Rough Colony Research Articles

Evolution toward extremely high imipenem resistance in Mycobacterium abscessus outbreak strains.

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem). M. abscessus produces a β-lactamase (BlaMab) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a cystic fibrosis center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC > 512 µg/mL). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased blaMab mRNA level. Concurrent increase in transcription of the preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 µg/mL). A frameshift mutation in msp2 responsible for the rough colony morphology and a SNP in ATP-dependent helicase hrpA cooccurred with the second increase in MIC (from 32 to 256 µg/mL). Increased BlaMab expression and enzymatic activity may have been due to altered regulation of the ppiA-blaMab operon by the mutated HrpA alone or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating M. abscessus, particularly imipenem-resistant strains.

Characterization of a Salmonella enterica serovar Typhimurium lineage with rough colony morphology and multidrug resistance

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of salmonellosis, and the emergence of multidrug-resistant pathovariants has become a growing concern. Here, we investigate a distinct rough colony variant exhibiting a strong biofilm-forming ability isolated in China. Whole-genome sequencing on 2,212 Chinese isolates and 1,739 publicly available genomes reveals the population structure and evolutionary history of the rough colony variants. Characterized by macro, red, dry, and rough (mrdar) colonies, these variants demonstrate enhanced biofilm formation at 28 °C and 37 °C compared to typical rdar colonies. The mrdar variants exhibit extensive multidrug resistance, with significantly higher resistance to at least five classes of antimicrobial agents compared to non-mrdar variants. This resistance is primarily conferred by an IncHI2 plasmid harboring 19 antimicrobial resistance genes. Phylogenomic analysis divides the global collections into six lineages. The majority of mrdar variants belong to sublineage L6.5, which originated from Chinese smooth colony strains and possibly emerged circa 1977. Among the mrdar variants, upregulation of the csgDEFG operons is observed, probably due to a distinct point mutation (−44G > T) in the csgD gene promoter. Pangenome and genome-wide association analyses identify 87 specific accessory genes and 72 distinct single nucleotide polymorphisms associated with the mrdar morphotype.

Antimicrobial susceptibility analysis of isepamicin combination treatments in Mycobacterium abscessus species

This study evaluated the antimicrobial potency of the combination of isepamicin (ISP) for Mycobacterium abscessus species (MABS). 34 clinical MABS strains were isolated from clinical samples. Of them, 11 (32.4 %) were M. abscessus subsp. abscessus (Mab), 22 (64.7 %) were M. abscessus subsp. massiliense (Mma), and one (2.9 %) was M. abscessus subsp. bolletii (Mbo). We compared susceptibility to sitafloxacin (STFX)-ISP and clarithromycin (CLR)-ISP combinations with those of the antimicrobial agents alone, and synergistic effects were observed in 41.2 % and 17.6 % when treated with STFX-ISP and CLR-ISP. By hierarchical cluster analysis, the isolates divided into treatment-sensitive and treatment-resistant groups. Non-Mma or rough colony isolates were significantly likely to belong to the treatment-sensitive group (p = 0.024, p < 0.001, respectively). These results suggest that the ISP-containing combination could be a new therapeutic strategy for MABS, especially in cases of non-Mma: treatment-refractory subspecies, and rough morphotypes: high-virulence morphotypes.

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.

A Case-Based Review of LVAD Infection by MABC

Abstract We present a review of literature and report a case of left ventricular assist device (LVAD) infection and postsurgical wound infection with Mycobacterium abscessus complex (MABC) following exposure to sewage and tap water, respectively. The initial infectious process involved the driveline and the deep pocket and progressed to involve LVAD. Prior to availability of culture results, the patient was started on linezolid followed by surgical debridement of driveline site and change of exit site. The debrided tissue grew a smooth and a rough colony variant of MABC. After 2 weeks of imipenem intravenous (IV), tigecycline IV, azithromycin oral (PO), and tedizolid PO, the patient presented to us with worsening of wound status. Antimicrobial therapy was changed to amikacin IV, imipenem IV, and cefoxitin IV along with bedside debridement, removal of 3 sutures, and placement of wound vacuum device. Amikacin IV, imipenem IV, and cefoxitin IV were then replaced with Recarbio (imipenem/cilastatin/relebactam) IV plus amoxicillin PO with significant improvement. The patient underwent LVAD removal and heart transplantation after 5 weeks and was discharged 15 days later. Following showering at home, the patient developed signs of extensive infection involving the surgical wound (chest and abdominal walls). Tigecycline IV and azithromycin IV were started. Imipenem/cilastatin/ relebactam IV plus amoxicillin PO were replaced by bedaquiline PO because of the continued financial burden to the patient. The patent was discharged on bedaquiline PO and azithromycin PO to complete 18 months of antimicrobial therapy at which time he was enjoying a very active lifestyle as he did prior to the diagnosis of advanced heart failure. He continues to do well 2 years 9 months after heart transplantation and more than a year after stopping all antimycobacterial therapy.

Evolution towards extremely high β-lactam resistance in Mycobacterium abscessus outbreak strains.

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem, meropenem). M. abscessus produces a β-lactamase (BlaMab) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a CF center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC >512 μg/ml). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased bla Mab mRNA level. Concurrent increase in transcription of preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 μg/ml). A frameshift mutation in msp2 responsible for the rough colony morphology, and a SNP in ATP-dependent helicase hrpA co-occurred with the second increase in MIC (from 32 to 256 μg/ml). Increased BlaMab expression and enzymatic activity may have been due to altered regulation of the ppiA-bla Mab operon by the mutated HrpA alone, or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating M. abscessus, particularly imipenem resistant strains.

Uncommon Salmonella Infantis Variants with Incomplete Antigenic Formula in the Poultry Food Chain, Italy.

Uncommon Salmonella Infantis variants displaying only flagellar antigens phenotypically showed identical incomplete antigenic formula but differed by molecular serotyping. Although most formed rough colonies, all shared antimicrobial resistances and the presence of usg gene with wild-type Salmonella Infantis. Moreover, they were undistinguishable wild-type Salmonella Infantis by whole-genome sequencing.

Thinking inside the box: Restoring the propolis envelope facilitates honey bee social immunity.

When wild honey bee colonies (Apis mellifera) nest in hollow tree cavities, they coat the rough cavity walls with a continuous layer of propolis, a substance comprised primarily of plant resins. Studies have shown that the resulting "propolis envelope" leads to both individual- and colony-level health benefits. Unfortunately, the smooth wooden boxes most commonly used in beekeeping do little to stimulate propolis collection. As a result, most managed bees live in hives that are propolis-poor. In this study, we assessed different surface texture treatments (rough wood boxes, boxes outfitted with propolis traps, and standard, smooth wood boxes) in terms of their ability to stimulate propolis collection, and we examined the effect of propolis on colony health, pathogen loads, immune gene expression, bacterial gene expression, survivorship, and honey production in both stationary and migratory beekeeping contexts. We found that rough wood boxes are the most effective box type for stimulating propolis deposition. Although the use of rough wood boxes did not improve colony survivorship overall, Melissococcus plutonius detections via gene expression were significantly lower in rough wood boxes, and viral loads for multiple viruses tended to decrease as propolis deposition increased. By the end of year one, honey bee populations in migratory rough box colonies were also significantly larger than those in migratory control colonies. The use of rough wood boxes did correspond with decreased honey production in year one migratory colonies but had no effect during year two. Finally, in both stationary and migratory operations, propolis deposition was correlated with a seasonal decrease and/or stabilization in the expression of multiple immune and bacterial genes, suggesting that propolis-rich environments contribute to hive homeostasis. These findings provide support for the practical implementation of rough box hives as a means to enhance propolis collection and colony health in multiple beekeeping contexts.

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.

Characterisation of Variants of Cyclic di-GMP Turnover Proteins Associated with Semi-Constitutive rdar Morphotype Expression in Commensal and Uropathogenic Escherichia coli Strains.

Expression of rdar (red, dry, and rough) colony morphology-based biofilm formation in Escherichia coli is highly variable. To investigate the molecular mechanisms of semi-constitutive rdar morphotype formation, we compared their cyclic di-GMP turnover protein content and variability to the highly regulated, temperature-dependent morphotype of the historical and modern ST10 isolates E. coli MG1655 and Fec10, respectively. Subsequently, we assessed the effects of cyclic di-GMP turnover protein variants of the EAL phosphodiesterases YcgG and YjcC and the horizontally transferred diguanylate cyclase DgcX on biofilm formation and motility. The two YcgG variants with truncations of the N-terminal CSS signaling domain were oppositely effective in targeting downregulation of rdar biofilm formation compared to the full-length reference protein. Expression of the C-terminal truncated variants YjcCFec67 and YjcCTob1 showed highly diminished apparent phosphodiesterase activity compared to the reference YjcCMG1655. For YjcCFec101, substitution of the C-terminus led to an apparently inactive enzyme. Overexpression of the diguanylate cyclase DgcX contributed to upregulation of cellulose biosynthesis but not to elevated expression of the major biofilm regulator csgD in the "classical" rdar-expressing commensal strain E. coli Fec10. Thus, the c-di-GMP regulating network is highly complex with protein variants displaying substantially different apparent enzymatic activities.

ArfA and SarA small GTPases involved in growth, patulin biosynthesis and virulence of Penicillium expansum

Although the Sar/Arf GTPase belonging to a small family of GTPases contributes to growth of fungi, its function in postharvest fungi has not been characterized. Here, ArfA and SarA gene deletion mutants were generated in Penicillium expansum. We found that ΔArfA and ΔSarA exhibited attenuated colony growth and rough colony morphology, as well as insufficient spore development. ΔArfA also increased hyphal branches. Stress experiments showed that knocking out the ArfA or SarA genes affected cell wall integrity of the P. expansum strain. In addition, ΔArfA and ΔSarA significantly downregulated the expression of genes controlling spore development (wetA, abaA and brlA) and MAPK pathway regulators (Fus3, Kss1, Hog1 and Mpk1). Absence of ArfA and SarA reduced patulin production, and the related gene expression was significantly limited. ΔArfA showed attenuated virulence in infected fruit and downregulated PG and PL gene expression. However, ΔSarA had no significant effect on fungal virulence. Taken together, both ArfA and SarA contribute to the growth, development and patulin biosynthesis in P. expansum. ArfA is also involved in the virulence of P. expansum in fruit.

Impacts of a DUF2207 Family Protein on Streptococcus mutans Stress Tolerance Responses and Biofilm Formation.

Locus SMU.243 in Streptococcus mutans was annotated as a member of the DUF2207 family proteins highly conserved in all bacteria but with unknown function. To investigate its role in S. mutans physiology, a SMU.243-deficient mutant was constructed using allelic exchange mutagenesis, and the impacts of SMU.243 deletion on bacterial growth, stress tolerance response, and biofilm formation were analyzed. Compared to the wild-type UA159, S. mutans lacking SMU.243 displayed a reduced growth rate and a reduced overnight culture density (p < 0.01) when grown at low pH and in the presence of methyl viologen. Relative to the parent strain, the deficient mutant also had a reduced survival rate following incubation in a buffer of pH 2.8 (p < 0.01) and in a buffer containing hydrogen peroxide at 58 mM after 60 min (p < 0.001) and had a reduced capacity in biofilm formation especially in the presence of sucrose (p < 0.01). To study any ensuing functional/phenotypical links between SMU.243 and uppP, which is located immediately downstream of SMU.243 and encodes an undecaprenyl pyrophosphate phosphatase involved in recycling of carrier lipid undecaprenyl phosphate, a uppP deficient mutant was generated using allelic exchange mutagenesis. Unlike the SMU.243 mutant, deletion of uppP affected cell envelope biogenesis and caused major increases in susceptibility to bacitracin. In addition, two variant morphological mutants, one forming rough colonies and the other forming mucoid, smooth colonies, also emerged following the deletion of uppP. The results suggest that the SMU.243-encoded protein of the DUF2207 family in S. mutans plays an important role in stress tolerance response and biofilm formation, but unlike the downstream uppP, does not seem to be involved in cell envelope biogenesis, although the exact roles in S. mutans' physiology awaits further investigation.

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.

Phenotypic and genetic features of a novel clinically isolated rough morphotype Candida auris.

Candida auris is a newly emerging pathogenic fungus of global concern and has been defined by the World Health Organization (WHO) as a member of the critical group of the most health-threatening fungi. This study reveals and reports for the first time that a rough morphotype C. auris strain causes urinary tract infections in non-intensive care unit (ICU) inpatients. Furthermore, the morphology, the scanning electronmicroscopy (SEM), Whole-genome resequencing and RNA sequencing of C. auris possessing rough morphotype colonies compared to their smooth morphotype counterparts. The newly identified phenotypic variation of C. auris appears round, convex, dry, and burr-like with a rough texture. SEM shows that rough type C. auris has a rough and uneven colony surface with radial wrinkles and irregular spore arrangement. Cells of the rough morphotype C. auris naturally aggregate into clusters with tight connections in the liquid, and it seems that the cell division is incomplete. A genome-wide analysis of the rough type C. auris confirmed its genetic association with the smooth type of C. auris prevalent in China (Shenyang) two years ago; however, single nucleotide polymorphism (SNP) mutations of five genes (ACE2, IFF6, RER2, UTP20, and CaO19.5847) were identified more recently. RNA-seq revealed IFF2/HYR3, DAL5, PSA31, and SIT1 were notably up-regulated, while multiple cell wall-associated genes (ALS1, MNN1, PUL1, DSE1, SCW11, PGA38, RBE1, FGR41, BGLI, GIT3, CEP3, and SAP2) were consistently down-regulated in rough morphotype C. auris. The rough phenotypic variation of C. auris is likely to be related to the structural and functional changes in cell wall proteins. This novel rough morphotype C. auris will provide a basis for further studies concerning the evolutionary characteristics of C. auris.

First detection of Trichothecium roseum causing leaf spots on tomato in Germany.

Tomato (Solanum lycopersicum L.) is one of the most popular vegetables in Germany and the demand for organically grown tomatoes in particular is growing steadily. In seasonal low-cost organic tomato cultivation, the leaf mould disease caused by Fulvia fulva is an increasing problem (Meyer et al. 2021). In 2020-2021, during a survey for F. fulva infected tomato leaves in Germany, untypical, circular, and dark brown necrotic spots were observed. To identify the pathogen, symptomatic leaves were collected from three different geographic locations in the north and south of Germany. Specimens were observed under a stereomicroscope and immediately processed. After surface disinfection of symptomatic leaf material, the leaf tissues were placed on potato dextrose agar (PDA) and subsequently incubated at 25 °C in the dark allowing mycelium to grow. Single spore isolates from each location were prepared for morphological and molecular analyses, as well as for pathogenicity determination. Three single spore isolates (JKI-GFP-22-010 - 012) showed moderate to rapid growth rates that reached 4 cm in diameter after 7 days. Based solely on morphology (S1), they were identified as Trichothecium roseum (Pers.) Link (Link 1809) by having pink dense mycelium with circular and rough edge colonies. The conidiophores were simple or branched (105 - 254 × 2 - 4 μm). Conidia were 2-celled, smooth, thick-walled, hyaline, ellipsoid to pyriform with slanting and truncate basal mark, and were produced in clusters, (10 - 18 × 7 - 9.5 μm). To afford the genetic data of these fungi, fungal DNA was extracted using a DNAeasy® Kit Plant Mini Kit (QIAGEN®, Germany) and then subjected to PCR using the primer sets as follows, ITS1/ITS4 (White et al. 1990) and LROR/LR6 (Vilgalys et al. 1990). The consensus sequences generated in this study were deposited in GenBank under the accession numbers ON787818 - 20 and ON787821 - 23, for ITS and LSU, respectively, and compared via NCBI-BLAST. Initial BLAST searches of both ITS and LSU regions revealed that the isolates share 99 - 100% homology with T. roseum. The morphology and phylogeny (S2) supported assignment of all isolates to T. roseum. To fulfil Koch's postulate, the pathogenicity of selected isolates (JKI-GFP-22-010 and 012) were tested by spraying spore suspensions (105 conidia ml-1) on adaxial surface of 21-day-old tomato plants cv. 'Moneymaker' (n=10). Plants sprayed with sterile water served as controls. All tomato plants were grown under greenhouse conditions (average 25 °C, 98% relative humidity). After 21 days, symptoms were identical to those of the originally infected leaves associated with T. roseum. As the disease progressed, necrotic lesions reached approximately 35 mm in diameter. The fungus was re-isolated and identified as T. roseum. No symptoms developed in the control plants. Trichothecium roseum has been detected infecting fruit, particularly on tomatoes, e.g. in Argentina (Dal et al. 2008), Brazil (Inácio et al. 2011), Korea (Yun et al. 2013), and Pakistan (Hamid et al. 2014). To our knowledge, this is the first report of T. roseum infecting tomato in Germany. As the pathogen was found in several locations in the south and north, it is likely already well established in Germany. To date, it seems to play a minor role in pathogenicity of tomatoes but should be monitored, especially with regard to climate change as there are now several recent reports from the temperate region. The disease development and interaction between susceptible host and pathogen should be concerned.

Ferritin from Mycobacterium abscessus is involved in resistance to antibiotics and oxidative stress.

Mycobacterium abscessus subsp. massiliense (Mycma) is a rapidly growing Mycobacterium belonging to the M. abscessus complex that is often associated with lung and soft tissue infection outbreaks. Mycma is resistant to many antimicrobials, including those used for treating tuberculosis. Therefore, Mycma infections are difficult to treat and may lead to high infectious complication rates. Iron is essential for bacterial growth and establishment of infection. During infection, the host reduces iron concentrations as a defense mechanism. To counteract the host-induced iron deficiency, Mycma produces siderophores to capture iron. Mycma has two ferritins (encoded by mycma_0076 and mycma_0077) modulated by different iron concentrations, which allow the survival of this pathogen during iron scarcity. In this study, we constructed knockout (Mycma 0076KO) and complemented (Mycma 0076KOc) gene strains for mycma_0076 to understand the function of 0076 ferritin. Deletion of mycma_0076 in Mycma led to the transition in colony morphology from smooth to rough, alteration of the glycopeptidolipids spectra, increased permeability of the envelope, reduction in biofilm formation, increased susceptibility to antimicrobials and hydrogen peroxide-induced oxidative stress, and decreased internalization by macrophages. This study shows that Mycma_0076 ferritin in Mycma is involved in resistance to oxidative stress and antimicrobials, and alteration of cell envelope architecture. KEY POINTS: • Deletion of the mycma_0076 gene altered colony morphology to rough; • Mycma 0076KO changed GPL profile; • Absence of Mycma_0076 ferritin results in increased susceptibility to antimicrobials and oxidative stress in Mycma. Legend: a In wild-type M. abscessus subsp. massiliensestrain, iron is captured from the environment by carboxymycobactins and mycobactins (1). Iron-dependent regulator (IdeR) proteins bind to ferrous iron (Fe+2) in the bacterial cytoplasm leading to the activation of the IdeR-Fe+2 complex (2). The activated complex binds to the promoter regions of iron-dependent genes, called iron box, which in turn help in the recruitment of RNA polymerase to promote transcription of genes such as mycma_0076 and mycma_0077 ferritin genes (3). Mycma_0076 and Mycma_0077 ferritins bind to excess iron in the medium and promote Fe2+ oxidation into ferric iron (Fe3+) and store iron molecules to be released under iron scarcity conditions. (4) Genes related to biosynthesis and transport of glycopeptidolipids (GPL) are expressed normally and the cell envelope is composed of different GPL species (colored squares represented on the cell surface (GPLs). Consequently, WT Mycma present smooth colony phenotype (5). b In Mycma 0076KO strain, the lack of ferritin 0076 causes overexpression of mycma_0077 (6), but does not restore wild-type iron homeostasis and thus may result in free intracellular iron, even in the presence of miniferritins (MaDps). The excess iron potentiates oxidative stress (7) by generating hydroxyl radicals through Fenton Reaction. During this process, through an unknown mechanism, that could involve Lsr2 (8), the expression of GPL synthesis locus is regulated positively and/or negatively, resulting in alteration of GPL composition in the membrane (as represented by different colors of squares on the cell surface), resulting in a rough colony phenotype (9). The changes of GPL can increase cell wall permeability, contributing to antimicrobial susceptibility (10).

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.

Deciphering Colonies of Phenotypic Switching-Derived Morphotypes of the Pathogenic Yeast Candida tropicalis.

Phenotypic switching generates fungal colonies with altered morphology and allows pathogens to adapt to changing environments. This study investigated the structure and genetic factors of switched morphotypes colonies in Candida tropicalis. Morphotypes of C. tropicalis comprised the clinical strain 49.07 that exhibited smooth colony phenotype and switched (crepe and rough) morphotypes that showed colonies with marked structural variations, including wrinkled surface, depressions areas, and irregular edges (structured morphology). The morphotypes were analyzed for the presence and distribution of the extracellular matrix (ECM) at the ultrastructural level-SEM. The composition of the ECM and the percentage of hyphae in colonies were evaluated. The expression of EFG1 (Enhanced filamentous growth protein 1), WOR1 (White-opaque regulator 1), and BCR1 (Biofilm and cell wall regulator 1) in the morphotypes was measured by RT-qPCR. Colonies of the switched variants exhibited distinct arrangements of ECM compared to the smooth phenotype (clinical strain). In addition, rough variant colonies showed higher amounts of total carbohydrates and proteins in ECM (p < 0.05). Switched (crepe and rough) colonies exhibited a higher percentage of hyphae throughout their development (p < 0.05). The mRNA expression levels of EFG1, WOR1, and BCR1 in the rough morphotype were significantly higher than they were in the smooth morphotype. In addition, there was a positive correlation between the expression of these genes and filamentation (hyphae formation) of the rough morphotype (r2 > 0.9472, p < 0.05). Structural variations in switched morphotypes colonies of C. tropicalis seem to be associated with increased hyphae growth and the amount and distribution of ECM. Switched colonies have distinct expressions of the EFG1, WOR1, and BCR1 master regulators genes.

Control of Salmonella in mung bean sprouts by antagonistic spore-forming Bacilli

Sprouting seeds are considered part of a healthy and nutritious diet, and accordingly, their consumption increases globally. However, edible sprouts are occasionally linked to outbreaks of foodborne diseases, with Salmonella enterica being a frequent contaminant. Biocontrol was recently proposed as a possible approach to combat sprout contamination during production. The purpose of this study was to identify sprout-derived Salmonella-antagonists as a basis for the development of new biocontrol agents. To this end, spore-forming bacteria were isolated from sprouted mung bean seeds, based on either in vitro anti-Salmonella activity or rough colony morphology and tested in planta for inhibition of mung bean sprout colonization. Artificially contaminated seeds (2 log CFU/g) treated with selected Bacillus strains (7 log CFU/ml) demonstrated extensive inhibition of Salmonella growth after 4-days of sprouting, resulting in 3.3–5.4 log CFU/g reduction compared to control (untreated). The potent antagonistic activity of the selected Bacillus isolates was confirmed for different S. enterica serovars. Interestingly, some of the antagonistic strains that demonstrated activity in vitro were not active in planta and vice versa, indicating that in vitro testing may not be the best strategy for screening potential biocontrol strains in sprouts. Confocal microscopy imaging demonstrated that in the absence of Bacillus, Salmonella readily proliferates and colonizes the entire sprouting seed. Treatment with an antagonistic Bacillus isolate markedly inhibited the colonization of Salmonella. Scanning electron microscopy of Bacillus-treated sprouts demonstrated the colonization of the sprout with biofilm-like structures, indicating the activation of the biofilm formation mechanism during sprout colonization. The identified Bacillus isolates provide a basis for the future development of novel biocontrol agents against Salmonella and perhaps other foodborne pathogens in edible sprouts.