Investigation of miRNA and cytokine expressions in latent tuberculosis infection and active tuberculosis.

BACKGROUND: In tuberculsosis (TB), miRNA has been used as a biomarker to distinguish between healthy individuals and TB patients. The aim of this study was to investigate (i) the association of the miRNA and cytokine expression levels, the course of tuberculosis infection, clinical forms and response to treatment, and (ii) the effects of genotypic features of bacteria on the course of tuberculosis and the relationship between miRNA and cytokine expressions and bacterial genotypes. METHODS: A total of 200 cases (100: culture positive active tuberculosis, 50: quantiferon positive latent tuberculosis infection and 50: quantiferon negative healthy controls) were included in the study. For the tuberculosis group at the time of admission and after treatment, for the latent tuberculosis infection and healthy control groups at the time of admission, miRNA and cytokine expressions were determined. Genotyping of M.tuberculosis isolates was performed by spoligotyping method. RESULTS: While, in the comparison of miRNA expressions between the pretreatment patient group and the healthy control group, there was a statistically significant decrease in the expression of miR-454-3p, miR-15a-5p, miR-590-5p, miR-381, and miR-449a in the Pulmonary TB group, there was no significant change in miRNA expression in extrapulmonary TB patients. When the cytokine expressions of the patient group and the healthy control group were compared before treatment, the expressions of all cytokines in the patient group decreased. However, the only cytokine that showed a significantly lower expression was IL12A in PTB patients. DISCUSSION: There is no significant relationship between the clinical course of the disease, cytokine and miRNA expression, and the genotype of the bacteria.

In vitro effects of ciprofloxacin, levofloxacin and moxifloxacin on Mycobacterium tuberculosis isolates.

INTRODUCTION: Increased tuberculosis prevalence, and isolation of multidrug resistant (MDR) Mycobacterium tuberculosis strains frequently as causative organisms from tuberculosis infections are resulted in increasing need of new anti-tuberculosis drugs. Nowadays, fluoroquinolones known to have fewer side effects than the other drugs used in treatment of tuberculosis are sometimes assessed even as first-line anti-tuberculosis drugs due to their in vitro and in vivo strong activity. It was aimed in this study to investigate phenotypically the fluoroquinolone susceptibility in MDR and non-MDR M. tuberculosis isolates. MATERIALS AND METHODS: A total of 126 MDR and non-MDR M. tuberculosis isolates from mycobacteriology laboratory of two hospitals in the Aegean Region of Turkey were included in the study. Ciprofloxacin (CIP), levofloxacin (LEV) and moxifloxacin (MXF) susceptibilities were assessed by agar proportion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. RESULT: Twelve (15.2%), 5 (6.3%) and 4 (5.1%) of the MDR M. tuberculosis strains were resistant to CIP, LEV, MXF, respectively [resistance breakpoints (µg/mL); CIP (> 2), LEV (> 1), MXF (> 0.5)] while non-MDR strains were susceptible to CIP, LEV, MXF. CONCLUSIONS: Consequently, although high fluoroquinolone susceptibilities were evaluated as a pleasing data in this study, to preserve their efficiency for many years steadily, quinolone usage and resistance increment in MDR M. tuberculosis isolates should be monitored elaborately.

Analyses of Plasmids Harbouring Quinolone Resistance Determinants in Enterobacteriaceae Members.

The aim of this study was to explore the plasmid characteristics of eight clinical Enterobacteriaceae strains containing extended broad spectrum beta-lactamases and plasmid-mediated quinolone resistance. Plasmids were transferred by conjugation or transformation and resistance determinants were investigated by PCR. We showed that at least one plasmid harbouring qnrB or qnrS determinant was transferred by conjugation in five isolates. QepA determinant was confirmed to be on a non-conjugative plasmid. We found at least one beta-lactamase gene in seven of the eight clinical isolates having plasmid-mediated quinolone resistance, which indicated that these two resistance determinants were mostly on the same conjugative plasmids.

[Molecular epidemiology of human Mycobacterium bovis infection in Aegean Region, Turkey]. / Ege Bölgesi'nde insan Mycobacterium bovis enfeksiyonlarinin moleküler epidemiyolojisi.

Mycobacterium bovis, which has a broader host spectrum as opposed to Mycobacterium tuberculosis that generally causes disease in humans, mainly leads to chronic progressive pulmonary disease in a large number of domestic and wild mammals, particularly cattle. The term "zoonotic tuberculosis (TB)" is used to describe M.bovis infection in humans. "Zoonotic TB" can not be differentiated clinically, radiologically or pathologically from TB caused by M.tuberculosis. The aim of this study was to evaluate the role of M.bovis in epidemiology of human TB in Aegean Region, Turkey and to determine M.bovis genotypes responsible for human TB. Thirteen M.bovis isolates identified by spoligotyping from 482 M.tuberculosis complex isolates obtained from clinical specimens sent for routine mycobacteriological examination to the Mycobacteriology Laboratory in Medical Microbiology Department of Ege University Faculty of Medicine Hospital between 2009 and 2014 were included in the study. Drug susceptibility tests of the isolates were performed by the BACTEC MGIT 960 automated system. It was determined that 9 (63.6%) of the 13 spoligotyped M.bovis isolates in this study were ST685 (SB0288), 1 (7.7%) was ST 1118 (SB0989) and 1 (7.7%) was ST820 (SB0856), for two isolates there were no registered data in SpolDB4 and Mbovis.org databases. All the isolates were susceptible to first-line antituberculosis drugs. It was determined that 13 M.bovis isolates examined in the study accounted for 2.7% of the 482 M.tuberculosis complex isolates spoligotyped in the same period. In this study, it was determined that 8 (%61.5) of 13 patients was male, 5 (38.5%) of them was female, 9 (69.2%) of the 13 patients had pulmonary TB and 4 (30.8%) had extra pulmonary TB. Seven of nine patients with pulmonary TB and two of the four patients with extrapulmonary TB were living in the rural area, and two patients with pulmonary TB had occupational exposure. Although ST683 (SB0140) is widely seen in the world among human isolates, it was not detectedin this study and other studies conducted in Turkey. In contrast, ST685 (SB0288) and ST1118 (SB0989), which have been reported very few in the world, found to be predominant in this study. This result suggested that they may be unique spoligotypes emerging in Anatolia. In conclusion, collaborative molecular epidemiological studies are needed in conjunction with researchers working in medicine and veterinary fields to determine precisely the importance of zoonotic TB in human TB in our country, to determine the route of transmission to humans and risk factors for zoonotic TB infections, to identify the dominant types between humans and animals and to understand the phylogeographic relationships of the strains in our country.

Genotype distribution of Mycobacterium tuberculosis in theAegean Region and associated demographic factors

Background/aim: The Aegean Region is the second-ranking region in Turkey according to the Human Development Index and population density and it hosts 1/8 of Turkey's population. Izmir is the largest city of the region, receiving internal migration both from inside and outside the region. The tuberculosis incidence in Izmir is lower than overall in Turkey: 17.7/100,000 in 2011. Our aims were to determine genotypes of Mycobacterium tuberculosis isolates; to explore possible associations between genotypes with case-demographic data, clinical presentation, and antimicrobial susceptibility patterns; and to determine variations in genotype distribution of strains isolated in Ege University Hospital, Izmir. Materials and methods: Forty-nine M. tuberculosis isolates from 49 patients in 1996-2000 and 421 M. tuberculosis isolates from 421 patients in 2009-2014 were spoligotyped. Drug susceptibility testing and demographic data of the 421 isolates were investigated. Chi-square, Student's t, and Mann-Whitney U tests were used for analyses. Results: Among the 470 M. tuberculosis strains, 132 different spoligopatterns were identified and 46 different clusters for 384 strains were determined. The most predominant spoligotypes were ST53 (n = 116; 24.7%) and ST41 (n = 38; 8.1%), followed by ST50 (5.7%), ST284 (4.7%), and ST4 (4.3%), respectively. ST53 was the most predominant type in both sexes. Multidrug resistance (MDR) was determined in 12 isolates, of which six were ST1.Conclusion: As a consequence of worldwide migration and increasing status of HIV-infected hosts, the increasing prevalence of Beijing strains with higher MDR rates may threaten disease control programs. With its increasing trend, ST284 could replace ST41 in the following years in this region.

[Investigation of the virulence factors of multidrug-resistant Acinetobacter baumannii isolates]. / Çok ilaca dirençli Acinetobacter baumannii izolatlarinda virülans faktörlerinin arastirilmasi

Acinetobacter baumannii is an opportunistic human pathogen which causes life-threatening nosocomial infections such as ventilator-associated pneumonia, bacteremia, meningitis, urinary tract and wound infections. Treatment options are very limited for infections caused by multi-drug resistant (MDR) A.baumannii strains. Until recently, the majority of studies related to A.baumannii have focused on antibiotic resistance, treatment protocols and epidemiological data, however, there have been few studies addressing the virulence factors of this organism. The features such as biofilm formation, serum resistance, motility, efflux pumps and iron acquisition mechanisms help the bacterium to survive in adverse environmental conditions and facilitate the development of an infection. The aim of the present study was to investigate the basic characteristics that contribute to the virulence of clinically important MDR A.baumannii isolates. Sixty-five ciprofloxacin-imipenem-trimethoprim/sulfamethoxazole-resistant A.baumannii strains isolated from various clinical specimens between December 2011 and March 2012 at Ege University Faculty of Medicine, Department of Medical Microbiology were included in the study. The clonal relationship of the isolates was analyzed by PCR using Enterobacterial repetitive intergenic consensus (ERIC)-2 primer. Biofilm formation, serum resistance, twitching and swarming motility, efflux pump and siderophore production were sought in representatives of each clone. Investigated MDR A.baumannii isolates were classified into seven main clusters, and the largest cluster included 86% of the strains. The virulence-associated features were investigated in 16 representative strains, including sub-groups. Twelve, three and one of the examined strains were determined to be strong, intermediate and weak biofilm producers, respectively. Siderophore production was not encountered in any of the isolates. Of the sixteen strains, two, one and thirteen isolates were found to be resistant, moderately susceptible and susceptible to bactericidal effect of serum, respectively. In our study, swarming motility was observed in seven strains while twitching motility was observed in only one strain. Swarming was simultaneously detected with twitching in one isolate. The presence of an efflux pump was investigated with ciprofloxacin in 16 representative strains but none of them were positive. However, eflux pump was determined in two of the five doxycycline resistant strains. Biofilm production was the most commonly observed characteristic among the examined strains. In addition, serum resistance, swarming and an efflux pump which has a spectrum including tetracyclines, were also determined among features associated with virulence. While the biofilm production was encountered at the members of all clones, serum resistance was found only in the representatives of the most dominant clone. Motility and the presence of an efflux pump were not associated with a particular clone. MDR A.baumannii strains are among the most important agents of nosocomial infections in our hospital and all over the world. Revealing the characteristics that play a role in the pathogenesis of these isolates, will contribute to infection control measures and to the investigation of new treatment options.

Tigecycline susceptibility in multidrug resistant Acinetobacter isolates from Turkey.

The present study aimed to evaluate antimicrobial activity of tigecydcline against 84 multidrug resistant (MDR) Acinetobacter spp. strains by disc diffusion and E-test methods. The results of disc diffusion test were compared according to two different interpretation ways. In addition, E-test results and the disc diffusion results that interpreted by both the methods were checked for compatibility. According to the disc diffusion test, 3 strains (3.57%) were found resistant to tigecycline when considering breakpoints suggested by Food and Drug Administration (FDA). On the other hand, none of the strains was found resistant to the evaluation criteria recommended by Jones etal. (2007). Considering E-test results of tigecycline, MIC, and MICG, values of tigecycline for Acinetobacter spp. were 0.75 and 1 mg/l, respectively. Based on FDA defined breakpoints for Enterobacteriaceae, any resistant isolate was detected. In conclusion, although there are some differences in the results, tigecycline was found quite effective on Acinetobacter spp. isolates with reference to the both disc diffusion and the E-test methods.

Effects of disinfectants and ciprofloxacin on quorum sensing genes and biofilm of clinical Pseudomonas aeruginosa isolates.

BACKGROUND: Pseudomonas aeruginosa is one of the most virulent bacteria and quorum sensing (QS) genes have an importance on virulence factors such as biofilm that provide resistance against disinfectants and antibiotics. OBJECTIVE: This study aimed to determine the minimum inhibitory concentrations of the disinfectants, to investigate the effects of disinfectants and ciprofloxacin on biofilm production mature biofilm of clinical P. aeruginosa isolates, and it was aimed to investigate the effects of the agents on the expression levels of several QS-related genes in the isolates. METHODS: Minimum inhibitory concentration (MIC) levels of polyhexamethylene biguanide (PHMB), chlorhexidine (CHX), quaternary ammonium compounds (QAC), glutaraldehyde (GLU) and ciprofloxacin (CIP) against clinical P. aeruginosa isolates were evaluated by microdilution method. Effects of the agents on the biofilm producing capacities of clonally unrelated nine strains were investigated by spectrophotometric method. Alterations in the expression of QS-related genes (lasI, lasR, rhlI and rhlR) were investigated by qPCR in three isolates that were CIP-susceptible and strong biofilm producer. RESULTS: According to microdilution method results, three isolates were found as resistant, one isolate was found as intermediate susceptible and five isolates were found as susceptible to CIP, and CHX (7.81-31.25 µg/mL) had the lowest MIC against P. aeruginosa. CHX inhibited biofilm production levels of eight of nine isolates, and GLU and CIP inhibited six of nine isolates in the presence of agents at MIC levels. GLU inhibited the mature biofilm levels of three of nine isolates at MIC and MIC/4 levels and four of nine isolates at MIC/2 levels. Expression levels of QS-related genes were reduced or induced in the presence of different disinfectants. CONCLUSIONS: More efforts are required to decrease the risk of ineffective and low-dose application of disinfectants and antimicrobials against bacteria. Targeting of QS-related genes may be a reasonable strategy for the inhibition of virulence factors in P. aeruginosa.

Investigation of the Antimicrobial Susceptibility Profile, Virulence Genes, and Epidemiologic Relationship of Clinical Salmonella Isolates.

OBJECTIVES: The objectives of this study were to investigate the epidemiologic relationship, prevalence of the beta-lactamase and virulence genes of clinical ampicillin-resistant Salmonella enterica. MATERIALS AND METHODS: In vitro ampicillin susceptibilities of 117 Salmonella enterica isolates obtained between 2011-2012 from Ege University Hospital, Bacteriology Laboratory of Medical Microbiology Department were examined using disc diffusion assays in accordance with the CLSI guidelines. The MIC levels in the ampicillin-resistant bacteria were determined using the broth microdilution method. The resistant strains were serotyped by the Public Health Institution. Epidemiologic relations of resistant strains were evaluated using ERIC-PCR. The presence of beta-lactamase genes and virulence factors were detected using PCR. RESULTS: The 117 S. enterica strains had ten isolates that were resistant to ampicillin, and the MIC range of ampicillin was found as 512-128 µg/mL. Ampicillin-resistant strains were susceptible to nalidixic acid, ciprofloxacin, cefotaxime, sulfamethoxazole/trimethoprim. Four different serotypes were identified and isolates were grouped into seven clusters. Five isolates carried blaTEM , and two carried the blaCTX-M gene. However, it was determined that blaSHV and blaPER genes did not exist in these strains. Virulence genes invA, pipD, and sopB were found in all isolates. sifA, pefA, and sopE genes were found in seven, four, and three isolates, respectively. CONCLUSION: Our data suggest that the rate of ampicillin resistance in S. enterica isolates was 8.5% in the two year period, but this ratio was generally lower than rates abroad. blaCTX-M and blaTEM genes could be responsible for ampicillin resistance. The blaSHV gene, which is highly prevalent in our country, was not found in any strains. sopB and pipD genes, which might be associated with beta-lactam resistance, were found in all strains. It is also noteworthy that the three isolates containing the sopE gene, which is associated with epidemic cases, were of the same serotypes and epidemiologic clusters.

Antibiotic Resistance Profiles and Genotypes of Acinetobacter baumannii Isolates and In Vitro Interactions of Various Antibiotics in Combination with Tigecycline and Colistin.

OBJECTIVES: The aim of this study was to determine the antibiotic resistance profile, clonal relation and efficacy of antibiotic combinations in nosocomial multidrug resistant (MDR) Acinetobacter baumannii. MATERIALS AND METHODS: Antibiotic susceptibilities of 84 MDR A. baumannii against tigecycline (TGC), colistin (CL), amikacin (AK), ciprofloxacin (CIP), meropenem (MR), moxifloxacin (MXF), rifampicin (RF) were determined by microdilution method. Clonal relationship was investigated by genotyping using AP-PCR and antibiotyping. Interactions of antibiotic combinations were tested against clonally unrelated strains by the checkerboard (CB) method. The efficacy of the best combinations was also assesed on a selected isolate by the time-kill (TK) method. RESULTS: CIP, RF, MXF, MR, AK resistance was found as 90.47%; 47.62%; 22.62%; 58.33%; 50% respectively; however; CL and TGC were not ascertained. The isolates were distinguished as 25 different antibiotypes and 15 varied molecular patterns. The best synergistic effect was detected in combinations of CL with RF (100%) and MR (100%), in combinations of TGC with RF (53%) against clonally unrelated 15 MDR A. baumannii isolates by the CB method. While CL-RF and CL-MR showed synergy by TK method like CB, on the other hand TGC-RF indicated additive interactions by TK. CONCLUSION: In this study, both synergy tests showed that CL in combination with RF would be a good option in MDR A. baumannii.

DNA-wrapped multi-walled carbon nanotube modified electrochemical biosensor for the detection of Escherichia coli from real samples.

This paper introduces DNA-wrapped multi-walled carbon nanotube (MWCNT)-modified genosensor for the detection of Escherichia coli (E. coli) from polymerase chain reaction (PCR)-amplified real samples while Staphylococcus aureus (S. aureus) was used to investigate the selectivity of the biosensor. The capture probe specifically recognizing E. coli DNA and it was firstly interacted with MWCNTs for wrapping of single-stranded DNA (ssDNA) onto the nanomaterial. DNA-wrapped MWCNTs were then immobilised on the surface of disposable pencil graphite electrode (PGE) for the detection of DNA hybridization. Electrochemical behaviors of the modified PGEs were investigated using Raman spectroscopy and differential pulse voltammetry (DPV). The sequence selective DNA hybridization was determined and evaluated by changes in the intrinsic guanine oxidation signal at about 1.0V by DPV. Numerous factors affecting the hybridization were optimized such as target concentration, hybridization time, etc. The designed DNA sensor can well detect E. coli DNA in 20min detection time with 0.5pmole of detection limit in 30µL of sample volume.

Development, characterization, and in vivo assessment of mucoadhesive nanoparticles containing fluconazole for the local treatment of oral candidiasis.

This study aimed to develop a suitable buccal mucoadhesive nanoparticle (NP) formulation containing fluconazole for the local treatment of oral candidiasis. The suitability of the prepared formulations was assessed by means of particle size (PS), polydispersity index, and zeta potential measurements, morphology analysis, mucoadhesion studies, drug entrapment efficiency (EE), in vitro drug release, and stability studies. Based on the optimum NP formulation, ex vivo drug diffusion and in vitro cytotoxicity studies were performed. Besides, evaluation of the antifungal effect of the optimum formulation was evaluated using agar diffusion method, fungicidal activity-related in vitro release study, and time-dependent fungicidal activity. The effect of the optimum NP formulation on the healing of oral candidiasis was investigated in an animal model, which was employed for the first time in this study. The zeta potential, mucoadhesion, and in vitro drug release studies of various NP formulations revealed that chitosan-coated NP formulation containing EUDRAGIT(®) RS 2.5% had superior properties than other formulations. Concerning the stability study of the selected formulation, the formulation was found to be stable for 6 months. During the ex vivo drug diffusion study, no drug was found in receptor phase, and this is an indication of local effect. The in vitro antifungal activity studies showed the in vitro efficacy of the NP against Candida albicans for an extended period. Also, the formulation had no cytotoxic effect at the tested concentration. For the in vivo experiments, infected rabbits were successfully treated with local administration of the optimum NP formulation once a day. This study has shown that the mucoadhesive NP formulation containing fluconazole is a promising candidate with once-a-day application for the local treatment of oral candidiasis.