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 determination of the relationship between p97/VCP, small VCP-interacting protein, two ERAD proteins and steroidogenesis in Leydig cell lines.

BACKGROUND: In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3ß-HSD in human and mouse. METHODS AND RESULTS: HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3ß-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3ß-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells. CONCLUSION: We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3ß-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.

The effectiveness of enterprise stent use on the treatment of intracranial atherosclerosis disease.

OBJECTIVE: To examine the clinical outcome of Enterprise stent in patients with severe and symptomatic intracranial atherosclerosis. MATERIAL AND METHOD: Twenty-five patients who underwent Enterprise stenting between January 2012 and March 2019 were included in this study. Exclusion criteria were previous intracranial stenting and inadequate follow-up. Technical success rates of the procedures were recorded. Clinical outcome was evaluated with pre- and post-treatment modified Rankin Scale scores. The patients were monitored for 18 months clinically and for 14.3 months radiologically. RESULTS: The mean age of the 15 males and 10 females was 61.6 ± 8.19. Of these 25 patients, 6 (24%) were in the anterior system and 19 (76%) were in posterior system. The mean degree of pre-treatment stenosis was 86.4% ± 7 with the mean lesion length of 12.5 ± 7.5 mm. The residual stenosis rate was 23.8% ± 8.81. Technical success rate was 100%. There were two major complications within the first 30 days (8%). Late major complications (after 30 days) occurred in one case (4%). Stent restenosis was detected in two patients (8%). No intracranial bleeding or mortality was observed. CONCLUSION: In this single-center study, we achieved high technical success and tolerable complication rates. Enterprise stent may be a good treatment alternative for severe intracranial stenosis especially in patients resistant to medical treatment when correct patient selection is made. However, further randomized controlled studies, including more cases should be carried out.

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.

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.

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.

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.

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.

Analysis of the effect of smoking on the buccal microbiome using next-generation sequencing technology.

PURPOSE: This study aimed to investigate the effect of smoking on the buccal microbiome and to analyse the descriptive ability of each of the seven hypervariable regions in their 16S rRNA genes. METHODOLOGY: Microbiome compositions of 40 buccal swab samples collected from smokers (n =20) and non-smokers (n =20) were determined using 16S rRNA sequencing. Seven different 16S rRNA hypervariable regions (V2, V3, V4, V6-7, V8 and V9) in each sample were amplified using the Ion Torrent 16S Metagenomics kit and were sequenced on the Ion S5 instrument. RESULTS: Seven hypervariable regions in the 16S rRNA gene were successfully sequenced for all samples tested. The data obtained with the V2 region was found to be informative but the consensus data generated according to a number of operational taxonomic unit reads gathered from all seven hypervariable regions gave the most accurate result. At the phylum level, no statistically significant difference was found between smokers and non-smokers whereas relative abundances of Veillonella atypica, Streptococcus australis, Prevotella melaninogenica, Prevotella salivae and Rothia mucilaginosa showed significant increases in the smoker group (P-adj=0.05). Alpha diversity results did not show a significant difference between the two groups; however, beta diversity analysis indicated that samples of smoker and non-smoker groups had a tendency to be clustered within themselves. CONCLUSION: The results of the current study indicate that smoking is a factor influencing buccal microbiome composition. In addition, sequencing of all seven hypervariable regions yielded more accurate results than those with any of the single variable regions.