molecular Types of Clinical Isolates of Klebsiella Pneumoniae

10.21203/rs.3.rs-331542/v1 ◽

2021 ◽

Author(s):

Mitra Ahmadi ◽

Payam Behzadi ◽

Reza Ranjbar

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Molecular Typing ◽

Virulence Genes ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Aminoglycoside Resistance ◽

Wide Range ◽

Resistance Patterns ◽

Esbl Producers

Abstract Background Klebsiella pneumoniae is armed with a wide range of antibiotic resistance mechanisms which mostly challenges effective treatment. Due to this fact, the aims of the current study were to identify the clinical strains of K . pneumoniae as well as to determine their phenotypes and molecular characterization related to antimicrobial resistance and virulence genes. Methods In this investigation, specimens from a hospital and different laboratories located in Shahr-e-Qods, Tehran, Iran were collected during a period of nine-month (December 2018 to August 2019). The isolated strains of K. pneumoniae were then identified through standard microbial and biochemical assays. Additionally, disk diffusion, combined disk, modified Hodge test and PCR were performed for antibiotic resistance of the strains and virulence genes profiling, respectively. The molecular typing was accomplished by ERIC-PCR. Results Eighty-four isolates of K. pneumoniae were identified and subjected to the study. Fifty- two percent of the isolated strains of K. pneumoniae were detected as multidrug resistant (MDR) pathotypes with the highest resistance to ceftriaxone (65%) and the lowest resistance to colistin (23%). Twenty-seven (52%) out of 52 (100%) MDR pathotypes of isolated K. pneumoniae were identified as ESBL producers. According to Modified Hodge Test (MHT) results, out of 24 resistant strains of isolated K. pneumoniae to imipenem and meropenem, 15 pathotypes (62.5%) were detected as KPC producers. The gene of blaCTX (encoding carbapenemase) with 96% ranked first, while the blaKPC gene with the prevalence of 71% ranked second among ESBL producers. The aminoglycoside resistance gene of Aac6-Ib showed the highest frequency with the prevalence percentage of 90%. The virulence genes of mrkD (94%) and magA (11%) were the highest and lowest among isolates, respectively. According to ERIC-PCR results the isolated strains of K. pneumoniae were divided into four clusters in which the cluster 4 was predominant group. Conclusions The high prevalence of antibiotic resistance and virulence genes in conjunction with a significant relationship between the strains reveals a high pathogenic capacity of the isolated pathotypes of K. pneumoniae . These findings emphasize the choose of more effective antibiotic regimens for treatment of infections caused by K. pneumoniae. Keywords: Klebsiella pneumoniae , antibiotic resistance, ESBL, virulence genes, molecular typing.

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The Characteristic of Virulence, Biofilm and Antibiotic Resistance of Klebsiella pneumoniae

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17176278 ◽

2020 ◽

Vol 17 (17) ◽

pp. 6278

Author(s):

Guoying Wang ◽

Guo Zhao ◽

Xiaoyu Chao ◽

Longxiang Xie ◽

Hongju Wang

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Resistance Mechanism ◽

Current Medical Research ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Current Medical ◽

Tolerance Mechanisms ◽

Rapid Spread ◽

Tract Infections

Klebsiella pneumoniae is an important gram-negative opportunistic pathogen that causes a variety of infectious diseases, including urinary tract infections, bacteremia, pneumonia, and liver abscesses. With the emergence of multidrug-resistant (MDR) and hypervirulent K. pneumoniae (hvKP) strains, the rapid spread of these clinical strains in geography is particularly worrying. However, the detailed mechanisms of virulence and antibiotic resistance in K. pneumoniae are still not very clear. Therefore, studying and elucidating the pathogenic mechanisms and drug resistance mechanism of K. pneumoniae infection are important parts of current medical research. In this paper, we systematically summarized the virulence, biofilm, and antibiotic tolerance mechanisms of K. pneumoniae, and explored the application of whole genome sequencing and global proteomics, which will provide new clues for clinical treatment of K. pneumoniae.

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MrkD Gene as a Regulator of Biofilm Formation with Correlation to Antibiotic Resistance among Clinical Klebsiella pneumoniae Isolates from Menoufia University Hospitals

10.51429/ejmm29318 ◽

2020 ◽

Vol 29 (3) ◽

pp. 137-144

Author(s):

Asmaa M. Elbrolosy ◽

Naira A. Eissa ◽

Nahed A. Al-Rajhy ◽

Esraa El-Sayed A. El-Mahdy ◽

Rasha G. Mostafa

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Diffusion Method ◽

Clinical Samples ◽

Resistance Pattern ◽

University Hospitals ◽

Pcr Assay ◽

Disk Diffusion Method ◽

Microbiological Methods

Background: Klebsiella pneumoniae (K. pneumoniae) is a common pathogen involved in a diverse array of life-threatening infections. Increasing frequent acquisition of antibiotic resistance by K. pneumoniae has given rise to multidrug-resistant pathogen mostly at the hospital level. Objectives: To assess the prevalence and antibiotic resistance pattern of the clinical K. pneumoniae isolates at Menoufia University Hospitals (MUHs) as well as to explore the role of mrkD gene as a regulator of biofilm formation. Methodology: A total of 340 different clinical samples were obtained from 270 patients who were admitted to MUHs and those from Outpatient clinics during the period from April 2018 to September 2019. 84 K. pneumoniae isolates were identified by the standard microbiological methods and vitek-2 system. The antimicrobial resistance pattern was determined by disk diffusion method. The biofilm-forming ability of all K. pneumoniae isolates was demonstrated phenotypically by the modified Congo red agar method (MCRA) and PCR assay verified the presence of mrkD gene as a genetic determinant of biofilm formation. Results: Klebsiella spp. represented 34.7% of the collected isolates and the predominant spp. was K. pneumoniae (91.3%). The highest resistance rates were for ceftriaxone (69%) followed by aztreonam (67.9%), 66.7% for each of piperacillin and ceftazidime, while the least resistance rate was for fosfomycin (8.3%). Biofilm production was detected among 83.3% of the isolates by MCRA method. A highly significant statistical difference was noted between biofilm- and non- biofilm - producing K. pneumoniae isolates regarding resistance to cefepieme and amikacin (P <0.001) and similarly regarding resistance to aztreonam, imipenem, meropenem, ertapenem and tobramycin (P<0.05). Conventional PCR assay showed that, 92% of the isolates harbored mrkD gene with a highly significant association with biofilm formation. Conclusion: The increasing prevalence and remarkable ability to acquire antibiotic resistance among K. pneumoniae isolates together with biofilm formation should alert even more regarding the hazard of this pathogen in hospital settings.

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Evaluation of efflux pump activity and biofilm formation in multidrug resistant clinical isolates of Pseudomonas aeruginosa isolated from a Federal Medical Center in Nigeria

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00417-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Florence Chijindu Ugwuanyi ◽

Abraham Ajayi ◽

David Ajiboye Ojo ◽

Adeyemi Isaac Adeleye ◽

Stella Ifeanyi Smith

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Efflux Pump ◽

Medical Center ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Diffusion Method ◽

Clinical Samples ◽

Biochemical Tests ◽

Pump Activity

Abstract Background Pseudomonas aeruginosa an opportunistic pathogen, is widely associated with nosocomial infections and exhibits resistance to multiple classes of antibiotics. The aim of this study was to determine the antibiotic resistance profile, biofilm formation and efflux pump activity of Pseudomonas strains isolated from clinical samples in Abeokuta Ogun state Nigeria. Methods Fifty suspected Pseudomonas isolates were characterized by standard biochemical tests and PCR using Pseudomonas species -specific primers. Antibiotic susceptibility testing was done by the disc diffusion method. Efflux pump activity screening was done by the ethidium bromide method and biofilm formation assay by the tissue plate method. Genes encoding biofilm formation (pslA & plsD) and efflux pump activity (mexA, mexB and oprM) were assayed by PCR. Results Thirty-nine Pseudomonas spp. were identified of which 35 were Pseudomonas aeruginosa and 4 Pseudomonas spp. All 39 (100%) Pseudomonas isolates were resistant to ceftazidime, cefuroxime and amoxicillin-clavulanate. Thirty-six (92%), 10(25.6%), 20 (51.2%), 11(28%) and 9(23%) of the isolates were resistant to nitrofurantoin, imipenem, gentamicin, cefepime and aztreonam respectively. All the isolates had the ability to form biofilm and 11 (28%) of them were strong biofilm formers. They all (100%) harboured the pslA and pslD biofilm encoding genes. Varied relationships between biofilm formation and resistance to ciprofloxacin, ofloxacin, cefixime, gentamicin, imipenem, and aztreonam were observed. Only 23(59%) of the Pseudomonas isolates phenotypically exhibited efflux pump activity but mexA gene was detected in all 39 (100%) isolates while mexB and oprM genes were detected in 91%, 92%, and 88% of strong, moderate and weak biofilm formers respectively. Conclusion Multidrug resistance, biofilm and efflux pump capabilities in Pseudomonas aeruginosa have serious public health implications in the management of infections caused by this organism.

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Virulent and multidrug‐resistant Klebsiella pneumoniae from clinical samples in Balochistan

International Wound Journal ◽

10.1111/iwj.13550 ◽

2021 ◽

Author(s):

Sareeen Fatima ◽

Faiza Liaqat ◽

Ali Akbar ◽

Muhammad Sahfee ◽

Abdul Samad ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Multidrug Resistant ◽

Clinical Samples

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Growing Menace of Antibacterial Resistance in Clinical Isolates ofPseudomonas aeruginosain Nepal: An Insight of Beta-Lactamase Production

BioMed Research International ◽

10.1155/2016/6437208 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Shamshul Ansari ◽

Rabindra Dhital ◽

Sony Shrestha ◽

Sangita Thapa ◽

Ram Puri ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Opportunistic Pathogen ◽

Resistance Rate ◽

Diffusion Method ◽

Beta Lactamase ◽

Beta Lactam ◽

Beta Lactam Antibiotics ◽

Microbiological Methods ◽

Immunosuppressed Patients

Introduction. Pseudomonas aeruginosais the most frequently isolated organism as it acts as the opportunistic pathogen and can cause infections in immunosuppressed patients. The production of different types of beta-lactamases renders this organism resistant to many commonly used antimicrobials. Therefore, the aim of this study was to document the antibiotic resistance rate inPseudomonas aeruginosaisolated from different clinical specimens.Methods. Pseudomonas aeruginosarecovered was identified by standard microbiological methods. Antibiotic susceptibility testing was performed by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines and all the suspected isolates were tested for the production of ESBLs, MBLs, and AmpC.Results.Out of total (178) isolates, 83.1% were recovered from the inpatient department (IPD). Majority of the isolates mediated resistance towards the beta-lactam antibiotics, while nearly half of the isolates were resistant to ciprofloxacin. Most of the aminoglycosides used showed resistance rate up to 75% but amikacin proved to be better option. No resistance to polymyxin was observed. ESBLs, MBLs, and AmpC mediated resistance was seen in 33.1%, 30.9%, and 15.7% isolates, respectively.Conclusions. Antibiotic resistance rate and beta-lactamase mediated resistance were high. Thus, regular surveillance of drug resistance is of utmost importance.

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