Colony Morphotypes Research Articles

Use of epigenetically modified bacteriophage and dual beta-lactams to treat a Mycobacterium abscessus sternal wound infection

Nontuberculous mycobacterium (NTM) infections are challenging to manage and are frequently non-responsive to aggressive but poorly-tolerated antibiotic therapies. Immunosuppressed lung transplant patients are susceptible to NTM infections and poor patient outcomes are common. Bacteriophages present an alternative treatment option and are associated with favorable clinical outcomes. Similarly, dual beta-lactam combinations show promise in vitro, but clinical use is sparse. We report here a patient with an uncontrolled Mycobacterium abscessus infection following a bilateral lung transplant and failed antibiotic therapy. Both smooth and rough colony morphotype strains were initially present, but treatment with two phages that kill the rough strain – including epigenetic-modification to overcome restriction – resulted in isolation of only the smooth strain. The rough and smooth strains have similar antibiotic susceptibilities suggesting that the phages specifically eliminated the rough strain. Dual beta-lactam therapy with meropenem and ceftazidime-avibactam provided further clinical improvement, and the phages act synergistically with meropenem in vitro.

Single-colony MALDI mass spectrometry imaging reveals spatial differences in metabolite abundance between natural and cultured Trichodesmium morphotypes.

Trichodesmium, a globally significant N2-fixing marine cyanobacterium, forms extensive surface blooms in nutrient-poor ocean regions. These blooms consist of a dynamic assemblage of Trichodesmium species that form distinct colony morphotypes and are inhabited by diverse microorganisms. Trichodesmium colony morphotypes vary in ecological niche, nutrient uptake, and organic molecule release, differentially impacting ocean carbon and nitrogen biogeochemical cycles. Here, we assessed the poorly studied spatial abundance of metabolites within and between three morphologically distinct Trichodesmium colonies collected from the Red Sea. We also compared these results with two morphotypes of the cultivable Trichodesmium strain IMS101. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) coupled with liquid extraction surface analysis (LESA) tandem mass spectrometry (MS2), we identified and localized a wide range of small metabolites associated with single-colony Trichodesmium morphotypes. Our untargeted MALDI-MSI approach revealed 80 unique features (metabolites) shared between Trichodesmium morphotypes. Discrimination analysis showed spatial variations in 57 shared metabolites, accounting for 62% of the observed variation between morphotypes. The greatest variations in metabolite abundance were observed between the cultured morphotypes compared to the natural colony morphotypes, suggesting substantial differences in metabolite production between the cultivable strain IMS101 and the naturally occurring colony morphotypes that the cultivable strain is meant to represent. This study highlights the variations in metabolite abundance between natural and cultured Trichodesmium morphotypes and provides valuable insights into metabolites common to morphologically distinct Trichodesmium colonies, offering a foundation for future targeted metabolomic investigations.IMPORTANCEThis work demonstrates that the application of spatial mass spectrometry imaging at single-colony resolution can successfully resolve metabolite differences between natural and cultured Trichodesmium morphotypes, shedding light on their distinct biochemical profiles. Understanding the morphological differences between Trichodesmium colonies is crucial because they impact nutrient uptake, organic molecule production, and carbon and nitrogen export, and subsequently influence ocean biogeochemical cycles. As such, our study serves as an important initial assessment of metabolite differences between distinct Trichodesmium colony types, identifying features that can serve as ideal candidates for future targeted metabolomic studies.

Inhibition of fusarium graminearum growth and spore germination by a streptomyces amritsarensis strain capable of killing and growing on microcystis scum.

Developing energy-saving and ecofriendly strategies for treating harvested Microcystis biomass. Streptomyces amritsarensis HG-16 was first reported to effectively kill various morphotypes of natural Microcystis colonies at very high cell densities. Concurrently, HG-16 grown on lysed Microcystis maintained its antagonistic activity against plant pathogenic fungus Fusarium graminearum. It could completely inhibit spore germination and destroy mycelial structure of F. graminearum. Transcriptomic analysis revealed that HG-16 attacked F. graminearum in a comprehensive way: interfering with replication, transcription, and translation processes, inhibiting primary metabolisms, hindering energy production and simultaneously destroying stress-resistant systems of F. graminearum. The findings of this study provide a sustainable and economical option for resource reclamation from Microcystis biomass: utilizing Microcystis slurry to propagate HG-16, which can subsequently be employed as a biocontrol agent for managing F. graminearum.

High Biofilm-Forming Multidrug-Resistant Salmonella Infantis Strains from the Poultry Production Chain.

The ability of Salmonella species to adhere to surfaces and form biofilms, leading to persistent environmental reservoirs, might represent a direct link between environmental contamination and food processing contamination. The purpose of this study was to investigate the biofilm-forming ability of 80 multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL) producing Salmonella enterica serovar Infantis strains isolated from the broiler food chain production through whole genome sequencing (WGS), PCR, and morphotype association assays. Biofilm formation was quantified by testing the strains at two different temperatures, using 96-well polystyrene plates. The rough and dry colony (rdar) morphotype was assessed visually on Congo red agar (CRA) plates. Based on our results, all tested S. Infantis strains produced biofilm at 22 °C with an rdar morphotype, while at 37 °C, all the isolates tested negative, except one positive. Most isolates (58.75%) exhibited strong biofilm production, while 36.25% showed moderate production. Only 5 out of 80 (6.25%) were weak biofilm producers. WGS analysis showed the presence of the fim cluster (fimADF) and the csg cluster (csgBAC and csgDEFG), also described in S. Typhimurium, which are responsible for fimbriae production. PCR demonstrated the presence of csgD, csgB, and fimA in all 80 S. Infantis strains. To our knowledge, this is the first study comparing the effects of two different temperatures on the biofilm formation capacity of ESBL producing S. Infantis from the broiler production chain. This study highlights that the initial biofilm components, such as curli and cellulose, are specifically expressed at lower temperatures. It is important to emphasize that within the broiler farm, the environmental temperature ranges between 18-22 °C, which is the optimum temperature for in vitro biofilm formation by Salmonella spp. This temperature range facilitates the expression of biofilm-associated genes, contributing to the persistence of S. Infantis in the environment. This complicates biosecurity measures and makes disinfection protocols on the farm and in the production chain more difficult, posing serious public health concerns.

Genome Sequence Comparisons between Small and Large Colony Phenotypes of Equine Clinical Isolates of Arcanobacterium hippocoleae.

Arcanobacterium hippocoleae is a Gram-positive fastidious bacterium and is occasionally isolated from the reproductive tract of apparently healthy mares (Equus caballus) or from mares with reproductive tract abnormalities. Apart from a few 16S rRNA gene-based GenBank sequences and one recent report on complete genome assembly, detailed genomic sequence and clinical experimental data are not available on the bacterium. Recently, we observed an unusual increase in the detection of the organism from samples associated with mare reproductive failures in Atlantic Canada. Two colony morphotypes (i.e., small, and large) were detected in culture media, which were identified as A. hippocoleae by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Here, we report the whole genome sequencing and characterization of the morphotype variants. The genome length of the large phenotypes was between 2.42 and 2.43, and the small phenotype was 1.99 Mbs. The orthologous nucleotide identity between the large colony phenotypes was ~99%, and the large and small colony phenotypes was between 77.86 and 78.52%, which may warrant the classification of the two morphotypes into different species. Phylogenetic analysis based on 16S rRNA genes or concatenated housekeeping genes grouped the small and large colony variants into two different genotypic clusters. The UvrA protein, which is part of the nucleotide excision repair (NER) system, and 3-isopropoylmalate dehydratase small subunit protein expressed by the leuD gene were identified as potential virulence factors in the large and small colony morphotypes, respectively. However, detailed functional studies will be required to determine the exact roles of these and other identified hypothetical proteins in the cellular metabolism and potential pathogenicity of A. hippocoleae in mares.

Loss of LpqM proteins in Mycobacterium abscessus is associated with impaired intramacrophage survival.

Mycobacterium abscessus causes persistent infections in patients with underlying pulmonary diseases, resulting in progressive lung function deterioration. The rough (R) morphotype is well-established as associated with chronic and more aggressive infections in patients. In this study, we individually and simultaneously deleted the MAB_1470c and MAB_1466c genes in M. abscessus S, without observing changes in colony morphotypes. However, these mutants exhibited a severe impairment in their ability to survive within human macrophages, highlighting the critical role of these two lipoproteins in M. abscessus virulence.

Colony phase variation switch modulates antimicrobial tolerance and biofilm formation in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii causes one of the most difficult-to-treat nosocomial infections. Polycationic drugs like polymyxin B or colistin and tetracycline drugs such as doxycycline or minocycline are commonly used to treat infections caused by carbapenem-resistant A. baumannii. Here, we show that a subpopulation of cells associated with the opaque/translucent colony phase variation by A. baumannii AB5075 displays differential tolerance to subinhibitory concentrations of colistin and tetracycline. Using a variety of microscopic techniques, we demonstrate that extracellular polysaccharide moieties mediate colistin tolerance to opaque A. baumannii at single-cell level and that mushroom-shaped biofilm structures protect opaque bacteria at the community level. The colony switch phenotype is found to alter several traits of A. baumannii, including long-term survival under desiccation, tolerance to ethanol, competition with Escherichia coli, and intracellular survival in the environmental model host Acanthamoeba castellanii. Additionally, our findings suggest that extracellular DNA associated with membrane vesicles can promote colony switching in a DNA recombinase-dependent manner.IMPORTANCEAs a WHO top-priority drug-resistant microbe, Acinetobacter baumannii significantly contributes to hospital-associated infections worldwide. One particularly intriguing aspect is its ability to reversibly switch its colony morphotype on agar plates, which has been remarkably underexplored. In this study, we employed various microscopic techniques and phenotypic assays to investigate the colony phase variation switch under different clinically and environmentally relevant conditions. Our findings reveal that the presence of a poly N-acetylglucosamine-positive extracellular matrix layer contributes to the protection of bacteria from the bactericidal effects of colistin. Furthermore, we provide intriguing insights into the multicellular lifestyle of A. baumannii, specifically in the context of colony switch variation within its predatory host, Acanthamoeba castellanii.

Machine learning identification of Pseudomonas aeruginosa strains from colony image data.

When grown on agar surfaces, microbes can produce distinct multicellular spatial structures called colonies, which contain characteristic sizes, shapes, edges, textures, and degrees of opacity and color. For over one hundred years, researchers have used these morphology cues to classify bacteria and guide more targeted treatment of pathogens. Advances in genome sequencing technology have revolutionized our ability to classify bacterial isolates and while genomic methods are in the ascendancy, morphological characterization of bacterial species has made a resurgence due to increased computing capacities and widespread application of machine learning tools. In this paper, we revisit the topic of colony morphotype on the within-species scale and apply concepts from image processing, computer vision, and deep learning to a dataset of 69 environmental and clinical Pseudomonas aeruginosa strains. We find that colony morphology and complexity under common laboratory conditions is a robust, repeatable phenotype on the level of individual strains, and therefore forms a potential basis for strain classification. We then use a deep convolutional neural network approach with a combination of data augmentation and transfer learning to overcome the typical data starvation problem in biological applications of deep learning. Using a train/validation/test split, our results achieve an average validation accuracy of 92.9% and an average test accuracy of 90.7% for the classification of individual strains. These results indicate that bacterial strains have characteristic visual 'fingerprints' that can serve as the basis of classification on a sub-species level. Our work illustrates the potential of image-based classification of bacterial pathogens and highlights the potential to use similar approaches to predict medically relevant strain characteristics like antibiotic resistance and virulence from colony data.

Immobilization of rough morphotype Mycolicibacterium neoaurum R for androstadienedione production.

Enhance the androstadienedione (Androst-1,4-diene-3,17-dione, ADD) production of rough morphotype Mycolicibacterium neoaurum R by repeated-batch fermentation of immobilized cells. M. neoaurum R was a rough colony morphotype variant, obtained from the routine plating of smooth M. neoaurum strain CICC 21097. M. neoaurum R showed rougher cell surface and aggregated in broth. The ADD production of M. neoaurum R was notably lower than that of M. neoaurum CICC 21097 during the free cell fermentation, but the yield gap could be erased after proper cell immobilization. Subsequently, repeated-batch fermentation of immobilized M. neoaurum R was performed to shorten the production cycle and enhance the bio-production efficiency of ADD. Through the optimization of the immobilization carriers and the co-solvents for phytosterols, the ADD productivity of M. neoaurum R immobilized by semi-expanded perlite reached 0.075g/L/h during the repeated-batch fermentation for 40days. The ADD production of the rough-type M. neoaurum R was notably enhanced by the immobilization onto semi-expanded perlite. Moreover, the ADD batch yields of M. neoaurum R immobilized by semi-expanded perlite were maintained at high levels during the repeated-batch fermentation.

Population heterogeneity in Mycobacterium smegmatis and Mycobacterium abscessus.

Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in M. smegmatis - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions. M. abscessus has two recognized morphotypes, smooth and rough. Interestingly, M. tuberculosis exists in only a rough form. The shift from smooth to rough in both M. smegmatis and M. abscessus was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle preparations compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among M. smegmatis and M. abscessus variants. Deletion of the global regulator lsr2 shifted the M. smegmatis intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse outcomes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.

Insights from colony formation: The necessity to consider morphotype when assessing the effect of antibiotics on cyanobacteria

Colonial cyanobacteria have been identified as the primary contributor to the global occurrence of cyanobacterial harmful algal blooms (cyanoHABs), which are further intensified by the presence of “pseudo-persistent” antibiotics. Nevertheless, the impact of antibiotics on the growth and size of colonial cyanobacteria remains unclear. In this study, the response of cyanobacterium Microcystis to varying doses of antibiotics was assessed (0, 0.1, 0.5, 1, 10, and 50 μg L-1) by comparing the unicellular and colonial morphotypes. Interestingly, the morphological structure of cyanobacteria plays a significant role in their reaction to antibiotics. In comparison to the unicellular morphotype, the colonial morphotype exhibited a greater promotion in growth rate (11 %—22 %) to low doses of antibiotics and was less inhibited (-121 %—-62 %) under high doses. Furthermore, antibiotics may affect the size of cyanobacterial colonies by disrupting the secretion of algal organic matter, which also exhibited a two-phase pattern. This work sheds light on the significance of methodology research involving both unicellular and colonial cyanobacteria. Future research and lake management should prioritize studying the morphological traits of cyanobacteria under different levels of antibiotic exposure. This approach may lead to novel strategies for predicting cyanoHABs under antibiotic pollution more effectively.

Bioinformatic and functional characterization of cyclic-di-GMP metabolic proteins in Vibrio alginolyticus unveils key diguanylate cyclases controlling multiple biofilm-associated phenotypes.

The biofilm lifestyle is critical for bacterial survival and proliferation in the fluctuating marine environment. Cyclic diguanylate (c-di-GMP) is a key second messenger during bacterial adaptation to various environmental signals, which has been identified as a master regulator of biofilm formation. However, little is known about whether and how c-di-GMP signaling regulates biofilm formation in Vibrio alginolyticus, a globally dominant marine pathogen. Here, a large set of 63 proteins were predicted to participate in c-di-GMP metabolism (biosynthesis or degradation) in a pathogenic V. alginolyticus strain HN08155. Guided by protein homology, conserved domains and gene context information, a representative subset of 22 c-di-GMP metabolic proteins were selected to determine which ones affect biofilm-associated phenotypes. By comparing phenotypic differences between the wild-type and mutants or overexpression strains, we found that 22 c-di-GMP metabolic proteins can separately regulate different phenotypic outputs in V. alginolyticus. The results indicated that overexpression of four c-di-GMP metabolic proteins, including VA0356, VA1591 (CdgM), VA4033 (DgcB) and VA0088, strongly enhanced rugose colony morphotypes and strengthened Congo Red (CR) binding capacity, both of which are indicators of biofilm matrix overproduction. Furthermore, rugose enhanced colonies were accompanied by increased transcript levels of extracellular polysaccharide (EPS) biosynthesis genes and decreased expression of flagellar synthesis genes compared to smooth colonies (WTpBAD control), as demonstrated by overexpression strains WTp4033 and ∆VA4033p4033. Overall, the high abundance of c-di-GMP metabolic proteins in V. alginolyticus suggests that c-di-GMP signaling and regulatory system could play a key role in its response and adaptation to the ever-changing marine environment. This work provides a robust foundation for the study of the molecular mechanisms of c-di-GMP in the biofilm formation of V. alginolyticus.

Multiple Mycobacterium abscessus O-acetyltransferases influence glycopeptidolipid structure and colony morphotype

Mycobacterium abscessus causes severe lung infections. Clinical isolates can have either smooth (S) or rough (R) colony morphotypes; of these, S but not R variants have abundant cell wall glycopeptidolipids (GPL) consisting of a peptidolipid core substituted by a 6-deoxy-talose (6-dTal) and rhamnose residues. Deletion of gtf1, encoding the 6-dTal transferase, results in the S-to-R transition, mycobacterial cord formation, and increased virulence, underscoring the importance of 6-dTal in infection outcomes. However, since 6-dTal is di-O-acetylated, it is unclear whether the gtf1 mutant phenotypes are related to the loss of the 6-dTal or the result of the absence of acetylation. Here, we addressed whether M. abscessus atf1 and atf2, encoding two putative O-acetyltransferases located within the gpl biosynthetic locus, transfer acetyl groups to 6-dTal. We found deletion of atf1 and/or atf2 did not drastically alter the GPL acetylation profile, suggesting there are additional enzymes with redundant functions. We subsequently identified two paralogs of atf1 and atf2, MAB_1725c and MAB_3448. While deletion of MAB_1725c and MAB_3448 had no effect on GPL acetylation, the triple atf1-atf2-MAB_1725c mutant did not synthetize fully-acetylated GPL and the quadruple mutant was totally devoid of acetylated GPL. Moreover, both triple and quadruple mutants accumulated hyper-methylated GPL. Finally, we show deletion of atf genes resulted in subtle changes in colony morphology, but had no effect on M. abscessus internalization by macrophages. Overall, these findings reveal the existence of functionally redundant O-acetyltransferases and suggest that O-acetylation influences the glycan moiety of GPL by deflecting biosynthetic flux in M. abscessus.

The problem of Mycobacterium abscessus complex: multi-drug resistance, bacteriophage susceptibility and potential healthcare transmission

ObjectivesMycobacterium abscessus complex is responsible for 2.6–13.0% of all non-tuberculous mycobacterial pulmonary infections and these are notoriously difficult to treat due to the complex regimens required, drug resistance and adverse effects. Hence, bacteriophages have been considered in clinical practice as an additional treatment option. Here, we evaluated antibiotic and phage susceptibility profiles of M. abscessus clinical isolates. Whole-genome sequencing (WGS) revealed the phylogenetic relationships, dominant circulating clones (DCCs), the likelihood of patient-to-patient transmission and the presence of prophages. MethodsAntibiotic susceptibility testing was performed using CLSI breakpoints (n = 95), and plaque assays were used for phage susceptibility testing (subset of n = 88, 35 rough and 53 smooth morphology). WGS was completed using the Illumina platform and analysed using Snippy/snp-dists and Discovery and Extraction of Phages Tool (DEPhT). ResultsAmikacin and Tigecycline were the most active drugs (with 2 strains resistant to amikacin, and one strain with Tigecycline MIC of 4 μg/mL). Most strains were resistant to all other drugs tested, with Linezolid and Imipenem showing the least resistance, at 38% (36/95) and 55% (52/95), respectively. Rough colony morphotype strains were more phage-susceptible than smooth strains (77%—27/35 versus 48%—25/53 in the plaque assays, but smooth strains are not killed efficiently by those phages in liquid infection assay). We have also identified 100 resident prophages, some of which were propagated lytically. DCC1 (20%—18/90) and DCC4 (22%—20/90) were observed to be the major clones and WGS identified 6 events of possible patient-to-patient transmission. DiscussionMany strains of M. abscessus complex are intrinsically resistant to available antibiotics and bacteriophages represent an alternative therapeutic option, but only for strains with rough morphology. Further studies are needed to elucidate the role of hospital-borne M. abscessus transmission.

WS07.03 A long-term study of Pseudomonas aeruginosa early patient isolate adaptation to the hypoxic environment

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which causes chronic lung infections in people with cystic fibrosis (CF) and is a major contributor to morbidity and mortality. Despite this, the mechanism driving its adaptation towards chronic colonisation in the CF lung is not yet fully understood. This work focuses on the adaptations of P. aeruginosa to hypoxia, one of the important environmental pressures present in the lung. Long term hypoxia has yet to be examined as a driver of adaptation towards the persistence of this bacterium in CF patients. We have studied the effect of long term adaptation to 6% oxygen for up to 28 days. Interestingly, distinctive colony morphotypes developed throughout the experiments, including small colony variants (SCV). One of the observed morphotypes appeared exclusively under low-oxygen pressure. Importantly, the colonies with visibly different morphologies were more common in hypoxia adapted cultures, comprising up to 98% of the population, while they never exceeded 35% in normoxia adapted cultures. Proteomic analysis showed changes in the abundance of over 700 proteins within 5 days of adaptation, including those involved in respiration and carbon metabolism, quorum sensing and phenazine biosynthesis. Interestingly, comparable changes in several pathways were consistent across both conditions while phenotype differences in motility, pyocyanin production, and protease production between the different colony morphotypes in samples taken over time were observed. Both the SCV and the normal colony variants (NCV) from the hypoxia adapted strains showed decreases in motility, pyocyanin production and protease production. A Congo red assay showed increased exopolysaccharide production in both hypoxia adapted SCV and NCVs. Overall, these results confirm that hypoxia indeed promotes the appearance of many phenotypes in P. aeruginosa that are associated with persistence in the lung and with poor patient outcomes.

An initial genomic blueprint of the healthy human oesophageal microbiome.

The oesophageal microbiome is thought to contribute to the pathogenesis of oesophageal cancer. However, investigations using culture and molecular barcodes have provided only a low-resolution view of this important microbial community. We therefore explored the potential of culturomics and metagenomic binning to generate a catalogue of reference genomes from the healthy human oesophageal microbiome, alongside a comparison set from saliva. Twenty-two distinct colonial morphotypes from healthy oesophageal samples were genome-sequenced. These fell into twelve species clusters, eleven of which represented previously defined species. Two isolates belonged to a novel species, which we have named Rothia gullae. We performed metagenomic binning of reads generated from UK samples from this study alongside reads generated from Australian samples in a recent study. Metagenomic binning generated 136 medium or high-quality metagenome-assembled genomes (MAGs). MAGs were assigned to 56 species clusters, eight representing novel Candidatus species, which we have named Ca. Granulicatella gullae, Ca. Streptococcus gullae, Ca. Nanosynbacter quadramensis, Ca. Nanosynbacter gullae, Ca. Nanosynbacter colneyensis, Ca. Nanosynbacter norwichensis, Ca. Nanosynococcus oralis and Ca. Haemophilus gullae. Five of these novel species belong to the recently described phylum Patescibacteria . Although members of the Patescibacteria are known to inhabit the oral cavity, this is the first report of their presence in the oesophagus. Eighteen of the metagenomic species were, until recently, identified only by hard-to-remember alphanumeric placeholder designations. Here we illustrate the utility of a set of recently published arbitrary Latinate species names in providing user-friendly taxonomic labels for microbiome analyses.Our non-redundant species catalogue contained 63 species derived from cultured isolates or MAGs. Mapping revealed that these species account for around half of the sequences in the oesophageal and saliva metagenomes. Although no species was present in all oesophageal samples, 60 species occurred in at least one oesophageal metagenome from either study, with 50 identified in both cohorts. Recovery of genomes and discovery of new species represents an important step forward in our understanding of the oesophageal microbiome. The genes and genomes that we have released into the public domain will provide a base line for future comparative, mechanistic and intervention studies.

In Vitro Synergistic Effects of Omadacycline with Other Antimicrobial Agents against Mycobacterium abscessus.

The clinical importance of Mycobacterium abscessus species (MABS) infections has been increasing. However, the standard treatment regimens recommended in the current guidelines often result in unfavorable outcomes. Therefore, we investigated the in vitro activity of omadacycline (OMC), a novel tetracycline, against MABS to explore its potential as a novel therapeutic option. The drug susceptibilities of 40 Mycobacterium abscessus subsp. abscessus (Mab) clinical strains obtained from the sputum of 40 patients from January 2005 to May 2014 were investigated. The MIC results for OMC, amikacin (AMK), clarithromycin (CLR), clofazimine (CLO), imipenem (IPM), rifabutin (RFB), and tedizolid (TZD) alone and their combined effects (with OMC) were examined using the checkerboard method. Additionally, we studied the differences in the effectiveness of the antibiotic combinations based on the colony morphotype of Mab. The MIC50 and MIC90 of OMC alone were 2 and 4 μg/mL, respectively. The combinations of OMC with AMK, CLR, CLO, IPM, RFB, and TZD showed synergy against 17.5%, 75.8%, 25.0%, 21.1%, 76.9%, and 34.4% of the strains, respectively. Additionally, OMC combined with CLO (47.1% versus 9.5%, P = 0.023) or TZD (60.0% versus 12.5%, P = 0.009) showed significantly higher synergy against strains with rough morphotypes than those with smooth morphotypes. In conclusion, the checkerboard analyses revealed that the synergistic effects of OMC were observed most frequently with RFB, followed by CLR, TZD, CLO, IPM, and AMK. Furthermore, OMC tended to be more effective against rough-morphotype Mab strains.

Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara.

Bacteria colonize specific niches in the animal gut. However, the genetic basis of these associations is often unclear. The proteobacterium Frischella perrara is a widely distributed gut symbiont of honey bees. It colonizes a specific niche in the hindgut and causes a characteristic melanization response. Genetic determinants required for the establishment of this association, or its relevance for the host, are unknown. Here, we independently isolated three point mutations in genes encoding the DNA-binding protein integration host factor (IHF) in F. perrara. These mutants abolished the production of an aryl polyene metabolite causing the yellow colony morphotype of F. perrara. Inoculation of microbiota-free bees with one of the mutants drastically decreased gut colonization of F. perrara. Using RNAseq, we found that IHF affects the expression of potential colonization factors, including genes for adhesion (type 4 pili), interbacterial competition (type 6 secretion systems), and secondary metabolite production (colibactin and aryl polyene biosynthesis). Gene deletions of these components revealed different colonization defects depending on the presence of other bee gut bacteria. Interestingly, one of the T6SS mutants did not induce the scab phenotype anymore despite colonizing at high levels, suggesting an unexpected role in bacteria-host interaction. IHF is conserved across many bacteria and may also regulate host colonization in other animal symbionts.

Unusual prophages in Mycobacterium abscessus genomes and strain variations in phage susceptibilities.

Mycobacterium abscessus infections are relatively common in patients with cystic fibrosis and are clinically challenging, with frequent intrinsic resistance to antibiotics. Therapeutic treatment with bacteriophages offers some promise but faces many challenges including substantial variation in phage susceptibilities among clinical isolates, and the need to personalize therapies for individual patients. Many strains are not susceptible to any phages or are not efficiently killed by lytic phages, including all smooth colony morphotype strains tested to-date. Here, we analyze a set of new M. abscessus isolates for the genomic relationships, prophage content, spontaneous phage release, and phage susceptibilities. We find that prophages are common in these M. abscessus genomes, but some have unusual arrangements, including tandemly integrated prophages, internal duplications, and they participate in active exchange of polymorphic toxin-immunity cassettes secreted by ESX systems. Relatively few strains are efficiently infected by any mycobacteriophages, and the infection patterns do not reflect the overall phylogenetic relationships of the strains. Characterization of these strains and their phage susceptibility profiles will help to advance the broader application of phage therapies for NTM infections.

Glycopeptidolipid Defects Leading to Rough Morphotypes of Mycobacterium abscessus Do Not Confer Clinical Antibiotic Resistance.

Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. Within chronically infected patients, M. abscessus isolates undergo molecular changes leading to increased virulence and antibiotic resistance. Specifically, mutations in glycopeptidolipid (GPL) synthesis genes, leading to the rough phenotype, are associated with invasive, nonremitting infections and a severe clinical course. It has been unclear whether GPL defects confer antibiotic resistance independently of other molecular changes. We used transposon technology to isolate a rough (GPL-defective; Tn MABS_4099cZeoR) mutant and compare it to a fully isogenic parent strain (ATCC 19977) bearing wild-type zeocin resistance (WTZeoR). Antibiotic susceptibility profiles of Tn_4099cZeoR and WTZeoR were tested and compared using the Sensititre RAPMYCOI antimicrobial susceptibility test plate. MICs were evaluated within clinically relevant values according to the Clinical and Laboratory Standards Institute (CLSI) standards. We found that M. abscessus with rough colony morphotype (Tn_4009c) had comparable antibiotic susceptibility to its smooth isogenic WT counterpart. Small differences (a 1:2 dilution) in MICs were found for imipenem, cefoxitin, and tigecycline, yet those small differences did not change the clinical susceptibility report for these antibiotics, as they fell within the same CLSI cutoffs for resistance. While small alternations in susceptibility to imipenem, cefoxitin, and tigecycline were noted, we conclude that the GPL mutations in M. abscessus did not confer clinically significant antibiotic resistance. Increased antibiotic resistance in the clinical setting may occur in an unrelated and parallel manner to GPL mutations. IMPORTANCE Mycobacterium abscessus chronically infects patients with preexisting lung diseases, leading to progressive deterioration in pulmonary function. The common perception among clinicians is that the rough phenotype is associated with progressive disease and severe clinical course, manifested as a widespread inflammatory response and resistance to antibacterials. However, as clinical isolates accumulate hundreds of mutations over the prolonged course of infection, it is unclear whether the rough phenotype per se is responsible for the antibiotic resistance seen in late-stage infections, or whether the resistance is related to other genetic changes in the bacteria. Previous studies mostly compared rough and smooth clinical isolates. Here, for the first time, we compared WT smooth bacteria to a specific rough, GPL-associated, otherwise-isogenic mutant. We determined that the rough morphotype had essentially identical antibiotic susceptibilities as the parent strain. The mechanistic basis for the antibiotic resistance observed in rough clinical isolates is therefore most probably related to other genetic determinants.