Culturing the Human Oral Microbiota, Updating Methodologies and Cultivation Techniques.

Recent years have been marked by a paradigm shift in the study of the human microbiota, with a re-emergence of culture-dependent approaches. Numerous studies have been devoted to the human microbiota, while studies on the oral microbiota still remain limited. Indeed, various techniques described in the literature may enable an exhaustive study of the microbial composition of a complex ecosystem. In this article, we report different methodologies and culture media described in the literature that can be applied to study the oral microbiota by culture. We report on specific methodologies for targeted culture and specific culture techniques and selection methodologies for cultivating members of the three kingdoms of life commonly found in the human oral cavity, namely, eukaryota, bacteria and archaea. This bibliographic review aims to bring together the various techniques described in the literature, enabling a comprehensive study of the oral microbiota in order to demonstrate its involvement in oral health and diseases.

Evaluation of Culture Top transport systems for assessing the bacterial diversity of microbiota by culturomics as compared to a routine transport system.

In recent years, metagenomics and then culturomics, which consists of the multiplication of media and culture conditions and the rapid identification of all bacterial colonies, have generated renewed interest in the human microbiota, and diseases associated with modifications in its composition in particular. The sample transport media included in diverse swab transport systems and the storage conditions are among the factors that influence the results of the culturomics. In this study, we compared the results of culturomics from paired skin, oral and rectal swabs from intensive care unit (ICU) patients using Culture Top sample transport medium as compared to our routine one. From 152 clinical samples, we were able to isolate and identify 45 600 colonies, belonging to 338 different bacterial species. The transport system Culture Top identified 282 different bacterial species, while 244 were identified by our routine system. Of these, 188 different bacterial species were commonly identified using both transport systems, while 94 (27.8 %) and 56 (16.5 %) were only identified using Culture Top and our routine system, respectively (P<0.001), but there was no significant difference in bacterial diversity at the genus or phylum level, or in terms of their type of respiration and cell wall. In conclusion, the Culture Top transport system appears to be complementary to our routine system, although it seems slightly superior in terms of isolated bacterial species.

A review of in vitro attempts to develop the axenic culture of Treponema pallidum and genomics-based suggestions to achieve this elusive goal.

To date, the axenic culture of Treponema pallidum remains a challenge in the field of microbiology despite countless attempts. Here, we conducted a comprehensive bibliographic analysis using several databases and search engines, namely Pubmed, Google scholar, Google, Web of Science and Scopus. Numerous unsuccessful empiric studies have been conducted and evaluated using as criteria dark-field microscopic observation of motile spiral shaped cells in the culture and virulence of the culture through rabbit infectivity. All of these studies failed to induce rabbit infectivity, even when deemed positive after microscopic observation leading to the misnomer of avirulent T. pallidum. In fact, this criterion was improperly chosen because not all spiral shaped cells are T. pallidum. However, these studies led to the formulation of culture media particularly favourable to the growth of several species of Treponema, including Oral Microbiology and Immunology, Zürich medium (OMIZ), Oral Treponeme Enrichment Broth (OTEB) and T-Raoult, thus allowing the increase in the number of cultivable strains of Treponema. The predicted metabolic capacities of T. pallidum show limited metabolism, also exhibited by other non-cultured and pathogenic Treponema species, in contrast to cultured Treponema species. The advent of next generation sequencing represents a turning point in this field, as the knowledge inferred from the genome can finally lead to the axenic culture of T. pallidum.

SCA Medium: A New Culture Medium for the Isolation of All Candida auris Clades.

Candida auris is an emerging multidrug-resistant yeast causing nosocomial infections and associated with high mortality in immunocompromised patients. Rapid identification and characterisation are necessary for diagnosis and containing its spread. In this study, we present a selective culture medium for all C. auris clades. This medium is sensitive with a limit of detection ranging between 101 and 102 CFU/mL. The 100% specificity of SCA (specific C. auris) medium is confirmed on a set of 135 Candida strains, 50 bacterial species and 200 human stool samples. Thus, this medium specifically selects for C. auris isolation from clinical samples, allowing the latter to study its phenotypic profile.

A Listeria monocytogenes clone in human breast milk associated with severe acute malnutrition in West Africa: A multicentric case-controlled study.

BACKGROUND: Severe acute malnutrition (SAM) is a major public health problem affecting children under the age of five in many low- and middle-income countries, and its resolution would contribute towards achieving the several sustainable development goals. The etiology of SAM is pluri-factorial, including delayed maturation of the gut microbiota, suboptimal feeding practices and dysfunctional breastfeeding. The recent serendipitous detection of Listeria monocytogenes in the breast milk of Malian women, in contrast to French women, suggests a possible association with SAM. METHODOLOGY/ PRINCIPAL FINDINGS: To investigate the possible association of L. monocytogenes carriage in breast milk and SAM, a case-control study was performed in Senegal, with subjects recruited from two areas. Using 16S amplicon sequencing, a culture independent method, 100% (152/152) of the mothers were positive for L. monocytogenes in their breast milk while qPCR analysis gave lower recovery rates. Interestingly, after enrichment in Fraser broth and seeding on PALCALM agar, all 10 isolated strains were isolated from the milk of 10 mothers who had SAM children which also had a significantly increased relative abundance of L. monocytogenes (0.34 (SD 0.35) vs 0.05 (SD 0.07) in controls, p<0.0001). The high genomic similarity between these strains and Malian breast milk strains from a previous study supports the hypothesis of endemic clone carriage in West Africa. Moreover, the in vitro growth inhibition of L. monocytogenes using breast milk samples was obtained from only 50% of the milk of mothers who had SAM children, in contrast to control samples which systematically inhibited the growth of L. monocytogenes with a higher inhibition diameter (15.7 mm (SD 2.3) in controls versus 3.5 mm (SD 4.6) in SAM, p = 0.0001). Lactobacillus and Streptococcus isolated from the breast milk of controls inhibit L. monocytogenes in a species-dependent manner. CONCLUSIONS/SIGNIFICANCE: Our study reveals a previously unsuspected carriage of L. monocytogenes in the breast milk of West African women, which is associated with SAM. The inhibitory effect of human selected lactic acid bacterial species against L. monocytogenes might provide new therapeutic and inexpensive options to prevent and treat this neglected public health issue.

Vitreoscilla massiliensis sp. nov., Isolated From the Stool of an Amazonian Patient.

Strain SN6T is a non-motile and non-spore-forming gram-negative bacterium which was isolated from the stool sample of an Amazonian patient. The optimum growth was observed at 37 °C, pH 7, and 0-5 g/l of NaCl. Based on the 16S rRNA gene sequence similarity, the strain SN6T exhibited 97.5% identity with Vitreoscilla stercoraria strain ATCC_15218 (L06174), the phylogenetically closest species with standing in nomenclature. The predominant fatty acid was hexadecenoic acid (31%). The genomic DNA G + C content of the strain SN6T was 49.4 mol %. After analysis of taxonogenomic data, phenotypic and biochemical characteristics, we concluded that strain SN6T represents a new species of the genus Vitreoscilla for which the name Vitreoscilla massiliensis sp.nov is proposed. The type strain is SN6T (=CSUR P2036 = LN870312 = DSM 100958).

Running after ghosts: are dead bacteria the dark matter of the human gut microbiota?

The human gut microbiota has been explored by a wide range of culture-dependent and culture-independent methods, revealing that many microbes remain uncharacterized and uncultured. In this work, we aimed to confirm the hypothesis that some of the species present in the human gut microbiota remain uncultured not because of culture limitations, but because all members of such species are dead before reaching the end of the gastro-intestinal tract.We evaluate this phenomenon by studying the microbial viability and culturability of the human gut microbiota from the fresh fecal materials of eight healthy adults. For the first time, we applied fluorescence-activated cell sorting (FACS) combined with 16S metagenomics analysis and microbial culturomics.We identified a total of 1,020 bacterial OTUs and 495 bacterial isolates through metagenomics and culturomics, respectively. Among the FACS metagenomics results, only 735 bacterial OTUs were alive, comprising on average 42% of known species and 87% of relative abundance per individual. The remaining uncultured bacteria were rare, dead, or injured.Our strategy allowed us to shed light on the dark matter of the human gut microbiota and revealed that both metagenomics and culturomics approaches are needed for greater insight into the diversity and richness of bacteria in the human gut microbiota. Further work on culture is needed to enhance the repertoire of cultured gut bacteria by targeting low abundance bacteria and optimizing anaerobic sample conditioning and processing to preserve the viability of bacteria.

Intra- and inter-laboratory comparison of mDixon and FastFung broths for Malassezia antifungal susceptibility testing.

BACKGROUND: Malassezia spp. antifungal susceptibility testing (AFST) capacities are limited by the lack of efficient and standardised AFST procedure, mainly because of the fastidious cultivation of these yeast. OBJECTIVES: This study aimed to compare the FastFung broth (FFB) to modified Dixon broth (mDIXB) for the in vitro AFST of Malassezia spp. Fluconazole, ketoconazole, voriconazole and terbinafine MICs against a 19 Malassezia strains, including 6 M furfur, 4 M pachydermatis, 5 M sympodialis and 4 M slooffiae. METHODS: The essential agreement (EA) between the two assays, and the intra- and inter-laboratory agreement of each assay were assessed. RESULTS: The MIC data obtained in our study were comparable to those reported in the literature. FFB showed to enhance Malassezia growth and displayed 100% (±2-fold dilution) EAs demonstrating similar performances to mDIXB. In addition, the MIC data obtained by using the FFB were reproducible between laboratories with EAs ranging from 94.7% to 100%. CONCLUSIONS: Therefore, FFB is a suitable alternative to mDXB for Malassezia spp. AFST.

Methanobrevibacter smithii Archaemia in Febrile Patients With Bacteremia, Including Those With Endocarditis.

BACKGROUND: The spectrum of infections caused by methanogens remains to be described. We searched for methanogens in the blood of febrile patients using specific tools. METHODS: Blood culture samples routinely collected in patients with fever were prospectively screened by specific PCR assays for methanogens. Positive samples were observed by autofluorescence and electron microscopy, analyzed by metagenomics and cultured using previously developed methods. Blood culture bottles experimentally inoculated were used as controls. The presence of methanogens in vascular and cardiac tissues was assessed by indirect immunofluorescence, fluorescent in situ hybridization and PCR-based investigations. RESULTS: PCR detection attempted in 7,716 blood samples, was negative in all 1,312 aerobic bottles and 810 bacterial culture-negative anaerobic bottles. PCRs were positive in 27/5,594 (0.5%) bacterial culture-positive anaerobic bottles collected from 26 patients. Sequencing confirmed Methanobrevibacter smithii associated with staphylococci in 14 patients, Enterobacteriaceae in nine patients and streptococci in three patients. Metagenomics confirmed M. smithii in five samples, and M. smithii was isolated in broth from two samples; the genomes of these two isolates were sequenced. Blood cultures experimentally inoculated with Enterobacteriaceae, Staphylococcus epidermidis or Staphylococcus hominis yielded hydrogen, but no methane, authentifying observational data. Three patients diagnosed with infectious mitral endocarditis, were indisputably diagnosed by microscopy, PCR-based detections and culture: we showed M. smithii microscopically and by a specific PCR followed by sequencing method in two of three cardiovascular tissues. CONCLUSIONS: Using appropriate laboratory methods, M. smithii is demonstrated as causing archaemia and endocarditis in febrile patients who are coinfected by bacteria.

FastFung: A novel medium for the culture and isolation of fastidious fungal species from clinical samples.

We developed a novel culture medium, referred to FastFung medium as suitable for the culture of clinical fungi, including fastidious ones, for both research and diagnostic studies. It is based on Schædler agar supplemented with many essential components for the growth of fastidious fungi. It also contains selective antibacterial agents for the inhibition of contaminant bacteria growth. In this preliminary study, the FastFung medium was compared to the gold standard Sabouraud medium for 98 fungal and 20 bacterial strains. The fungal strain positive culture rate was 100% vs. 95% and the bacterial strain inhibition was 100% vs. 20%, for the FastFung and Sabouraud media, respectively. When compared to the Sabouraud medium on 120 clinical samples, the FastFung medium displayed both a higher fungal colonies count, and a lower culture contamination rate. Storage at 4 °C for 4 weeks did not alter the FastFung culture medium performances for the six isolates of Candida, Cryptococcus, and Penicillium tested. These encouraging results suggest future development of using the FastFung medium in clinical mycology and in mycobiome characterization. Further prospective evaluation aiming at assessing whether implementing the FastFung medium in the routine workflow simplifies and strengthen fungal isolation capacities in the clinical laboratory is warranted.

Comparison of Three Skin Sampling Methods and Two Media for Culturing Malassezia Yeast.

Malassezia is a lipid-dependent commensal yeast of the human skin. The different culture media and skin sampling methods used to grow these fastidious yeasts are a source of heterogeneity in culture-based epidemiological study results. This study aimed to compare the performances of three methods of skin sampling, and two culture media for the detection of Malassezia yeasts by culture from the human skin. Three skin sampling methods, namely sterile gauze, dry swab, and TranswabTM with transport medium, were applied on 10 healthy volunteers at 5 distinct body sites. Each sample was further inoculated onto either the novel FastFung medium or the reference Dixon agar for the detection of Malassezia spp. by culture. At least one colony of Malassezia spp. grew on 93/300 (31%) of the cultures, corresponding to 150 samplings. The positive culture rate was 67%, 18%, and 15% (P < 10-3), for samples collected with sterile gauze, TranswabTM, and dry swab, respectively. The positive culture rate was 62% and 38% (P < 0.003) by using the FastFung and the Dixon media, respectively. Our results showed that sterile gauze rubbing skin sampling followed by inoculation on FastFung medium should be implemented in the routine clinical laboratory procedure for Malassezia spp. cultivation.

Proof of Concept of Culturomics Use of Time of Care.

Culturomics, a high throughput culture method with rapid identification of the colonies by Matrix Assisted Laser Desorption Ionization/Time Of Flight Mass Spectrometry (MALDI-TOF MS), has demonstrated its contribution to the exploration of the gut microbiota over the past 10 years. However, the cost, work time and workload, considerably limit its use on a large scale or emergency context. Here, by testing two different stool samples, including a stool sample from a patient requiring rapid immunotherapy treatment, we tested a new fast culturomic protocol using two pre-incubation media, blood culture bottle and YCFA modified medium. Both media were supplemented with 2 ml of rumen fluid filtered at 0.2 µm and 2 ml of defibrinated and sterile sheep blood. Unlike the standard culturomics, subculturing of blood culture bottle were performed at reduced incubation time (3 h, 6 h, 9 h, 24 h) and at a longer incubation time (3 days, 7 days, and 10 days) at 37°C. By testing 5,200 colonies per MALDI-TOF MS and obtaining a comparable number of cultured bacterial species (131 to 143) in a stool sample, this new protocol reduced the number of colonies tested by 57%, working time by 78.6% and cost by 72.2%. In addition, we highlighted that the proportion of strict anaerobic species has increased by 24%, known to be the preferential targets for biotherapy, including Faecalibacterium prausnitzii, Akkermansia muciniphila, Christensenella minuta, and Phascolarctobacterium faecium. Finally, this work showed that some bacterial species grew earlier but disappeared with prolonged incubation times.

Dual RNase and ß-lactamase Activity of a Single Enzyme Encoded in Archaea.

ß-lactam antibiotics have a well-known activity which disturbs the bacterial cell wall biosynthesis and may be cleaved by ß-lactamases. However, these drugs are not active on archaea microorganisms, which are naturally resistant because of the lack of ß-lactam target in their cell wall. Here, we describe that annotation of genes as ß-lactamases in Archaea on the basis of homologous genes is a remnant of identification of the original activities of this group of enzymes, which in fact have multiple functions, including nuclease, ribonuclease, ß-lactamase, or glyoxalase, which may specialized over time. We expressed class B ß-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The ß-lactamase activity was inhibited by ß-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to have been transferred to the Flavobacteriaceae group especially the Elizabethkingia genus, in which the expressed gene shows a more specialized activity on thienamycin, but no glyoxalase activity. The expressed class C-like ß-lactamase gene, from Methanosarcina sp., also shows hydrolysis activity on nitrocefin and is more closely related to DD-peptidase enzymes. Our findings highlight the need to redefine the nomenclature of ß-lactamase enzymes and the specification of multipotent enzymes in different ways in Archaea and bacteria over time.

State of the Art in the Culture of the Human Microbiota: New Interests and Strategies.

The last 5 years have seen a turning point in the study of the gut microbiota with a rebirth of culture-dependent approaches to study the gut microbiota. High-throughput methods have been developed to study bacterial diversity with culture conditions aimed at mimicking the gut environment by using rich media such as YCFA (yeast extract, casein hydrolysate, fatty acids) and Gifu anaerobic medium in an anaerobic workstation, as well as media enriched with rumen and blood and coculture, to mimic the symbiosis of the gut microbiota. Other culture conditions target phenotypic and metabolic features of bacterial species to facilitate their isolation. Preexisting technologies such as next-generation sequencing and flow cytometry have also been utilized to develop innovative methods to isolate previously uncultured bacteria or explore viability in samples of interest. These techniques have been applied to isolate CPR (Candidate Phyla Radiation) among other, more classic approaches. Methanogenic archaeal and fungal cultures present different challenges than bacterial cultures. Efforts to improve the available systems to grow archaea have been successful through coculture systems. For fungi that are more easily isolated from the human microbiota, the challenge resides in the identification of the isolates, which has been approached by applying matrix-assisted laser desorption ionization-time of flight mass spectrometry technology to fungi. Bacteriotherapy represents a nonnegligible avenue in the future of medicine to correct dysbiosis and improve health or response to therapy. Although great strides have been achieved in the last 5 years, efforts in bacterial culture need to be sustained to continue deciphering the dark matter of metagenomics, particularly CPR, and extend these methods to archaea and fungi.

Cross-reactivity between tumor MHC class I-restricted antigens and an enterococcal bacteriophage.

Intestinal microbiota have been proposed to induce commensal-specific memory T cells that cross-react with tumor-associated antigens. We identified major histocompatibility complex (MHC) class I-binding epitopes in the tail length tape measure protein (TMP) of a prophage found in the genome of the bacteriophage Enterococcus hirae Mice bearing E. hirae harboring this prophage mounted a TMP-specific H-2Kb-restricted CD8+ T lymphocyte response upon immunotherapy with cyclophosphamide or anti-PD-1 antibodies. Administration of bacterial strains engineered to express the TMP epitope improved immunotherapy in mice. In renal and lung cancer patients, the presence of the enterococcal prophage in stools and expression of a TMP-cross-reactive antigen by tumors correlated with long-term benefit of PD-1 blockade therapy. In melanoma patients, T cell clones recognizing naturally processed cancer antigens that are cross-reactive with microbial peptides were detected.

Klenkia terrae resistant to DNA extraction in germ-free mice stools illustrates the extraction pitfall faced by metagenomics.

Over the past decade, metagenomics has become the preferred method for exploring complex microbiota such as human gut microbiota. However, several bias affecting the results of microbiota composition, such as those due to DNA extraction, have been reported. These bias have been confirmed with the development of culturomics technique. In the present study, we report the contamination of a gnotobiotic mice unit with a bacterium first detected by gram staining. Scanning electron microscopy and transmission electron microscopy permitted to detect a bacterium with a thick cell wall. However, in parallel, the first attempt to identify and culture this bacterium by gene amplification and metagenomics of universal 16S rRNA failed. Finally, the isolation in culture of a fastidious bacterium not detected by using universal PCR was successfully achieved by using a BCYE agar plate with CO2 atmosphere at 30 °C. We performed genome sequencing of this bacterium using a strong extraction procedure. The genomic comparison allowed us to classify this bacterium as Klenkia terrae. And finally, it was also detected in the stool and kibble that caused the contamination by using specific qPCR against this bacterium. The elucidation of this contamination provides additional evidence that DNA extraction could be a bias for the study of the microbiota. Currently, most studies that strive to analyze and compare the gut microbiota are based on metagenomics. In a gnotobiotic mice unit contaminated with the fastidious Actinobacteria Klenkia terrae, standard culture, 16S rRNA gene amplification and metagenomics failed to identify the micro-organism observed in stools by gram-staining. Only a procedure based on culturomics allowed us to identify this bacterium and to elucidate the mode of contamination of the gnotobiotic mice unit through diet.

The Antioxidants Glutathione, Ascorbic Acid and Uric Acid Maintain Butyrate Production by Human Gut Clostridia in The Presence of Oxygen In Vitro.

Uncontrolled oxidative stress, reported in Salmonella and HIV infections, colorectal cancer or severe acute malnutrition, has been associated with anaerobic gut microbiome alteration, impaired butyrate production, mucosal immunity dysregulation and disruption of host-bacterial mutualism. However, the role of major antioxidant molecules in the human body, such as glutathione, ascorbic acid and uric acid, has been neglected in this context. Here, we performed an in vitro metabolomics study of the 3 most odorous anaerobic microbes isolated from the human gut in our laboratory (Clostridium sporogenes, Clostridium subterminale and Romboutsia lituseburensis) when grown in anaerobiosis or in aerobiosis with these 3 antioxidant molecules via gas and liquid chromatography-mass spectrometry (GC/MS and LC/MS). There was no growth or volatile organic compound production in aerobic cultures without the 3 antioxidant molecules. In anaerobiosis, the major metabolic products of the bacteria were thiols, alcohols and short-chain fatty acid esters. The production of alkanes, cycloheptatriene and, paradoxically, increased butyrate production, was observed in the cultures grown in aerobiosis with the 3 antioxidant molecules. The qualitative shift suggests specific molecular mechanisms that remain to be elucidated. The increased production of butyrate, but also isobutyrate and isovalerate in vitro suggests that these 3 antioxidant molecules contributed to the maintenance and active resilience of host-bacterial mutualism against mucosal oxygen and uncontrolled oxidative stress in vivo.

Draft genome and description of Negativicoccus massiliensis strain Marseille-P2082, a new species isolated from the gut microbiota of an obese patient.

Strain Marseille-P2082, an anaerobic, non-motile, asporogenous, Gram-negative, coccoid bacterium was isolated from the faeces of a 33 year-old obese French woman before bariatric surgery. The isolate exhibits 98.65% 16S rRNA gene nucleotide sequence similarity with Negativicoccus succinicivorans strain ADV 07/08/06-B-1388T, its current closest phylogenetic neighbour with standing in nomenclature. However, the dDDH relatedness between the new isolate and N. succinicivorans type strain ADV 07/08/06-B-1388T is 52.5 ± 2.7%. Strain Marseille-P2082 has a genome of 1,360,589 bp with a 51.1% G+C content. Its major fatty acids were identified as C18:1n9, C18:0 and C16:0. Based on its phenotypic, genomic and phylogenetic characteristics, strain Marseille-P2082T [= CSURP2082 (Collection de Souches de l'Unité des Rickettsies) = DSM 100853] is proposed as the type strain of the novel species Negativicoccus massiliensis sp. nov. The 16S rRNA gene sequence and whole-genome shotgun sequence have been deposited in EMBL-EBI under accession numbers LN876651 and LT700188, respectively.

Culture of Methanogenic Archaea from Human Colostrum and Milk.

Archaeal sequences have been detected in human colostrum and milk, but no studies have determined whether living archaea are present in either of these fluids. Methanogenic archaea are neglected since they are not detected by usual molecular and culture methods. By using improved DNA detection protocols and microbial culture techniques associated with antioxidants previously developed in our center, we investigated the presence of methanogenic archaea using culture and specific Methanobrevibacter smithii and Methanobrevibacter oralis real-time PCR in human colostrum and milk. M. smithii was isolated from 3 colostrum and 5 milk (day 10) samples. M. oralis was isolated from 1 milk sample. For 2 strains, the genome was sequenced, and the rhizome was similar to that of strains previously isolated from the human mouth and gut. M. smithii was detected in the colostrum or milk of 5/13 (38%) and 37/127 (29%) mothers by culture and qPCR, respectively. The different distribution of maternal body mass index according to the detection of M. smithii suggested an association with maternal metabolic phenotype. M. oralis was not detected by molecular methods. Our results suggest that breastfeeding may contribute to the vertical transmission of these microorganisms and may be essential to seed the infant's microbiota with these neglected critical commensals from the first hour of life.