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Didier Raoult - One of the best experts on this subject based on the ideXlab platform.
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Koch Postulate: Why Should we Grow Bacteria?
Archives of Medical Research, 2017Co-Authors: Jean-christophe Lagier, Gregory Dubourg, Sophie Amrane, Didier RaoultAbstract:Understanding infectious diseases has long relied on the Koch postulate, which consists of the pure culture of Microorganisms. The advent of molecular methods in clinical microbiology has led the phasing out of culture as a diagnostic tool and metagenomics has become the technique most commonly used to assess the impact of commensal microbes on human health. However, culturing microbes has led to substantial advances, even recently, in infectious diseases involving Fastidious Microorganisms, as evidenced by the Tropheryma whipplei or Bartonella species. This allows their genomes to be sequenced and, consequently, new diagnostic tools to be acquired, experimental model to be created and antibiotic susceptibility testing to be performed. In addition, extensive culture focused on isolation of human commensals, know as the culturomics approach, has increased the number of bacteria isolated from humans by more than 35% over the past five years. As strains belonging to the same species can have different impacts on human health, it would appear necessary to pursue efforts to constitute an exhaustive bank of isolates in order to establish further proof of concepts. This review discusses recent examples, including the influence of Akkermansia muciniphila and Faecalibacterium prausnitzii, as well as the secretion of lugdunin by Staphylococcus lugdunensis which is efficient against the nasal carriage of Staphylococcus aureus. Finally, responses to some anti-cancer therapies and the treatment of Clostridium difficile infections through the use of fecal microbiota transplantation clearly suggest that culturing marks the beginning of bacteriotherapy.
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Cell Extract-Containing Medium for Culture of Intracellular Fastidious Bacteria
2016Co-Authors: Sudhir Singh, Malgorzata Kowalczewska, Sophie Edouard, Carole Eldin, Céline Perreal, Pascal Weber, Saïd Azza, Didier RaoultAbstract:The culture of Fastidious Microorganisms is a critical step in infectious disease studies. As a proof-of-concept experiment, we evaluated an empirical medium containing eukaryotic cell extracts for its ability to support the growth of Coxiella burnetii. Here, we demonstrate the exponential growth of several bacterial strains, including the C. burnetiiNine Mile phase I and phase II strains, and C. burnetii isolates from humans and animals. Low-oxygen-tension conditions and the presence of small hydro-philic molecules and short peptides were critical for facilitating growth. Moreover, bacterial antigenicity was conserved, reveal-ing the potential for this culture medium to be used in diagnostic tests and in the elaboration of vaccines against C. burnetii. We were also able to grow the majority of previously tested intracellular and Fastidious bacterial species, including Tropheryma whipplei,Mycobacterium bovis, Leptospira spp., Borrelia spp., and most putative bioterrorism agents. However, we were unable to culture Rickettsia africae and Legionella spp. in this medium. The versatility of this medium should encourage its use as a re-placement for the cell-based culture systems currently used for growing several facultative and putative intracellular bacterial species. Microbial culture constitutes the foundation of modern infec-tious disease study methodology. The isolation and culture of a disease-causing agent are a keystone of Koch’s postulates, and the isolation of Microorganisms in pure culture is crucial for di-agnosis in medical microbiology. Microbial culture facilitates th
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BLOOD CULTURE NEGATIVE ENDOCARDITIS
Infective Endocarditis, 2016Co-Authors: Pierre-edouard Fournier, George Watt, Paul N. Newton, Cristiane Lamas, Pierre Tattevin, Didier RaoultAbstract:Despite progress in blood culture media and automated growth detection systems, blood culture-negative endocarditis (BCNE) still represents 5–69.7 % of all endocarditis cases. Under the BCNE acronym are grouped diverse clinical entities: (i) the most common are cases of bacterial endocarditis caused by usual pathogens in which the negativity of blood cultures is explained by the early antibiotic treatment started prior to blood sampling; (ii) endocarditis caused by Fastidious Microorganisms that require prolonged incubation and/or specific media, including Brucella sp., defective streptococci (Abiotrophia sp., Gemella sp.), HACEK bacteria, Propionibacterium acnes, fungi; (iii) endocarditis caused by strictly (Coxiella burnetii, Tropheryma whipplei) or facultative (Bartonella sp.) intracellular bacteria; and (iv) endocarditis that complicate cancers (marantic endocarditis), auto-immune diseases (lupus, Behcet, rheumatoid arthritis) or allergy to pork. Various diagnostic strategies, combining systematic serological, molecular, biochemical and/or histological assays, have improved the diagnostic yield of BCNE but this condition remains challenging.
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Cell Extract-Containing Medium for Culture of Intracellular Fastidious Bacteria
Journal of clinical microbiology, 2013Co-Authors: Sudhir Singh, Malgorzata Kowalczewska, Sophie Edouard, Carole Eldin, Céline Perreal, Pascal Weber, Saïd Azza, Didier RaoultAbstract:ABSTRACT The culture of Fastidious Microorganisms is a critical step in infectious disease studies. As a proof-of-concept experiment, we evaluated an empirical medium containing eukaryotic cell extracts for its ability to support the growth of Coxiella burnetii. Here, we demonstrate the exponential growth of several bacterial strains, including the C. burnetii Nine Mile phase I and phase II strains, and C. burnetii isolates from humans and animals. Low-oxygen-tension conditions and the presence of small hydrophilic molecules and short peptides were critical for facilitating growth. Moreover, bacterial antigenicity was conserved, revealing the potential for this culture medium to be used in diagnostic tests and in the elaboration of vaccines against C. burnetii. We were also able to grow the majority of previously tested intracellular and Fastidious bacterial species, including Tropheryma whipplei, Mycobacterium bovis, Leptospira spp., Borrelia spp., and most putative bioterrorism agents. However, we were unable to culture Rickettsia africae and Legionella spp. in this medium. The versatility of this medium should encourage its use as a replacement for the cell-based culture systems currently used for growing several facultative and putative intracellular bacterial species.
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Use of Shell-Vial Cell Culture Assay for Isolation of Bacteria from Clinical Specimens: 13 Years of Experience
Journal of clinical microbiology, 2005Co-Authors: Frédérique Gouriet, Michel Drancourt, Florence Fenollar, Jean-yves Patrice, Didier RaoultAbstract:The spectrum of infectious diseases is wide and changing. The reliable diagnosis of infectious disease remains a difficult problem even for specialized laboratories. Isolation of new Microorganisms will allow the description of clinical features of new diseases and the characterization of new pathogens, enabling genetic descriptions, physiological analyses, improvement of diagnostic tools, and antibiotic susceptibility testing for bacteria. Therefore, the isolation of infecting bacteria is not only a mean of diagnosis but also a basis for the evaluation of much needed improved diagnostic assays and a route to enhance understanding of the diversity and the epidemiology of infections (21). The successful isolation of Fastidious Microorganisms is often based on cell culture (21). The success of this technique is based on two critical points. First, the ratio of Microorganisms to cells should be as high as possible (21). Second, centrifugation enhances the adhesion of intracellular Microorganisms to the cells (52). In 1976, a centrifugation-cell microculture system, called the shell-vial assay, was first used for the diagnosis of viral disease due to cytomegalovirus and allowed early antigen detection (22). This diagnostic tool is becoming one of the most commonly used techniques in clinical virology laboratories since it reduces turnaround time (19). For a long time, Rickettsia spp., which are strict intracellular gram-negative bacteria, were isolated using embryonated egg and/or animal inoculations (44). As the National Reference Center for Rickettsiosis in France, our laboratory started to adapt the shell-vial assay for bacteria culture in 1986 (32). This technique, due to the highly infectious nature of such bacteria, is routinely performed in a biosafety level 3 laboratory and has allowed us to isolate first Rickettsia sp. (32) and second Coxiella burnetii (45) from various specimens. For several years, our laboratory has developed the facilities and experience for cultivating Fastidious Microorganisms, using this versatile cell culture system. This has allowed us to receive a large panel of samples from France and abroad, and, notably, this approach has allowed the isolation and the establishment of the first strain of the bacillus of Whipple's disease, Tropheryma whipplei, from a cardiac valve (39). From September 1991 to August 2003, our laboratory attempted to routinely achieve isolation of Microorganisms from various clinical samples, arthropods, and animals by the shell-vial assay. Using this technique, we were able to isolate not only rickettsial species (28, 29, 32, 51) and Coxiella burnetii (45) but also Fastidious bacteria, such as Francisella tularensis (13), Legionella pneumophila (27), Brucella melitensis (48), Chlamydia trachomatis (35), and T. whipplei (39). In this study, we report our complete 13-year experience of shell-vial culture assay.
Buket Cicioglu Aridogan - One of the best experts on this subject based on the ideXlab platform.
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Comparison of the BACTEC blood culture system versus conventional methods for culture of normally sterile body fluids
Advances in Therapy, 2007Co-Authors: Emel Sesli Çetin, Selçuk Kaya, Mustafa Demirci, Buket Cicioglu AridoganAbstract:This study compared the BACTEC™ blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md) with conventional culture methods for recovery and time to detection of significant isolates from normally sterile body fluids. A total of 412 specimens were included in the study. Half of the specimens were inoculated directly into the automated blood culture system. The remaining specimens were centrifuged at 3000 rpm for 10 min and were inoculated onto conventional media. Clinically significant Microorganisms were isolated from 41 specimens (10%) by both culture systems; however, for 62 specimens (14.9%), growth was detected only with the BACTEC system. No isolates were detected with only conventional culture methods. A significant difference was noted between the blood culture system and routine culture methods for recovery of pathogenic Microorganisms that were from sterile body fluids. The most frequently isolated Microorganisms recovered only with the blood culture system were gram-positive cocci; gram-negative bacilli were the most frequently isolated Microorganisms that were recovered with both culture methods. Streptococcus pneumoniae, Streptococcus viridans , Aeromonas hydrophila , and Brucella were recovered only with the blood culture system. Furthermore, the mean time to detection of significant pathogens was significantly less with the blood culture system than with conventional media. The BACTEC blood culture system was found to improve the yield of clinically significant isolates from normally sterile body fluids with reduced time to detection; it may be advantageous for isolation of Fastidious Microorganisms, such as Brucella and S pneumoniae , especially from cerebrospinal and synovial fluid specimens.
Mi-kyung Lee - One of the best experts on this subject based on the ideXlab platform.
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Vaginal Candida and Microorganisms Related to Sexual Transmitted Diseases in Women with Symptoms of Vaginitis
Korean Journal of Clinical Microbiology, 2012Co-Authors: Kye Hyun Kim, Mi-kyung LeeAbstract:Background: The female genital tract is equipped to deal with a variety of foreign substances including a wide array of Microorganisms. It is important to consider Candida-bacterial interactions in balance between healthy colonization versus vaginitis. The objectives of this study were to evaluate the association between microorganism distribution and vaginitis, and to investigate the possibility of an interaction between vaginal Candida and other Microorganisms in female genital tract. Methods: A total of 516 vaginal secretions were collected between October 2008 and June 2010 from patients with suspected vaginitis. Identification of Candida species and detection of 6 Fastidious Microorganisms (Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasma urealyticum) were performed using a VITEK 2 system (bioMerieux, Inc., Hazelwood, MO, USA) and multiplex PCR (Seegene, Biotechnology, Inc., Seoul, Korea), respectively. Results: M. genitalium, U. urealyticum, and C. trachomatis were more often detected in association with vaginal candidiasis. A statistically significant association between Candida and M. genitalium was observed (P
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Clinical Implications of Multiplex PCR Detection of Fastidious Microorganisms in Vaginitis Patients.
Korean Journal of Clinical Microbiology, 2011Co-Authors: Mi-kyung LeeAbstract:Background: Bacterial vaginitis (BV) and Trichomonas vaginitis are the most frequently recurring infectious diseases in women. Therefore, accurate tests for post-treatment follow-up are required. A multiplex PCR assay allows for the simultaneous detection of multiple pathogens in a single specimen. In this study, we assessed the clinical implications of multiplex PCR detection of Fastidious Microorganisms causing vaginitis. Methods: A total of 216 vaginitis patients who presented to Chung-Ang University Yongsan Hospital with more than one positive result on multiplex PCR (Trichomonas vaginalis (TV), Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Mycoplasma genitalium (MG), Mycoplasma hominis (MH)) were retrospectively enrolled in this study. Each patient’s clinical symptoms, initial treatment and follow-up for BV, and other related test results were also retrospectively reviewed. Results: The most commonly reported symptom was abnormal discharge, followed by pruritis (73.1%), lower abdominal pain (38.4%), urination difficulties (13%), and others such as fever. According to the multiplex PCR results, there were 116 cases (35.8%) of MH, 86 cases (26.5%) of UU, 62 cases (19.1%) of CT, and 84 cases (38.9%) were mixed infections. Among those patients with single infections, treatment changed for 63 cases (65.6%) while treatment remained unchanged for 17 (17.7%) after PCR results were reported. Conclusion: The diagnosis of BV using multiplex PCR is clinically effective and the results of which can be incorporated in antibiotic selection for patients with multiple sexually transmitted diseases (STD). Multiplex PCR may be especially helpful in the diagnosis of patients in whom the differentiation of STD pathogens is difficult using traditional methods. (Korean J Clin Microbiol 2011;14:30-35)
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Detection of Cryptic Microorganisms in Patients with Chronic Prostatitis by Multiplex Polymerase Chain Reaction
Korean Journal of Urology, 2007Co-Authors: Tae-hyoung Kim, Mi-kyung Lee, Tae Heung Kim, Hye Ryoun Kim, Soon Chul Myung, Young Sun KimAbstract:Purpose: Chronic prostatitis frequently occurs in men of all ages. Recent studies suggest that Fastidious Microorganisms may play a role in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). The aim of this study was to evaluate the usefulness and significance of multiplex polymerase chain reaction (PCR) in the diagnosis of CP/CPPS. Materials and Methods: First voided urine (FVU) and/or expressed prostatic secretions (EPS) were collected from 92 patients. Multiplex PCR, using Dual Specificity Oligo (DSO TM ) primers, was used to test for Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Ureaplasma urealyticum (UU). Results: Multiplex PCR can be easily analyzed via visual comparison. Nine (39.1%) of the 23 CP/CPPS IIIa and 12 (17.4%) of the 69 IIIb patients had positive multiplex PCR, with a total of 27 Microorganisms isolated, including CT, MH, MG, UU, TV and NG in 9, 7, 4, 4, 2 and 1 case, respectively. Co-infections with 2 or 3 organisms occurred in 5 cases. For the samples collected from 32 patients for both FVU and EPS, 68.7% gave the same results. Conclusions: Multiplex PCR, using DSO TM primers, can be useful for the simple detection of Fastidious Microorganisms in CP/CPPS. To achieve reliable results with multiplex PCR, feasible guidelines and standardization are of major importance. Further studies will be required to define the usefulness of molecular tests for CP/CPPS in clinical practice. (Korean J Urol 2007;48:304-309)
Canan Demir - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of the Bactec microbial detection system for culturing miscellaneous sterile body fluids.
Research in microbiology, 2005Co-Authors: Fusun Zeynep Akcam, Guler Yayli, Ersin Uskun, Onur Kaya, Canan DemirAbstract:This study sought to evaluate the efficacy of the Bactec blood culture system for culturing sterile body fluids, in comparison with traditional culture methods. A total of 906 specimens were cultured using both conventional media and the Bactec detection system. Differences in numbers of isolates determined by each system were compared. Clinically significant Microorganisms were isolated from 15.3% (139) of 906 specimens by both the Bactec system and conventional culture, whereas for 8.8% (80) of the specimens a positive culture was obtained by Bactec only. Of the 80 specimens in which growth was detected only in the Bactec blood culture system, 11.0% were comprised of cerebrospinal fluid, 9.9% peritoneal fluid, 4.9% pleural fluid, 4.5% synovial fluid and 9.1% bone marrow materials. Strains of Brucella melitensis, Neisseria meningitidis, Pseudomonas fluorescens and Rothia dentocariosa were cultured only by the Bactec system. In conclusion, the Bactec blood culture system might be advantageous for isolation of Fastidious Microorganisms such as Brucella, especially from cerebrospinal and peritoneal fluid specimens.
Emel Sesli Çetin - One of the best experts on this subject based on the ideXlab platform.
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Comparison of the BACTEC blood culture system versus conventional methods for culture of normally sterile body fluids
Advances in Therapy, 2007Co-Authors: Emel Sesli Çetin, Selçuk Kaya, Mustafa Demirci, Buket Cicioglu AridoganAbstract:This study compared the BACTEC™ blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md) with conventional culture methods for recovery and time to detection of significant isolates from normally sterile body fluids. A total of 412 specimens were included in the study. Half of the specimens were inoculated directly into the automated blood culture system. The remaining specimens were centrifuged at 3000 rpm for 10 min and were inoculated onto conventional media. Clinically significant Microorganisms were isolated from 41 specimens (10%) by both culture systems; however, for 62 specimens (14.9%), growth was detected only with the BACTEC system. No isolates were detected with only conventional culture methods. A significant difference was noted between the blood culture system and routine culture methods for recovery of pathogenic Microorganisms that were from sterile body fluids. The most frequently isolated Microorganisms recovered only with the blood culture system were gram-positive cocci; gram-negative bacilli were the most frequently isolated Microorganisms that were recovered with both culture methods. Streptococcus pneumoniae, Streptococcus viridans , Aeromonas hydrophila , and Brucella were recovered only with the blood culture system. Furthermore, the mean time to detection of significant pathogens was significantly less with the blood culture system than with conventional media. The BACTEC blood culture system was found to improve the yield of clinically significant isolates from normally sterile body fluids with reduced time to detection; it may be advantageous for isolation of Fastidious Microorganisms, such as Brucella and S pneumoniae , especially from cerebrospinal and synovial fluid specimens.