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Didier Raoult - One of the best experts on this subject based on the ideXlab platform.
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fluorescence in situ hybridization a complementary molecular tool for the clinical diagnosis of infectious diseases by intracellular and Fastidious Bacteria
Fems Microbiology Reviews, 2019Co-Authors: Elsa Prudent, Didier RaoultAbstract:Many obligate or facultative intracellular Bacteria pose a critical problem in clinical microbiology diagnosis as a result of their Fastidious growth or lack of growth in conventional culture media. Molecular diagnosis is based on the analysis and demonstration of nucleic acids (DNA and RNA). In the field of infectiology, it combines laboratory medicine with the technology of molecular genetics to identify infectious pathogens. Fluorescence in situ hybridization (FISH) is used for the detection and localization of nucleotide sequences in various samples while preserving cell integrity. For more than 30 years, FISH methods have in constant evolution with the development of rRNA-targeted probes and synthetic molecules, such as PNA, which have contributed to the development of this technique in various fields by research and diagnostic laboratories. We describe here a panel of infectious diseases due to intracellular Bacteria for which FISH diagnosis has proven its effectiveness. FISH techniques were applied in cases of blood-culture-negative endocarditis, respiratory infections, gastrointestinal diseases, mycoBacterial infections, highly pathogenic microorganisms and other Fastidious Bacteria such as spirochetes. FISH has been proven to be applicable to various samples and for diverse infectious diseases, it can be used as a complementary tool for the diagnosis of infectious diseases by intracellular and Fastidious Bacteria.
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Cell Extract-Containing Medium for Culture of Intracellular Fastidious Bacteria
2016Co-Authors: Sudhir Singh, Sophie Edouard, Malgorzata Kowalczewska, 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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Acquired Resistance to Trimethoprim- Sulfamethoxazole during Whipple Disease and Expression of the Causative Target Gene
2016Co-Authors: Nawal Bakkali, Florence Fenollar, Silpak Biswas, Jeanmarc Rolain, Didier RaoultAbstract:Background. Whipple disease is a chronic infection caused by Tropheryma whipplei. Trimethoprim-sulfamethoxazole is recommended for treatment of Whipple disease but is associated with treatment failure. T. whipplei is resistant in vitro to trimethoprim, because the gene targeted by this agent is missing. Methods. Apatient experienced clinical failure during treatment with trimethoprim-sulfamethoxazole. The gene encoding the enzyme putatively believed to be dihydropteroate synthase (DHPS), the target of sulfamethoxazole, was amplified and sequenced for 20 T. whipplei strains from our laboratory and for isolates recovered from a case patient at the time of diagnosis and the time of treatment failure. An Escherichia coli knockout strain for this gene was complemented with the sequences from a susceptible strain and from isolates recovered from the case patient. Susceptibilities of complemented E. coli to sulfamethoxazole were tested. Results. The target gene was identified among genes encoding a unique trifunctional enzyme in which DHPS is combined with the 2 preceding enzymes of the folate biosynthesis pathway. Changes in the amino acid sequence of putative DHPS were detected in the case patient. Gene complementation showed that the gene encoding putative DHPS restored the folate biosynthesis pathway and susceptibility to sulfamethoxazole, whereas themutated sequence was associated with sulfamethoxazole resistance. Conclusions. Antibiotic susceptibility of Fastidious Bacteria such asT. whipplei can be evaluated bymeans of gen
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Cell Extract-Containing Medium for Culture of Intracellular Fastidious Bacteria
Journal of clinical microbiology, 2013Co-Authors: Sudhir Singh, Sophie Edouard, Malgorzata Kowalczewska, 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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Uveitis caused by Fastidious Bacteria
Acta Ophthalmologica, 2009Co-Authors: Bahram Bodaghi, Michel Drancourt, Didier Raoult, Nathalie Cassoux, Phuc LehoangAbstract:Purpose The etiologic evaluation of uveitis is frequently unsuccessful if only noninvasive methods are used. We conducted a prospective study to evaluate systematic screening for pathogens of uveitis. Methods All patients with uveitis referred to the participating tertiary ophthalmology departments from January 2001 to September 2007 underwent intraocular and serum specimen collection. The standardized protocol for laboratory investigations included universal polymerase chain reaction (PCR)-based detection of any Bacteria and mycoses, specific PCR-based detection of Fastidious (difficult-to-grow) Bacteria and herpes viruses, and culture of vitreous fluid. Sera were tested for Fastidious Bacteria. Results Among the 1321 included patients, infection was diagnosed in 147 (11.1%) patients: 78 (53%) were caused by Fastidious Bacteria that included spirochetes, Bartonella species, intracellular Bacteria (Chlamydia species, Rickettsia species, Coxiella burnetii), and Tropheryma whipplei; 18 by herpes viruses; and 9 by fungi. Bartonella quintana, Coxiella burnetii, Paracoccus yeei, Aspergillus oryzae, and Cryptococcus albidus were found to be associated with uveitis for the first time, to our knowledge. Conclusion We recommend applying a 1-step diagnostic procedure that incorporates intraocular, specific microbial PCR with serum analyses in tertiary centers to determine the etiology of uveitis.
David N. Fredricks - One of the best experts on this subject based on the ideXlab platform.
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effects of Bacterial vaginosis associated Bacteria and sexual intercourse on vaginal colonization with the probiotic lactobacillus crispatus ctv 05
Sexually Transmitted Diseases, 2011Co-Authors: Benjamin Ngugi, David N. Fredricks, Anke Hemmerling, Elizabeth A Bukusi, Gideon Kikuvi, Joseph Gikunju, Stephen Shiboski, Craig R CohenAbstract:Objective Several Fastidious Bacteria have been associated with Bacterial vaginosis (BV), but their role in lactobacilli recolonization failure is unknown. We studied the effect of seven BV-associated Bacterial species and two Lactobacillus species on vaginal colonization with L. crispatus CTV-05 (LACTIN-V).
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temporal variability of human vaginal Bacteria and relationship with Bacterial vaginosis
PLOS ONE, 2010Co-Authors: Sujatha Srinivasan, Tina L. Fiedler, David N. Fredricks, Katherine K Thomas, Caroline Mitchell, Jeanne M Marrazzo, Congzhou Liu, Kathy AgnewAbstract:Background: Little is known about short-term Bacterial fluctuations in the human vagina. This study used PCR to assess the variability in concentrations of key vaginal Bacteria in healthy women and the immediate response to antibiotic treatment in women with Bacterial vaginosis (BV). Methodology/Principal Findings: Twenty-two women assessed for BV using Amsel’s criteria were evaluated daily for 7 or 14 days, then at 2, 3 and 4 weeks, using a panel of 11 bacterium-specific quantitative PCR assays. Participants with BV were treated with 5 days of intravaginal metronidazole. Participants without BV had vaginal biotas dominated by lactobacilli, whose levels fluctuated with menses. With onset of menstruation, quantities of Lactobacillus jensenii and Lactobacillus crispatus decreased and were found to be inversely related to Gardnerella vaginalis concentrations (p,0.001). Women with BV had a variety of Fastidious Bacteria whose concentrations dropped below detection thresholds 1–5 days after starting metronidazole. Recurrent BV was characterized by initial profound decreases of BV-associated Bacteria after treatment followed by subsequent increases at relapse. Conclusions/Significance: The microbiota of the human vagina can be highly dynamic. Healthy women are colonized with Lactobacillus species, but levels can change dramatically over a month. Marked increases in G. vaginalis were observed during menses. Participants with BV have diverse communities of Fastidious Bacteria that are depleted by vaginal metronidazole therapy. Women with recurrent BV initially respond to antibiotic treatment with steep declines in Bacterial concentrations, but these Bacteria later reemerge, suggesting that antibiotic resistance in these Bacteria is not an important factor mediating BV recurrence.
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detection of Fastidious vaginal Bacteria in women with hiv infection and Bacterial vaginosis
Infectious Diseases in Obstetrics & Gynecology, 2009Co-Authors: Caroline Mitchell, David N. Fredricks, Carla C Moreira, Kathleen Paul, Angela M Caliendo, Jaclynn Kurpewski, Jessica Ingersoll, Susan CuuvinAbstract:Background. Fastidious Bacteria have been associated with Bacterial vaginosis (BV) using PCR methods. We assessed the prevalence of these Bacteria in HIV-1 infected women and their relationship with vaginal pH and shedding of HIV-1 RNA. Methods. 64 cervicovaginal lavage (CVL) samples were collected from 51 women. Vaginal microbiota were characterized using 8 bacterium-specific quantitative PCR assays. Results. Women with the Fastidious Bacteria Bacterial Vaginosis Associated Bacterium (BVAB) 1, 2, and 3 showed a trend to increased HIV-1 shedding (OR 2.59–3.07, P = .14–.17). Absence of Lactobacillus crispatus (P < .005) and presence of BVAB2 (P < .001) were associated with elevated vaginal pH. BVAB1, 2, and 3 were highly specific indicators of BV in HIV-infected women, with specificities of 89%–93%. Conclusions. Fastidious Bacteria (BVAB 1, 2, and 3) remain specific indicators of BV in HIV-infected women, and BVAB2 may contribute to the elevated vaginal pH that is a hallmark of this syndrome.
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comparison of oral and vaginal metronidazole for treatment of Bacterial vaginosis in pregnancy impact on Fastidious Bacteria
BMC Infectious Diseases, 2009Co-Authors: Caroline Mitchell, Jane Hitti, Kathy J Agnew, David N. FredricksAbstract:Bacterial vaginosis (BV) is a common condition that is associated with preterm birth and acquisition of complex communities of vaginal Bacteria that include several Fastidious species. Treatment of BV in pregnancy has mixed effects on the risk of preterm delivery, which some hypothesize is due to variable antibiotic efficacy for the Fastidious Bacteria. Both oral and intravaginal metronidazole can be used to treat Bacterial vaginosis in pregnancy, but little is known about the impact of different routes of antibiotic administration on concentrations of Fastidious vaginal Bacteria. This was a sub-study of a larger randomized trial of oral versus vaginal metronidazole for treatment of BV in pregnancy. Fifty-three women were evaluated, including 30 women who received oral metronidazole and 23 who received intravaginal metronidazole. Bacterial taxon-specific quantitative PCR assays were used to measure concentrations of Bacterial vaginosis associated bacterium (BVAB) 1, 2, and 3, Gardnerella vaginalis, Atopobium species, Leptotrichia/Sneathia species, Megasphaera species, and Lactobacillus crispatus before and after antibiotic treatment. Concentrations of Leptotrichia and Sneathia spp. and the Fastidious Clostridia-like bacterium designated BVAB1 decreased significantly with oral (p = .002, p = .02) but not vaginal therapy (p = .141, p = .126). The Fastidious bacterium BVAB3 did not significantly decrease with either treatment. Concentrations of Atopobium spp., reportedly resistant to metronidazole in vitro, dropped significantly with oral (p = .002) and vaginal (p = .001) treatment. There was no significant difference in the magnitude of change in Bacterial concentrations between oral and vaginal treatment arms for any of the Bacterial species. Lactobacillus crispatus concentrations did not change. Both oral and vaginal metronidazole therapy in pregnant women result in a significant decrease in concentrations of most BV-associated anaerobic Bacteria, with the exception that Leptotrichia, Sneathia and BVAB1 do not significantly decrease with vaginal metronidazole therapy. These data suggest that the route of antibiotic administration has a minor impact on Bacterial eradication in pregnant women with BV. This trial is registered with ClinicalTrials.gov, number NCT00153517
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changes in vaginal Bacterial concentrations with intravaginal metronidazole therapy for Bacterial vaginosis as assessed by quantitative pcr
Journal of Clinical Microbiology, 2009Co-Authors: Tina L. Fiedler, David N. Fredricks, Katherine K Thomas, Caroline Mitchell, Jeanne M MarrazzoAbstract:Several Fastidious Bacteria have been associated with Bacterial vaginosis (BV) using broad-range Bacterial PCR methods such as consensus sequence 16S rRNA gene PCR, but their role in BV remains poorly defined. We describe changes in vaginal Bacterial concentrations following metronidazole therapy for BV. Vaginal swabs were collected from women with BV diagnosed using Amsel clinical criteria, and vaginal fluid was assessed by Gram stain to generate Nugent scores. Follow-up swabs were collected 1 month after a 5-day course of vaginal 0.75% metronidazole gel and analyzed for 24 subjects with cured BV and 24 subjects with persistent BV. Changes in Bacterial concentrations were measured using eight bacterium-specific 16S rRNA gene quantitative PCR assays. DNA from several Fastidious BV-associated Bacteria (BVAB) were present at high concentrations in the vagina prior to treatment. Successful antibiotic therapy resulted in 3- to 4-log reductions in median Bacterial loads of BVAB1 (P = 0.02), BVAB2 (P = 0.0004), BVAB3 (P = 0.03), a Megasphaera-like bacterium (P < 0.0001), Atopobium species (P < 0.0001), Leptotrichia/Sneathia species (P = 0.0002), and Gardnerella vaginalis (P < 0.0001). Median posttreatment Bacterial levels did not change significantly in subjects with persistent BV except for a decline in levels of BVAB3. The presence or absence of BV is reflected by vaginal concentrations of BV-associated Bacteria such as BVAB1, BVAB2, Leptotrichia/Sneathia species, Atopobium species, Gardnerella vaginalis, and a Megasphaera-like bacterium, suggesting that these Bacteria play an important role in BV pathogenesis and may be suitable markers of disease and treatment response.
Thierry Bégué - One of the best experts on this subject based on the ideXlab platform.
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Early prosthetic joint infection due to Ureaplasma urealyticum: Benefit of 16S rRNA gene sequence analysis for diagnosis
Elsevier, 2019Co-Authors: Caroline Rouard, Sabine Pereyre, Sophie Abgrall, Christelle Guillet-caruba, Pierre Diviné, Nadège Bourgeois-nicolaos, Sandrine Roy, Véronique Mangin D’ouince, Cécile Bébéar, Thierry BéguéAbstract:Detection of the most frequently Bacteria involved in prosthetic joint infection (PJI) is usually performed by conventional cultures. We report a case of early PJI due to Ureaplasma urealyticum, diagnosed by 16S rRNA gene sequence analysis, which highlights the interest of molecular methods if Fastidious Bacteria are involved in PJI. Keywords: Prosthetic joint infection, Ureaplasma urealyticum, 16S rRNA gene, Molecular diagnosi
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Early prosthetic joint infection due to Ureaplasma urealyticum: Benefit of 16S rRNA gene sequence analysis for diagnosis
Journal of Microbiology Immunology and Infection, 2019Co-Authors: Caroline Rouard, Sabine Pereyre, Sophie Abgrall, Christelle Guillet-caruba, Pierre Diviné, Nadège Bourgeois-nicolaos, Sandrine Roy, Cécile Bébéar, Véronique Mangin D'ouince, Thierry BéguéAbstract:Detection of the most frequently Bacteria involved in prosthetic joint infection (PJI) is usually performed by conventional cultures. We report a case of early PJI due to Ureaplasma urealyticum, diagnosed by 16S rRNA gene sequence analysis, which highlights the interest of molecular methods if Fastidious Bacteria are involved in PJI.
Michel Drancourt - One of the best experts on this subject based on the ideXlab platform.
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planctomycetes as host associated Bacteria a perspective that holds promise for their future isolations by mimicking their native environmental niches in clinical microbiology laboratories
Frontiers in Cellular and Infection Microbiology, 2020Co-Authors: Odilon D Kabore, Sylvain Godreuil, Michel DrancourtAbstract:Traditionally recognized as environmental Bacteria, Planctomycetes have just been linked recently to human pathology as opportunistic pathogens, arousing a great interest for clinical microbiologists. However, the lack of appropriate culture media limits our future investigations as no Planctomycetes have ever been isolated from patients' specimens despite several attempts. Several Planctomycetes have no cultivable members and are only recognized by 16S rRNA gene sequence detection and analysis. The cultured representatives are slow-growing Fastidious Bacteria and mostly difficult to culture on synthetic media. Accordingly, the provision of environmental and nutritional conditions like those existing in the natural habitat where yet uncultured/refractory Bacteria can be detected might be an option for their potential isolation. Hence, we systematically reviewed the various natural habitats of Planctomycetes, to review their nutritional requirements, the physicochemical characteristics of their natural ecological niches, current methods of cultivation of the Planctomycetes and gaps, from a perspective of collecting data in order to optimize conditions and the protocols of cultivation of these Fastidious Bacteria. Planctomycetes are widespread in freshwater, seawater, and terrestrial environments, essentially associated to particles or organisms like macroalgae, marine sponges, and lichens, depending on the species and metabolizable polysaccharides by their sulfatases. Most Planctomycetes grow in nutrient-poor oligotrophic environments with pH ranging from 3.4 to 11, but a few strains can also grow in quite nutrient rich media like M600/M14. Also, a seasonality variation of abundance is observed, and bloom occurs in summer-early autumn, correlating with the strong growth of algae in the marine environments. Most Planctomycetes are mesophilic, but with a few Planctomycetes being thermophilic (50°C to 60°C). Commonly added nutrients are N-acetyl-glucosamine, yeast-extracts, peptone, and some oligo and macro-elements. A biphasic host-associated extract (macroalgae, sponge extract) conjugated with a diluted basal medium should provide favorable results for the success of isolation in pure culture.
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colonization of hospital water networks by gemmata massiliana a new planctomycetes bacterium
Current Microbiology, 2015Co-Authors: R. Aghnatios, Michel DrancourtAbstract:Planctomycetes have been isolated from various hydric environments. These Fastidious Bacteria are overlooked by routine 16S rRNA gene-based PCR detection in hospital laboratories, and their presence has not been documented in the health-care environment. Using a specific culture protocol, we recently isolated a new, non-filterable Planctomycetes species, Gemmata massiliana, from one hospital water network. The goal of the study was to monitor the presence of G. massiliana in two hospital water networks. We developed a G. massiliana-specific real-time PCR system and monitored the presence of the Planctomycetes for 12 months in two hospital water networks, in filtered water collected at the intensive care unit and in non-filtered water collected from dental chairs, tanks, and usage points. Four of 180 (2.2 %) filtered water samples tested positive versus 23 of 204 (11.3 %) non-filtered points (p 0.05). However, this organism was detected significantly more frequently during April and September than the 10 other months. Because G. massiliana is deeply entrenched in the hospitalized patient’s environment, evaluating this organism as a new opportunistic, health-care-associated pathogen is warranted.
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current and past strategies for Bacterial culture in clinical microbiology
Clinical Microbiology Reviews, 2015Co-Authors: Jean Christophe Lagier, Isabelle Pagnier, Oleg Mediannikov, Sophie Edouard, Michel DrancourtAbstract:SUMMARY A pure Bacterial culture remains essential for the study of its virulence, its antibiotic susceptibility, and its genome sequence in order to facilitate the understanding and treatment of caused diseases. The first culture conditions empirically varied incubation time, nutrients, atmosphere, and temperature; culture was then gradually abandoned in favor of molecular methods. The rebirth of culture in clinical microbiology was prompted by microbiologists specializing in intracellular Bacteria. The shell vial procedure allowed the culture of new species of Rickettsia. The design of axenic media for growing Fastidious Bacteria such as Tropheryma whipplei and Coxiella burnetii and the ability of amoebal coculture to discover new Bacteria constituted major advances. Strong efforts associating optimized culture media, detection methods, and a microaerophilic atmosphere allowed a dramatic decrease of the time of Mycobacterium tuberculosis culture. The use of a new versatile medium allowed an extension of the repertoire of archaea. Finally, to optimize the culture of anaerobes in routine bacteriology laboratories, the addition of antioxidants in culture media under an aerobic atmosphere allowed the growth of strictly anaerobic species. Nevertheless, among usual Bacterial pathogens, the development of axenic media for the culture of Treponema pallidum or Mycobacterium leprae remains an important challenge that the patience and innovations of cultivators will enable them to overcome.
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Uveitis caused by Fastidious Bacteria
Acta Ophthalmologica, 2009Co-Authors: Bahram Bodaghi, Michel Drancourt, Didier Raoult, Nathalie Cassoux, Phuc LehoangAbstract:Purpose The etiologic evaluation of uveitis is frequently unsuccessful if only noninvasive methods are used. We conducted a prospective study to evaluate systematic screening for pathogens of uveitis. Methods All patients with uveitis referred to the participating tertiary ophthalmology departments from January 2001 to September 2007 underwent intraocular and serum specimen collection. The standardized protocol for laboratory investigations included universal polymerase chain reaction (PCR)-based detection of any Bacteria and mycoses, specific PCR-based detection of Fastidious (difficult-to-grow) Bacteria and herpes viruses, and culture of vitreous fluid. Sera were tested for Fastidious Bacteria. Results Among the 1321 included patients, infection was diagnosed in 147 (11.1%) patients: 78 (53%) were caused by Fastidious Bacteria that included spirochetes, Bartonella species, intracellular Bacteria (Chlamydia species, Rickettsia species, Coxiella burnetii), and Tropheryma whipplei; 18 by herpes viruses; and 9 by fungi. Bartonella quintana, Coxiella burnetii, Paracoccus yeei, Aspergillus oryzae, and Cryptococcus albidus were found to be associated with uveitis for the first time, to our knowledge. Conclusion We recommend applying a 1-step diagnostic procedure that incorporates intraocular, specific microbial PCR with serum analyses in tertiary centers to determine the etiology of uveitis.
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high prevalence of Fastidious Bacteria in 1520 cases of uveitis of unknown etiology
Medicine, 2008Co-Authors: Michel Drancourt, C Terrada, Pierre Berger, John Conrath, Bahram Bodaghi, Phuc LehoangAbstract:Abstract:The etiologic evaluation of uveitis is frequently unsuccessful when noninvasive methods are used. We conducted a prospective study to evaluate systematic screening for pathogens of uveitis. All patients with uveitis referred to the participating tertiary ophthalmology departments from Janua
Jeanmarc Rolain - One of the best experts on this subject based on the ideXlab platform.
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Acquired Resistance to Trimethoprim- Sulfamethoxazole during Whipple Disease and Expression of the Causative Target Gene
2016Co-Authors: Nawal Bakkali, Florence Fenollar, Silpak Biswas, Jeanmarc Rolain, Didier RaoultAbstract:Background. Whipple disease is a chronic infection caused by Tropheryma whipplei. Trimethoprim-sulfamethoxazole is recommended for treatment of Whipple disease but is associated with treatment failure. T. whipplei is resistant in vitro to trimethoprim, because the gene targeted by this agent is missing. Methods. Apatient experienced clinical failure during treatment with trimethoprim-sulfamethoxazole. The gene encoding the enzyme putatively believed to be dihydropteroate synthase (DHPS), the target of sulfamethoxazole, was amplified and sequenced for 20 T. whipplei strains from our laboratory and for isolates recovered from a case patient at the time of diagnosis and the time of treatment failure. An Escherichia coli knockout strain for this gene was complemented with the sequences from a susceptible strain and from isolates recovered from the case patient. Susceptibilities of complemented E. coli to sulfamethoxazole were tested. Results. The target gene was identified among genes encoding a unique trifunctional enzyme in which DHPS is combined with the 2 preceding enzymes of the folate biosynthesis pathway. Changes in the amino acid sequence of putative DHPS were detected in the case patient. Gene complementation showed that the gene encoding putative DHPS restored the folate biosynthesis pathway and susceptibility to sulfamethoxazole, whereas themutated sequence was associated with sulfamethoxazole resistance. Conclusions. Antibiotic susceptibility of Fastidious Bacteria such asT. whipplei can be evaluated bymeans of gen
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use of maldi tof mass spectrometry for identification of Bacteria that are difficult to culture
Journal of Microbiological Methods, 2013Co-Authors: Silpak Biswas, Jeanmarc RolainAbstract:Rapid and reliable detection and identification of Bacterial species are necessary for diagnosis and efficient treatment. Until recently, Bacterial identification in clinical laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The identification of anaerobic Bacteria, Fastidious and slow growing Bacteria using conventional methods is time consuming, expensive and complicated. Many anaerobes grow poorly or are nonreactive in most diagnostic systems. Unambiguous diagnosis of active tuberculosis is a time-consuming process, requiring as long as 12 weeks for positive identification of the organism. This long time frame presents challenges for case identification. Early identification of pathogenic Bacteria is very important for the disease control. Recently, bacteriologists have focused their attention on the use of mass spectrometry (MS) for Bacterial identification, especially Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF). Use of MALDI-TOF-MS is described in this review, with a special emphasis on the successful identification of groups of Bacteria, which are difficult to culture. MALDI-TOF-MS is a powerful, rapid, precise, and cost-effective method for identification of intact Bacteria, compared to conventional phenotypic techniques or molecular biology. Our review suggests that identification of anaerobes, Fastidious Bacteria and slow growing Bacteria, has been improved by the arrival of MALDI-TOF-MS in clinical laboratories.
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acquired resistance to trimethoprim sulfamethoxazole during whipple disease and expression of the causative target gene
The Journal of Infectious Diseases, 2008Co-Authors: Nawal Bakkali, Florence Fenollar, Silpak Biswas, Jeanmarc Rolain, Didier RaoultAbstract:Background. Whipple disease is a chronic infection caused by Tropheryma whipplei. Trimethoprim-sulfamethoxazole is recommended for treatment of Whipple disease but is associated with treatment failure. T. whipplei is resistant in vitro to trimethoprim, because the gene targeted by this agent is missing. Methods. A patient experienced clinical failure during treatment with trimethoprim-sulfamethoxazole. The gene encoding the enzyme putatively believed to be dihydropteroate synthase (DHPS), the target of sulfamethoxazole, was amplified and sequenced for 20 T. whipplei strains from our laboratory and for isolates recovered from a case patient at the time of diagnosis and the time of treatment failure. An Escherichia coli knockout strain for this gene was complemented with the sequences from a susceptible strain and from isolates recovered from the case patient. Susceptibilities of complemented E. coli to sulfamethoxazole were tested. Results. The target gene was identified among genes encoding a unique trifunctional enzyme in which DHPS is combined with the 2 preceding enzymes of the folate biosynthesis pathway. Changes in the amino acid sequence of putative DHPS were detected in the case patient. Gene complementation showed that the gene encoding putative DHPS restored the folate biosynthesis pathway and susceptibility to sulfamethoxazole, whereas the mutated sequence was associated with sulfamethoxazole resistance. Conclusions. Antibiotic susceptibility of Fastidious Bacteria such as T. whipplei can be evaluated by means of gene complementation techniques. Mutations in the target gene of sulfamethoxazole appear during treatment.