Clinical Microbiological Research Research Articles

Comparison of the diversity of anaerobic-cultured gut bacterial communities on different culture media using 16S rDNA sequencing

The gut microbiome is a dense and diverse community of different microorganisms that deeply influence human physiology and that have important interactions with pathogens. For the correct antibiotic treatment of infections, with its twin goals of effective inhibition of the pathogen and limitation of collateral damage to the microbiome, the identification of infectious organisms is key. Microbiological culturing is still the mainstay of pathogen identification, and anaerobic species are among the most demanding bacterial communities to culture. This study aimed to evaluate the impact of growth media on the culture of an-aerobic bacteria from human stool samples. Stool samples from eight human subjects were cultured each on a yeast extract cysteine blood agar (HCB) and a modified peptone-yeast extract-glucose (MPYG) plate and subjected to Illumina NGS analysis after DNA extraction and amplification. The results showed tight clustering of sequencing samples belonging to the same human subject. Various differences in bacterial richness and evenness could be observed between the two media, with HCB plates supporting the growth of a more diverse microbial community, and MPYG plates improving the growth rates of certain taxa. No statistical significance was observed between the groups. This study highlights the importance of choosing the appropriate growth media for anaerobic bacterial culture and adjusting culture conditions to target specific pathological conditions. HCB plates are suitable for standard microbiological diagnostics, while MPYG plates may be more appropriate for targeting specific conditions. This work emphasizes the role of next-generation sequencing in supporting future research in clinical microbiology.

P-333 Combining deep shotgun metagenomic sequencing and culturomics for in-depth characterization of a potential endometrial microbiome

Abstract Study question Does the endometrium host a microbiome, and if so, are these taxa distinct from the vaginal/cervical microbiome? Summary answer Multifaceted analysis of endometrial tissue reveals no distinct microbial taxa compared to the vaginal/cervical samples, questioning whether detected microbiota are endometrium-related or result from cross-contamination. What is known already Recent studies suggest a role for the endometrial microbiota in reproductive health, yet the existence of an endometrial microbiome and its specific bacterial taxa remain enigmatic. The female reproductive tract, with a bacterial abundance gradient, is predominantly Lactobacillus-dominated in the lower region. Inconsistent findings on Lactobacillus dominance in endometrial samples are often ascribed to cross-contamination and methodological constraints, highlighting the need for precise methodologies in studying the low-biomass endometrial environment. Study design, size, duration Fifteen women, undergoing a diagnostic hysteroscopy and endometrial biopsy, as part of the subfertility work-up, were included in this study. Microbiota profiles of endometrial biopsies were compared to paired vaginal and cervical swabs from nine patients using 16S amplicon sequencing. Furthermore, the microbial composition of six additional endometrium biopsies was characterized using both a culturomics-based approach and deep metagenomic sequencing. Participants/materials, setting, methods Endometrial biopsies (n = 15) were taken with a sterile Pipelle de Cornier and snap-frozen in liquid nitrogen, followed by -80 °C storage and 16S amplicon sequencing (n = 9) targeting the V4-region or deep shotgun metagenomic sequencing (n = 6) (Prebiomics, Italy). Simultaneously, in nine women, paired cervical and vaginal swabs (Copan Diagnostics, Italy) were taken and preserved in eNAT medium. In six participants, an additional endometrial biopsy was collected and stored in Amies medium for immediate culturomics. Main results and the role of chance We observed a high similarity in the microbial composition of endometrial biopsies, cervical, and vaginal samples using 16 amplicon sequencing, but also some taxa were only found in the endometrial samples. Notably, the presence of Gemmobacter, a waterborne genus, was only found in endometrial biopsies and could be a source of contamination from water used during hysteroscopy to flush the uterus. Most endometrial microbiome profiles were Lactobacillus dominated, with exceptions observed in three patients. No significant differences for alpha diversity measures (richness and evenness) of the microbiota composition were observed between the different niches. For the first time we applied both culturomics and deep shotgun metagenomic sequencing for the characterization of the microbial composition in the same endometrial biopsies. Commonly identified genera using both techniques include Lactobacillus, Prevotella, Bifidobacterium, Anaerococcus, and Cuticubacterium. Remarkably, at the species level, the two approaches revealed almost no overlapping species within each biopsy, underscoring the importance of combining multiple methodologies in examining low-biomass niches like the endometrium. Limitations, reasons for caution A limitation of this study is the low-biomass nature of endometrial biopsies which are prone to cross-contamination with the lower female reproductive tract. This challenges downstream analysis, as it becomes difficult to distinguish between a relevant microbiological signal and contaminating noise. Wider implications of the findings This study emphasizes the need to consider a multifaceted microbiome approach when exploring low-biomass environments, like the endometrium. The critical consideration of potential cross-contamination by the lower female reproductive tract and its implications for downstream analysis is paramount in clinical microbiology research and demands meticulous handling. Trial registration number not applicable

Prevalence of Hepatitis C Infection and its Genotypes in Suspected Hemodialysis Patients, Southwest of Iran

Background: Hemodialysis patients are more prone to Hepatitis C Virus (HCV) infection due to the need for long-term hemodialysis and blood transfusions. Objectives: The present study aimed to determine the HCV infection burden, viral load, and genotype pattern in hemodialysis patients referred to a research center from 2011 to 2018. Methods: Among 131 hemodialysis patients with suspected HCV infection, referred to Prof. Alborzi Clinical Microbiology Research Center, Shiraz, Iran, from 2011 to 2018, the HCV rate was assessed with the enzyme-linked immunosorbent assay and the HCV RNA load and genotypes by one-step TaqMan real-time PCR. Results: The prevalence of HCV-Ab positivity was 29% among hemodialysis patients, of whom 21 (57%) were HCV RNA-positive. In the rest of the hemodialysis patients who were HCV-Ab-negative, the HCV RNA was detected in five (12%) patients. Genotype 3 (Gt-3) was the most prevalent one detected in 50% of the patients whose genotypes were determined. Also, the HCV viral load in HCV-seropositive patients was generally higher than that in HCV-seronegative ones. Conclusions: This study showed that high HCV infection and different genotype patterns among hemodialysis patients compared to the general population are the main predictors of HCV infection, which indicates healthcare facility transmission because of inappropriate infection management practices.

Introducing clinical nanorachaeaology: Isolation by co-culture of Nanopusillus massiliensis sp. nov.

Nanoarchaeota, obligate symbiont of some environmental archaea with reduced genomes, have been described in marine thermal vent environments, yet never detected in hosts, including humans. Here, using laboratory tools geared towards the detection of nanoarchaea including PCR-sequencing, WGS, microscopy and culture. We discovered a novel nanoarchaea, Nanopusillus massiliensis, detected in dental plate samples by specific PCR-based assays. Combining fluorescent in situ hybridization (FISH) with scanning electron microscopy disclosed close contacts between N. massiliensis and the archaea Methanobrevibacter oralis in these samples. Culturing one sample yielded co-isolation of M. oralis and N. massiliensis with a 606,935-bp genome, with 23.6% GC encoded 16 tRNA, 3 rRNA and 942 coding DNA sequences, of which 400 were assigned to clusters of orthologous groups. The discovery of N. massiliensis, made publicly available in collection, extended our knowledge of human microbiota diversity, opening a new field of research in clinical microbiology here referred to as clinical nanoarchaeology.

Evaluation of the approaches and knowledge level of tuberculosis diagnosis and treatment of infectious disease research assistants and specialist physicians

This study aims to evaluate approaches and knowledge level for tuberculosis (TB) diagnosis and treatment among infectious diseases and clinical microbiology research assistants and specialist physicians. This was a descriptive study on the research assistants and specialist physicians. A questionnaire consisting of 24 questions prepared by the researchers was used. Data were analyzed using SPSS, version 22.0. In this study, 116 physicians participated. The average age of participants was 41.07 ± 8.65 years, and 64.7% were female. The proportion of physicians with no pulmonary and non-pulmonary TB experience was calculated as 6.9% and 3.4%, respectively. Acid-fast-bacilli (AFB) tests were most frequently used in the diagnosis of pulmonary TB. On the other hand, the tuberculin skin test (TST) + chest radiography (CR) was most frequently used for latent TB in immunosuppressed subjects (91.4% and 69%, respectively). The most common non-pulmonary TB clinic form encountered was lymphadenitis (88.8%); the most common treatment problem was drug side effects (75%); and the most common drug resistance was to isoniazid (25.9%). It was determined that physicians encountered HIV and TB coinfection in 62.9% of patients, and standard TB treatment was applied for most patients (48.3%). The most frequent yearly TST + CR + AFB trials (69%) were performed in the screenings of health workers who were in contact with TB patients. It was observed that 44% of physicians used a negative pressurized chamber and/or isolation chamber in the institution, and 72.4% used a N95/FFP3 mask during examination. It was determined that the participants were mostly correct about TB. Infectious diseases and clinical microbiology physicians continue to encounter TB patients. This group of physicians often has a good level of knowledge and experience with TB, although special training for this disease is low.

Comparison of HCV viral load and its genotype distributions in HCV mono- and HIV/HCV co-infected illicit drug users

BackgroundBecause of shared modes of transmission, patients with hepatitis C virus (HCV) infection are often co-infected with other types of hepatitis viruses and/or HIV. We studied HCV viral load and its genotype patterns among HCV mono- and HCV/HIV co-infected Illicit Drug Users in Fars province-Iran.MethodsTotally, 580 HCV seropositive IDUs referred to Prof. Alborzi Clinical Microbiology Research Center, Shiraz, Iran, without receiving any anti-HCV treatment, were enrolled. After their HCV infections were reconfirmed by one step rapid diagnostic test, HCV RNA level and HCV genotypes were determined by Taq-man real-time PCR assays. Their HIV serostatus was determined and seropositive patients were excluded from the group.In addition, 104 HIV/HCV co-infected IDUs referred from Shiraz Behavioral Diseases Consultation Center (SBDC) were assessed for HCV RNA level and HCV genotype patterns, as well.ResultsThe overall estimated HIV prevalence was 6.7% (39/580) among HCV seropositive IDUs. Genotype 1, the most prevalent genotype in both groups, was detected in 69% and 49% of co- and mono-infected IDUs, respectively. Median HCV viral load was significantly higher in HIV/HCV co-infected patients, compared with that among HCV mono-infected counterparts.ConclusionsGiven the higher baseline HCV viral load and GT1 attributed to poorer treatments response, HCV treatment must be more considered among HCV/HIV co-infected IDUs, compared to those mono-infected with HCV.

Report of China’s research growth and development in clinical microbiology

Background: To investigate the academic status of clinical microbiology in China through survey of articles in international clinical microbiology journals and compare the outputs of clinical microbiology research of three major regions in China respectively—Mainland (ML), Taiwan (TW), and Hong Kong (HK). Methods: All articles of clinical microbiology were selected from “infectious disease”, “microbiology” and “virology” categories from Journal Citation Reports (JCR) in Web of Science. Articles from the ML, TW and HK were retrieved from PubMed. The number of total articles, clinical trials, case reports, impact factors (IF) and articles published in each journal were conducted for analyzing. Results: A total of 13,109 articles from 52 clinical microbiology journals were written by Chinese authors, 8,151 of which are from ML, 3,213 from TW, and 1,745 from HK. And the number of clinical trials and case reports in China is 1,272. Accumulated IF of articles from ML (31,061.1) was much higher than that of TW (11,934.48) and HK (7,301.624), while the average IF of articles from HK is the highest. Conclusions: The total number of articles from China especially ML increased significantly and steadily in the last decade whereas the amount of articles in HK and TW had been level off. The number of articles published from the ML is dominant through three regions in China, while the quality of articles is best in HK.

Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry: A New Guide to Infectious Disease

Pediatric Infections Research Center, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran Corresponding author: Ali Amanati, Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran. Fax: +98-7136325655, E-mail: ali_amanati_1356@yahoo.com

Susceptibility pattern of Candida albicans isolated from Iranian patients to antifungal agents.

Background and Purpose:Candidiasis is a major fungal infection, and Candida albicans is the major cause of infections in humans. The Clinical and Laboratory Standards Institute (CLSI) developed new breakpoints for antifungal agents against C. albicans. In this multi-center study, we aimed to determine the drug susceptibility profile of C. albicans, isolated from Iranian population according to new species-specific CLSI.Materials and Methods:Clinical samples were cultured on Sabouraud dextrose agar and were incubated at room temperature for seven days. The isolates were transferred to Professor Alborzi Clinical Microbiology Research Center, Shiraz, Iran. C. albicans were identified by using API 20C AUX system. Broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of amphotericin B, caspofungin, voriconazole, fluconazole, posaconazole, itraconazole, and ketoconazole, based on CLSI document M27-S4 and new breakpoints for some azoles and caspofungin.Results:Overall, 397 C. albicans were isolated from patients admitted to ten university hospitals in Iran. The MIC90 of the isolates to amphotericin B, caspofungin, voriconazole, fluconazole, posaconazole, itraconazole, and ketoconazole were 0.125, 0.125, 0.125, 1, 0.064, 0.5, and 0.125 µg/ml, and rates of resistance were 0.5%, 0.3%, 3.8%, 2.8%, and 2.5% for amphotericin B, caspofungin, voriconazole, fluconazole, and itraconazole, respectively.Conclusion:According to our data, fluconazole is the drug of choice for management of patients at risk for systemic candidiasis throughout the region, since it is cost-effective with low side effects.

Do you kiss your mother with that mouth? An authentic large-scale undergraduate research experience in mapping the human oral microbiome.

Clinical microbiology testing is crucial for the diagnosis and treatment of community and hospital-acquired infections. Laboratory scientists need to utilize technical and problem-solving skills to select from a wide array of microbial identification techniques. The inquiry-driven laboratory training required to prepare microbiology graduates for this professional environment can be difficult to replicate within undergraduate curricula, especially in courses that accommodate large student cohorts. We aimed to improve undergraduate scientific training by engaging hundreds of introductory microbiology students in an Authentic Large-Scale Undergraduate Research Experience (ALURE). The ALURE aimed to characterize the microorganisms that reside in the healthy human oral cavity—the oral microbiome—by analyzing hundreds of samples obtained from student volunteers within the course. Students were able to choose from selective and differential culture media, Gram-staining, microscopy, as well as polymerase chain reaction (PCR) and 16S rRNA gene sequencing techniques, in order to collect, analyze, and interpret novel data to determine the collective oral microbiome of the student cohort. Pre- and postsurvey analysis of student learning gains across two iterations of the course (2012–2013) revealed significantly higher student confidence in laboratory skills following the completion of the ALURE (p < 0.05 using the Mann-Whitney U-test). Learning objectives on effective scientific communication were also met through effective student performance in laboratory reports describing the research outcomes of the project. The integration of undergraduate research in clinical microbiology has the capacity to deliver authentic research experiences and improve scientific training for large cohorts of undergraduate students.

Phylogenetic Analysis of Torque Teno Virus in Hepatitis C Virus Infected Patients in Shiraz

Torque teno virus (TTV) was the first human Circoviridae detected in a Japanese patient with unknown hepatitis in 1997. Subsequently, several studies performed to evaluate different aspects of Torque teno virus pathogenesis. The present study aimed to determine dominant genotype of Torque teno virus in chronic hepatitis disease using 5΄-UTR sequence among patients infected by hepatitis C virus in Shiraz - Iran. The study conducted in 240 patients with chronic hepatitis C from Prof. Alborzi Clinical Microbiology Research Center. The presence of Torque teno virus DNA and its genotype in plasma was assessed by nested polymerase chain reaction using two primer sets for 5΄-UTR and N22 regions. Phylogenetic analysis was performed based on 5΄-UTR region. DNA of Torque teno virus was detected in 220 out of 240 (92 %) patients with chronic hepatitis C by the use of 5΄-UTR primer based PCR method and in 12 out of 240 (5%) by the use of N22 primer. Based on phylogenetic analysis it was shown that the Dominant genotype in this study was 11. Genotypes 1, 3, 17, and 22 were also detected. Some sequences could not be classified to a specific genotype. The prevalence of Torque teno virus DNA in patients with chronic hepatitis C disease by the use of 5΄-UTR primer appeared to be higher compared to that revealed by N22 primer. We observed five genotypes among hepatitis C chronic patients in our study.

What Happened to Research in Clinical Microbiology in the United States?

The title of this article is loaded with negative innuendo—the question is not “What is the state of research in clinical microbiology in the United States” but, rather, “What has happened to happened to research in clinical microbiology in the United States.” We begin with the premise