Unraveling the world of halophilic and halotolerant bacteria in cheese by combining cultural, genomic and metagenomic approaches.

Halophilic/halotolerant bacteria are generally assumed to live in natural environments, although they may also be found in foods such as cheese and seafood. These salt-loving bacteria have been occasionally characterized in cheese, and studies on their ecological and technological functions are still scarce. We therefore selected 13 traditional cheeses to systematically characterize these microorganisms in their rinds via cultural, genomic and metagenomic methods. Using different salt-based media, we identified 35 strains with unique 16S rRNA and rpoB gene sequences, whose whole genome was sequenced. Twenty are Gram-positive species including notably Brevibacterium aurantiacum (6) and Staphylococcus equorum (3), which are also frequently added as starters. ANI and pan-genomic analyses confirm the high genetic diversity of B. aurantiacum and reveal the presence of two subspecies in S. equorum, as well as the genetic proximity of several cheese strains to bovine isolates. Additionally, we isolated 15 Gram-negative strains, potentially defining ten new species of halophilic/halotolerant cheese bacteria, in particular for the genera Halomonas and Psychrobacter. The use of all the genomes sequenced in this study as a reference to complement those existing in the databases allowed us to study the representativeness of 66 species of halophilic/halotolerant bacteria in 74 cheese rind metagenomes. While Gram-positive strains may flourish in the different types of technologies, Gram-negative species are particularly abundant in cheeses with high moisture, such as washed-rind cheeses. Finally, analyses of co-occurrences reveal assemblies, including the frequent coexistence of several species of the same genus, forming moderately complex ecosystems with functional redundancies that probably ensure stable cheese development.

Characterization of lactic acid bacteria isolated from a traditional Ivoirian beer process to develop starter cultures for safe sorghum-based beverages.

The present study aimed to characterize lactic acid bacteria involved in the different processing steps of tchapalo, a traditional Ivoirian beverage, for their potential application as starter cultures in food and beverages. Lactic acid bacteria (LAB) were therefore isolated and enumerated at different steps of the process on MRS and BEA agars. Of the 465 isolates, 27 produced bacteriocins that inhibit Lactobacillus delbrueckii F/31 strain. Of those, two also inhibited Listeria innocua ATCC 33090, while two others displayed inhibitory activity against L.innocua ATCC 33090, E. faecalis CIP 105042, E. faecalis ATCC 29212, Streptococcus sp. clinical LNSP, E. faecalis CIP 105042 and E. faecium ATCC 51558. The dominant species involved in tchapalo LAB fermentation, as determined by 16S rRNA gene sequencing, were Lactobacillus fermentum (64%), followed by Pediococcus acidilactici (14%). Two strains representing the two dominant species, L. fermentum S6 and P. acidilactici S7, and two potential bacteriocin producers, Weissella confusa AB3E41 and Enterococcus faecium AT1E22, were selected for further characterization. First, genome analysis showed that these strains do not display potential harmful genes such as pathogenic factors or transmissible antibiotic resistance genes. Furthermore, phylogenetic analyses were performed to assess evidence of eventual links to groups of strains with particular properties. They revealed that (i) L. fermentum S6 and P. acidilactici S7 are closely related to strains that ferment plants, (ii) E. faecium AT1E22 belongs to the environmental clade B of E. faecium, while W. confusa is quite similar to other strains also isolated from plant fermentations. Further genome analysis showed that E. faecium AT1E22 contains the Enterocin P gene probably carried by a megaplasmid, whereas no evidence of a bacteriocin gene was found in W. confusa AB3E41. The metabolic and the first step of the probiotic potentials of the different strains were analyzed. Lactobacillus fermentum S6 and P. acidilactici S7 are good candidates to develop starter cultures, and E. faecium AT1E22 should be further tested to confirm its potential as a probiotic strain in the production of sorghum wort.

Coupling next-generation sequencing to dominant positive screens for finding antibiotic cellular targets and resistance mechanisms in Escherichia coli.

In order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.

The initial state of the human gut microbiome determines its reshaping by antibiotics.

Microbiome studies have demonstrated the high inter-individual diversity of the gut microbiota. However, how the initial composition of the microbiome affects the impact of antibiotics on microbial communities is relatively unexplored. To specifically address this question, we administered a second-generation cephalosporin, cefprozil, to healthy volunteers. Stool samples gathered before antibiotic exposure, at the end of the treatment and 3 months later were analysed using shotgun metagenomic sequencing. On average, 15 billion nucleotides were sequenced for each sample. We show that standard antibiotic treatment can alter the gut microbiome in a specific, reproducible and predictable manner. The most consistent effect of the antibiotic was the increase of Lachnoclostridium bolteae in 16 out of the 18 cefprozil-exposed participants. Strikingly, we identified a subgroup of participants who were enriched in the opportunistic pathogen Enterobacter cloacae after exposure to the antibiotic, an effect linked to lower initial microbiome diversity and to a Bacteroides enterotype. Although the resistance gene content of participants' microbiomes was altered by the antibiotic, the impact of cefprozil remained specific to individual participants. Resistance genes that were not detectable prior to treatment were observed after a 7-day course of antibiotic administration. Specifically, point mutations in beta-lactamase blaCfxA-6 were enriched after antibiotic treatment in several participants. This suggests that monitoring the initial composition of the microbiome before treatment could assist in the prevention of some of the adverse effects associated with antibiotics or other treatments.

Differences in antibiotic-induced oxidative stress responses between laboratory and clinical isolates of Streptococcus pneumoniae.

Oxidants were shown to contribute to the lethality of bactericidal antibiotics in different bacterial species, including the laboratory strain Streptococcus pneumoniae R6. Resistance to penicillin among S. pneumoniae R6 mutants was further shown to protect against the induction of oxidants upon exposure to unrelated bactericidal compounds. In the work described here, we expanded on these results by studying the accumulation of reactive oxygen species in the context of antibiotic sensitivity and resistance by including S. pneumoniae clinical isolates. In S. pneumoniae R6, penicillin, ciprofloxacin, and kanamycin but not the bacteriostatic linezolid, erythromycin, or tetracycline induced the accumulation of reactive oxygen species. For the three bactericidal compounds, resistance to a single molecule prevented the accumulation of oxidants upon exposure to unrelated bactericidal antibiotics, and this was accompanied by a reduced lethality. This phenomenon does not involve target site mutations but most likely implicates additional mutations occurring early during the selection of resistance to increase survival while more efficient resistance mechanisms are being selected or acquired. Bactericidal antibiotics also induced oxidants in sensitive S. pneumoniae clinical isolates. The importance of oxidants in the lethality of bactericidal antibiotics was less clear than for S. pneumoniae R6, however, since ciprofloxacin induced oxidants even in ciprofloxacin-resistant S. pneumoniae clinical isolates. Our results provide a clear example of the complex nature of the mode of action of antibiotics. The adaptive approach to oxidative stress of S. pneumoniae is peculiar, and a better understanding of the mechanism implicated in response to oxidative injury should also help clarify the role of oxidants induced by antibiotics.

Age-related prevalence of Methanomassiliicoccus luminyensis in the human gut microbiome.

A 16S rDNA sequence-based investigation of methanogenic Archaea in the human stools found Methanobrevibacter smithii in 99.2% and Methanosphaera stadtmanae in 32.6%. The recently described Methanomassiliicoccus luminyensis found by others to be representative of a new order of methanogenic Archaea was found in 4% of stool specimens. The prevalence of M. luminyensis significantly increased with age, contrary to M. smithii and M. stadtmanae.

Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces.

During attempts to obtain novel, human-associated species of the domain Archaea, a coccoid micro-organism, designated strain B10(T), was isolated in pure culture from a sample of human faeces collected in Marseille, France. On the basis of its phenotypic characteristics and 16S rRNA and mcrA gene sequences, the novel strain was classified as a methanogenic archaeon. Cells of the strain were non-motile, Gram-staining-positive cocci that were approximately 850 nm in diameter and showed autofluorescence at 420 nm. Cells were lysed by 0.1% (w/v) SDS. With hydrogen as the electron donor, strain B10(T) produced methane by reducing methanol. The novel strain was unable to produce methane when hydrogen or methanol was the sole energy source. In an atmosphere containing CO(2), strain B10(T) could not produce methane from formate, acetate, trimethylamine, 2-butanol, 2-propanol, cyclopentanol, 2-pentanol, ethanol, 1-propanol or 2,3-butanediol. Strain B10(T) grew optimally with 0.5-1.0% (w/v) NaCl, at pH 7.6 and at 37 °C. It required tungstate-selenite for growth. The complete genome of the novel strain was sequenced; the size of the genome was estimated to be 2.05 Mb and the genomic DNA G+C content was 59.93 mol%. In phylogenetic analyses based on 16S rRNA gene sequences, the highest sequence similarities (98.0-98.7%) were seen between strain B10(T) and several uncultured, methanogenic Archaea that had been collected from the digestive tracts of a cockroach, a chicken and mammals. In the same analysis, the non-methanogenic 'Candidatus Aciduliprofundum boonei' DSM 19572 was identified as the cultured micro-organism that was most closely related to strain B10(T) (83.0% 16S rRNA gene sequence similarity). Each of the three treeing algorithms used in the analysis of 16S rRNA gene sequences indicated that strain B10(T) belongs to a novel order that is distinct from the Thermoplasmatales. The novel strain also appeared to be distinct from Methanosphaera stadtmanae DSM 3091(T) (72.9% 16S rRNA gene sequence similarity), another methanogenic archaeon that was isolated from human faeces and can use methanol in the presence of hydrogen. Based on the genetic and phenotypic evidence, strain B10(T) represents a novel species of a new genus for which the name Methanomassiliicoccus luminyensis gen. nov., sp. nov. is proposed. The type strain of the type species is B10(T) ( = DSM 24529(T) = CSUR P135(T)).

Tungsten-enhanced growth of Methanosphaera stadtmanae.

BACKGROUND: The methanogenic Archaea Methanosphaera stadtmanae has been detected in the human gut microbiota by both culture and culture-independent methods. Its growth reaches an exponential phase after 5 to 7-day culture in medium 322 (10% vol). Our recent successful isolation of Methanomassiliicoccus luminyensis, a tungstate-selenite-requiring Archaea sharing similar metabolism characteristics with M. stadtmanae prompted us to study the effects of tungsten and selenium on M. stadtmanae growth. FINDINGS: Addition of 0.2 mg/L sodium tungstate to medium 322 yielded, 48 hours after inoculation, a growth rate equivalent to that obtained after 6 days with control culture as measured by methane monitoring and optical density measurement. Addition of 50 µg/mL sodium selenate had no effect on M. stadtmanae growth. Quantitative real-time PCRs targeting the M. stadtmanae 16S rRNA confirmed these data. CONCLUSIONS: These data provide new information regarding the poorly known nutritional requirements of the human gut colonizing organismsM. stadtmanae. Adding sodium tungstate to basal medium may facilitate phenotypic characterization of this organism and additionally aid the isolation of new Archaea from complex host microbiota.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of Archaea: towards the universal identification of living organisms.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) identification of Archaea has been limited to some environmental extremophiles belonging to distant taxa. We developed a specific protocol for MALDI-TOF-MS identification of Archaea and applied it to seven environmental human-associated Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, and the recently described Methanomassiliicoccus luminyensi Archaea. After mechanical lyse, we observed a unique protein profile for each organisms comprising 7-24 peaks ranging from 3,015 to 10,632 Da with a high quality score of 7.38 ± 1.26. Profiles were reproducible over successive experiments performed at 1, 2, and 3-week growth durations and unambiguously distinguished the Archaea from all of the 3,995 bacterial spectra in the Brüker database. After the incorporation of the determined profiles into a local database, archaeal isolates were blindly identified within 10 min with an identification score of 1.9-2.3. The MALDI-TOF-MS-based clustering of these archaeal organisms was consistent with their 16S rDNA sequence-based phylogeny. These data prove that MALDI-TOF-MS profiling could be used as a first-line technique for the identification of human Archaea. In complement to previous reports for animal cells, Bacteria and giant viruses, MALDI-TOF-MS therefore appears as a universal method for the identification of living unicellular and multicellular organisms.

The antimicrobial resistance pattern of cultured human methanogens reflects the unique phylogenetic position of archaea.

OBJECTIVES: Methanogenic archaea are constant members of the human oral and digestive microbiota retrieved, in particular, from periodontitis lesions. The objective of the study was to determine their susceptibility to antimicrobials. METHODS: Using the macrodilution method in Hungate tubes with optical microscope observation combined with monitoring methane production, we determined the antibiotic resistance characteristics of eight methanogenic archaea. RESULTS: Methanobrevibacter smithii strains were resistant to ampicillin, streptomycin, gentamicin, rifampicin, ofloxacin, tetracycline and amphotericin B, with MICs ≥ 100 mg/L; these strains were also highly resistant to vancomycin (MIC ≥ 50 mg/L). They were moderately resistant to chloramphenicol (MIC ≤ 25 mg/L), and were susceptible to bacitracin (MIC ≤ 4 mg/L), metronidazole, ornidazole and squalamine (MIC ≤ 1 mg/L). The susceptibility of Methanosphaera stadtmanae was the same as M. smithii, except for chloramphenicol (MIC ≤ 4 mg/L), and Methanobrevibacter oralis yielded the same data as M. smithii, except for bacitracin (MIC ≤ 25 mg/L). The antibiotic susceptibility pattern of 'Methanomassiliicoccus luminyensis', which was recently isolated from human faeces, was identical to that of M. smithii. CONCLUSIONS: Human methanogenic archaea are highly resistant to antibiotics, being susceptible only to molecules that are also effective against both bacteria and eukarya. Methanogenic archaea are good candidates to test for antimicrobial activity against members of this unique domain of life. Further studies to develop new molecules specifically targeting archaea as potential causes of infection are warranted.

Archaea as emerging organisms in complex human microbiomes.

In this work, we review the state of knowledge of Archaea associated with the human microbiome. These prokaryotes, initially discovered in extreme environments, were named Archaea because these environments were thought to be the most primitive on Earth. Further research revealed that this terminology is misleading because these organisms were later found in various non-extreme environments, including the human host. Further examination of the human microbiome has enabled the isolation of three archaeal species, Methanobrevibacter smithii, Methanosphaera stadtmanae and Methanobrevibacter oralis, which are associated with oral, intestinal and vaginal mucosae in humans. Moreover, molecular studies including metagenomic analyses detected DNA sequences indicative of the presence of additional methanogenic and non-methanogenic Archaea in the human intestinal tract. All three culturable Archaea are strict anaerobes, although their potential role in human diseases remains to be established. Future research aims to detect and culture additional human mucosa-associated Archaea and to look for their presence in additional human tissues, to establish their role in human infections involving complex flora.

Cyt toxin expression reveals an inverse regulation of insect and plant virulence factors of Dickeya dadantii.

The plant pathogenic bacteria Dickeya dadantii is also a pathogen of the pea aphid Acyrthosiphon pisum. The genome of the bacteria contains four cyt genes, encoding homologues of Bacillus thuringiensis Cyt toxins, which are involved in its pathogenicity to insects. We show here that these genes are transcribed as an operon, and we determined the conditions necessary for their expression. Their expression is induced at high temperature and at an osmolarity equivalent to that found in the plant phloem sap. The regulators of cyt genes have also been identified: their expression is repressed by H-NS and VfmE and activated by PecS. These genes are already known to regulate plant virulence factors, but in an opposite way. When tested in a virulence assay by ingestion, the pecS mutant was almost non-pathogenic while hns and vfmE mutants behaved in the same way as the wild-type strain. Mutants of other regulators of plant virulence, GacA, OmpR and PhoP, that do not control Cyt toxin production, also showed reduced pathogenicity. In an assay by injection of bacteria, the gacA strain was less pathogenic but, surprisingly, the pecS mutant was slightly more virulent. These results show that Cyt toxins are not the only virulence factors required to kill aphids, and that these factors act at different stages of the infection. Moreover, their production is controlled by general virulence regulators known for their role in plant virulence. This integration could indicate that virulence towards insects is a normal mode of life for D. dadantii.

High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol.

BACKGROUND: The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. METHODOLOGY/PRINCIPAL FINDINGS: Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae) yielded a sequence similarity of 99-100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. CONCLUSIONS/SIGNIFICANCE: In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome.