Clonal spread of trimethoprim-sulfamethoxazole-resistant Stenotrophomonas maltophilia isolates in a tertiary hospital.

Aim: The aims of this study were to: (i) determine antibiotic susceptibility of clinical Stenotrophomonas maltophilia isolates, (ii) investigate the presence of different classes of integrons and sul genes responsible for sulphonamide resistance, (iii) assess the molecular epidemiology of the isolates by determining their clonal relatedness, and (iv) investigate the potential sources of infection by collecting environmental samples when necessary. Methods: 99 S. maltophilia isolates from clinical specimens of hospitalized patients were screened by PCR for sul1, sul2, sul3 genes, and integron-associated integrase genes: intI1, intI2, and intI3. PFGE was used to determine the clonal relatedness of the isolates. Results: Susceptibility rates for trimethoprim-sulfamethoxazole, levofloxacin, and ceftazidime were 90.9%, 91.9%, and 53.5% respectively. All trimethoprim-sulfamethoxazole-resistant isolates were positive for intI1 and sul1. PFGE analysis revealed that 24 of the isolates were clonally related, clustering in seven different clones. Five of the nine trimethoprim-sulfamethoxazole-resistant isolates were clonally related. The first isolate in this clone was from a wound sample of a patient in the infectious diseases clinic, and the other four were isolated from the bronchoalveolar lavage samples of patients in the thoracic surgery unit. The patient with the first isolate neither underwent bronchoscopy nor stayed in the thoracic surgery unit. Although clustering was observed in bronchoalveolar lavage samples, no S. maltophilia growth was detected in environmental samples. Conclusion: The findings demonstrated that the sul1 gene carried by class 1 integrons plays an important role in trimethoprim-sulfamethoxazole resistance in S. maltophilia isolates. PFGE analysis revealed a high degree of genetic diversity. However, detection of clonally related isolates suggests the acquisition from a common source and/or cross-transmission of this microorganism between the patients.

Molecular characterization of the multi-drug resistant Myroides odoratimimus isolates: a whole genome sequence-based study to confirm carbapenem resistance.

The bacteria belonging to the Myroides genus are opportunistic pathogens causing community or hospital-acquired infections that result in treatment failure due to antibiotic resistance. This study aimed to investigate molecular mechanisms of antibiotic resistance, clonal relatedness, and the biofilm forming capacity of the 51 multi-drug resistant Myroides odoratimimus. All isolates were screened for blaKPC, blaOXA, blaVIM, blaIMP, blaMUS, blaTUS, blaNDM, and blaB genes by using PCR amplification. Whole genome sequencing (WGS) was applied on three randomly selected isolates for further investigation of antibiotic resistance mechanisms. Clonal relatedness was analyzed by Pulsed-field gel electrophoresis (PFGE) and the microtiter plate method was used to demonstrate biofilm formation. All isolates were positive for biofilm formation. PCR analysis resulted in a positive for only the blaMUS-1 gene. WGS identified blaMUS-1, erm(F), ere(D), tet(X), and sul2 genes in all strains tested. Moreover, the genomic analyses of three strains revealed that genomes contained a large number of virulence factors (VFs). PFGE yielded a clustering rate of 96%. High clonal relatedness, biofilm formation, and multi-drug resistance properties may lead to the predominance of these opportunistic pathogens in hospital environments and make them cause nosocomial infections.

Molecular epidemiological investigation of carbapenem resistant Klebsiella pneumoniae isolated from intensive care unit patients of six geographical regions of Turkey.

INTRODUCTION: Klebsiella pneumonia causes serious infections in hospitalized patients. In recent years, carbapenem-resistant infections increased in the world. The molecular epidemiological investigation of carbapenem-resistant K. pneumoniae isolates was aimed in this study. METHODOLOGY: Fifty carbapenem-resistant K. pneumoniae isolates from six geographical regions of Turkey between September 2019-2020 were included in the study. The disk diffusion method was used for the antibiotic susceptibility testing. The microdilution confirmed colistin susceptibility. Genetic diversity was investigated by MLST (Multi-Locus Sequence Typing). RESULTS: The resistance rates were as follows: 49 (98%) for meropenem, 47 (94%) imipenem, 50 (100%) ertapenem, 30 (60%) colistin and amoxicillin-clavulanate, 49 (98%) ceftriaxone, 48 (96%) cefepime, 50 (100%) piperacillin-tazobactam, 47 (94%) ciprofloxacin, 40 (80%) amikacin, 37 (74%) gentamicin. An isolate resistant to colistin by disk diffusion was found as susceptible to microdilution. ST 2096 was the most common (n:16) sequence type by MLST. ST 101 (n:7), ST14 (n:6), ST 147 and ST 15 (n:4), ST391 (n:3), ST 377 and ST16 (n:2), ST22, ST 307, ST 985, ST 336, ST 345, and ST 3681 (n:1) were classified in other isolates. In Istanbul and Ankara ST2096 was common. Among Turkey isolates, the most common clonal complexes (CC) were CC14 (n:26) and CC11 (n = 7). CONCLUSIONS: In Turkey, a polyclonal population of CC14 throughout the country and inter-hospital spread were indicated. The use of molecular typing tools will highlight understanding the transmission dynamics.

Whole-Genome Sequence Analysis of Carbapenem-Heteroresistant Klebsiella pneumoniae and Escherichia coli Isolates.

Carbapenem-heteroresistant isolates can be misclassified as susceptible by in vitro susceptibility tests, leading to treatment failure. The underlying mechanisms of heteroresistance, where the bacterial isolate harbors both resistant and susceptible subpopulations, are poorly understood. The aim of the current study was to clarify molecular mechanisms responsible for carbapenem heteroresistance. Whole-genome shotgun sequencing was performed for both resistant and susceptible subpopulations of three Klebsiella pneumoniae and one Escherichia coli blood isolates, which were identified as carbapenem-heteroresistant by the population analysis profile method. The software from the Center for Genomic Epidemiology was used to identify genomic similarities, antibiotic resistance genes, Multilocus Sequence Typing (MLST), and core-genome MLST(cgMLST). Both susceptible and resistant subpopulations of the E. coli strain had the same MLST profiles. MLST1/2 and cgMLST for E. coli were 46/736 and 119473, respectively. The susceptible and resistant subpopulations of each K. pneumoniae strain exhibited identical MLST profiles. The genetic background for antimicrobial resistance in three K. pneumoniae strains was almost similar between the colonies inside and outside the inhibition zone of each strain, however, there were remarkable differences between the three strains. The blaKPC-2 and blaOXA-48 genes were responsible for carbapenem resistance for E. coli and K. pneumoniae strains, respectively. This is the first study, which has demonstrated similar genotypic and resistant gene profiles in the resistant and susceptible subpopulations of each strain. Additional metabolic and transcriptomic investigations are needed to understand the mechanisms responsible for carbapenem heteroresistance.

The effect of pre-pregnancy obesity on gut and meconium microbiome and relationship with fetal growth.

OBJECTIVE: To investigate the effect of pre-pregnancy obesity on maternal and newborn microbiomes and fetal growth. METHODS: Individuals who gained body weight in accordance with the recommendations during pregnancy and normal gestastional age are included in the study and were separated into two groups, normal (n = 20) and obese (n = 20), based on their body mass index (BMI) value of pre-pregnancy. Maternal stool samples collected during the first trimester of pregnancy and meconium samples collected at birth were evaluated using 16S rRNA gene-based microbiome analysis. RESULTS: The stool samples of mothers who were obese before pregnancy harbored a higher (59.9 versus 52.3%) relative abundance of Firmicutes and a lower (7.1 versus 4.1%) relative abundance of Proteobacteria than the stool samples of mothers with normal body weight pre-pregnancy. In contrast, in the meconium samples of mothers who were obese pre-pregnancy, compared to those of mothers who had a normal body weight pre-pregnancy, the phylum Firmicutes was less (56.0 versus 69.0%) abundant and Proteobacteria (9.0 versus 8.5%) was more abundant. There was a negative correlation between pre-pregnancy BMI, birth weight, weight/height ratio and alpha diversity indices (Shannon and Chao1). CONCLUSIONS: Pre-pregnancy obesity can affect pregnant and newborn gut microbiota, which might related to fetal growth of the newborn.

The effect of gestational weight gain on serum total oxidative stress, total antioxidant capacity and gut microbiota.

This study aimed to investigate the effect of gestational weight gain on total oxidative stress (TOS), total antioxidant capacity (TAC), oxidative stress index (OSI), dietary antioxidant intake, and the gut microbiome. The study was carried out on 40 pregnant women divided as follows: a) normal prepregnancy weight and gestational weight gain of 11.5-16.0 kg (n=10) b) normal prepregnancy weight and gestational weight gain of >16.0 kg (n=10) c) obese before pregnancy and gestational weight gain of 5-9 kg (n=10) and d) obese before pregnancy and gestational weight gain of >9.0 kg (n=10). Serum TOS and TAC levels, dietary antioxidant intake, and microbiome diversity of the gut microbiome were evaluated during the third trimester of pregnancy. A positive correlation was found between body mass index (BMI) in the third trimester and serum TOS levels and OSI. In women with normal prepregnancy weight, an increase in the Firmicutes and Bacteroidetes phyla was observed when gestational weight gain was above the recommended values (p<0.05). In women who were obese before pregnancy, an increase only in the Bacteroidetes phylum was observed when gestational weight gain was above the recommended values (p<0.05). A positive correlation was found between Firmicutes/Bacteroidetes and OSI, and a negative correlation was found between Firmicutes/Bacteroidetes and dietary antioxidant intake (p<0.05). Prepregnancy body weight, high serum TOS level, and dietary antioxidant intake are determinant factors for microbial diversity, with increased serum TOS levels caused by increased gestational weight gain.

Respiratory viruses in the healthy middle ear and middle ear with otitis media with effusion.

To investigate the presence of respiratory viruses in the middle ear cavity of the individuals with a healthy middle ear and the children with otitis media with effusion (OME). A total of 72 middle ear samples were collected from 25 children with OME (Group 1) and 47 individuals with no middle ear disease (Group 2). Multiplex real-time polymerase chain reaction was used to investigate the presence of 20 different respiratory viruses. Virus results were compared with bacteriomes of the same populations. At least one respiratory virus was detected in 56% of the patients in Group 1 and 12.8% of the individuals in Group 2. The viral co-infection rate for Group 1 and 2 was 8% and 2.1%, respectively. In Group 1, adenovirus was the most frequently detected virus with a rate of 24%, either alone (16%) or concurrent with other viruses (8%), followed by influenza B (12%), rhinovirus, and bocavirus (8%) each. Parainfluenza 4, coronavirus OC43, and RSV A/B were detected in 4% of the sample each. In Group 2, rhinovirus was detected in two samples (4.3%) followed by adenovirus, coronavirus OC43, coronavirus E299, and coronavirus NL63 with a rate of 2.1% each. The detection rate of respiratory viruses was significantly higher in children aged 6 to 11 years. There was no positive association between virus and bacteria found in the middle ear cavity. The current study has provided comprehensive data indicating the presence of diverse respiratory viruses in the healthy middle ear cavity. Our results also suggest that respiratory viruses might have a contribution to OME pathogenesis.

Microbiological investigation of samples collected from healthy middle ears during cochlear implant surgery.

This study aimed to investigate the bacteriome in microscopically healthy middle ear mucosa using Next-generation sequencing (NGS) technology. A total of 60 middle ear washing fluids of pediatric and adult were obtained from 47 patients (35 children and 12 adults). Both children and adults with normal middle ears harbored diverse bacteriome. Seventeen different genera with a mean relative abundance of more than 1% were detected in all samples. Both in adult and children, the most abundant genus was Propionibacterium followed by Streptococcus, Staphylococcus, and Ralstonia. The species Propionibacterium acnes and Corynebacterium tuberculostearicum were significantly more abundant in the adult group. Although there were differences in the prevalence and relative abundance of some bacteria observed from adult and child groups, no specific genus or species was detected only in children or adults.

Early Detection and Control of Methicillin resistant Staphylococcus aureus Outbreak in an Intensive Care Unit.

BACKGROUND: Although Methicillin resistant Staphylococcus aureus (MRSA) is one of the major pathogens of healthcare associated infections, we had only sporadic cases in our intensive care unit (ICU) for years. AIMS: To investigate the sudden increase in the number of MRSA cases in ICU. STUDY DESIGN: Descriptive study. METHODS: From the 5th December 2016 to 26th January 2017, we detected 11 new MRSA cases in ICU. Screening of 73 ICU healthcare workers (HCWs) and screening of 13 patients was performed for outbreak investigation. Nine clinical isolates available in stocks and eight screening MRSA isolates were included in molecular studies. PFGE, spa-mecA-mecC-PVL in-house multiplex PCR assay and spa typing, SCCmec typing were performed for all isolates. Sequence type of the representative strain was determined by Multi-Locus Sequence typing (MLST). RESULTS: All strains were mecA positive, PVL negative, and have the same antimicrobial susceptibility pattern except for two strains. All clinical, two patient screening and three nasal isolates of HCWs showed the same pulsotype, named clone A. The spa type of outbreak isolates is t030 and the SCCmec type is SCCmecIII; the MLST type of representative strain is ST239 (PFGE pulsotype A, ST239-SCCmecIII-t030). Unrelated three isolates had PFGE pulsotype B-SCCmecI-t030, PFGE pulsotype C-SCCmecIII-t459, PFGE pulsotype D-SCCmecIII. CONCLUSION: Molecular typing techniques are the cornerstones for the investigation of outbreaks. Infection control measures, such as enhancing cleaning procedures, promoting hand hygiene, should be enforced in the ICU unit. All patients, including those who have already been discharged to other departments, must be put on contact isolation. HCWs carrying the MRSA strains could be offered decolonization.

Mycobiome in the Middle Ear Cavity with and Without Otitis Media with Effusion.

Objective: No data have yet been published revealing the composition and the diversity of fungal communities (mycobiome) in the human middle ear cavity. The presented study investigated the mycobiome in the middle ear cavities of individuals with healthy middle ears and patients with otitis media with effusion. Methods: A total of 77 middle ear and four adenoid samples were collected from 47 individuals (35 children and 12 adults) in Group 1 and from 20 children in Group 2. The mycobiome profile was analyzed with nuclear ribosomal internal transcribed spacer 2 (ITS2) based metabarcoding using an Illumina MiSeq metagenomics kit. Results: ITS2-based metabarcoding detected 14 different genera and 17 different species with a mean relative abundance of ≥1% in the samples analyzed. Mycobiome profile was similar between the adenoid tissue and the middle ear cavity, between Groups 1 and Group 2, and between children and adults. Fusarium, Stemphylium, Candida, and Cladosporium were the most abundant genera detected in all samples. The mean relative abundances of the genera Candida and Fusarium were remarkably higher in Group 2 compared to Group 1. Conclusion: The species Candida glaebosa, Candida cretensis, Aspergillus ruber, Penicillium desertorum, and Rhizopus arrhizus were significantly more abundant in patients with otitis media with effusion (OME), raising the possibility that they affect the pathogenesis of OME.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Blood Culture Isolates from Three Hospitals in Turkey.

We aimed to investigate the clonal relationships, common sequence types, and carbapenemase genes in 177 non-repetitive blood culture isolates of Acinetobacter baumannii collected from patients at three university hospitals in Turkey in 2016. Molecular epidemiological characteristics of the isolates were examined using pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) (Pasteur scheme-cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB). Multiplex PCR was used to investigate the carbapenemase genes, including blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like, blaIMP, blaVIM, and blaNDM. PFGE genotyping yielded 92 pulsotypes with a clustering ratio of 69.7%. As per a ≥85% similarity coefficient, 159 (90.9%) isolates were found to be clonally related. The blaOXA-23-like and blaOXA-58-like genes were identified in 100% and 28.2% of the isolates, respectively. The blaNDM gene was identified in two isolates. The MLST analysis included 54 isolates with different pulsotypes, and 29 sequence types (STs). Most of the isolates (n = 36) belonged to the clonal complex (CC)2, one isolate belonged to CC1, and one isolate belonged to CC164. Sixteen new STs (ST1235-ST1250) were identified. Identifying both global ST2 and a large number of new STs, revealed high genetic diversity in A. baumannii isolates in the study population.

A Healthcare-Associated Outbreak of Urinary Tract Infections Due to Myroides odoratimimus.

Myroides spp. are low-grade opportunistic pathogens. Outbreaks due to Myroides spp. have rarely been described in the literature to date. We report a healthcare-associated outbreak of urinary tract infections (UTIs), caused by Myroides odoratimimus, in a Turkish hospital. As of March 2019 until May 2019, 6 strains of M. odoratimimus were isolated from the urine samples of patients, all of whom were hospitalized in intensive care units. After identification and antibiotic susceptibility testing using the VITEK 2 system, MALDI-TOF-MS and 16S rRNA-based sequencing methods were performed for confirmation and species-level identification. Pulsed-field gel electrophoresis (PFGE) was performed in order to investigate the clonal relatedness of the isolates. All the patients were immunocompromised and underwent urinary catheterization. None of the patients had urinary neoplasm, surgery, or calculi. VITEK 2 and MALDI-TOF-MS systems revealed that the isolates belonged to the Myroides genus; however, the aforementioned systems neglected to identify the isolates at the species level. The isolates were all successfully identified as M. odoratimimus through 16S rRNA-based sequencing. The isolates were resistant to every antibiotic tested. All isolates had an indistinguishable PFGE pattern, thus indicating cross-transmission between cases. Although M. odoratimimus is rarely isolated from human specimens, clinicians should be aware of its ability to cause UTIs and infectious outbreaks.

[The Identification of Acinetobacter baumannii Blood Isolates by MALDI-TOF MS, ARDRA and blaOXA-51-like Gene-Specific Real-Time PCR]. / Acinetobacter baumannii Kan Izolatlarinin MALDI-TOF MS, ARDRA ve blaOXA-51-benzeri Gene Özgül Gerçek Zamanli Polimeraz Zincir Reaksiyonu ile Tanimlanmasi.

The Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex consists of phenotypically very similar nosocomial species; A.baumannii, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii and Acinetobacter djikshoorniae and one environmental species A.calcoaceticus. The rapid and accurate identification of the members of Acb complex is critical as these nosocomial pathogens can show differences in antimicrobial susceptibility and clinical outcomes. The conventional phenotypic methods are slow, unreliable and less efficient for the differentiation of Acb complex species, including the A.baumannii species within the Acb complex. Although various molecular methods are available, such as amplified ribosomal DNA restriction analysis (ARDRA) and blaOXA-51-like gene specific PCR, they are usually inconvenient for the routine diagnostic laboratories. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers an opportunity for rapid, cost-effective and convenient bacterial identification in routine diagnostic procedures conducted in clinical laboratory. In this study, we aimed to evaluate the diagnosis performance of MALDI-TOF MS system to identify blood isolates of A.baumannii. A total of 180 nonduplicate carbapenem resistant Acb complex (strain numbers; TR1-TR60) and A.baumannii (TR61-TR180) blood isolates were collected from the intensive care units of the three university hospitals in Turkey from January 2016 to December 2016. All isolates were evaluated by using blaOXA-51-like gene specific real time (Rt-PCR) analysis, ARDRA (restriction enzymes-AluI, CfoI, MboI, MspI, RsaI) method and MALDI-TOF MS (VITEK® MS, bioMérieux, France) system. All the strains except TR10, TR31, TR35 and TR52 were identified as A.baumannii by ARDRA method. Out of 177 of all the isolates, presence of blaOXA-51-like gene was found except for TR10, TR31 and TR52 isolates. However, TR31 without the presence of blaOXA-51-like gene was identified as A.pittii using the ARDRA. Totally 176 isolates which were identified as A.baumannii by both of the methods, ARDRA and Rt-PCR- blaOXA-51-like, were accepted as a reference for the evaluation of the diagnosis performance capacity of the MALDI-TOF MS. Overall, for all 176 isolates tested, the sensitivity obtained with the MALDI-TOF MS were 99.4% with 75% specificity. The accuracy value of the method was determined as 98.9% for the identification of A.baumannii to the species level. MALDI-TOF MS is increasingly used in diagnostic microbiology for the routine identification of bacteria to the genus, species or subspecies level with high rates of sensitivity and specificity. In future, by expanding the database, MALDI-TOF MS system would possibly become the ideal method for routine diagnostic laboratories that could potentially identify more species and even determine some characteristics of antimicrobial resistance and virulence determinants.

Mutations Observed in the SARS-CoV-2 Spike Glycoprotein and Their Effects in the Interaction of Virus with ACE-2 Receptor.

Coronaviruses (CoVs) classified in the Coronaviridae family infect a very large spectrum of vertebrate group. Seven CoVs that cause human disease consist of Alpha-CoVs, which are HCoV-229E, and NL63 and beta-CoVs, which are MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-HKU1, and SARS-CoV-2. SARS-CoV-2 is an enveloped, positive-polarity, single-stranded RNA virus responsible for a new Coronavirus disease 2019 (COVID-19). The mutagenic ability of the SARS-CoV-2 directs its evolution and genome variability, thus allowing viruses to escape from host immunity and develop drug resistance. Tracing viral mutations is also important for the development of new vaccines, antiviral drugs, and diagnostic systems. During replication in the host cell, genomic mutations occur in the virus and these mutations are transferred to new generations. For this reason, systematic monitoring of mutations in the SARS-CoV-2 genome allows observation of the national and international molecular epidemiology of the virus. SARS-CoV-2 spike (S) glycoprotein is vital in the binding of the virus to the host cell receptor that is angiotensin converting-enzyme 2 (ACE2), membrane fusion, vaccine studies and immune response to the virus. Therefore, mutations in the gene encoding the S glycoprotein and especially the possible variations in the receptor binding domain (RBD) in S gene are important issues to be emphasized. In this article, information about the mutations observed in the SARS-CoV-2 S glycoprotein and their possible effects are presented.

A healthcare-associated outbreak of urinary tract infections due to Myroides odoratimimus.

Myroides spp. are low-grade opportunistic pathogens. There were only a few outbreaks due to Myroides spp. described in the literature to date. We report a healthcare-associated outbreak of urinary tract infections caused by Myroides odoratimimus in a Turkish hospital. From March to May 2019, six strains of M. odoratimimus were isolated from the urine samples of patients hospitalized in the intensive care units (ICUs). After identification and antibiotic susceptibility testing with VITEK 2 system, MALDI-TOF-MS and 16S rRNA based sequencing methods were performed for confirmation and species level identification. Pulsed-field gel electrophoresis (PFGE) was used to investigate clonal relatedness of the isolates. All the patients were immunocompromised and underwent urinary catheterization. None of them had urinary neoplasm, surgery or calculi. VITEK 2 and MALDI-TOF-MS systems revealed that the isolates belong to the Myroides genus but lacked to identify the isolates at the species level. 16S rRNA based sequencing successfully identified all the isolates as M. odoratimimus. The isolates were resistant to all antibiotics tested. All isolates had indistinguishable PFGE pattern indicating cross-transmission between cases. Although M. odoratimimus is rarely isolated from human specimens, clinicians should be aware of its ability to cause UTIs and outbreaks.

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Background/aim: Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure. Materials and methods: Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA.DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB's Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used. Results: Optimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion: The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

[Determination of the Subspecies of Francisella tularensis Isolated in Turkey by Molecular Methods]. / Türkiye'de Izole Edilen Francisella tularensis Alt Türlerinin Moleküler Yöntemlerle Belirlenmesi.

Francisella tularensis is a gram-negative, coccobasillus, facultative intracellular bacteria and causes a zoonotic disease, tularemia in humans. F.tularensis has four subspecies, which have different virulences for humans as F.tularensis subsp. tularensis, F.tularensis subsp. holarctica, F.tularensis subsp. mediasiatica and F.tularensis subsp. novicida. F.tularensis subsp. tularensis is the most virulent subspecies and mortality rate is high in human cases. F.tularensis subsp. holarctica, which has been reported in our country to date, has lower virulence than that of subsp. tularensis, and causes rare lethality among untreated patients. According to the erythromycin resistance and the properties of glucose-glycerol fermentation, F.tularensis subsp. holarctica has three biovar as biovar I, biovar II and biovar japonica. F.tularensis subsp. mediasiatica has been reported only in a few central asian countries and its virulence is similar to the F.tularensis subsp. holarctica F.tularensis subsp. novicida is avirulent for immunocompetent individuals but has been observed to cause infection in immunocompromised individuals. The aim of this study was to determine the F.tularensis subspecies in 259 F.tularensis strains isolated from clinical specimens, drinking water and a rodent sample and 517 F.tularensis PCR-positive DNA isolated from clinical specimens between years 2009 and 2014. Conventional PCR was performed using primers specific for the RD1 (Region Difference) region of F.tularensis. Subspecies were differentiated depending on the difference in PCR amplification product size. In our study, F.tularensis subsp. holarctica was detected in 764 samples yielding 922 base pair (bp) amplification product. The DNA samples obtained from one water and 11 lymph aspirates were determined as F.tularensis subsp. holarctica biovar japonica. The DNA sequence analysis of the amplification product of the RD1 region of the isolate from water sample was determined. The 1136 bp nucleotide sequence obtained from the DNA sequence analysis was 100% similar to F.tularensis subsp. holarctica biovar japonica (FCS075 strain-accesion number AF469618) when compared with GenBank data. The whole genome sequence of this isolate was also determined and recorded to GenBank with accesion number CP007148. None of the samples used in our study belonged to other sub-species. F.tularensis subsp. holarctica biovar japonica positive 11 lymph aspirate samples were sent to our center from Ankara (n= 1), Kayseri (n= 1) and Afyon (n= 9) provinces. The results of the current study revealed that F.tularensis subsp. holarctica biovar japonica caused a tularemia outbreak in a village in Afyon province at first time and it was observed sporadically in two other different provinces.

Optimization and validation of a real-time polymerase chain reaction protocol for the diagnosis of human brucellosis.

Due to limitations in commercial diagnostic methods, this study aimed to develop a reliable real-time polymerase chain reaction (Rt-PCR) assay for early diagnosis of brucellosis. Optimization of the Rt-PCR method was performed on serum samples spiked by Brucella melitensis with different densities ranging from 101 to 108 colony-forming units (cfu)/mL; each density was prepared in ten samples. The limit of detection was investigated by using Thermo DNA extraction kit with Maxima SYBR Green Rt-PCR and two TaqMan probe-based Rt-PCR protocols performed by QuantiTect and TEMPase multiplex PCR master mixes in two thermal cyclers, which were Rotor-Gene and Bio-Rad. The validation of the optimized protocol was carried on 20 brucellosis-negative samples and 20 samples spiked with B. melitensis by using a combination of Thermo DNA extraction kit with TEMPase PCR master mix. SYBR Green Rt-PCR yielded positive results on all samples having ≥ 104 cfu/mL of B. melitensis in both thermal cyclers. Its limit of detection was 112 DNA copies per reaction. The positivity of both probe-based Rt-PCR protocols was 100% and 80% on the samples having 103 cfu/mL and 102 cfu/mL of B. melitensis, respectively. The limit of detection of probe-based protocols was defined as 4 DNA copies per reaction. The optimized Rt-PCR protocol showed high-level accuracy, precision, specificity, and sensitivity, each having a rate of 100%. The current study indicated that the TaqMan probe-based Rt-PCR protocol optimized and validated with serum samples can be reliably used for early diagnosis of brucellosis.

Molecular characterization of the influenza A(H1N1)pdm09 isolates collected in the 2015-2016 season and comparison of HA mutations detected in Turkey since 2009.

Influenza A(H1N1)pdm09 pandemic virus causing the 2009 global outbreak moved into the post-pandemic period, but its variants continued to be the prevailing subtype in the 2015-2016 influenza season in Europe and Asia. To determine the molecular characteristics of influenza A(H1N1)pdm09 isolates circulating during the 2015-2016 season in Turkey, we identified mutations in the hemagglutinin (HA) genes and investigated the presence of H275Y alteration in the neuraminidase genes in the randomly selected isolates. The comparison of the HA nucleotide sequences revealed a very high homology (>99.5%) among the studied influenza A(H1N1)pdm09 isolates, while a relatively low homology (96.6%-97.2%), was observed between Turkish isolates and the A/California/07/2009 vaccine virus. Overall 14 common mutations were detected in HA sequences of all 2015-2016 influenza A(H1N1)pdm09 isolates with respect to the A/California/07/2009 virus, four of which located in three different antigenic sites. Eleven rare mutations in 12 HA sequences were also detected. Phylogenetic analysis revealed that all characterized influenza A(H1N1)pdm09 isolates formed a single genetic cluster, belonging to the genetic subclade 6B.1, defined by HA amino acid substitutions S84N, S162N, and I216T. Furthermore, all isolates showed an oseltamivir-sensitive genotype, suggesting that Tamiflu (Oseltamivir) could still be the drug of choice in Turkey.

The bacteriome of otitis media with effusion: Does it originate from the adenoid?

OBJECTIVE: The aim of this study was to evaluate the composition and the diversity of bacteriome in middle ear effusion (MEE) and adenoid specimens of pediatric patients having otitis media with effusion (OME). MATERIALS AND METHODS: Sample collection from children with OME followed by next generation sequencing. Seventeen adenoid and 43 middle ear effusion specimens from 25 children having OME were evaluated. Microbiome analysis was performed via Ion 16S rRNA metagenomics kit. RESULTS: Twenty-two different bacterial species were identified from all of the samples analyzed. There were variations in the prevalence and relative abundance of the bacteriome observed between adenoid and MEE samples. MEE microbiome was significantly dominated by Alloicoccus otitis (44%), Turicella otitidis (6%), and Staphylococcus auricularis (3%). Whereas, Rothia mucilaginosa (39%), R. dentocariosa (11%), S. aureus (5%), Veillonella rogosae (2%), Granulicatella elegans (2%), Granulicatella adiacens (2%), Eikenella corrodens (1%), and Prevotella nanceiensis (1%) had significantly higher relative abundance in adenoid samples. Overall, there was no statistically significant difference in alpha diversity of MEE and adenoid samples, whereas adenoid samples constituted a cluster in the beta diversity graph. CONCLUSION: Bacteriome of MEE is mostly dominated by A. otitis yet accompanied by other bacteria with lower relative abundances suggests that OME is likely to be a polymicrobial process. Despite similarities, significant differences in relative abundances of several predominant species between bacteriome in the MEE and adenoid put the theory that OME in children is originated from the adenoids under question.