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During April 5-July 8, 2022, the World Health Organization (WHO) received reports of 1,010 probable cases of severe acute hepatitis of unknown etiology (SAHUE) in young children from 35 countries [...]

A large proportion of bacteria, from a multitude of environments, are not yet able to be grown in the laboratory, and therefore microbiological and molecular biological investigations of these bacteria are challenging. A way to circumvent this challenge is to analyze the metagenome, the entire collection of DNA molecules that can be isolated from a particular environment or sample. This collection of DNA molecules can be sequenced and assembled to determine what is present and infer functional potential, or used as a PCR template to detect known target DNA and potentially unknown regions of DNA nearby those targets; however assigning functions to new or conserved hypothetical, functionally cryptic, genes is difficult. Functional metagenomics allows researchers to determine which genes are responsible for selectable phenotypes, such as resistance to antimicrobials and metabolic capabilities, without the prerequisite needs to grow the bacteria containing those genes or to already know which genes are of interest. It is estimated that a third of the resident species of the human oral cavity is not yet cultivable and, together with the ease of sample acquisition, makes this metagenome particularly suited to functional metagenomic studies. Here we describe the methodology related to the collection of saliva samples, extraction of metagenomic DNA, construction of metagenomic libraries, as well as the description of functional assays that have previously led to the identification of new genes conferring antimicrobial resistance., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Tn 916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn 916 /Tn 1545 family of CTns. Tn 916 -like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn 916 -like elements also carry accessory, antimicrobial resistance genes; most commonly the tetracycline resistance gene, tet (M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet (M) and tet (L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn 916 -like elements indicated that a partial Tn 916 -like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn 916 -like transposons; demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn 916 -like element cloned in TT31 likely represents an ancestral Tn 916 .

Antimicrobial resistance is common in the microbial inhabitants of the human oral cavity. Antimicrobials are commonly encountered by oral microbes as they are present in our diet, both naturally and anthropogenically, and also used in oral healthcare products and amalgam fillings. We aimed to determine the presence of genes in the oral microbiome conferring reduced susceptibility to common antimicrobials. From an Escherichia coli library, 12,277 clones were screened and ten clones with reduced susceptibility to triclosan were identified. The genes responsible for this phenotype were identified as fabI, originating from a variety of different bacteria. The gene fabI encodes an enoyl-acyl carrier protein reductase (ENR), which is essential for fatty acid synthesis in bacteria. Triclosan binds to ENR, preventing fatty acid synthesis. By introducing the inserts containing fabI, ENR is likely overexpressed in E. coli, reducing the inhibitory effect of triclosan. Another clone was found to have reduced susceptibility to cetyltrimethylammonium bromide and cetylpyridinium chloride. This phenotype was conferred by a UDP-glucose 4-epimerase gene, galE, homologous to one from Veillonella parvula. The product of galE is involved in lipopolysaccharide production. Analysis of the E. coli host cell surface showed that the charge was more positive in the presence of galE, which likely reduces the binding of these positively charged antiseptics to the bacteria. This is the first time galE has been shown to confer resistance against quaternary ammonium compounds and represents a novel, epimerase-based, global cell adaptation, which confers resistance to cationic antimicrobials.