Rough Morphotype Research Articles

Easily applicable modifications to electroporation conditions improve the transformation efficiency rates for rough morphotypes of fast-growing mycobacteria

Electroporation is the most widely used and efficient method to transform mycobacteria. Through this technique, fast- and slow-growing mycobacteria with smooth and rough morphotypes have been successfully transformed. However, transformation efficiencies differ widely between species and strains. In this study, the smooth and rough morphotypes of Mycobacteroides abscessus and Mycolicibacterium brumae were used to improve current electroporation procedures for fast-growing rough mycobacteria. The focus was on minimizing three well-known and challenging limitations: the mycobacterial restriction-modification systems, which degrade foreign DNA; clump formation of electrocompetent cells before electroporation; and electrical discharges during pulse delivery, which were reduced by using salt-free DNA solution. Herein, different strategies are presented that successfully address these three limitations and clearly improve the electroporation efficiencies over the current procedures. The results demonstrated that combining the developed strategies during electroporation is highly recommended for the transformation of fast-growing rough mycobacteria.

Biofilm Formation by Salmonella enterica Strains.

Biofilm formation by five different Salmonella enterica strains was assessed qualitatively and quantitatively under different incubation conditions. The strains exhibited different adherence abilities to test tubes. The isolates revealed Red Dry and Rough (RDAR) and Brown Dry and Rough (BDAR) morphotypes when cultured on Congo Red Agar (CRA). The pellicles formed by the tested strains ranged from strong to fragile when incubated in LB without NaCl at 27 °C. Smooth and White (SAW) morphotype on CRA and very weak pellicles were observed when the bacterial strains were incubated at 37 °C. The effect of temperature and media on biofilm formation by the tested strains was significant. Among the five Salmonella isolates, S. enteritidis TM 6 and S. enteritidis TM 68 formed strong biofilms when incubated in LB without NaCl at 27 °C for 24 h and consequently selected to be analysed under scanning electron microscope (SEM). Scanning electron micrographs revealed that S. enteritidis TM 6 formed more complex colonies when compared to those formed by S. enteritidis TM 68. As far as we know, this is the first study that provides quantitative and qualitative data for 5 Salmonella enterica isolates in different media mimicking four different nutritional conditions attwo different temperatures after 24 and 48 h. The strains included two serovars S. bredeney and S. anatum, which are rarely accounted for. Additionally, the studies that described S. enteritidis biofilms under SEM are extremely limited, which makes it among the first comprehensive studies that screened for S. enteritidis biofilms.

O-Methylation of the Glycopeptidolipid Acyl Chain Defines Surface Hydrophobicity of Mycobacterium abscessus and Macrophage Invasion.

Mycobacterium abscessus, an emerging pathogen responsible for severe lung infections in cystic fibrosis patients, displays either smooth (S) or rough (R) morphotypes. The S-to-R transition is associated with reduced levels of glycopeptidolipid (GPL) production and is correlated with increased pathogenicity in animal and human hosts. While the structure of GPL is well established, its biosynthetic pathway is incomplete. In addition, the biological functions of the distinct structural parts of this complex lipid remain elusive. Herein, the fmt gene encoding a putative O-methyltransferase was deleted in the M.abscessus S variant. Subsequent biochemical and structural analyses demonstrated that methoxylation of the fatty acyl chain of GPL was abrogated in the Δfmt mutant, and this defect was rescued upon complementation with a functional fmt gene. In contrast, the introduction of fmt derivatives mutated at residues essential for methyltransferase activity failed to complement GPL defects, indicating that fmt encodes an O-methyltransferase. Unexpectedly, phenotypic analyses showed that Δfmt was more hydrophilic than its parental progenitor, as demonstrated by hexadecane-aqueous buffer partitioning and atomic force microscopy experiments with hydrophobic probes. Importantly, the invasion rate of THP-1 macrophages by Δfmt was reduced by 50% when compared to the wild-type strain. Together, these results indicate that Fmt O-methylates the lipid moiety of GPL and plays a substantial role in conditioning the surface hydrophobicity of M.abscessus as well as in the early steps of the interaction between the bacilli and macrophages.

A TLR2-Activating Fraction From Mycobacterium abscessus Rough Variant Demonstrates Vaccine and Diagnostic Potential.

Mycobacterium abscessus is a prevalent pathogenic mycobacterium in cystic fibrosis (CF) patients and one of the most highly drug resistant mycobacterial species to antimicrobial agents. It possesses the property to transition from a smooth (S) to a rough (R) morphotype, thereby influencing the host innate immune response. This transition from the S to the R morphotype takes place in patients with an exacerbation of the disease and a persistence of M. abscessus. We have previously shown that the exacerbation of the Toll-like receptor 2 (TLR2)-mediated inflammatory response, following this S to R transition, is essentially due to overproduction of bacilli cell envelope surface compounds, which we were able to extract by mechanical treatment and isolation by solvent partition in a fraction called interphase. Here, we set up a purification procedure guided by bioactivity to isolate a fraction from the R variant of M. abscessus cells which exhibits a high TLR2 stimulating activity, referred to as TLR2-enriched fraction (TLR2eF). As expected, TLR2eF was found to contain several lipoproteins and proteins known to be stimuli for TLR2. Vaccination with TLR2eF showed no protection toward an M. abscessus aerosol challenge, but provided mild protection in ΔF508 mice and their FVB littermates when intravenously challenged by M. abscessus. Interestingly however, antibodies against TLR2eF compounds were detected during disease in CF patients. In conclusion, we show the potential for compounds in TLR2eF as vaccine and diagnostic candidates, in order to enhance diagnosis, prevent and/or treat M. abscessus-related infections.

3-D printed polyvinyl alcohol matrix for detection of airborne pathogens in respiratory bacterial infections

Novel sampling matrices were manufactured using 3D printing for the detection of respiratory pathogens in expired air. A specific configuration of the matrices was designed using Computer-Aided Design software. Polyvinyl alcohol (PVA) was printed using fused deposition modelling to create a multilayer matrix to enhance the capture of bacteria. The performance of these matrices was compared with gelatine filters that have been used for this work to date. PVA matrices (60 mm diameter) were contaminated with bacteria either by direct inoculation, or by aerosol exposure using an Omron A3 nebuliser. Rough and smooth morphotypes of Mycobacterium abscessus, M. smegmatis and M. bovis BCG, were used in this study to contaminate the matrices. PVA matrices and gelatine sampling filters were contaminated to compare recovery rates for quantitative analyses. These were dissolved in water, bacteria pelleted and DNA extracted followed by a Mycobacterium-specific quantitative Polymerase Chain Reaction (qPCR).The results showed that 3D printed PVA matrices are very effective to capture the bacteria. 3D printed PVA matrix and gelatine filters yielded results of the same order of magnitude for mycobacterial analyses, however, PVA matrix offers several advantages over the latter material. 3D printed PVA is considered as an economic and time-effective matrix as it is cheaper than gelatine filters. PVA is sufficiently robust to be handled and loaded into the surgical masks for sampling, compared to the brittle gelatine filters that required supportive frames. PVA is a synthetic material and it is suitable for DNA-based analyses, whilst gelatine is derived from animal collagen, and carries a high bacterial DNA background that interferes with the target DNA analysis. Furthermore, PVA dissolves in distilled water without requiring chemicals or enzymes, such as the case for gelatine hydrolysis. To summarise, 3D printed PVA sampling matrix is considered a promising tool used for microbiological diagnostic purposes.

Life in near-anoxic conditions: A case study of the ichnology and infaunal ecology of Silurian graptolitic black shales from Sardinia, Italy

A trace fossil assemblage from the Silurian (Llandovery) black shales of the Genna Muxerru Formation is reported for the first time. The ichnofossil record is abundant, well-preserved and comprises Alcyonidiopsis, Cochlichnus, Phymatoderma, Planolites (large morphotype), Planolites (rough morphotype) and a ‘Small chondritid’. Sedimentological and ichnological evidence indicates that the bioturbated black shales were deposited in outer shelf (or deeper) settings with severe depletion in oxygen. The seafloor provided a high food supply for the infauna. The palaeoenvironment of the Genna Muxerru Formation is an ancient analogue of modern oxygen-minimum zones. Burrow fills are apparently massive in macroscopic view, whereas observations under the petrographic and stereoscopic microscope show that a lifestyle dominated, i.e. that of pellet-filling sediment-feeders. As such, the Genna Muxerru Formation provides not only a new ethologic reference for low-oxygen settings but also a methodological benchmark for ichnological analysis of black shales. Bioturbation in black shales may be commoner than expected but its recognition requires microscopic observation.

The rough colony morphotype of Mycobacterium avium exhibits high virulence in human macrophages and mice

Introduction. The incidence of Mycobacterium avium complex (MAC) pulmonary disease (MAC PD), a refractory chronic respiratory tract infection, is increasing worldwide. MAC has three predominant colony morphotypes: smooth opaque (SmO), smooth transparent (SmT) and rough (Rg).Aim. To determine whether colony morphotypes can predict the prognosis of MAC PD, we evaluated the virulence of SmO, SmT and Rg in mice and in human macrophages.Methodology. We compared the characteristics of mice and human macrophages infected with the SmO, SmT, or Rg morphotypes of M. avium subsp. hominissuis 104. C57BL/6 mice and human macrophages derived from peripheral mononuclear cells were used in these experiments.Results. In comparison to SmO- or SmT-infected mice, Rg-infected mice revealed severe pathologically confirmed pneumonia, increased lung weight and increased lung bacterial burden. Rg-infected macrophages revealed significant cytotoxicity, increased bacterial burden, secretion of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CCL5 and CCL3), and formation of cell clusters. Rg formed larger bacterial aggregates than SmO and SmT. Cytotoxicity, bacterial burden and secretion of IL-6, CCL5 and CCL3 were induced strongly by Rg infection, and were decreased by disaggregation of the bacteria.Conclusion. M. avium Rg, which is associated with bacterial aggregation, has the highest virulence among the predominant colony morphotypes.

Glycopeptidolipid Genotype Correlates With the Severity of Mycobacterium abscessus Lung Disease.

Smooth and rough colony morphotypes of Mycobacterium abscessus are associated with virulence, but some isolates form both smooth and rough colonies, impeding successful morphotype identification. Reportedly, smooth/rough morphotypes are also related to the glycopeptidolipid (GPL) genotype. However, the accuracy of GPL genotyping to discriminate morphotypes and the relationship between GPL genotype and clinical characteristics of M abscessus lung disease have not been verified. A retrospective analysis of colony morphology, GPL genotype, and clinical data from 182 patients with M abscessus lung disease was conducted. Of 194 clinical isolates, 126 (65.0%), 15 (7.7%), and 53 (27.3%) exhibited rough, smooth, and mixed colony morphotypes, respectively. Glycopeptidolipid genotyping indicated that 86.7% (13 of 15) of smooth isolates belonged to the GPL-wild type (WT) group, whereas 98.4% (124 of 126) of rough isolates belonged to the GPL-mutant type (MUT) group. Therefore, GPL genotyping accurately distinguished between smooth and rough morphotypes. Mixed colony morphotypes were also divided into GPL-WT (18.9%) and GPL-MUT (81.1%) groups. Further analysis revealed that patients infected with the GPL-MUT group presented with significantly worse baseline clinical characteristics and exacerbated episodes of lung disease. Glycopeptidolipid genotyping accurately distinguishes smooth and rough colony morphotypes. Patients infected with the GPL-MUT genotype exhibit worse clinical characteristics and are at a higher risk of exacerbated lung disease.

Serum immunoglobulin a antibodies to glycopeptidolipid core antigen for Mycobacteroides abscessus complex lung disease.

Mycobacteroides abscessus complex (MABC) exhibits smooth morphotypes, expressing glycopeptidolipid (GPL), and rough morphotypes, expressing diminished GPL, on the MABC cell wall. Few reports have focused on the relationship between anti-GPL-core immunoglobulin A (IgA) antibody and colony morphology in MABC lung disease. This study aimed to test GPL core antigen in patients with MABC lung disease to investigate the relationship between coinfection/contamination in other nontuberculous mycobacteria species and colony morphology variant in MABC isolates. Patients with MABC lung disease and contamination diagnosed between 2012 and 2017 at our hospital were enrolled retrospectively. Of the assessed patients, 43 patients with MABC lung disease and 13 with MABC contamination were included. There was a significant difference in anti-GPL-core IgA antibody levels between them (P = 0.02). Forty-three patients with MABC lung disease were divided into two groups as positive and negative antibodies groups. A significant increase in the positive anti-GPL-core IgA antibody was observed in coexistence with both Mycobacterium avium complex (MAC) (P = 0.02) and the isolate of the smooth variant (P = 0.03) in MABC. Anti-GPL-core IgA antibodies in patients with MABC are greatly influenced by MAC coexistence, and colony morphology variant of the MABC isolate.

Insight into the biology of Mycobacterium mucogenicum and Mycobacterium neoaurum clade members

Nontuberculous mycobacteria, NTM, are of growing concern and among these members of the Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clades can cause infections in humans and they are resistant to first-line anti-tuberculosis drugs. They can be isolated from different ecological niches such as soil, tap water and ground water. Mycobacteria, such as Mmuc and Mneo, are classified as rapid growing mycobacteria, RGM, while the most familiar, Mycobacterium tuberculosis, belongs to the slow growing mycobacteria, SGM. Modern “omics” approaches have provided new insights into our understanding of the biology and evolution of this group of bacteria. Here we present comparative genomics data for seventeen NTM of which sixteen belong to the Mmuc- and Mneo-clades. Focusing on virulence genes, including genes encoding sigma/anti-sigma factors, serine threonine protein kinases (STPK), type VII (ESX genes) secretion systems and mammalian cell entry (Mce) factors we provide insight into their presence as well as phylogenetic relationship in the case of the sigma/anti-sigma factors and STPKs. Our data further suggest that these NTM lack ESX-5 and Mce2 genes, which are known to affect virulence. In this context, Mmuc- and Mneo-clade members lack several of the genes in the glycopeptidolipid (GLP) locus, which have roles in colony morphotype appearance and virulence. For the M. mucogenicum type strain, MmucT, we provide RNASeq data focusing on mRNA levels for sigma factors, STPK, ESX proteins and Mce proteins. These data are discussed and compared to in particular the SGM and fish pathogen Mycobacterium marinum. Finally, we provide insight into as to why members of the Mmuc- and Mneo-clades show resistance to rifampin and isoniazid, and why MmucT forms a rough colony morphotype.

1341. Development of a Series of High-Throughput Screens to Identify Leads for Nontuberculous Mycobacteria Drug Design

BackgroundNontuberculous mycobacteria (NTM), particularly Mycobacterium avium complex and Mycobacterium abscessus complex, cause significant morbidity and mortality in patients with impaired host immunity or pre-existing structural lung conditions. NTM infections are increasing at an alarming rate worldwide and there is a dearth of progress in regard to the development of efficacious and tolerable drugs to treat such infections. Traditional drug discovery screens do not account for the diverse physiological conditions, microenvironments, and compartments that the bacilli encounter during human infection. In order to help populate the NTM drug pipeline, and explore the disconnect between in vitro activity, in vivo activity, and clinical outcomes, we are developing a high throughput in vitro assay platform that will more closely model the unique infection-relevant conditions encountered by NTM.MethodsWe are developing and validating a suite of in vitro assays that screen compounds for activity against extracellular planktonic bacteria, extracellular bacteria within biofilms, intracellular bacteria, and nutrient-starved non-replicating bacteria.ResultsWe are using both the smooth and rough morphotypes of M. abscessus and M. avium. We have validated high throughput assays to pharmaceutical standards for replicating and non-replicating M. abscessus. We have also tested a panel of 18 known anti-mycobacterial compounds. Assay development is currently underway to test compounds for activity against NTM in biofilm and inside macrophages as well.ConclusionTo enhance hit identification for scaffolds to use as starting points for NTM drug development, focused libraries of compounds that have undergone significant preclinical profiling and/or compounds with known activity against M. tuberculosis (TB) will be screened. Such a “piggyback” approach usurps advances made in TB drug development and leverages them for NTM drug discovery. This will help expedite novel drug development, reduce attrition rate, and offer a shorter route to clinical use as it exploits the prior investment in medicinal chemistry, pharmacology, and toxicology.Disclosures All authors: No reported disclosures.

Influence of Phenotypic Dissociation in Bacillus subtilis Strain ET-1 on Iturin A Production.

Iturin A is a very important cyclic lipopeptide produced by several B. subtilis strains and has large commercial and therapeutic application potentials but its production on industrial scale has not been realized yet. In the present study, we have observed that the strain ET-1 of Bacillus subtilis, a producer of Iturin A, can present at least three different colony morphologies, which we arbitrarily called Rough, Smooth, and Mucoid morphotypes (R-, S-, and M-form). Performing HPLC analysis, a significant difference between the amounts of Iturin A produced by the three morphotypes was found. The morphotype R-form showed the highest productivity with yields about 10 and 100 times higher than morphotypes S and M, respectively. The results show that the production of Iturin A by B. subtilis could be strongly influenced by the phenotypic heterogeneity of cells within the inoculum. Indeed, we have observed that, pasteurizing the inoculum before seeding in order to improve the homogeneity removing the phenotypes less able to synthesize the Iturin A, its yields in a bench-scale production could be significantly improved. This can represent an important control factor also at industrial scale to improve the Iturin A yields, the robustness, the replicability, and consequently the cost-effectiveness of fermentation processes.

Phenotypic switching in Candida tropicalis alters host-pathogen interactions in a Galleria mellonella infection model

Candida tropicalis is a human pathogen associated with high mortality rates. We have reported a switching system in C. tropicalis consisting of five morphotypes – the parental, switch variant (crepe and rough), and revertant (crepe and rough) strains, which exhibited altered virulence in a Galleria mellonella model. Here, we evaluate whether switching events may alter host-pathogen interactions by comparing the attributes of the innate responses to the various states. All switched strains induced higher melanization in G. mellonella larvae than that induced by the parental strain. The galiomicin expression was higher in the larvae infected with the crepe and rough morphotypes than that in the larvae infected with the parental strain. Hemocytes preferentially phagocytosed crepe variant cells over parental cells in vitro. In contrast, the rough variant cells were less phagocytosed than the parental strain. The hemocyte density was decreased in the larvae infected with the crepe variant compared to that in the larvae infected with the parental strain. Interestingly, larvae infected with the revertant of crepe restored the hemocyte density levels that to those observed for larvae infected with the parental strain. Most of the switched strains were more resistant to hemocyte candidacidal activity than the parental strain. These results indicate that the switch states exhibit similarities as well as important differences during infection in a G. mellonella model.

Genotypic and phenotypic characteristics associated with biofilm formation in Escherichia coli and Salmonella spp. isolated from ulam in Terengganu

Nowadays, the foodborne outbreaks associated with fresh produces, including ulam, are increasing worldwide. The biofilm formation or bacterial attachment to plant surface is the initial step towards the contamination in fresh produce. The biofilm phenotype of bacteria grown on congo red agar is termed as red, dry and rough (rdar) morphotype. The binding of congo red dye with both biological proteins and inert surfaces is due to the presence of curli fimbriae and cellulose as the main extracellular components. The objective of this study was to determine the rdar morphotypes, biofilm ability and the role of csgA gene of Escherichia coli and Salmonella spp. isolated from ulam or Malaysian herbs. A total of 29 isolates, including 23 E. coli and 6 Salmonella spp. were analyzed for their ability to produce biofilm by colony morphology test, microtiter plate biofilm assay and qualitative biofilm test (pellicle formation). The presence of the csgA gene of E. coli was identified by PCR, which demonstrated the potential gene that able to produce curli fimbriae. Results revealed that 16 (69.6%) E. coli isolates were categorized as strong biofilm producers, 2 (8.7%) as moderate biofilm producers, 3 (13%) as weak biofilm producers, whereas 2 (8.7%) as negative biofilm producers (did not produce biofilm). While 4 (66.7%) Salmonella spp. isolates were identified as strong biofilm producers, 1 (16.7%) as moderate biofilm producers and 1 (16.7%) as negative biofilm producers. Majority of the E. coli strains (69.6%) were identified as strong biofilm producers and able to express rdar morphotypes. The ability of the of E. coli and Salmonella spp. isolates to form biofilm reveals the ability of these isolates to persist on the fresh vegetables and become hosts for the disease transmission to humans or/and animals.

Lsr2 Is an Important Determinant of Intracellular Growth and Virulence in Mycobacterium abscessus

Mycobacterium abscessus, a pathogen responsible for severe lung infections in cystic fibrosis patients, exhibits either smooth (S) or rough (R) morphotypes. The S-to-R transition correlates with inhibition of the synthesis and/or transport of glycopeptidolipids (GPLs) and is associated with an increase of pathogenicity in animal and human hosts. Lsr2 is a small nucleoid-associated protein highly conserved in mycobacteria, including M. abscessus, and is a functional homolog of the heat-stable nucleoid-structuring protein (H-NS). It is essential in Mycobacterium tuberculosis but not in the non-pathogenic model organism Mycobacterium smegmatis. It acts as a master transcriptional regulator of multiple genes involved in virulence and immunogenicity through binding to AT-rich genomic regions. Previous transcriptomic studies, confirmed here by quantitative PCR, showed increased expression of lsr2 (MAB_0545) in R morphotypes when compared to their S counterparts, suggesting a possible role of this protein in the virulence of the R form. This was addressed by generating lsr2 knock-out mutants in both S (Δlsr2-S) and R (Δlsr2-R) variants, demonstrating that this gene is dispensable for M. abscessus growth. We show that the wild-type S variant, Δlsr2-S and Δlsr2-R strains were more sensitive to H2O2 as compared to the wild-type R variant of M. abscessus. Importantly, virulence of the Lsr2 mutants was considerably diminished in cellular models (macrophage and amoeba) as well as in infected animals (mouse and zebrafish). Collectively, these results emphasize the importance of Lsr2 in M. abscessus virulence.

DncV Synthesizes Cyclic GMP-AMP and Regulates Biofilm Formation and Motility in Escherichia coli ECOR31.

Cyclic dinucleotides (cDNs) act as intracellular second messengers, modulating bacterial physiology to regulate the fundamental life style transition between motility and sessility commonly known as biofilm formation. Cyclic GMP-AMP (cGAMP), synthesized by the dinucleotide cyclase DncV, is a newly discovered cDN second messenger involved in virulence and chemotaxis in Vibrio cholerae O1 biovar El Tor. Here we report a novel role for horizontally transferred DncV in cGAMP production and regulation of biofilm formation and motility in the animal commensal strain Escherichia coli ECOR31. ECOR31 expresses a semiconstitutive temperature-independent rdar (red, dry, and rough) morphotype on Congo red agar plates characterized by the extracellular matrix components cellulose and curli fimbriae which requires activation by the major biofilm regulator CsgD and cyclic di-GMP signaling. In contrast, C-terminal His-tagged DncV negatively regulates the rdar biofilm morphotype and cell aggregation via downregulation of csgD mRNA steady-state level. Furthermore, DncV sequentially promotes and inhibits adhesion to the abiotic surface after 24 h and 48 h of growth, respectively. DncV also suppresses swimming and swarming motility posttranscriptional of the class 1 flagellum regulon gene flhD Purified DncV produced different cDNs, cyclic di-GMP, cyclic di-AMP, an unknown product(s), and the dominant species 3'3'-cGAMP. In vivo, only the 3'3'-cGAMP concentration was elevated upon short-term overexpression of dncV, making this work a first report on cGAMP production in E. coli Regulation of rdar biofilm formation and motility upon overexpression of untagged DncV in combination with three adjacent cotransferred gene products suggests a novel temperature-dependent cGAMP signaling module in E. coli ECOR31.IMPORTANCE The ability of bacteria to sense and respond to environmental signals is critical for survival. Bacteria use cyclic dinucleotides as second messengers to regulate a number of physiological processes, such as the fundamental life style transition between motility and sessility (biofilm formation). cGAMP, which is synthesized by a dinucleotide cyclase called DncV, is a newly discovered second messenger involved in virulence and chemotaxis in the Vibrio cholerae biovar El Tor causing the current 7th cholera pandemic. However, to what extent cGAMP exists and participates in physiological processes in other bacteria is still unknown. In this study, we found an elevated cGAMP level to possibly regulate biofilm formation and motility in the animal commensal E. coli strain ECOR31. Thus, we detected a novel role for cGAMP signaling in regulation of physiological processes other than those previously reported in proteobacterial species.

The afc antifungal activity cluster, which is under tight regulatory control of ShvR, is essential for transition from intracellular persistence of Burkholderia cenocepacia to acute pro-inflammatory infection.

The opportunistic pathogen Burkholderia cenocepacia is particularly life-threatening for cystic fibrosis (CF) patients. Chronic lung infections with these bacteria can rapidly develop into fatal pulmonary necrosis and septicaemia. We have recently shown that macrophages are a critical site for replication of B. cenocepacia K56-2 and the induction of fatal pro-inflammatory responses using a zebrafish infection model. Here, we show that ShvR, a LysR-type transcriptional regulator that is important for biofilm formation, rough colony morphotype and inflammation in a rat lung infection model, is also required for the induction of fatal pro-inflammatory responses in zebrafish larvae. ShvR was not essential, however, for bacterial survival and replication in macrophages. Temporal, rhamnose-induced restoration of shvR expression in the shvR mutant during intramacrophage stages unequivocally demonstrated a key role for ShvR in transition from intracellular persistence to acute fatal pro-inflammatory disease. ShvR has been previously shown to tightly control the expression of the adjacent afc gene cluster, which specifies the synthesis of a lipopeptide with antifungal activity. Mutation of afcE, encoding an acyl-CoA dehydrogenase, has been shown to give similar phenotypes as the shvR mutant. We found that, like shvR, afcE is also critical for the switch from intracellular persistence to fatal infection in zebrafish. The closely related B. cenocepacia H111 has been shown to be less virulent than K56-2 in several infection models, including Galleria mellonella and rats. Interestingly, constitutive expression of shvR in H111 increased virulence in zebrafish larvae to almost K56-2 levels in a manner that absolutely required afc. These data confirm a critical role for afc in acute virulence caused by B. cenocepacia that depends on strain-specific regulatory control by ShvR. We propose that ShvR and AFC are important virulence factors of the more virulent Bcc species, either through pro-inflammatory effects of the lipopeptide AFC, or through AFC-dependent membrane properties.

A proteomics approach for the identification of species-specific immunogenic proteins in the Mycobacterium abscessus complex

The Mycobacterium abscessus complex can cause fatal pulmonary disease, especially in cystic fibrosis patients. Diagnosing M. abscessus complex pulmonary disease is challenging. Immunologic assays specific for M. abscessus are not available. In this study seven clinical M. abscessus complex strains and the M. abscessus reference strain ATCC19977 were used to find species-specific proteins for their use in immune assays. Six strains showed rough and smooth colony morphotypes simultaneously, two strains only showed rough mophotypes, resulting in 14 separate isolates. Clinical isolates were submitted to whole genome sequencing. Proteomic analysis was performed on bacterial lysates and culture supernatant of all 14 isolates. Species-specificity for M. abscessus complex was determined by a BLAST search for proteins present in all supernatants. Species-specific proteins underwent in silico B- and T-cell epitope prediction. All clinical strains were found to be M. abscessus ssp. abscessus. Mutations in MAB_4099c as a likely genetic basis of the rough morphotype were found in six out of seven clinical isolates. 79 proteins were present in every supernatant, of which 12 are exclusively encoded by all members of M. abscessus complex plus Mycobacterium immunogenum. In silico analyses predicted B- and T-cell epitopes in all of these 12 species-specific proteins.

Biofilm-Forming Capacity of Five Salmonella Strains and Their Fate on Postharvest Mini Cucumbers

Salmonella enterica is one of the pathogens that is frequently identified as the cause of fresh produce-related outbreaks. Biofilm formation is a factor that can contribute to pathogen survival on produce surface. The goal of our current research was to investigate the survival of five S. enterica strains representing different serotypes (i.e., Typhimurium, Enteritidis, Daytona, Poona, and Newport) on whole mini cucumbers stored at refrigeration (4°C) and room temperature (22°C). We also determined the strains survival on glass slides and in phosphate-buffered saline at 4 and 22°C, as well as the ability to form biofilms on a solid-liquid interphase. A rapid decrease in cell density (>4-log reduction over 8 days) of all five tested strains was observed on glass slides, while a slower die-off (<1-log reduction in 8 days) was observed in PBS. No significant difference in the die-off rate was observed among the five strains at 4 or 22°C. The die-off rate on the surface of mini cucumbers at 4°C was significantly slower ( P < 0.02) for Salmonella Enteritidis LMFS-S-JF-005 compared with the remaining four strains. At 22°C, Salmonella Poona S306 was able to grow by more than 1.5 log units on whole mini cucumbers over a period of 8 days, while the cell density of the other four strains remained at the same level compared with day 0. At this temperature, Salmonella Poona S306 was also able to form significantly stronger biofilms on a solid-liquid interphase ( P < 0.01) and was the only strain that presented a red, dry, and rough morphotype on Congo red agar plates, indicating the formation of both curli fimbriae and cellulose. These results revealed that the fate of Salmonella on mini cucumbers is strain specific, which highlighted the need for tailored mitigation strategies, such as the effective control of temperature and moisture for limiting the survival or growth of high-risk Salmonella strains between harvest and consumption of fresh produce.

Mycobacterium decipiens sp. nov., a new species closely related to the Mycobacterium tuberculosis complex.

Two mycobacterial strains with close similarity to the Mycobacterium tuberculosis complex (MTBC) were isolated from cutaneous lesions of patients in the USA and Italy. At the phenotypic level, similarities to the MTBC included slow growth rate, rough morphotype of the unpigmented colonies and nearly identical high-performance liquid chromatography profiles of mycolic acids. In contrast to the MTBC, the strains were niacin- and nitrate-negative, and catalase-positive both at 68 °C and in semi-quantitative tests. The clinical isolates were more closely related to M. tuberculosis than to any other known mycobacterium and scored positive with commercial DNA probes (Hologic AccuProbe M. tuberculosis). Both average nucleotide identity and genome-to-genome distance suggested the strains are different from the MTBC. Therefore, given the distinguishing phenotypic and genomic-scale differences, we submit that the strains belong to a new species we have named Mycobacteriumdecipiens with type strain TBL 1200985T (=ATCC TSD-117T=DSM 105360T).