Assessment of antibiotic resistance pattern in Acinetobacter bumannii carrying bla oxA type genes isolated from hospitalized patients

AbstractPlease cite this article as: Goudarzi H, Douraghi M, Ghalavand Z, Goudarzi M. Assessment of antibiotic resistance pattern in Acinetobacter baumannii carrying bla oxA type genes isolated from hospitalized patients. Novel Biomed 2013;1(2):54-61.Introduction: Acinetobacter baumannii is a Gram-negative coccobacillus and one of the most opportunistic pathogens responsible for serious infections in hospitalized patients.Methods: During a 12 month study, 221 clinical isolates and 22 environmental Acinetobacter baumannii isolates were collected. In vitro susceptibility of Acinetobacter baumannii isolates to 13 antimicrobial agents amikacin; cefepime; ceftazidime; ciprofloxacin; meropenem; piperacillin/tazobactam; sulfamethoxazole/ trimethoprim; imipenem; tigecycline; colistin; gentamycin; ceftriaxone; levofloxacin was performed by the disk diffusion method and Minimum Inhibitory Concentration(MICs) of imipenem; levofloxacin and cefepime.was done by the E-test according to Clinical and Laboratory Standards Institute (CLSI) criteria. blaOXA-23, blaOXA-24, blaOXA-58, blaOXA-51genes were detected by polymerase chain reaction and sequencing.Results: The result of antimicrobial susceptibility test of clinical isolates by the disk diffusion method revealed that that all strains of Acinetobacter baumannii were resistant to piperacillin/tazobactam. The rates of resistance to the majority of antibiotics tested varied between 69% and 100 %, with the exception of tigecycline and colistin. Of 221 isolates tested 99(44.8%) were XDR. All strains carry a blaOXA-51-like gene. blaOXA-23gene was the most prevalence among blaOXA-types.Conclusion: colistin and tigecycline can be effective drugs for treatment of Acinetobacter baumannii infections. Continuous Surveillance for Acinetobacter baumannii multidrug-resistant strains is necessary to prevent the further spread of resistant isolates

Evaluation the cytotoxic effect of cytotoxin-producing Klebsiella oxytoca isolates on the HEp-2 cell line by MTT assay

Background: The cytotoxic effects on epithelial cells of the human are not observed in other strains of Klebsiella spp and are only observed in K. oxytoca strains. MTT assay was used to evaluate cytotoxic activity. In this study, colorimetric method was used to evaluate the cytotoxic effect of cytotoxin-producing isolates on Hep-2 cell line and determines the percentage of surviving cells. Materials and methods: In this study, we collected a total of 75 K. oxytoca strains isolate and we detected the production of toxins and their cytotoxic effects on HEp-2 cells. Colorimetric method such as MTT assay was used to evaluate the cytotoxic effect of cytotoxin-producing isolates on Hep-2 cell line and determines the percentage of surviving cells. Results: Nine isolates had cytotoxic effects on HEp-2 cells. The results of MTT assay showed that the isolated strains were different from the control stain in terms of toxinogenicity and cytotoxic effects on HEp-2 cells at the studied dilutions (1:3, 1:6, 1:12, 1:24, 1:48, and 1:96). Conclusions: In the current study, Percentage of Hep-2 surviving cells exposed to 1:3, 1:6, 1:12, 1:24, 1:48, and 1:96 supernatant dilutions of cytotoxin-producing Klebsiella oxytoca isolates was different

Molecular identification of Pseudomonas aeruginosa recovered from cystic fibrosis patients

Objective. Precise identification of various morphotypes of Pseduomonas aeruginosa which developed during cystic fibrosis (CF) is of prime importance. We aimed to identify the isolates of P. aeruginosa recovered from CF patients at the genus and species level through primers targeting oprI and oprL genes via PCR. Methods. Sputum samples or throat swabs were taken from 100 CF patients and plated on cetrimide agar. All suspected colonies were primarily screened for P. aeruginosa by a combination of phenotypic tests. Molecular identification of colonies was per- formed using specific primers for oprI and oprL genes.Results. Based on phenotypic tests, P. aeruginosa isolates were recovered from 40% of CF patients. Forty isolates yielded ampli- con of oprI gene using genus-specific primers confirming the identity of fluorescent pseudomonads. However, 37 of 40 isolates yielded amplicon of oprL gene using species-specific primers, verifying the identity of P. aeruginosa. Conclusion. This study showed that the species-specific PCR tar- geting oprL gene can be used as accurate test for identification of highly adaptable P. aeruginosa in CF patients. This procedure may provide a simple and reliable method for identification of various morphotypes

Identification of Klebsiella pneumoniae Carbapenemase-producing Klebsiella oxytoca in Clinical Isolates in Tehran Hospitals, Iran by Chromogenic Medium and Molecular Methods

Objectives: Production of carbapenemase, especially Klebsiella pneumoniae carbapenemases (KPC), is one of the antibiotic resistance mechanisms of Enterobacteriaceae such as Klebsiella oxytoca. This study aimed to investigate and identify KPC-producing K. oxytoca isolates using molecular and phenotypic methods. Methods: A total of 75 isolates of K. oxytoca were isolated from various clinical samples, and were verified as K. oxytoca after performing standard microbiological tests and using a polymerase chain reaction (PCR) method. An antibiotic susceptibility test was performed using a disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines. CHROMagar KPC chromogenic culture media was used to examine and confirm the production of the carbapenemase enzyme in K. oxytoca isolates; in addition, PCR was used to evaluate the presence of blaKPC gene in K. oxytoca strains. Results: Of a total of 75 K. oxytoca isolates, one multidrug resistant strain was isolated from the urine of a hospitalized woman. This strain was examined to assess its ability to produce carbapenemase enzyme; it produced a colony with a blue metallic color on the CHROMagar KPC chromogenic culture media. In addition, the blaKPC gene was confirmed by PCR. After sequencing, it was confirmed and deposited in GenBank. Conclusion: To date, many cases of KPC-producing Enterobacteriaceae, in particular K. pneumoniae, have been reported in different countries; there are also some reports on the identification of KPC-producing K. oxytoca. Therefore

Identification of cytotoxin-producing Klebsiella oxytoca strains isolated from clinical samples with cell culture assays

BACKGROUND: Klebsiella oxytoca is an opportunistic pathogen which damages intestinal epithelium through producing cytotoxin tilivalline. This toxin plays a role in the pathogenesis of bacteria and is the main virulence factor which leads to antibiotic-associated hemorrhagic colitis progress. MATERIALS AND METHODS: In this study, we collected a total of 75 K. oxytoca strains isolated from the stool, urine, blood, wounds, and sputum and evaluated them in terms of the production of toxins; we detected their cytotoxic effects on HEp-2 cells. RESULTS: Of all the isolates, five K. oxytoca strains isolated from the stool cultures, two strains isolated from the blood cultures, one strains isolated from the wound cultures, and one strains isolated from the urine cultures had cytotoxic effects on HEp-2 cells. The strains isolated from sputum cultures had no cytotoxic effects on HEp-2 cells. CONCLUSIONS: In the current study, the majority of strains isolated from the stool of the patients included cytopathic effects on HEp-2 cells. Copyright © 2017 Elsevier Ltd. All rights reserved

Molecular typing of cytotoxin-producing Klebsiella oxytoca isolates by 16S-23S internal transcribed spacer PCR

Cytotoxin is one of the important pathogenic factors, which plays a role in the virulence of Klebsiella oxytoca. The aim of this study was to investigate molecular typing of clinical isolates of the cytotoxin-producing K. oxytoca using internal transcribed spacer (ITS)PCR. A total of 75 isolates of K. oxytoca were isolated from clinical samples; they were verified as K. oxytoca by standard microbiological tests and PCR. Production of toxin determines the cytotoxic effects on HEp-2 cells. The genetic diversity of isolates of the cytotoxin-producing K. oxytoca were defined by ITS-PCR. Of all the isolates investigated, five K. oxytoca strains isolated from stool cultures, two strains from blood samples, one strain from a wound and one strain isolated from urine had cytotoxic effects on HEp-2 cells. The ITS-PCR patterns showed genetic diversity among cytotoxin-producing isolates. The ITS-PCR method had good discriminatory power; performance of this method and interpretation of the results were easy and repeatable. Five genetic diversity patterns were identified by ITS-PC

Molecular typing of cytotoxin-producing Klebsiella oxytoca isolates by 16S-23S internal transcribed spacer PCR

Cytotoxin is one of the important pathogenic factors, which plays a role in the virulence of Klebsiella oxytoca. The aim of this study was to investigate molecular typing of clinical isolates of the cytotoxin-producing K. oxytoca using internal transcribed spacer (ITS)PCR. A total of 75 isolates of K. oxytoca were isolated from clinical samples; they were verified as K. oxytoca by standard microbiological tests and PCR. Production of toxin determines the cytotoxic effects on HEp-2 cells. The genetic diversity of isolates of the cytotoxin-producing K. oxytoca were defined by ITS-PCR. Of all the isolates investigated, five K. oxytoca strains isolated from stool cultures, two strains from blood samples, one strain from a wound and one strain isolated from urine had cytotoxic effects on HEp-2 cells. The ITS-PCR patterns showed genetic diversity among cytotoxin-producing isolates. The ITS-PCR method had good discriminatory power; performance of this method and interpretation of the results were easy and repeatable. Five genetic diversity patterns were identified by ITS-PCR

Isolation and characterization of a multidrug-resistant Clostridioides difficile toxinotype V from municipal wastewater treatment plant

Purpose: Wastewater treatment plant (WWTP) is regarded as a potential source for transmission of Clostridioides difficile from urban areas into the surface water, through feces of human and animals. The aim of this study was to screen and characterize the C. difficile bacteria in inlet and outlet wastewater of different WWTPs in Tehran, Iran. Methods: Totally, 72 samples were collected from three different WWTPs (inlet site and outlet sites) during a year. C. difficile was isolated and characterized in terms of toxins, toxinotype, resistance profile and genes, and colonization factors using PCR. Results: One C. difficile toxinotype V was isolated from the outlet samples. The isolate was susceptible to vancomycin but resistant to metronidazole, tetracycline, ciprofloxacin, and moxifloxacin using MIC Test Strips. The isolated C. difficile was toxigenic (tcdA, tcdB, cdtA, cdtB positive and CPE positive) and had tcdC-A genotype. No mutations were found in fliC and fliD. The slpA sequence type was 078 � 01. The C. difficile was positive for tetM, int, but negative for vanA, nim, and tndX genes. Mutations were not observed in gyrA and gyrB genes. Conclusions: This study provided evidence of presence of a multidrug-resistant C. difficile toxinotype V in one of the municipal WWTP. The transmission of such isolate to the environment and reuse of treated wastewater by human pose a threat to human health and dissemination of antibiotic resistant bacteria which are untreatable. © 2020, Springer Nature Switzerland AG

Clonal Relationship and Resistance Profiles Among ESBL-Producing Escherichia coli

AmpC β-lactamases hydrolyze all β-lactams except cefepime and carbapenems. The study of AmpC-producing E. coli has high priority for the infection control committee. This research is aimed to investigate the resistant urinary AmpC-generating E. coli isolates and identify their genetic variety. Some 230 E. coli isolates from patients suffering urinary tract infection symptoms were studied in 2017–2018 to assess their susceptibility toward antimicrobial agents. AmpC gene was evaluated by PCR and molecular typing using the 10-loci MLVA method. MLVA images were examined by BioNumerics 6.6 software through the use of the UPGMA algorithms. Thirty-eight AmpC-generating E. coli isolates were detected. The most abundant determinant was blaCIT and blaEBC, blaFOX, and blaDHA had the next ranks, respectively. Six major clusters and a singleton were identified by MLVA. AmpC beta-lactamases in urinary isolates of E. coli in the hospital under study and high rate of additional resistance to gentamicin, cotrimoxazole and ciprofloxacin. The most frequent gene determinant of AmpC beta-lactamase was blaCIT and vary depending on time and geographical location