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Anaerobic clinical microbiology remains a challenge due to specialist culture requirements, coupled with the increase in and spread of antimicrobial resistance. The normal human microbiota is primarily composed of anaerobic bacteria, and is now recognised as a source of life-threatening anaerobic infection. More recent metataxonomic and metagenomic sequencing has extended interest in the potential role of the microbiota in a plethora of other aspects of human health, from obesity to mental health. In addition, the successful use of faecal microbiota transplants for the treatment of clostridial infection raises potential unchartered long-term consequences and possibilities.

In conjunction with Anaerobe 2019 and Anaerobe 2021, this collection will provide scientific insights into the future impact of anaerobic bacteria in human health and disease, addressing the implications of recent microbiota studies as well as the continued threat of emerging and re-emerging anaerobic infection.

This collection is open for submissions – please submit your article here, stating that your manuscript is part of the Anaerobe collection.

Collection Contents

11 results

    • Clinical utility of 16S rRNA PCR in pleural infection

      Pleural infections cause major morbidity and mortality, particularly amongst paediatric and elderly populations. The aetiology is broad, but pleural culture fails to yield a causative pathogen in approximately 40 % of cases. Alternative pathogen identification methods are therefore required. The aim of the study was to investigate the yield from and impact on patient care when performing 16S rRNA PCR on culture-negative pleural fluid specimens and to determine whether any individual laboratory parameters were associated with a positive 16S rRNA PCR result. We conducted a study on 90 patients with suspected pleural infection, who had a culture-negative pleural fluid specimen, which underwent 16S rRNA PCR analysis between August 2017 and June 2019. This study was undertaken at a large NHS Trust in London, UK. Thirty-one per cent of culture-negative pleural fluid specimens tested by 16S rRNA PCR yielded a positive PCR result. Our data demonstrated that 16S rRNA PCR detected a significantly higher proportion of (<0.0001) and fastidious, slow-growing and anaerobic pathogens (=0.0025) compared with culture-based methods. Of the 25 16S rRNA PCR results that were positive for a causative pathogen, 76 % had a direct impact on clinical management. No single laboratory variable was found to be associated with a positive 16S rRNA PCR result. The findings from our real-world evaluation highlight the importance of 16S rRNA PCR in confirming pleural infection when the aetiology is unknown, and its direct, positive impact on clinical management.

    • antivirulence activity of baicalin against

      is an enteric pathogen that causes a serious toxin-mediated colitis in humans. Bacterial exotoxins and sporulation are critical virulence components that contribute to pathogenesis, and disease transmission and relapse, respectively. Therefore, reducing toxin production and sporulation could significantly minimize pathogenicity and disease outcome in affected individuals.

      This study investigated the efficacy of a natural flavone glycoside, baicalin, in reducing toxin synthesis, sporulation and spore germination in .

      Hypervirulent isolates BAA 1870 or 1803 were cultured in brain heart infusion broth with or without the subinhibitory concentration (SIC) of baicalin, and incubated at 37 °C for 24 h under strictly anaerobic conditions. The supernatant was harvested after 24 h for determining toxin production by ELISA. In addition, a similar experiment was performed wherein samples were harvested for assessing total viable counts, and heat-resistant spore counts at 72 h of incubation. Furthermore, spore germination and spore outgrowth kinetics, with or without baicalin treatment, was measured in a plate reader by recording optical density at 600 nm. Finally, the effect of baicalin on toxin, sporulation and virulence-associated genes was investigated using real-time quantitative PCR.

      The SIC of baicalin significantly reduced toxin synthesis, sporulation and spore outgrowth when compared to control. In addition, genes critical for pathogenesis were significantly down-regulated in the presence of baicalin.

      Our results suggest that baicalin could potentially be used to control , and warrant future studies .

    • phage kills enteroaggregative without human microbiome dysbiosis

      Bacteriophage therapy can be developed to target emerging diarrhoeal pathogens, but doing so in the absence of microbiome disruption, which occurs with antibiotic treatment, has not been established.

      Identify a therapeutic bacteriophage that kills diarrhoeagenic enteroaggregative (EAEC) while leaving the human microbiome intact.

      Phages from wastewater in Portland, OR, USA were screened for bacteriolytic activity by overlay assay. One isolated phage, , was classified by electron microscopy and genome sequencing. A mouse model of infection determined whether the phage was therapeutic against EAEC. 16S metagenomic analysis of anaerobic cultures determined whether a normal human microbiome was altered by treatment.

      virus , a member of the strictly lytic family , killed a case-associated EAEC isolate from a child in rural Tennessee in a dose-dependent manner, and killed EAEC isolates from Columbian children. A single dose of (multiplicity of infection: 100) 1 day post-infection reduced EAEC recovered from mouse faeces. also killed EAEC when cultured anaerobically in the presence of human faecal bacteria. While the addition of EAEC reduced the β-diversity of the human microbiota, that of the cultures with either faeces alone, faeces with EAEC and , or with just phage was not different statistically.

      killed EAEC isolate EN1E-0007 and , while not altering the diversity of normal human microbiota in anaerobic culture, and thus could be part of an effective therapy for children in developing countries and those suffering from EAEC-mediated traveller’s diarrhoea without causing dysbiosis.

    • strains of dairy-product origin adhere to mucin and survive digestive juices

      There is an ever present need to isolate and characterize indigenous bacterial strains with potential probiotic health benefits for humans.

      of dairy origin was focused because of its propensity to adhere to the intestinal glycoprotein, mucin.

      The lactobacillus strains were screened for mucin adhesion, resistance to low pH and bile, autoaggregation, hydrophobicity, and survival in an digestion model. The cholesterol-lowering and oxalate-degrading effects of selected strains were also determined. Safety was assessed for haemolytic, mucinolytic and gelatinase activity, biogenic amine production, antibiotic resistance and phenol resistance. Expression of the 32-mmub adhesion-related gene was also measured following strain exposure to simulated gastrointestinal tract (GIT) digestion.

      The selected mucin-adhesive strains were tolerant to acid (pH 3.0) and bile (0.25 %) and demonstrated >85 % survival following simulated human digestion in the presence of milk. The digestive treatment did not affect the adhesive potential of PL20, and PL27, regardless of the food matrix. The simulated digestion had less effect on their adhesion than on the type strain and it also did not correlate with the mmub gene expression level as determined by qPCR. The selected strains exhibited cholesterol removal (36–44 %) and degraded oxalate (66–55 %). Neither of these strains exhibited undesirable characteristics.

      These preliminary findings suggest a functionality in the two strains of with high colonization potential on GIT mucosal membranes and possible health-promoting effects. This prima facie evidence suggests the need for further studies to test these probiotic candidates as live biotherapeutic agents .

    • efficacy of sodium selenite in reducing toxin production, spore outgrowth and antibiotic resistance in hypervirulent

      This study investigated the efficacy of the essential mineral, selenium (sodium selenite), in reducing the toxin production, spore outgrowth and antibiotic resistance of .

      Two hypervirulent isolates were cultured in brain heart infusion broth with and without a sub-minimum inhibitory concentration (sub-MIC) of sodium selenite, and the supernatant and bacterial pellet were harvested for total toxin quantitation and RT-qPCR analysis of toxin-encoding genes, respectively. Additionally, isolates were cultured in brain heart infusion broth containing 0.5 or 1× the minimum inhibitory concentration (MIC) of either ciprofloxacin or vancomycin with or without sub-MICs of sodium selenite. Further, the effect of sodium selenite on germination and spore outgrowth was also determined by exposing spores to a sub-MIC of sodium selenite in a germination medium and measuring the germination and outgrowth by measuring the optical density at 600  nm.

      Sodium selenite significantly reduced toxin synthesis, cytotoxicity and spore outgrowth. Further, the expression of the toxin production genes, and , was downregulated in the presence of sodium selenite, while sodium selenite significantly increased the sensitivity of to ciprofloxacin , but not vancomycin, as revealed by decreased bacterial growth in samples containing ciprofloxacin+selenium compared to the antibiotic control. Although the sub-MIC of sodium selenite did not inhibit spore germination, it was capable of completely inhibiting spore outgrowth.

      Our results suggest that sodium selenite could potentially be used to control and indicate that future studies are warranted.

    • The clinical significance of carbapenem-resistant Klebsiella pneumoniae rectal colonization in critically ill patients: from colonization to bloodstream infection

      Purpose . To highlight the clinical significance of carbapenem-resistant Klebsiella pneumoniae (CRKP) rectal colonization by examining the risk factors for CRKP rectal colonization and subsequent bloodstream infection (BSI) in critically ill patients.

      Methodology . Prospective study of CRKP rectal colonization in an intensive care unit (ICU) during a 39-month period. CRKP strains isolated from both the blood cultures and corresponding rectal specimens (n=96) of patients were screened by PCR for the presence of antibiotic resistance-associated genes. Molecular analyses were conducted to investigate the clonal relatedness of CRKP strains from the rectal and blood specimens.

      Results . Among the 498 patients, 226 were rectally colonized by CRKP, 48 of whom developed a CRKP BSI. The median time from hospital admission to the detection of CRKP rectal colonization was 8 days, while the median time from colonization to BSI was 4 days. The duration of ICU stay, patient/nurse ratio and prior use of antianaerobic antimicrobials were associated with CRKP rectal colonization. No specific factor was associated with BSIs in the colonized patients. The bla KPC-2 gene was detected in all 96 strains, which were all classified as sequence type ST-258. Representative pairs (n=48) of CRKP strains colonizing and infecting the same patient shared the same pulsotype.

      Conclusion . Our results indicate that hospitalized patients become infected with their colonizing strains, supporting the strong association between colonization and BSI. Limiting antianaerobic antimicrobial administration, reducing the duration of ICU stay and maintaining a low patient/nurse ratio are possible strategies to restrict rectal CRKP colonization in ICUs.

    • Examining diabetic heel ulcers through an ecological lens: microbial community dynamics associated with healing and infection

      Purpose. While some micro-organisms, such as Staphylococcus aureus , are clearly implicated in causing tissue damage in diabetic foot ulcers (DFUs), our knowledge of the contribution of the entire microbiome to clinical outcomes is limited. We profiled the microbiome of a longitudinal sample series of 28 people with diabetes and DFUs of the heel in an attempt to better characterize the relationship between healing, infection and the microbiome.

      Methodology. In total, 237 samples were analysed from 28 DFUs, collected at fortnightly intervals for 6 months or until healing. Microbiome profiles were generated by 16S rRNA gene sequence analysis, supplemented by targeted nanopore sequencing.

      Result/Key findings. DFUs which failed to heal during the study period (20/28, 71.4 %) were more likely to be persistently colonized with a heterogeneous community of micro-organisms including anaerobes and Enterobacteriaceae (log-likelihood ratio 9.56, P=0.008). During clinically apparent infection, a reduction in the diversity of micro-organisms in a DFU was often observed due to expansion of one or two taxa, with recovery in diversity at resolution. Modelling of the predicted species interactions in a single DFU with high diversity indicated that networks of metabolic interactions may exist that contribute to the formation of stable communities.

      Conclusion. Longitudinal profiling is an essential tool for improving our understanding of the microbiology of chronic wounds, as community dynamics associated with clinical events can only be identified by examining changes over multiple time points. The development of complex communities, particularly involving Enterobacteriaceae and strict anaerobes, may be contributing to poor outcomes in DFUs and requires further investigation.

    • RelA/DTD-mediated regulation of spore formation and toxin production by type A strain SM101

      RelA is a global regulator for stationary phase development in the model bacterium . The gene forms a bicistronic operon with the downstream gene. In this study, we evaluated the significance of RelA and DTD proteins in spore formation and toxin production by an important gastrointestinal pathogen . Our β-glucuronidase assay showed that in strain SM101, forms a bicistronic operon with its downstream gene, and the promoter is expressed during both vegetative and sporulation conditions. By constructing double and single mutants in SM101, we found that: (1) RelA is required for maintaining the efficient growth capacity of SM101 cells during vegetative conditions; (2) both RelA and DTD are required for spore formation and enterotoxin (CPE) production by SM101; (3) RelA/DTD activate CodY, which is known to activate spore formation and CPE production in SM101 by activating a key sporulation-specific σ factor F; (4) as expected, RelA/DTD activate sporulation-specific σ factors (σ, σ, σ and σ) by positively regulating Spo0A production; and finally (5) RelA, but not DTD, negatively regulates phospholipase C (PLC) production by repressing gene expression. Collectively, our results demonstrate that RelA modulates cellular physiology such as growth, spore formation and toxin production by type A strain SM101, although DTD also plays a role in these pleiotropic functions in coordination with RelA during sporulation. These findings have implications for the understanding of the mechanisms involved in the infectious cycle of .

    • hydrogen sulfide enhances methyl mercaptan-induced pathogenicity in mouse abscess formation

      produces hydrogen sulfide (HS) from -cysteine. However, the role of HS produced by in periodontal inflammation is unclear. In this study, we identified the enzyme that catalyses HS production from -cysteine and analysed the role of HS using a mouse abscess model. The enzyme identified was identical to methionine γ-lyase (PG0343), which produces methyl mercaptan (CHSH) from -methionine. Therefore, we analysed HS and CHSH production by W83 and a PG0343-deletion mutant (ΔPG0343) with/without -cysteine and/or -methionine. The results indicated that CHSH is produced constitutively irrespective of the presence of -methionine, while HS was greatly increased by both W83 and ΔPG0343 in the presence of -cysteine. In contrast, CHSH production by ΔPG0343 was absent irrespective of the presence of -methionine, and HS production was eliminated in the absence of -cysteine. Thus, CHSH and HS production involves different substrates, -methionine or -cysteine, respectively. Based on these characteristics, we analysed the roles of CHSH and HS in abscess formation in mice by W83 and ΔPG0343. Abscess formation by W83, but not ΔPG0343, differed significantly in the presence and absence of -cysteine. In addition, the presence of -methionine did not affect the size of abscesses generated by W83 and ΔPG0343. Therefore, we conclude that HS produced by does not induce inflammation; however, HS enhances inflammation caused by CHSH. Thus, these results suggest the HS produced by plays a supportive role in inflammation caused by methionine γ-lyase.

    • Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from

      ‘Ene’-reductases have attracted significant attention for the preparation of chemical intermediates and biologically active products. To date, research has been focussed primarily on Old Yellow Enzyme-like proteins, due to their ease of handling, whereas 2-enoate reductases from clostridia have received much less attention, because of their oxygen sensitivity and a lack of suitable expression systems. A hypothetical 2-enoate reductase gene, , was identified in DSM 795. The encoded protein shares a high degree of homology to clostridial FMN- and FAD-dependent 2-enoate reductases, including the cinnamic acid reductase proposed to be involved in amino acid metabolism in proteolytic clostridia. The gene was cloned and overexpressed in Successful expression depended on the use of strictly anaerobic conditions for both growth and enzyme preparation, since FldZ was oxygen-sensitive. The enzyme reduced aromatic enoates, such as cinnamic acid or -coumaric acid, but not short chain unsaturated aliphatic acids. The β,β-disubstituted nitroalkene, -1-nitro-2-phenylpropene, was reduced to enantiopure -1-nitro-2-phenylpropane with a yield of 90 %. By contrast, the β-disubstituted nitroalkene, -2-nitro-1-phenylpropene, was reduced with a moderate yield of 56 % and poor enantioselectivity (16 % ee for -2-nitro-1-phenylpropane). The availability of an expression system for this recombinant clostridial 2-enoate reductase will facilitate future characterisation of this unusual class of ‘ene’-reductases, and expand the biocatalytic toolbox available for enantioselective hydrogenation of carbon-carbon double bonds.

    • Discovery of a novel lantibiotic nisin O from A2-162, isolated from the human gastrointestinal tract

      A novel -like sequence was identified from the dominant human gut bacterium strain A2-162. This sequence was extended to reveal a putative lantibiotic operon with biosynthetic and transport genes, two sets of regulatory genes, immunity genes, three identical copies of a nisin-like gene with an unusual leader peptide, and a fourth putative gene. Comparison with other nisin clusters showed that the closest relationship was to nisin U. A2-162 demonstrated antimicrobial activity against when grown on solid medium in the presence of trypsin. Fusions of predicted structural sequences with the nisin A leader were expressed in containing the nisin A operon without . Expression of the leader sequence fused to the predicted structural produced a growth defect in that was dependent upon the presence of biosynthetic genes, but failed to produce antimicrobial activity. Insertion of the cluster into MG1614 gave an increased immunity to nisin A, but this was not replicated by the expression of . Nisin A induction of containing the cluster and genes allowed detection of the NsoA1 pre-peptide by Western hybridization. When this heterologous producer was grown with nisin induction on solid medium, antimicrobial activity was demonstrated in the presence of trypsin against , and . This research adds to evidence that lantibiotic production may be an important trait of gut bacteria and could lead to the development of novel treatments for intestinal diseases.

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