Diversity determination of CTX-M1 producing klebsiella pneumoniae using multilocus variable-number tandem repeat analysis, semnan, Iran

Background: CTX-M is the most prevalent and rapidly growing type of the extended-spectrum β-lactamase (ESBL) family and CTXM1 is the most common type of blaCTX-M. Objectives: The current study aimed at investigating the genetic diversity of CTX-M-1-producing Klebsiella pneumoniae circulating in Semnan, Iran evaluated by multilocus variable-number tandem repeat analysis (MLVA). Methods: A total of 110 isolates of K. pneumoniae were collected from different clinical samples. The antibiotic suceptibility and double disk synergy test were determined according to CLSI (the clinical and laboratory standards institute) guidelines. The polymerase chain reaction (PCR) method was performed to detect CTX-M-1. The eight loci for MLVA genotyping were selected along with the primers previously described. Results: Imipenem, with 84.7 susceptibility, was the most effective antibiotic against K. pneumoniae. Seventy (63.63) isolates had ESBL positive results and 42 (60 ) of them were positive for CTX-M-1 gene. Totally, 28 MLVA genotypes were discriminated, evaluation of diversity indexes (DIs) for eight loci showed that six different alleles were the most polymorphic and the most DI was 0.807. Conclusions: The findings of the current study demonstrated heterogeneity among CTX-M-1-producing K. pneumonia strains. The presence of CTX-M-1 in different MLVA types demonstrated that a certain clone is not responsible for spreading the isolates. © 2018, Author(s)

The complex genetic region conferring transferable antibiotic resistance in multidrug-resistant and extremely drug-resistant Klebsiella pneumoniae clinical isolates

Antibiotic resistance due to transferable resistance genes is one of the most important concerns in Klebsiella pneumoniae isolated from nosocomial infections. Eighty-eight K. pneumoniae isolates were confirmed through biochemical methods. In addition, antimicrobial susceptibility testing was performed using a disc-diffusion method. Extended-spectrum β-lactamase production among the isolates was screened using a double-disc synergism test, and the resistance genes were identified using PCR. The eight loci for multiple-locus variable number tandem repeat analysis (MLVA) genotyping were selected along with the primers. According to our findings, neomycin (5; 5.6) and carbapenems (10; 11.3) showed the most remarkable inhibitory effect but co-trimoxazole (46; 52.2) was the least effective antibiotic against K. pneumoniae isolates. blaCTX-M-1, qnrA, qnrB, qnrS, intI, intII, aac3 and aac6 were detected in 30 (34), 5 (5.6), 29 (32.9), 23 (26.1), 88 (100), 72 (81.8), 26 (29.5) and 28 (31.8) of the 88 isolates, respectively. But none of the K. pneumoniae isolates expressed the intIII gene. Using MLVA, 23 MLVA types and eight clusters were identified. Extended-spectrum β-lactamase-producing K. pneumoniae isolates were classified into two clonal complexes. Effective strategies for infection control should be applied to monitor and control the spread of multidrug-resistant isolates by the resistance genes located on the mobile genetic elements. © 202

Determination of investigation of the link between human and animal Brucella isolates in Iran using multiple-locus variable number tandem repeat method comprising 16 loci (MLVA-16)

Background: Epidemiological studies are important tools to assess the diversity of Brucella isolates and to estimate their epidemiological relationship among isolates from different geographical origins. In this study the MLVA16 (multiple-locus variable number tandem repeat analysis based on 16 loci) was employed to investigate the diversity of Brucella spp. Isolated from humans and animals for epidemiological purposes and to determine the most common Brucella genotypes in Iran. Methods: We designed a molecular-based study to evaluate the potential reservoirs of human brucellosis. After isolation and identification of 54 Brucella spp human and animal specimens from three regions of Iran, bacterial genomic DNA was extracted MLVA with three panel was used for the genotyping of isolates. The size of PCR products were analyzed and converted to repeat unit numbers using a published allele numbering system and data set was imported into Bionumerics. Results: Three isolates (5.55) were identified as Brucella abortus and 51 (94.44) as Brucella melitensis. Two isolates of Brucella abortus were from humans and one from an animal. Thirty-four Brucella melitensis isolates were from humans and 17 from animals. Using MLVA16-genotyping, 54 isolates with genetic similarity coefficient of 80 were divided into 46 genotypes and 22 genotypes were represented by a single isolate, while 4, 2, 1 and 2 genotypes were represented by 2, 3, 4 and 7 isolates, respectively. The most prevalent genotype was represented by 14 isolates. There were two other frequent genotypes each represented by seven isolates, among which only one was restricted to a geographic region. Discriminatory power for each locus was determined in this study and panel 2B shows the high discretionary power Bruce04 (0.837), Bruce30 (0.806), Bruce 09 (0.787), Bruce 07 (0.772), Bruce16 (0.766). Conclusion: MLVA16 analysis of 54 Brucella isolates showed high level polymorphism in their genotypes. Only two genotypes, each observed in seven isolates, were related to one another and only one of these genotypes were found in to two separate regions. © 202

Determination of the frequency of β-lactamase genes (bla SHV, bla TEM, bla CTX-M) and phylogenetic groups among ESBL-producing uropathogenic Escherichia coli isolated from outpatients

Escherichia coli accounts for 70-95 of community-acquired urinary tract infections (UTIs). Recently, there has been an increase in the prevalence of extended-spectrum β-lactamase (ESBL) in the community which required an accurate identification for better management. Therefore, the current study was performed to determine the antimicrobial resistance pattern, investigate ESBL phenotypes and genotypes (blaCTX-M, bla TEM and bla SHV genes) and determine the phylogenetic groups among ESBL-positive isolates from outpatients. One hundred and eighty-three positive urine samples were collected from 4450 outpatient clinic attendees. Antibiotic susceptibility was determined and ESBL phenotype screening was carried out using disk diffusion agar and combination disk techniques, respectively. The assessment of the presence of the blaCTX-M, bla TEM and blaSHV genes and phylogenetic grouping were performed using the polymerase chain reaction (PCR) method. Out of 183 E. coli isolates, 59 (32.2) showed a positive ESBL phenotype. The prevalence of ESBL-producing E. coli was higher in males (57.4). Fifty-seven of the ESBL-producing strains carried at least one of the β-lactamase genes (bla CTX-M, bla TEM, bla SHV). Phylotyping of multi-drug resistant isolates indicated that the isolates belonged to B2, A and D phylogroups. Analysis of resistance patterns among these phylogroups revealed that 74.4, 55.3 and 29.7 of the isolates in the B2 group were resistant to trimethoprim-sulfamethoxazole, ciprofloxacin and gentamicin, respectively. Most of the strains in the phylogroup B2 carried the bla CTX-M gene. All the ESBL-producing isolates were placed in one of the four phylogenetic groups. The presence of CTX-M and resistance to quinolones were more frequent in B2 strains than in non-B2 strains. © 2020 Azam Fattahi et al., published by De Gruyter, Berlin/Boston

Virulence factors and antimicrobial resistance in uropathogenic escherichia coli strains isolated from cystitis and pyelonephritis

Background/aim: The aim of this study was to investigate the prevalence of virulence genes as well as patterns of antibiotic resistance in cystitis and pyelonephritis uropathogenic Escherichia coli (UPEC) isolates. Materials and methods: Two hundred UPEC isolates were collected from hospitalized patients with pyelonephritis (n = 50) and cystitis (n = 150) in Shafa Hospital in Iran. Antimicrobial susceptibility and ESBL production were determined with confirmatory tests. Polymerase chain reaction assay was performed to determine the prevalence of virulence genes in UPEC strains. Results: Of a total 200 UPEC isolates, the highest and lowest resistance rates to antibiotics were for cephalexin (74) and nitrofurantoin (9), respectively. Of these isolates, 72 (36) and 128 (64) strains were ESBL-positive and ESBL-negative, respectively. The frequency of fimH, papC, and hly was 64, 38, and 12, respectively. The most commonly identified virulence gene in ESBL-positive and ESBL-negative strains was fimH 46 (23) and 86 (43), respectively. The hlyA gene was more prevalent among patients with pyelonephritis than cystitis. Conclusion: The frequency of virulence genes was not significantly different between pyelonephritis and cystitis UPEC strains in the studied patients, but the prevalence rates of hlyA and papC genes were higher among UPEC strains isolated from inpatients compared to outpatients; hence, they could be considered as useful targets for prophylactic interventions. © TUBİTAK

Evaluation of virulence factors and antibiotic resistance patterns in clinical urine isolates of klebsiella pneumoniae in Semnan, Iran

Background: Klebsiella pneumoniae as an opportunistic pathogen can be the cause of a range of nosocomial and community-acquired infections. Many virulence factors help these bacteria overcome an immune system and cause various diseases. K1 and K2 capsular antigens, also magA, wcaG, and rmpA are well-known K. pneumoniae virulence factors. Klebsiella pneumoniae has been revealed to have the ability to acquire resistance to many antibiotics, which cause treatment failure. Objectives: This study aimed at determining the prevalence of magA, wcaG, rmpA, Capsular type K1, Capsular type K2, TEM, and SHV in K. pneumoniae isolates. Methods: A total of 173 non-duplicate K. pneumoniae isolates were collected from two different hospitals in Semnan, Iran, from urine specimens. Klebsiella pneumoniae was identified by conventional bacteriological tests. Disk diffusion test was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Detection of virulence factors, TEM, and SHV gene was performed by specific primers. Results: Frequency of virulence factors was as follow: capsular type K2: 32.9, rmpA: 20.2, capsular type K1: 6.9, and wcaG: 16.2. Also, the SHV and TEM were observed in 46.8 and 33.5, respectively. Antibiotics resistance rates were as follow, imipenem: 7.5, ciprofloxacin: 16.1, levofloxacin: 17.3, amoxicillin-clavulanic acid: 30, trimethoprim-sulfamethoxazole: 32.9, cefepime: 34.1, nitrofurantoin: 35.8, amikacin: 36.4, aztreonam: 39.3, ceftazidime: 42.7. Conclusions: Frequency of some virulence factors including capsular type K2, rmpA, wcaG, and also resistant rate to imipenem, amikacin, and ceftazidime were significantly higher than similar studies. Presence of virulence factors accompanied by drug resistance should make bacteria an infectious agent and lead to treatment failure. © 2018, Author(s)

How phages overcome the challenges of drug resistant bacteria in clinical infections

Nowadays the most important problem in the treatment of bacterial infections is the appearance of MDR (multidrug-resistant), XDR (extensively drug-resistant) and PDR (pan drug-resistant) bacteria and the scarce prospects of producing new antibiotics. There is renewed interest in revisiting the use of bacteriophage to treat bacterial infections. The practice of phage therapy, the application of phages to treat bacterial infections, has been around for approximately a century. Phage therapy relies on using lytic bacteriophages and purified phage lytic proteins for treatment and lysis of bacteria at the site of infection. Current research indicates that phage therapy has the potential to be used as an alternative to antibiotic treatments. It is noteworthy that, whether phages are used on their own or combined with antibiotics, phages are still a promising agent to replace antibiotics. So, this review focuses on an understanding of challenges of MDR, XDR, and PDR bacteria and phages mechanism for treating bacterial infections and the most recent studies on potential phages, cocktails of phages, and enzymes of lytic phages in fighting these resistant bacteria. © 2020 Taati Moghadam et al

Molecular genotyping of Acinetobacter baumannii species isolated from patients in Tehran, Iran, by repetitive element PCR fingerprinting

Background & objective: Acinetobacter baumannii is an opportunistic pathogen with high pathogenic and antibiotic-resistance potential and is also considered as one of the main nosocomial agents, specifically in the intensive care units (ICUs). It is highly important to use molecular biology methods in the epidemiological studies, determine the source of infection, and understand the relationships and distributional patterns of pathogens. Therefore, the current study aimed to determining the similar molecular types in the A. baumannii species isolated from patients in Tehran, Iran, by the repetitive element PCR fingerprinting (REP-PCR) method. Methods: A total of 350 clinical samples were collected from patients admitted to different hospital in Tehran, assessed to identify Acinetobacter spp., based on the special culture media and biochemical test results. The resistance of isolates was evaluated against 11 different antibiotics. The cefepime and ceftazidime were assessed by the minimum inhibitory concentration (MIC) method, based on serial dilutions. The genome of isolated strains was extracted using the modified boiling method and amplified in REP-PCR technique using specific primers. Results: In the current study, out of 120 isolates of Acinetobacter spp., 100 (76.9) were identified as A. baumannii, mostly from ICUs and infectious diseases wards. The isolates of A. baumannii in the current study mostly showed antimicrobial resistance against cefepime and ceftazidime, and had the highest sensitivity to polymyxin B. About 70 of A. baumannii isolates in the current study were resistant to 3 or more antibiotics. According to dendrogram analyses, the patterns were classified to A-I with the maximum population (36) of group A. All genotypes of Acinetobacter spp. in the current study showed resistance against carbapenems and aminoglycosides. Conclusion: High similarities between the isolates in the current study indicated the high distribution of A. baumannii species in the hospitals of Tehran. © 2018, IRANIAN JOURNAL OF PATHOLOGY

Effects of sub-inhibitory concentration of antibiotic and heat stress on the expression of type II TA system genes in Brucella spp

Objective: Bacteria can react to stress conditions using the Toxin-Antitoxin (TA) system. This study investigated the expression of TA system genes under heat and antibiotic stresses in Brucella spp. Methods: To determine the effects of sub-inhibitory concentration (sub-MIC) of rifampin on bacterial survival and growth, a colony-forming unit was quantitated, and turbidity was assessed following the treatment of Brucella isolates, with ½ minimum inhibitory concentration (MIC) of antibiotic at different time intervals. Also, Brucella isolates were exposed to heat stress (42 °C) compared to the control (37 °C). Finally, the expression of TA system genes in Brucella isolates was evaluated one hour after treatment using the quantitative reverse transcription PCR (qRT-PCR) method. Results: Our results showed that the growth of the Brucella isolates reduced in the presence of the sub-MIC of antibiotics compared to the control. The results of the qPCR assay showed that, in the presence of rifampicin the expression of the TA system genes increased and, under the heat stress conditions, the expression of the TA system genes increased compared to controls expect brnT / brnA system. Conclusion: Although the exact role of the TA system in response to various stresses is not yet fully understood, our study provided information on the effectiveness of the type II TA system under heat and antibiotic stress conditions by examining the gene expression of type II systems in Brucella isolates. © 202