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bioRxiv2017

A semi-lethal CRISPR-Cas system permits DNA acquisition in Enterococcus faecalis

Antibiotic resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Enterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack CRISPR-Cas genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in E. faecalis is that MGEs that are targeted by native CRISPR-Cas systems can be transiently maintained. Here, we investigate the basis for this \"CRISPR tolerance.\" We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, DNA repair genes were not up-regulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient non-lethality and use this knowledge to develop a robust CRISPR-assisted genome editing scheme. Our results suggest that E. faecalis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.\n\nImportanceCRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently non-lethal. We uncover novel phenotypes associated with this \"CRISPR tolerance\" and, after determining its genetic basis, develop a genome editing platform in E. faecalis with negligible off-target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR-induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.

microbiology↗

bioRxiv2017

High-dimensional microbiome interactions shape host fitness

Gut bacteria can affect key aspects of host fitness, such as development, fecundity, and lifespan, while the host in turn shapes the gut microbiome. Microbiomes co-evolve with their hosts and have been implicated in host speciation. However, it is unclear to what extent individual species versus community interactions within the microbiome are linked to host fitness. Here we combinatorially dissect the natural microbiome of Drosophila melanogaster and reveal that interactions between bacteria shape host fitness through life history tradeoffs. We find that the same microbial interactions that shape host fitness also shape microbiome abundances, suggesting a potential evolutionary mechanism by which microbiome communities (rather than just individual species) may be intertwined in co-selection with their hosts. Empirically, we made germ-free flies colonized with each possible combination of the five core species of fly gut bacteria. We measured the resulting bacterial community abundances and fly fitness traits including development, reproduction, and lifespan. The fly gut promoted bacterial diversity, which in turn accelerated development, reproduction, and aging: flies that reproduced more died sooner. From these measurements we calculated the impact of bacterial interactions on fly fitness by adapting the mathematics of genetic epistasis to the microbiome. Host physiology phenotypes were highly dependent on interactions between bacterial species. Higher-order interactions (involving 3, 4, and 5 species) were widely prevalent and impacted both host physiology and the maintenance of gut diversity. The parallel impacts of bacterial interactions on the microbiome and on host fitness suggest that microbiome interactions may be key drivers of evolution.\n\nSignificanceAll animals have associated microbial communities called microbiomes that can influence the physiology and fitness of their host. It is unclear to what extent individual microbial species versus ecology of the microbiome influences fitness of the host. Here we mapped all the possible interactions between individual species of bacteria with each other and with the hosts physiology. Our approach revealed that the same bacterial interactions that shape microbiome abundances also shape host fitness traits. This relationship provides a feedback that may favor the emergence of co-evolving microbiome-host units.

microbiology↗

bioRxiv2017

Copy number variation in fungi and its implications for wine yeast genetic diversity and adaptation

In recent years, copy number (CN) variation has emerged as a new and significant source of genetic polymorphisms contributing to the phenotypic diversity of populations. CN variants are defined as genetic loci that, due to duplication and deletion, vary in their number of copies across individuals in a population. CN variants range in size from 50 base pairs to whole chromosomes, can influence gene activity, and are associated with a wide range of phenotypes in diverse organisms, including the budding yeast Saccharomyces cerevisiae. In this review, we introduce CN variation, discuss the genetic and molecular mechanisms implicated in its generation, how they can contribute to genetic and phenotypic diversity in fungal populations, and consider how CN variants may influence wine yeast adaptation in fermentation-related processes. In particular, we focus on reviewing recent work investigating the contribution of changes in CN of fermentation-related genes associated with the adaptation and domestication of yeast wine strains and offer notable illustrations of such changes, including the high levels of CN variation among the CUP genes, which confer resistance to copper, and the preferential deletion and duplication of the MALI and MAL3 loci, respectively, which are responsible for metabolizing maltose and sucrose. Based on the available data, we propose that CN variation is a substantial dimension of yeast genetic diversity that occurs largely independent of single nucleotide polymorphisms. As such, CN variation harbors considerable potential for understanding and manipulating yeast strains in the wine fermentation environment and beyond.

microbiology↗

bioRxiv2017

The impact of HIV-1 within-host evolution on transmission dynamics

The adaptive potential of HIV-1 is a vital mechanism to evade host immune responses and antiviral treatment. However, high evolutionary rates during persistent infection can impair transmission efficiency and alter disease progression in the new host, resulting in a delicate trade-off between within-host virulence and between-host infectiousness. This trade-off is visible in the disparity in evolutionary rates at within-host and between-host levels, and preferential transmission of ancestral donor viruses. Understanding the impact of within-host evolution for epidemiological studies is essential for the design of preventive and therapeutic measures. Herein, we review recent theoretical and experimental work that generated new insights into the complex link between within-host evolution and between-host fitness, revealing temporal and selective processes underlying the structure and dynamics of HIV-1 transmission.

microbiology↗

bioRxiv2017

Archaeal community structure and underlying ecological processes in swine manure slurry

The ecological processes underlying the observed patterns in community composition of archaea in swine manure slurry are poorly understood. We studied the archaeal communities from six swine manure slurry storage tanks using paired-end Illumina sequencing of the v3 hypervariable region of 16S rRNA gene. Across all samples, the archaeal community was dominated by methanogens related to Thermoplasmata, Methanomicrobia, and Methanobacteria classes. At the genus level, the archaeal community was dominated by a single uncultured lineage of archaea, vadinCA11, followed by methanogenic genera Methanobrevibacter, Methanosarcina, Methanosphaera, Methanogenium, Methanocorpusculum, Methanoculleus, and Methanomicrococcus. Significant phylogenetic signals were detected across relatively short phylogenetic distances, indicating that closely related archaeal operational taxonomic units (OTUs) tend to have similar niches. The standardized effect sizes of mean nearest taxon distance (SES.MNTD) showed that archaeal community was phylogenetically clustered, suggesting that environmental filtering deterministically influence the within-community composition of archaea. However, between-community analysis based on {beta}-nearest taxon index ({beta}NTI) revealed that both deterministic selection and stochastic processes operate simultaneously to govern the assembly of archaeal communities. These findings significantly enhance our understanding of archaeal community assembly and underlying ecological processes is swine manure slurry.

microbiology↗

bioRxiv2017

Drivers of antimicrobial resistance amongst intestinal Escherichia coli isolated from children in South Asia and sub-Saharan Africa

Antimicrobial resistance (AMR) dynamics are poorly understood in developing countries, where data on the prevalence of AMR in enteric bacteria are sparse, particularly among children and in the community setting. Here we use a combination of phenotyping, genomics and antimicrobial usage data to investigate patterns of AMR amongst atypical enteropathogenic E. coli (aEPEC) strains isolated from children <5 years old in seven countries (four in sub-Saharan Africa and three in South Asia) over a three-year period. We detected very high rates of AMR, with 65% of isolates displaying resistance to [≥]3 drug classes; the rates of AMR were the same amongst strains associated with diarrhea and strains that were carried asymptomatically. Whole genome sequencing identified a diversity of genetic mechanisms for AMR, which could explain >95% of observed phenotypic resistance. Analysis of AMR gene co-occurrence revealed clusters of acquired AMR genes that were frequently co-located on small plasmids and transposons, providing opportunities for acquisition of multidrug resistance in a single step. We used discriminant analysis to investigate potential drivers of AMR within the bacterial population, and found that genetic determinants of AMR were associated with geographical location of isolation but not with phylogenetic lineage of the E. coli strain or disease status of the human host. Comparison with antimicrobial usage data showed that the prevalence of resistance to newer drugs (fluoroquinolones and third-generation cephalosporins) was correlated with usage, which was generally higher in South Asia than Africa. In particular, fluoroquinolone resistance-associated mutations in gyrA were significantly associated with use of these drugs for treatment of diarrheic children. Notably resistance to older drugs such as trimethoprim, chloramphenicol and ampicillin, which are conferred by acquired AMR genes that were frequently clustered together in mobile genetic elements, were common in all locations despite differences in usage; this suggests that reversion to sensitivity is unlikely to occur even if these drugs are removed from circulation. This study provides much-needed insights into the frequencies of AMR in intestinal E. coli in community-based children in developing countries and to antimicrobial usage for diarrhea where the burden of infections is greatest.

microbiology↗

bioRxiv2017

Dynamic protein aggregation regulates bacterial dormancy depth critical for antibiotic tolerance

The ability of some bacteria within a population to tolerate antibiotic treatment is often attributed to prolonged bacterial infection1-3. Unlike antibiotic resistance, which generally results from genetic mutations or plasmid transfer4,5, antibiotic tolerance usually refers to the phenomenon that a subgroup of cells can survive high dose antibiotic treatment as a result of phenotypic heterogeneity6,7. Previous studies mainly associate antibiotic tolerance with cell dormancy, by hypothesizing that the lethal effects of antibiotics are disabled due to the extremely slow metabolic and proliferation rates in dormant bacteria 8,9. However, less is known about how surviving bacteria subsequently escape from the dormant state and resuscitate, which is equally important for disease recurrence. Here we monitored the process of bacterial antibiotic tolerance and regrowth at the single-cell level, and found that each individual survival cell shows different dormancy depth, which in return regulates whether and when it can resume growth after removal of antibiotic. The persister cells are considered to be in shallow dormancy depth, while the viable but non-culturable cells (VBNC cells) are in deep dormancy depth. We further implemented time-lapse fluorescent imaging and biochemical analysis to establish that dynamic endogenous protein aggregation is an important indicator of bacterial dormancy depth. For cells to leave the dormant state and resuscitate, clearance of cellular protein aggregates and recovery of proteostasis are required. Through additional mutagenesis studies, we found the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the timeline (whether and when) for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.

microbiology↗

bioRxiv2017

A new method to isolate algal species from mix algal culture

To meet the issues of energy and environment, algae cultivation for biofuel and CO2 sequestration is getting popular at the global level. Specific algal strains have been identified for production of biofuel, biomolecules and biomass. To start algae cultivation at lab or industrial scale, it is requirement to have isolated and identified algal culture for targeted products. Water sample for algae from aquatic system is usually consist of mix culture of algae and need to be processed for targeted isolated algal strains using reported techniques like streaking, spraying, serial dilution, and single-cell isolations. But none of these techniques is considered as efficient or popular except streaking on agar plate which involves a set of microbial techniques and may take months to make isolation properly. Here, a new method is proposed to make alginic acid solution using aquatic sample followed by pouring it in calcium chloride solution drop by drop which makes the beads with single or more algal species trapped in each bead. The trapped algal species in the beads are grown in 96 wells plate having single bead in each well with standard medium leading to microscopic verification of the isolated algal species to process further. A mix culture from a lake was subjected to isolation using proposed method and excellent results were obtained in one week duration.

microbiology↗

bioRxiv2017

The Escherichia coli CpxAR system does not sense surface contact

For proper biofilm formation, bacteria must have mechanisms in place to sense adhesion to surfaces. In Escherichia coli, the CpxAR and RcsCDB systems have been reported to sense surfaces. The CpxAR system is widely considered to be responsible for sensing attachment, to specifically hydrophobic surfaces. Here, using both single-cell and population-level analyses, we confirm RcsCDB activation upon surface contact, but find that the CpxAR system is not activated, in contrast to what had earlier been reported. Thus, the role of CpxAR in surface sensing and initiation of biofilm formation needs to be reconsidered.\n\nSignificance statementE. coli is capable of forming medically problematic biofilms, which are surface-associated microbial communities, protected by an exopolymeric matrix and with increased antibiotic tolerance. How these bacteria sense physical contact with a surface, which may lead to initiation of the biofilm formation process, is largely elusive. The signal transduction systems CpxAR and RcsCDB have previously been found to activate upon surface contact. Here, we confirm that RcsCDB is a surface sensing system, immediately responding to attachment. In contrast, using two different experimental approaches, we found that the CpxAR system does not perceive surface contact. Thus, contrary to the current view, the CpxAR system does not play a signaling role in the first step of biofilm initiation.

microbiology↗

bioRxiv2017

Prevalence, mechanisms and comparison of detection methods of fosfomycin resistance in E. coli from urinary tract infections

As numbers of bacterial isolates resistant to first line antibiotics rise there has been a revival in the use of older drugs such as fosfomycin. Fosfomycin is a cell wall inhibitor with a unique mode of action, increasingly used in the treatment of urinary tract infections. In this study, the prevalence of fosfomycin resistant E. coli in a panel of 1000 urine isolates was investigated. Three different clinically used fosfomycin susceptibility testing methods were assessed and genome sequencing used to characterise resistant isolates.\n\nOf the 1000 isolates, 676 were E. coli of which initial susceptibility testing with the MAST Uri(R)system suggested 81 (12%) were fosfomycin resistant. Of these, 62 were subsequently confirmed as being E. coli. However, using micro-broth dilution, agar dilution and E-test strips, a lower rate of 1.3% (8/62) of E. coli isolates were robustly identified as being truly fosfomycin resistant; a prevalence comparable with other similar studies. The use of E-test and 96-well breakpoint plates gave results that were inconsistent and hard to interpret. Resistant isolates of E. coli belonged to diverse MLST types and each had a unique set of chromosomal alterations in genes associated with fosfomycin resistance. Changes in GlpT and UhpT/UhpA transport systems were commonly identified, with 6/8 of the resistant isolates possessing amino-acid changes or deletions absent in susceptible strains. Fosfomycin resistant isolates were not multiply drug resistance and did not carry plasmidic fosfomycin resistance genes. Therefore, the use of fosfomycin may be unlikely to drive selection of a particular clone or movement of transferrable resistance genes.\n\nFosfomycin remains a viable option for the treatment of E. coli in uncomplicated UTIs, different susceptibility testing platforms can give very different results regarding the prevalence of fosfomycin resistance with false positives a potential problem that may unnecessarily limit use of this agent.

microbiology↗

bioRxiv2017

Amplicon sequencing of the 16S-ITS-23S rRNA operon with long-read technology for improved phylogenetic classification of uncultured prokaryotes

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions of environmental samples and to discover previously unknown lineages. Its unique structure of interspersed conserved and variable regions is an excellent target for PCR and allows for classification of reads at all taxonomic levels. However, the relatively few phylogenetically informative sites prevent confident phylogenetic placements of novel lineages that are deep branching relative to reference taxa. This problem is exacerbated when only short 16S rRNA gene fragments are sequenced. To resolve their placement, it is common practice to gather more informative sites by combining multiple conserved genes into concatenated datasets. This however requires genomic data which may be obtained through relatively expensive metagenome sequencing and computationally demanding analyses. Here we develop a protocol that amplifies a large part of 16S and 23S rRNA genes within the rRNA operon, including the ITS region, and sequences the amplicons with PacBio long-read technology. We tested our method with a synthetic mock community and developed a read curation pipeline that reduces the overall error rate to 0.18%. Applying our method on four diverse environmental samples, we were able to capture near full-length rRNA operon amplicons from a large diversity of prokaryotes. Phylogenetic trees constructed with these sequences showed an increase in statistical support compared to trees inferred with shorter, Illumina-like sequences using only the 16S rRNA gene (250 bp). Our method is a cost-effective solution to generate high quality, near full-length 16S and 23S rRNA gene sequences from environmental prokaryotes.

microbiology↗

bioRxiv2017

The first recorded incidence of Deinococcus radiodurans R1 biofilm formation and its implications in heavy metals bioremediation

Radiation tolerant Deinococcus radiodurans R1 is reported to be a potential bacterium for the treatment of low level active wastes. So far there are no reports on the biofilm producing capability of D. radiodurans and heavy metal biosorption. In this study, it was observed that a recombinant D. radiodurans strain with a plasmid harbouring gfp and kanR has formed significant biofilm (~10 m thick). Analysis of biofilm matrix components produced by D. radiodurans showed that the matrix consisted primarily of proteins and carbohydrates with a little amount of extracellular DNA (eDNA). Further, studies showed that D. radiodurans biofilm formation was enhanced at higher concentrations (up to 25 mM) of Ca2+. Further studies on D. radiodurans biofilm showed that Ca2+ enhanced significant biosorption of the heavy metals (Co, Ni). In the presence of 25 mM Ca2+, the D. radiodurans (Kanr) biofilm showed 35% and 25% removal of Co2+ and Ni2+ respectively. While in the absence of Ca2+, D. radiodurans biofilm showed relatively low biosorption of Co (7%) and Ni (3%). Ca2+ also significantly enhanced exopolysaccharide (EPS) production in the biofilm matrix. This infers that EPS could have mediated the heavy metal biosorption. This study signifies the potential use of D. radiodurans biofilm in the remediation of radioactive waste components.\n\nSignificance and Impact of this StudyThis is the first ever recorded study on the Deinococcus radiodurans R1 biofilm. This organism, being the most radioresistant micro-organism ever known, has always been speculated as a potential bacterium to develop a bioremediation process for radioactive heavy metal contaminants. However, the lack of biofilm forming capability proved to be a bottleneck in developing such technology. This study records the first incidence of biofilm formation in a recombinant D. radiodurans, serendipitously, and also discusses its implications in removal of heavy-metals, such as Co and Ni.

microbiology↗

bioRxiv2017

Cultivation and genomic analysis of Candidatus Nitrosocaldus islandicus, a novel obligately thermophilic ammonia-oxidizing Thaumarchaeon

Ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaea are the only known aerobic ammonia oxidizers in geothermal environments. Although molecular data indicate the presence of phylogenetically diverse AOA from the Nitrosocaldus clade, group 1.1b and group 1.1a Thaumarchaea in terrestrial high-temperature habitats, only one enrichment culture of an AOA thriving above 50 {degrees}C has been reported and functionally analyzed. In this study, we physiologically and genomically characterized a novel Thaumarchaeon from the deep-branching Nitrosocaldaceae family of which we have obtained a high ([~]85 %) enrichment from biofilm of an Icelandic hot spring (73 {degrees}C). This AOA, which we provisionally refer to as \"Candidatus Nitrosocaldus islandicus\", is an obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer, which stoichiometrically converts ammonia to nitrite at temperatures between 50 {degrees}C and 70 {degrees}C. Ca. N. islandicus encodes the expected repertoire of enzymes proposed to be required for archaeal ammonia oxidation, but unexpectedly lacks a nirK gene and also possesses no identifiable other enzyme for nitric oxide (NO) generation. Nevertheless, ammonia oxidation by this AOA appears to be NO-dependent as Ca. N. islandicus is, like all other tested AOA, inhibited by the addition of an NO scavenger. Furthermore, comparative genomics revealed that Ca. N. islandicus has the potential for aromatic amino acid fermentation as its genome encodes an indolepyruvate oxidoreductase (iorAB) as well as a type 3b hydrogenase, which are not present in any other sequenced AOA. A further surprising genomic feature of this thermophilic ammonia oxidizer is the absence of DNA polymerase D genes - one of the predominant replicative DNA polymerases in all other ammonia-oxidizing Thaumarchaea. Collectively, our findings suggest that metabolic versatility and DNA replication might differ substantially between obligately thermophilic and other AOA.

microbiology↗

bioRxiv2017

Growth and extended survival of Escherichia coli O157:H7 in soil organic matter

Enterohaemorrhagic Escherichia coli such as serotype O157:H7 are a leading cause of food-associated outbreaks. While the primary reservoir is associated with cattle, plant foods have been associated as sources of human infection. E. coli is able to grow in the tissue of food plants such as spinach. While fecal contamination is the primary suspect, soil has been underestimated as a potential reservoir. Persistence of bacterial populations in open systems is the product of growth, death, predation, and competition. Here we report that E. coli O157:H7 can grow using the soluble compounds in soil, and characterize the effect of soil growth in the stationary phase proteome. E. coli 933D (stxII-) was cultured in Soil Extracted Soluble Organic Matter (SESOM) and the culturable count determined for 24 d. The proteomes of exponential and stationary phase populations were characterized by 2D gel electrophoresis and protein spots were identified by MALDI-TOF mass spectrometry. While LB controls displayed a death phase, SESOM grown population remained culturable for 24 d, indicating an altered physiological state with superior longevity. This was not due to decreased cell density on entry to stationary phase as 24h SESOM populations concentrated 10-fold retained their longevity. Principal component analysis showed that stationary phase proteomes from SESOM and LB were different. Differences included proteins involved in stress response, motility, membrane and wall composition, nutrient uptake, translation and protein turnover, and anabolic and catabolic pathways, indicating an altered physiological state of soil-grown cells entering stationary phase. The results suggest that E. coli may be a soil commensal that in absence of predation and competition maintains stable populations in soil.

microbiology↗

bioRxiv2017

Molecular insights into Vibrio cholerae’s intra-amoebal host-pathogen interactions

Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the hosts osmoregulatory organelle. In this study, we deciphered the molecular mechanisms involved in the maintenance of V. choleraes intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrated that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, play a key role role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.

microbiology↗

bioRxiv2017

Mapping mutational effects along the evolutionary landscape of HIV envelope

The immediate evolutionary space accessible to HIV is largely determined by how single amino-acid mutations affect fitness. These mutational effects can shift as the virus evolves. However, the prevalence of such shifts in mutational effects remains unclear. Here we quantify the effects on viral growth of all amino-acid mutations to two HIV envelope (Env) proteins that differ at >100 residues. Most mutations similarly affect both Envs, but the amino-acid preferences of a minority of sites have clearly shifted. These shifted sites usually prefer a specific amino acid in one Env, but tolerate many amino acids in the other. Surprisingly, shifts are only slightly enriched at sites that have substituted between the Envs -- and many occur at residues that do not even contact substitutions. Therefore, long-range epistasis can unpredictably shift Env's mutational tolerance during HIV evolution, although the amino-acid preferences of most sites are conserved between moderately diverged viral strains.

microbiology↗

bioRxiv2017

Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure

The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy pathway in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.

microbiology↗

bioRxiv2017

ACI-1 class A beta-lactamase is widespread across human gut microbiomes due to transposons harboured by tailed prophages

Antibiotic resistance is increasing among pathogens at unprecedented rates and the human body contains a large pool of antibiotic resistance genes that can be spread among bacteria by mobile genetic elements. Acidaminococcus intestini, a bacterium found in the human gut that belongs to the class of Negativicutes, is the first gram-negative coccus shown to be resistant to beta-lactam antibiotics. Resistance is conferred by aci1, a gene encoding the ACI-1 class A beta-lactamase, but the evolutionary history of aci1 and its distribution across other Negativicutes and in the human gut microbiota remains obscure. We discovered that ACI-1 proteins are phylogenetically distinct from class A beta-lactamases of gram-positive Firmicutes and that the aci1 gene occurs in bacteria scattered across the Negativicutes clade, suggesting possible mobilization. In the reference A. intestini RyC-MR95 strain, we found that aci1 is surrounded by mobile DNA, transposon derived sequences directly flank aci1 and are likely the vehicle for its mobility. These transposon sequences reside within a prophage context consisting of two likely degraded tailed prophages, the first prophages to be characterised in A. intestini. We found aci1 in at least 56 (4.4%) out of 1,267 human gut metagenome samples, mostly hosted within A. intestini, and, where could be determined, mostly within a similar constellation of mobile elements to that found in the reference A. intestini genome. These human samples are from individuals in Europe, China and the USA, showing that aci1 is widely distributed globally. Additionally, we examined the nine different Negativicute genome assemblies that contain aci1, and found that only two of these strains show a similar mobile element context around aci1 to the reference A. intestini with transposons adjacent to a tailed prophage. However, in all nine cases aci1 is flanked by transposon derived sequences, and these sequences are diverse, suggesting the activity and degradation of multiple transposons. Overall, we show that ACI-1 proteins form a distinct class A beta lactamase family, and that the aci1 gene is present in human guts worldwide within Negativicute bacterial hosts, due to transposons, sometimes inserted into tailed prophages.

microbiology↗