Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia.

IMPORTANCE: Continued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes, which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.

Curtanaerobium respiraculi gen. nov., sp. nov., a novel anaerobic bacterium isolated from human bronchoalveolar lavage fluid.

Two related anaerobic strains, designated as SWB101512T and SWB19611, were isolated from the bronchoalveolar lavage fluid of two lung cancer patients. Cells were Gram-stain-positive, non-motile and non-spore-forming. Growth could be observed at 26-45 °C (optimum, 37 °C), pH 5.0-8.5 (optimum, pH 7.0) and with 0.5-2.0 % (v/w) NaCl (optimum, 1.0%). The 16S rRNA gene sequences of SWB101512T and SWB19611 showed the highest similarities to Denitrobacterium detoxificans DSM 21843T (91.1 and 91.3 %, respectively). The phylogenetic tree based on the 16S rRNA gene sequences and the core genome sequences demonstrated that the two strains clustered together and formed a distinct lineage within the family Eggerthellaceae. The DNA G+C contents of strains SWB101512T and SWB19611 were 62.0 and 61.9 mol%, respectively. The predominant cellular fatty acids of strains SWB101512T and SWB19611 were C16 : 0 DMA (27.8 and 28.8 %, respectively). The respiratory menaquinone in both strains was menaquinone 6 and the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two phospholipids, three glycolipids and three unidentified lipids. Based on evidence from phenotypic, chemotaxonomic and genomic analyses, a new genus and species belonging to the family Eggerthellaceae, named Curtanaerobium respiraculi gen. nov., sp. nov. is proposed. The type strain is SWB101512T (=GDMCC 1.2991T=JCM 35330T).

Dehalogenimonas etheniformans sp. nov., a formate-oxidizing, organohalide-respiring bacterium isolated from grape pomace.

A strictly anaerobic, organohalide-respiring bacterium, designated strain GPT, was characterized using a polyphasic approach. GPT is Gram-stain-negative, non-spore-forming and non-motile. Cells are irregular cocci ranging between 0.6 and 0.9 µm in diameter. GPT couples growth with the reductive dechlorination of 1,2-dichloroethane, vinyl chloride and all polychlorinated ethenes, except tetrachloroethene, yielding ethene and inorganic chloride as dechlorination end products. H2 and formate serve as electron donors for organohalide respiration in the presence of acetate as carbon source. Major cellular fatty acids include C16 : 0, C18 : 1ω9c, C16 : 1, C14 : 0 and C18 : 0. On the basis of 16S rRNA gene phylogeny, GPT is most closely related to Dehalogenimonas formicexedens NSZ-14T and Dehalogenimonas alkenigignens IP3-3T with 99.8 and 97.4 % sequence identities, respectively. Genome-wide pairwise comparisons based on average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization do not support the inclusion of GPT in previously described species of the genus Dehalogenimonas with validly published names. On the basis of phylogenetic, physiological and phenotypic traits, GPT represents a novel species within the genus Dehalogenimonas, for which the name Dehalogenimonas etheniformans sp. nov. is proposed. The type strain is GPT (= JCM 39172T = CGMCC 1.17861T).

Lutispora saccharofermentans sp. nov., a mesophilic, non-spore-forming bacterium isolated from a lab-scale methanogenic landfill bioreactor digesting anaerobic sludge, and emendation of the genus Lutispora to include species which are non-spore-forming and mesophilic.

A novel anaerobic, mesophilic, non-spore-forming bacterium (strain m25T) was isolated from methanogenic enrichment cultures obtained from a lab-scale methanogenic landfill bioreactor containing anaerobic digester sludge. Cells were Gram-stain-negative, catalase-positive, oxidase-negative, rod-shaped, and motile by means of a flagellum. The genomic DNA G+C content was 40.11 mol%. The optimal NaCl concentration, temperature and pH for growth were 2.5 g l-1, 35 °C and at pH 7.0, respectively. Strain m25T was able to grow in the absence of yeast extract on glycerol, pyruvate, arginine and cysteine. In the presence of 0.2 % yeast extract, strain m25T grew on carbohydrates and was able to use glucose, cellobiose, fructose, raffinose and galactose. The novel strain could utilize glycerol, urea, pyruvate, peptone and tryptone. The major fatty acids were iso-C15  :  0, C14  :  0, C16  :  0 DMA (dimethyl acetal) and iso-C15 : 0 DMA. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate was closely related to Lutispora thermophila EBR46T (95.02 % 16S rRNA gene sequence similarity). Genome relatedness was determined using both average nucleotide identity and amino acid identity analyses, the results of which both strongly supported that strain m25T belongs to the genus Lutispora. Based on its unique phylogenetic features, strain m25T is considered to represent a novel species within the genus Lutispora. Moreover, based on its unique physiologic features, mainly the lack of spore formation, a proposal to amend the genus Lutispora is also provided to include the non-spore-forming and mesophilic species. Lutispora saccharofermentans sp. nov. is proposed. The type strain of the species is m25T (=DSM 112749T=ATCC TSD-268T).

Phocaeicola oris sp. nov., an anaerobic bacterium isolated from the saliva of a patient with oral squamous cell carcinoma.

A Gram-stain-negative or -positive, strictly anaerobic, non-spore-forming and pleomorphic bacterium (designated 14-104T) was isolated from the saliva sample of a patient with oral squamous cell carcinoma. It was an acid-tolerant neutralophilic mesophile, growing at between 20 and 40 °C (with optimum growth at 30 °C) and pH between pH 3.0 and 7.0 (with optimum growth at pH 6.0-7.0). It contained anteiso-C15 : 0 and C15 : 0 as the major fatty acids. The genome size of strain 14-104T was 2.98 Mbp, and the G+C content was 39.6 mol%. It shared <87 % 16S rRNA sequence similarity, <71 % orthologous average nucleotide identity, <76 % average amino acid identity and <68 %% of conserved proteins with its closest relative, Phocaeicola abscessus CCUG 55929T. Reconstruction of phylogenetic and phylogenomic trees revealed that strain 14-104T and P. abscessus CCUG 55929T were clustered as a distinct clade without any other terminal node. The phylogenetic and phylogenomic analyses along with physiological and chemotaxonomic data indicated that strain 14-104T represents a novel species in the genus Phocaeicola, for which the name Phocaeicola oris sp. nov. is proposed. The type strain is 14-104T (=BCRC 81305T= NBRC 115041T).

Fecal microbiota in patients with a stoma decreases anaerobic bacteria and alters taxonomic and functional diversities.

Colorectal cancer (CRC) is one of the most common malignant diseases. Generally, stoma construction is performed following surgery for the resection of the primary tumor in patients with CRC. The association of CRC with the gut microbiota has been widely reported, and the gut microbiota is known to play an important role in the carcinogenesis, progression, and treatment of CRC. In this study, we compared the microbiota of patients with CRC between with and without a stoma using fecal metagenomic sequencing data from SCRUM-Japan MONSTAR-SCREEN, a joint industry-academia cancer research project in Japan. We found that the composition of anaerobes was reduced in patients with a stoma. In particular, the abundance of Alistipes, Akkermansia, Intestinimonas, and methane-producing archaea decreased. We also compared gene function (e.g., KEGG Orthology and KEGG pathway) and found that gene function for methane and short-chain fatty acids (SCFAs) production was underrepresented in patients with a stoma. Furthermore, a stoma decreased Shannon diversity based on taxonomic composition but increased that of the KEGG pathway. These results suggest that the feces of patients with a stoma have a reduced abundance of favorable microbes for cancer immunotherapy. In conclusion, we showed that a stoma alters the taxonomic and functional profiles in feces and may be a confounding factor in fecal microbiota analysis.

Stygiolobus caldivivus sp. nov., a facultatively anaerobic hyperthermophilic archaeon isolated from the Unzen hot spring in Japan.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KN-1T, was isolated from Unzen hot spring in Japan and characterized. The cells of KN-1T were irregular cocci with a diameter of 1.0-3.0 µm that grew at 55-87.5 °C (optimum: 75 °C) and pH 1.0-5.5 (optimum: 3.0). Chemolithoautotrophic growth of KN-1T occurred in the presence of S0 or H2 under oxic conditions. Under anoxic conditions, KN-1T grew with S0, ferric citrate and FeCl3 as electron acceptors. A phylogenetic analysis of 16S rRNA gene sequences showed that the species most closely related to KN-1T was Stygiolobus azoricus JCM 9 021T, with 98.9 % sequence identity, indicating that strain KN-1T belongs to the genus Stygiolobus. This genus has been considered to consist of obligate anaerobes since its description in 1991. However, KN-1T grew under oxic, microoxic and anoxic conditions. Moreover, KN-1Tutilized various complex substrates and some sugars as carbon or energy sources, which is also different from S. azoricus JCM 9 021T. The average nucleotide identity and amino acid identity values between KN-1T and S. azoricus JCM 9 021T were 79.4 and 76.1 %, respectively, indicating that KN-1T represents a novel species. Its main polar lipids were calditoglycerocaldarchaeol and caldarchaeol, and its DNA G+C content was 40.1 mol%. We also found that S. azoricus JCM 9021T grew under microoxic conditions in the presence of H2 as an electron donor, indicating that this genus does not comprise obligate anaerobes. Based on this polyphasic taxonomic analysis, we propose the novel species, Stygiolobus caldivivus sp. nov., whose type strain is KN-1T (=JCM 34 622T=KCTC 4 293T).

Meta-omics-aided isolation of an elusive anaerobic arsenic-methylating soil bacterium.

Soil microbiomes harbour unparalleled functional and phylogenetic diversity. However, extracting isolates with a targeted function from complex microbiomes is not straightforward, particularly if the associated phenotype does not lend itself to high-throughput screening. Here, we tackle the methylation of arsenic (As) in anoxic soils. As methylation was proposed to be catalysed by sulfate-reducing bacteria. However, to date, there are no available anaerobic isolates capable of As methylation, whether sulfate-reducing or otherwise. The isolation of such a microorganism has been thwarted by the fact that the anaerobic bacteria harbouring a functional arsenite S-adenosylmethionine methyltransferase (ArsM) tested to date did not methylate As in pure culture. Additionally, fortuitous As methylation can result from the release of non-specific methyltransferases upon lysis. Thus, we combined metagenomics, metatranscriptomics, and metaproteomics to identify the microorganisms actively methylating As in anoxic soil-derived microbial cultures. Based on the metagenome-assembled genomes of microorganisms expressing ArsM, we isolated Paraclostridium sp. strain EML, which was confirmed to actively methylate As anaerobically. This work is an example of the application of meta-omics to the isolation of elusive microorganisms.

Clinical and microbiological features of anaerobic implant-related infection in 80 patients after orthopedic surgery.

OBJECTIVES: Implant-related infection is a common complication after orthopedic surgery, but there is limited research focused on anaerobic infections. We retrospectively analyzed data from 80 patients with anaerobic implant-related infections in order to investigate the clinical features, bacterial distribution and antimicrobial resistant characteristics of this disease. METHODS: 80 patients who underwent implant-related infections with anaerobes were included. Pathogens were isolated and identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry with verification of 16s rRNA sequencing. Antimicrobial susceptibility testing (AST) was performed using Epsilometric test (E-test). RESULTS: Among the 80 patients, 61.2% (49/80) were infected with anaerobes alone, while 38.8% (31/80) were co-infected with anaerobes and other bacteria. Early infection cases involving anaerobe-alone infections were significantly higher compared to the co-infection group (P < 0.001), also exhibiting lower levels of neutrophils (P = 0.033) and ESR (P = 0.046). Anaerobe-alone infections in the prosthetic joint infection group represented a higher proportion compared with other implant-related infections (P = 0.031). Among all species of anaerobes identified, the top 3 were Cutibacterium acnes, Finegoldia magna and Peptostreptococcus anaerobius. Low MIC values to vancomycin was recorded in C. acnes strains and for amoxicillin/clavulanic acid and piperacillin/tazobactam in most F. magna strains. One of the C. acnes and F. magna strains appeared multi-drug resistant except to vancomycin. CONCLUSIONS: Anaerobe-alone infections have later first onset times and lower infection biomarker levels compared to co-infected patients. The first choice against C. acnes is vancomycin, while amoxicillin/clavulanic acid and piperacillin/tazobactam are recommended for F. magna.

The Urinary Microbiome in Postmenopausal Women with Recurrent Urinary Tract Infections.

PURPOSE: The etiology of postmenopausal recurrent urinary tract infection (UTI) is not completely known, but the urinary microbiome is thought to be implicated. We compared the urinary microbiome in menopausal women with recurrent UTIs to age-matched controls, both in the absence of acute infection. MATERIALS AND METHODS: This is a cross-sectional analysis of baseline data from 64 women enrolled in a longitudinal cohort study. All women were using topically applied vaginal estrogen. Women >55 years of age from the following groups were enrolled: 1) recurrent UTIs on daily antibiotic prophylaxis, 2) recurrent UTIs not on antibiotic prophylaxis and 3) age-matched controls without recurrent UTIs. Catheterized urine samples were collected at least 4 weeks after last treatment for UTI and at least 6 weeks after initiation of vaginal estrogen. Samples were evaluated using expanded quantitative urine culture (EQUC) and 16S rRNA gene sequencing. RESULTS: With EQUC, there were no significant differences in median numbers of microbial species isolated among groups (p=0.96), even when considering Lactobacilli (p=0.72). However, there were trends toward different Lactobacillus species between groups. With 16S rRNA sequencing, the majority of urine samples contained Lactobacillaceae, with nonsignificant trends in relative abundance among groups. Using a Bayesian analysis, we identified significant differences in anaerobic taxa associated with phenotypic groups. Most of these differences centered on Bacteroidales and the family Prevotellaceae, although differences were also noted in Actinobacteria and certain genera of Clostridiales. CONCLUSIONS: Associations between anaerobes within the urinary microbiome and postmenopausal recurrent UTI warrants further investigation.

Anaerobic Microbial Metabolism of Dichloroacetate.

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising "Candidatus Dichloromethanomonas elyunquensis" strain RM utilizes DCA as an energy source, and the transient formation of formate, H2, and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated "Candidatus Dichloromethanomonas elyunquensis" strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg-1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.IMPORTANCE Dichloroacetate (DCA) is ubiquitous in the environment due to natural formation via biological and abiotic chlorination processes and the turnover of chlorinated organic materials (e.g., humic substances). Additional sources include DCA usage as a chemical feedstock and cancer drug and its unintentional formation during drinking water disinfection by chlorination. Despite the ubiquitous presence of DCA, its fate under anoxic conditions has remained obscure. We discovered an anaerobic bacterium capable of metabolizing DCA, identified the enzyme responsible for DCA dehalogenation, and elucidated a novel DCA fermentation pathway. The findings have implications for the turnover of DCA and the carbon and electron flow in electron acceptor-depleted environments and the human gastrointestinal tract.

Anaerobic bacterial communities associated with oral carcinoma: Intratumoral, surface-biofilm and salivary microbiota.

It was estimated that more than 700 bacterial species inhabit the oral cavity of healthy humans. Anaerobes comprise a significant fraction of the oral bacteriome and play an important role in the formation of multi-species biofilms attached to various anatomical sites. Bacterial biofilms are also associated with pathologic laesions of the oral cavity, including oral squamous cell carcinoma (OSCC), and distinct oral taxa could also be detected within the tumors, i.e. in deep biopsy samples. These observations suggested that certain oral bacteria or oral bacterial communities may play a causative role in oral carcinogenesis, in addition to the well characterized risk factors of oral cancer. Alternatively, it was also proposed that a subset of oral bacteria may have a growth advantage in the unique microenvironment of OSCC. Recently, a series of studies analysed the OSCC-associated bacterial communities using metataxonomic, metagenomic and metatranscriptomic approaches. This review outlines the major differences between the community structure of microbiota in tumor biopsy, surface-biofilm and salivary or oral wash samples collected from OSCC patients, compared to corresponding samples from control persons. A special emphasis is given to the anaerobic bacteria Fusobacterium nucleatum and Fusobacterium periodonticum that were characterised repeatedly as "OSCC-associated" in independent studies. Predicted microbial functions and relevant in vivo experimental models of oral carcinogenesis will also be summarized.

Disruption of Cross-Feeding Inhibits Pathogen Growth in the Sputa of Patients with Cystic Fibrosis.

A critical limitation in the management of chronic polymicrobial infections is the lack of correlation between antibiotic susceptibility testing (AST) and patient responses to therapy. Underlying this disconnect is our inability to accurately recapitulate the in vivo environment and complex polymicrobial communities in vitro However, emerging evidence suggests that, if modeled and tested accurately, interspecies relationships can be exploited by conventional antibiotics predicted to be ineffective by standard AST. As an example, under conditions where Pseudomonas aeruginosa relies on cocolonizing organisms for nutrients (i.e., cross-feeding), multidrug-resistant P. aeruginosa may be indirectly targeted by inhibiting the growth of its metabolic partners. While this has been shown in vitro using synthetic bacterial communities, the efficacy of a "weakest-link" approach to controlling host-associated polymicrobial infections has not yet been demonstrated. To test whether cross-feeding inhibition can be leveraged in clinically relevant contexts, we collected sputa from cystic fibrosis (CF) subjects and used enrichment culturing to isolate both P. aeruginosa and anaerobic bacteria from each sample. Predictably, both subpopulations showed various antibiotic susceptibilities when grown independently. However, when P. aeruginosa was cultured and treated under cooperative conditions in which it was dependent on anaerobic bacteria for nutrients, the growth of both the pathogen and the anaerobe was constrained despite their intrinsic antibiotic resistance profiles. These data demonstrate that the control of complex polymicrobial infections may be achieved by exploiting obligate or facultative interspecies relationships. Toward this end, in vitro susceptibility testing should evolve to more accurately reflect in vivo growth environments and microbial interactions found within them.IMPORTANCE Antibiotic efficacy achieved in vitro correlates poorly with clinical outcomes after treatment of chronic polymicrobial diseases; if a pathogen demonstrates susceptibility to a given antibiotic in the lab, that compound is often ineffective when administered clinically. Conversely, if a pathogen is resistant in vitro, patient treatment with that same compound can elicit a positive response. This discordance suggests that the in vivo growth environment impacts pathogen antibiotic susceptibility. Indeed, here we demonstrate that interspecies relationships among microbiotas in the sputa of cystic fibrosis patients can be targeted to indirectly inhibit the growth of Pseudomonas aeruginosa The therapeutic implication is that control of chronic lung infections may be achieved by exploiting obligate or facultative relationships among airway bacterial community members. This strategy is particularly relevant for pathogens harboring intrinsic multidrug resistance and is broadly applicable to chronic polymicrobial airway, wound, and intra-abdominal infections.

The Surface Microbiome of Clinically Unaffected Skinfolds in Hidradenitis Suppurativa: A Cross-Sectional Culture-Based and 16S rRNA Gene Amplicon Sequencing Study in 60 Patients.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease of the skin associated with specific lesional dysbiotic features. We studied the microbiome of clinically unaffected typical HS sites (armpits, inguinal folds, and gluteal clefts) in 60 patients with HS and 17 healthy controls. A total of 192 samples obtained by swabbing were analyzed by bacterial cultures. Of these, 116 randomly selected samples were studied by 16S rRNA gene amplicon sequencing. Patients and controls showed similar characteristics, except for smoking (87% vs. 6%, respectively). HS skinfolds were characterized by an increased abundance of anaerobes, predominantly Prevotella, but also Actinomyces, Campylobacter ureolyticus, and Mobiluncus, contrasting with a lower abundance of skin commensals such as Staphylococcus epidermidis, a major component of the skin microbiome; Kocuria; and Micrococcus luteus. The following three independent factors were associated with an abundance of high anaerobes by multivariate analysis: samples originating from patients with HS patients (P = 2.1 × 10-4); body mass index (P = 5 × 10-5); and the sampling site, the gluteal cleft being the most anaerobic area, followed by inguinal folds and axilla (P = 3 × 10-6). The microbiome of clinically unaffected HS skinfolds is reminiscent, albeit to a minor extent, of the microbiome of chronic suppurative HS lesions and may fuel inflammation at a preclinical stage of the disease.

Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy.

Closthioamide (CTA) is a rare example of a thioamide-containing nonribosomal peptide and is one of only a handful of secondary metabolites described from obligately anaerobic bacteria. Although the biosynthetic gene cluster responsible for CTA production and the thioamide synthetase that catalyzes sulfur incorporation were recently discovered, the logic for peptide backbone assembly has remained a mystery. Here, through the use of in vitro biochemical assays, we demonstrate that the amide backbone of CTA is assembled in an unusual thiotemplated pathway involving the cooperation of a transacylating member of the papain-like cysteine protease family and an iteratively acting ATP-grasp protein. Using the ATP-grasp protein as a bioinformatic handle, we identified hundreds of such thiotemplated yet nonribosomal peptide synthetase (NRPS)-independent biosynthetic gene clusters across diverse bacterial phyla. The data presented herein not only clarify the pathway for the biosynthesis of CTA, but also provide a foundation for the discovery of additional secondary metabolites produced by noncanonical biosynthetic pathways.

An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters.

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.

Overuse of antianaerobic drug is associated with poor postchemotherapy prognosis of patients with hepatocellular carcinoma.

Overuse of antibiotic drugs alters the composition of gut microbiota and has detrimental effects on the host. In our study, we investigated association of gut flora and antibiotics in the prognosis of patients with liver cancer who have undergone chemotherapy by analyzing two independent clinical studies. We retrospectively subanalyzed a previously reported randomized controlled trial (RCT) on hepatic arterial infusion chemotherapy in patients with hepatocellular carcinoma (HCC) to investigate the association between use of antibiotics and prognosis. In the other study, we prospectively determined the abundance of specific bacterial genus in patients with HCC by sequencing 16S ribosomal RNA and assessed its association with survival. Subanalysis of the RCT data showed that, of 26 types of antibiotics used, administration of carbapenem before or during chemotherapy was associated with poor progression-free survival (PFS) and overall survival (OS) of patients with HCC (carbapenem + vs. -; median PFS, 78 days vs. 154 days, p = 0.0053; median OS, 177 days vs. 475 days, p = 0.0003). Multivariate analysis revealed that antianaerobic drug use is an independent predictor of poor prognosis. In the prospective study, the abundance of Blautia in fecal microbiota correlated positively with both PFS and OS of patients with HCC who underwent chemotherapy. Use of antibiotics targeting anaerobes is associated with a poor prognosis in patients with HCC who have undergone chemotherapy, whereas the intestinal anaerobic bacteria, Blautia is associated with a good prognosis. These findings might indicate the need for caution regarding overuse of broad-spectrum antibiotics targeting anaerobes in patients with HCC.

Susceptibility of endometrial isolates recovered from women with clinical pelvic inflammatory disease or histological endometritis to antimicrobial agents.

The CDC recommended outpatient treatment of pelvic inflammatory disease (PID) is an intramuscular dose of ceftriaxone plus 14 days of doxycycline, with or without metronidazole. European guidelines (2017) include moxifloxacin plus ceftriaxone as a first line regimen, particularly for women with Mycoplasma genitalium-associated PID. However, the susceptibility of bacteria recovered from the endometrium of women with PID to moxifloxacin is unknown. The in vitro antibiotic susceptibility of facultative and anaerobic bacteria recovered from endometrial biopsy samples were evaluated from 105 women having symptomatic PID and/or histologically confirmed endometritis. A total of 342 endometrial isolates from enrollment visits were identified using a combination of biochemical tests and sequencing. Isolates were tested for antimicrobial susceptibility using agar dilution against ceftriaxone, clindamycin, doxycycline, metronidazole and moxifloxacin according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Neisseria gonorrhoeae was susceptible to ceftriaxone with all isolates having an MIC of 0.03 µg/mL. All the other endometrial isolates were susceptible to ceftriaxone, except for Prevotella species, only half of which were susceptible. The in vitro susceptibility profile for BV-associated bacteria (Gardnerella vaginalis, Atopobium vaginae, Prevotella species, Porphyromonas species and anaerobic gram-positive cocci) revealed greater susceptibility to moxifloxacin compared to doxycycline. Moxifloxacin was superior to metronidazole for G. vaginalis and A. vaginae, and either metronidazole or moxifloxacin was needed to cover Prevotella species. Based on in vitro susceptibility testing, the combination of ceftriaxone plus moxifloxacin provides similar coverage of facultative and anaerobic pathogens compared to the combination of ceftriaxone, metronidazole and doxycycline. Head to head clinical studies of these treatment regimens are needed to evaluate clinical efficacy and eradication of endometrial pathogens following treatment.

Natural products from anaerobes.

Natural product discovery in the microbial world has historically been biased toward aerobes. Recent in silico analysis demonstrates that genomes of anaerobes encode unexpected biosynthetic potential for natural products, however, chemical data on natural products from the anaerobic world are extremely limited. Here, we review the current body of work on natural products isolated from strictly anaerobic microbes, including recent genome mining efforts to discover polyketides and non-ribosomal peptides from anaerobes. These known natural products of anaerobes have demonstrated interesting molecular scaffolds, biosynthetic logic, and/or biological activities, making anaerobes a promising reservoir for future natural product discovery.

Comparative genomic analysis of Geosporobacter ferrireducens and its versatility of anaerobic energy metabolism.

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H+ and Na+ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na+-dependent F-type ATPase and H+-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.