Phenotypic and Genotypic Characterization of ESBL-, AmpC-, and Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli Isolates

Objectives: Drug resistance among gram-negative bacteria is a worldwide challenge. Due to the importance of drug-resistant Klebsiella pneumoniae and Escherichia coli strains in hospital-acquired infections, we aimed to determine the phenotypic and genotypic characteristics of ESBL-, AmpC-, and carbapenemase-producing isolates obtained from hospitalized patients in Tehran and Ilam (Iran). Materials and Methods: In total, 90 K. pneumoniae isolates and 65 E. coli isolates were collected from various infections. Phenotypic identification of bacterial isolates was performed using standard methods. Phenotypic screening of ESBL, AmpC, and carbapenemase enzymes was carried out. Detection of ESBL, AmpC, and carbapenemase genes was also performed by the PCR method. Results: Phenotypic detection tests showed that 36 (40) K. pneumoniae and 23 (35.4) E. coli isolates were ESBL producers. Moreover, 18 (20) and 6 (9.2) K. pneumoniae and E. coli isolates were AmpC producers, respectively. Modified Hodge test results indicated that 39 (43.3) K. pneumoniae and 18 (27.7) E. coli isolates produced carbapenemase. Molecular tests showed that 40 of K. pneumoniae and 36.9 of E. coli isolates were ESBL positive. AmpC was detected in 24.4 and 13.8 of K. pneumoniae and E. coli isolates. Carbapenemase was detected in 34 (37.8) K. pneumoniae and 13 (20) E. coli isolates. -Conclusion: In this study, 3 K. pneumoniae isolates simultaneously carried ESBL, AmpC, and carbapenemase genes. Up-to-date strategies such as combination therapy or utilization of new antimicrobial agents might help to combat such drug-resistant organisms. © 2019 The Author(s) Published by S. Karger AG, Basel

The Prevalence of Shiga toxin-1 in non-Shigella dysenteriae isolates collected from diarrhea samples in patients, Ahvaz, Iran

BACKGROUND: Acute diarrhea is a major public health problem particularly in developing countries. Shigellosis is one of the substantial causative agents of microbial dysentery and still has a remarkable prevalence particularly in areas with poor hygienic infrastructures. The probable existence of the deadly Shiga toxin (Stx) protein in some Shigella strains would manifest life-threatening clinical symptoms of the infection. METHODS: The aim of this study was to determine the presence of Shigella toxin 1 (Stx1) in isolated from patients with diarrhea. Totally, 227 Shigella species including 60 S. flexneri, 157 S. sonnei, and 10 S. boydii were collected from diarrheal patients in tropical infectious diseases research center of Ahvaz, Iran, during 2013-2015. The isolates were collected mostly from the intensive care unit, infectious disease, and surgery settings. The isolates were identified and the polymerase chain reaction (PCR) was performed to detect the stx gene. RESULTS: The results indicated that none of them encode the stx1 gene. CONCLUSION: Isolates of this study were not capable of stx1 encoding. Future investigations should consider the relations between other Shigella species and Shigella toxin in Iran

The Prevalence of pilA and algD Virulence Genes in Pseudomonas aeruginosa Urinary Tract and Tracheal Isolates

P. aeruginosa is one of the bacteria opportunistic that played main role in pathogenicity of patient in urinary tract infection (UTI) and respiratory tract infections. So, the aim of this study was to evaluate the prevalence of virulence genes including algD and pilA among Pseudomonas aeruginosa in urinary tract infection and tracheal isolates. After DNA extraction of clinical isolates, polymerase chain reaction was performed, and the results highlighted algD in all isolates, while pilA was dominant in tracheal isolates. We concluded that pathogenicity of urinary tract infection isolates is more than tracheal isolates, but future studies should confirm this

Assessment of biofilm formation in Pseudomonas aeruginosa by antisense mazE-PNA

The hallmark patogenicity in Pseudomonas aeruginosa (P. aeruginosa) is biofilm formation that is not easy to eradicate, because it has variety mechanisms for antibiotic resistance. In addition, toxin-antitoxin (TA) system may play role in biofilm formation. The current study aimed to evaluate the role of TA loci in biofilm formation. Therefore, 18 P. aeruginosa clinical isolates were collected and evaluated for specific biofilm and TA genes. The analysis by RT-qPCR demonstrated that expression of mazE antitoxin in biofilm formation was increase. On the other hand, mazE antitoxin TA system was used as target for antisense PNA. mazE-PNA was able to influence in biofilm formation and was inhibit at 5,10 and 15 mu M concentrations biofilm formation in P aeruginosa. Therefore, it could be highlighted target for anti-biofilm target to eradicate P. aeruginosa biofilm producer. (C) 2017 Elsevier Ltd. All rights reserved

Genomic Diversity and Virulence Genes among Clinical Isolates of Pseudomonas aeruginosa

Background: Typing of nosocomial pathogens is necessary to determine the source of an outbreak. The aim was to determine the genomic variability among Pseudomonas aeruginosa (P. aeruginosa) by random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) methods. Methods: Fifty P. aeruginosa isolates were obtained from the hospitals. The source of these isolates were burn wound and urinary tract infections. After detection of P. aeruginosa by biochemical methods, chromosomal deoxyribonucleic acid (DNA) was extracted by a DNA extraction kit. ERIC-PCR and RAPD- PCR was done by standard methods. The polymerase chain reaction (PCR) products were run and visualized in 1.5 agarose gels stained with ethidium bromide. Results: Fifty P. aeruginosa isolates were analyzed by ERIC-PCR and RAPD-PCR methods. Multiple PCR fragment sizes generated by two PCR methods and PCR product size were between 200 - 3500 bp, and 10 and 7 different PCR patterns were detected by ERIC-PCR and RAPD-PCR, respectively. Eleven isolates were not detected by ERIC-PCR method. Fifteen isolates were typed to a single genotype by the RAPD-PCR method. Conclusions: We suggested that ERIC and RAPD PCR are equally suitable, inexpensive, fast, reproducible, and discriminatory as rapid DNA typing tools for effective epidemiological surveillance of P. aeruginosa isolates. Our results suggest that these DNA typing tools could be used in routine epidemiological surveillance, outbreak surveillance, and in the identification of the source of transmission of P. aeruginosa

Molecular characteristics, antimicrobial resistance profiles, and antibiotic resistance determinants in uropathogenic fluoroquinolone resistant-Escherichia coli isolates

The concern about multidrug resistance (MDR) in the bacterial pathogen is significant. The antimicrobial susceptibility testing performed in forty Escherichia coli isolates responsible for urinary tract infections (UTIs) to following antibiotics: ceftazidime, cefotaxime, ceftriaxone, aztreonam, co-trimoxazole, gentamicin, amikacin, nitrofurantoin, tobramycin, ciprofloxacin, and nalidixic acid performed by disk diffusion method. The presence of resistance sul1, aacC1, aadB, aphA6, qnrA, qnrB, qnrS, and ESBL genes were investigated in clinical isolates by PCR-based sequencing assay. Molecular fingerprinting of UTI isolates characterized by Pulsed-Field Gel Electrophoresis for clonal distribution and determined of predominant pulsotypes. The highest and least active agents against uropathogenic E. coli (UPEC) isolates were ciprofloxacin (100), and tobramycin (7.5). Fifty-percent (20/40) of the UPEC isolates were identified as ESBL-producers by phenotype. The most prevalent ESBL genes were related to bla TEM (100; 20/20). The presence of sul1, aadB, qnrB, and qnrS genes detected in 67.5 (n = 27), 5 (n = 2), 47.5 (n = 19), and 2.5 (n = 1) of E. coli isolates, respectively. None of isolates harbor of aacC1, aphA6, and qnrA genes. The results of molecular characterizations confirmed that 31 pulsotypes found among 40 UPEC isolates. Of 31 pulsotypes, 26 types were unique and five types found in more than one isolate. Only two isolates (2/40) were not typeable. A high prevalence of plasmid-mediated quinolone resistance and ESBL genes in E. coli detected in urine samples. Data in this study suggested that these multiple pulsotypes patterns showed no clonal association between UPEC isolates

Molecular analysis of uropathogenic E.coli isolates from Urinary tract infections in Ilam

The aim of the current study was to investigate the prevalence virulence genes profile in UPEC isolates in Ilam. For this purpose, a total of 8o UPEC isolates for patients with UTIs were collected during 6 months period. The multiplex polymerase chain reaction (Multiplex PCR) was used for detection of the papEF, fimH, iucD, hlyA, fyuA, and ompT genes. The prevalence of genes the fimH, papEF, iucD, fyuA, hlyA, hlyA and ompT were 87.5, 47.5, 60, 67.5, 27.5, 47.5 and 71.2, respectively. Among all of isolates, 27 gene profiles were obtained. The number isolates related to 4 virulence factor simultaneous that were 25 isolates. Considering the different antibiotic patterns, will be proposed develop and an appropriate program for antibiotics in each region and even in each hospital. Also, investigation about the profile of different virulence genes which capable of inducing various pathogens is will be done in the future

The rationale behind antibiotic resistance pattern in Klebsiella pneumoniae

Presently, there is an increase in antibiotic resistance in bacteria, due to relax prescription of antibiotics, especially in Iran. Undoubtedly, in toxin antitoxin (TA) system, a toxin neutralized by antitoxin, which known as a potent antimicrobial target; but there is no extensive survey on the prevalence of TA loci in large scale of Klebsiella pneumoniae. Therefore, this study aims to determine the prevalence of different TA loci in clinical and environmental K. pneumoniae isolates. For this reason, 48 K. pneumoniae clinical isolates and 49 K. pneumoniae environmental isolates were subjected for evaluation of different TA loci. The results of current study indicated that there is no association between antibiotic resistances and presence of TA loci in clinical and environmental K. pneumoniae. The role of TA loci as a potent target in antibiotic resistant K. pneumoniae has been complicated. Therefore, more studies should be performed to explain why TA loci are presented in K. pneumoniae and what is the rationale behind antibiotic resistant K. pneumoniae

Molecular characterization of AmpC β-lactamases among Klebsiella pneumoniae isolated from Ilam and Tehran hospitals, from Iran

The common use of beta lactam antibiotics for tratment of bacteria infections leads to increase the world wide microbial resistance by producing beta lactamase enzyme among clinical isolates. In recent years, the production of broad-spectrum beta-lactamase enzymes in clinical isolates, especially E.coli and Klebsiella bacteria are common. Typical Ampc enzymes (class C- ESBLs) confirm resistance to most oxyimino cephalosporins. The aim of this study is to determine the prevalence of AmpC type extended spectrum beta lactamases genes in clinical isolates of Klebsiella pneumoniae. 108 clinical sample of K.pneumoniae, isolated from hospitalized patients procured from two hospitals in Ilam and Tehran. To identify Ampc genes, PCR method was used. 95/3 percent of isolates were resistant to Cefoxitin, 49 isolates were positive for FOXM cluster genes, 35 were positive for DHAM cluster genes and 6 were positive for CITM cluster genes. Our results showed that among of clinical isolates of Klebsiella pneumoniae, prevalence broad-spectrum beta-lactamase enzymes and Ampc genes are relatively high