Rough Morphotype Research Articles

A dTDP-L-rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in Mycobacterium abscessus

Mycobacterium abscessus causes severe lung infections in cystic fibrosis patients and exhibits smooth (S) or rough (R) morphotypes. Disruption of glycopeptidolipid (GPL) production results in the S-to-R transition but the underlying molecular mechanisms of this transition remain incompletely understood. Herein, we characterized MAB_4111c in relation to GPL synthesis and investigated the effects of MAB_4111c deletion in M. abscessus pathogenicity. An enzymatic assay indicated that MAB_4111c, also designated Tle for Talose epimerase, is converting dTDP-L-Rhamnose into dTDP-6-deoxy-L-Talose. A tle deletion mutant was constructed in the S variant of M. abscessus and relative areas of Rhamnose and 6-deoxy-Talose and their methylated forms expressed as ratios of total monosaccharides, showed an altered GPL profile lacking 6-deoxy-Talose. Thus, Tle provides dTDP-6-deoxy-L-Talose, subsequently used by the glycosyltransferase Gtf1 to transfer 6-deoxy-Talose to the GPL backbone. Strikingly, the tle mutant exhibited a R morphotype, showed impaired sliding motility and biofilm formation, and these phenotypes were rescued upon functional complementation. Moreover, deletion of tle in M. abscessus results in increased pathogenicity and killing in zebrafish embryos. Together, our results underscore the importance of the dTDP-L-Rhamnose 4-epimerase activity in GPL biosynthesis and in influencing M. abscessus virulence.

Mycobacterium abscessus strain variability in preclinical drug development: does it really matter?

Abstract Background New treatment options for Mycobacterium abscessus infections are urgently needed. Since a correlation between MICs and clinical outcomes is not clearly established, potency of novel drugs needs to be evaluated using additional in vitro drug activity assays. Preclinical drug activity assays generally use the M. abscessus type strain ATCC 19977. However, M. abscessus complex entails a genetically and morphologically diverse group, and it is questionable whether drug activity observed against ATCC 19977 is representative of drug activity against clinical M. abscessus isolates. Objectives To assess whether the relationship between MIC and the quantitative antimycobacterial activity of amikacin, imipenem and clofazimine differs between the ATCC 19977 strain and clinical M. abscessus isolates. Methods Experiments were performed with M. abscessus ATCC 19977 and a subset of six clinical isolates covering the three M. abscessus subspecies and the smooth and rough morphotypes. Cultures were exposed to the drugs at 4-fold increasing, MIC-standardized concentrations, and the mycobacterial load was assessed over time. Results Concentration- and time-dependent activity of amikacin, imipenem and clofazimine against the six clinical isolates was similar. Only slight variations in drug activity were observed between ATCC 19977 and clinical isolates. Conclusions Time- and concentration-dependent drug activity against the ATCC 19977 strain seems indicative for in vitro drug behaviour against M. abscessus complex clinical isolates. Including one clinical smooth morphotype isolate alongside ATCC 19977 seems appropriate for reliable interpretation of this particular in vitro drug activity assay as part of the M. abscessus preclinical drug development pipeline.

The diversity of clinical Mycobacterium abscessus isolates in morphology, glycopeptidolipids and infection rates in a macrophage model.

Introduction. Mycobacterium abscessus (MABS) is a pathogenic bacterium that can cause severe lung infections, particularly in individuals with cystic fibrosis. MABS colonies can exhibit either a smooth (S) or rough (R) morphotype, influenced by the presence or absence of glycopeptidolipids (GPLs) on their surface, respectively. Despite the clinical significance of these morphotypes, the relationship between GPL levels, morphotype and the pathogenesis of MABS infections remains poorly understood.Gap statement. The mechanisms and implications of GPL production and morphotypes in clinical MABS infections are unclear. There is a gap in understanding their correlation with infectivity and pathogenicity, particularly in patients with underlying lung disease.Aim. This study aimed to investigate the correlation between MABS morphology, GPL and infectivity by analysing strains from cystic fibrosis patients' sputum samples.Methodology. MABS was isolated from patient sputum samples and categorized by morphotype, GPL profile and replication rate in macrophages. A high-content ex vivo infection model using THP-1 cells assessed the infectivity of both clinical and laboratory strains.Results. Our findings revealed that around 50 % of isolates displayed mixed morphologies. GPL analysis confirmed a consistent relationship between GPL content and morphotype that was only found in smooth isolates. Across morphotype groups, no differences were observed in vitro, yet clinical R strains were observed to replicate at higher levels in the THP-1 infection model. Moreover, the proportion of infected macrophages was notably higher among clinical R strains compared to their S counterparts at 72 h post-infection. Clinical variants also infected THP-1 cells at significantly higher rates compared to laboratory strains, highlighting the limited translatability of lab strain infection data to clinical contexts.Conclusion. Our study confirmed the general correlation between morphotype and GPL levels in smooth strains yet unveiled more variability within morphotype groups than previously recognized, particularly during intracellular infection. As the R morphotype is the highest clinical concern, these findings contribute to the expanding knowledge base surrounding MABS infections, offering insights that can steer diagnostic methodologies and treatment approaches.

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.

Transcriptome profiles of macrophages upon infection by morphotypic smooth and rough variants of Mycobacterium abscessus

Mycobacterium abscessus (Mab) infection can be deadly in patients with chronic lung diseases like cystic fibrosis (CF). In vitro and in vivo, Mab may adopt a smooth (S) or rough (R) morphotype, the latter linked to more severe disease conditions. In vitro studies revealed differences in pathogenicity and immune response to S and R morphotypes. We propose that in vivo both morphotypes exist and may transiently switch depending on the environment, having important pathogenic and immunologic consequences. This can be modeled by morphotypic S and R variants of Mab selected based on in vitro growth conditions. Here, we report the first analysis of early transcriptional events in mouse bone marrow derived macrophages (BMDMs) upon infection with media-selected interchangeable Mab-S and Mab-R morphotypes.The early transcriptional events after infection with both morphotypes showed considerable overlap of the pro-inflammatory genes that were differentially regulated compared to the uninfected macrophages. We also observed signature genes significantly differentially regulated in macrophages during infection of media-selected morphotypic Mab-S and Mab-R variants. In conclusion, media-selected Mab-S and Mab-R behave in a similar fashion to stable S and R types with respect to pathogenesis and immune response, serving as a useful model for environmentally influenced morphotype selection.

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.

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

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.

Clinical characteristics and drug susceptibility profiles of Mycobacterium abscessus complex infection at a medical school in Thailand

ObjectivesThis study investigated the differences in epidemiological and clinical data, and antimicrobial susceptibilities among different subspecies of Mycobacterium abscessus complex (MABSC) clinical isolates at a medical school in Thailand.MethodsA total of 143 MABSC clinical isolates recovered from 74 patients were genotypically analyzed for erm(41), rrl, and rrs mutations, and antimicrobial susceptibilities were determined using a broth microdilution method. Patient characteristics and clinical outcomes were reviewed from the medical records.ResultsSeventy-four patients were infected with 28/74 (37.8%) M. abscessus subspecies abscessus (MAB), 43/74 (58.1%) M. abscessus subsp. massiliense (MMA), and 3/74 (4.1%) M. abscessus subsp. bolletii (MBO). The clinical findings and outcomes were generally indistinguishable between the three subspecies. All three subspecies of MABSC clinical isolates exhibited high resistance rates to ciprofloxacin, doxycycline, moxifloxacin, TMP/SMX, and tobramycin. MAB had the highest resistance rates to clarithromycin (27.8%, 20/72) and amikacin (6.9%, 5/72) compared to MBO and MMA, with p < 0.001 and p = 0.004, respectively. In addition, the rough morphotype was significantly associated with resistance to amikacin (8.9%, 5/56), clarithromycin (26.8%, 15/56), and imipenem (76.8%, 43/56) (p < 0.001), whereas the smooth morphotype was resistant to linezolid (57.1%, 48/84) (p = 0.002). In addition, T28 of erm(41), rrl (A2058C/G and A2059C/G), and rrs (A1408G) mutations were detected in 87.4% (125/143), 16.1% (23/143), and 9.1% (13/143) of MABSC isolates, respectively.ConclusionsThree MABSC subspecies caused a variety of infections in patients with different underlying comorbidities. The drug susceptibility patterns of the recent circulating MABSC strains in Thailand were different among the three MABSC subspecies and two morphotypes.

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.

Novel Synergies and Isolate Specificities in the Drug Interaction Landscape of Mycobacterium abscessus.

Mycobacterium abscessus infections are difficult to treat and are often considered untreatable without tissue resection. Due to the intrinsic drug-resistant nature of the bacteria, combination therapy of three or more antibiotics is recommended. A major challenge in treating M. abscessus infections is the absence of a universal combination therapy with satisfying clinical success rates, leaving clinicians to treat infections using antibiotics lacking efficacy data. We systematically measured drug combinations in M. abscessus to establish a resource of drug interaction data and identify patterns of synergy to help design optimized combination therapies. We measured 191 pairwise drug combination effects among 22 antibacterials and identified 71 synergistic pairs, 54 antagonistic pairs, and 66 potentiator-antibiotic pairs. We found that commonly used drug combinations in the clinic, such as azithromycin and amikacin, are antagonistic in the lab reference strain ATCC 19977, whereas novel combinations, such as azithromycin and rifampicin, are synergistic. Another challenge in developing universally effective multidrug therapies for M. abscessus is the significant variation in drug response between isolates. We measured drug interactions in a focused set of 36 drug pairs across a small panel of clinical isolates with rough and smooth morphotypes. We observed strain-dependent drug interactions that cannot be predicted from single-drug susceptibility profiles or known drug mechanisms of action. Our study demonstrates the immense potential to identify synergistic drug combinations in the vast drug combination space and emphasizes the importance of strain-specific combination measurements for designing improved therapeutic interventions.

Biofilm and antimicrobial resistance profile of extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase producing Enterobacteriaceae in vegetables and salads

This study characterized 360 vegetables and salad samples for extended-spectrum beta-lactamase (ESBL)- and AmpC-beta-lactamase-producing Enterobacteriaceae. The isolated bacteria species were processed for beta-lactamase production, antimicrobial resistance (AMR) and other virulence potentials following standard microbiological and spectrometry methods. The ESBL and AmpC-beta-lactamase-producing bacteria were positive in 16/360 (4.4%) and 4/360 (1.1%) of the vegetable samples, respectively. Molecular identification of the bacterial isolates revealed them as follows: 37 E. coli, 6 Klebsiella pneumoniae and 4 Klebsiella variicola. The total number of ESBL-producing enterobacterial phenotypes was 38, while the AmpC-beta-lactamase-producing enterobacterial phenotype was 9. The beta-lactamase-producing enterobacterial morphotypes revealed that 16/47(34.0%) were rough and moist morphotypes, 25/47(53.2%) were rough and dry morphotypes, while 6/47(12.8%) were smooth and white morphotype. The biofilm profile included strong biofilm 41(87.2%), moderate biofilm 4(8.5%) and weak biofilm 2(4.3%), as all the isolates formed biofilm on LB broth. Fourteen multi-drug resistance (MDR) phenotypes were observed in the beta-lactamase-producing E. coli isolates. A total of 40/47(85.1%) isolates had ≥1 β-lactamase gene and other AMR genes, with the blaCTX-1 being the most prevalent. Our findings conclude that food vegetables are important reservoirs of beta-lactamase-producing enterobacteria and a potential health risk to consumers of unprocessed vegetables.

The cell envelope of Mycobacterium abscessus and its role in pathogenesis.

Mycobacterium abscessus is a nontuberculosis mycobacterium (NTM) that has shown an exponential rise in its ability to cause disease. Due to its ubiquitous presence in the environment, M. abscessus is widely implicated in secondary exacerbations of many nosocomial infections and genetic respiratory disorders, such as cystic fibrosis (CF). Contrary to other rapidly growing NTMs, the cell envelope of M. abscessus harbors several prominent features and undergoes modifications that are responsible for its pathogenesis. Compositional changes of the mycobacterial outer membrane (MOM) significantly decrease the presence of glycopeptidolipids (GPLs) and enable the transition from a colonizing, smooth morphotype into a virulent, rough morphotype. The GPLs are transported to the MOM by the Mycobacterial membrane proteins Large (MmpL), which further act as drug efflux pumps and confer antibiotic resistance. Lastly, M. abscessus possesses 2 type VII secretion systems (T7SS): ESX-3 and ESX-4, both of which have recently been implicated in host-pathogen interactions and virulence. This review summarizes the current knowledge of M. abscessus pathogenesis and highlights the clinically relevant association between the structure and functions of its cell envelope.

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.

Invasive Non-Typhoidal Salmonella Lineage Biofilm Formation and Gallbladder Colonization Vary But Do Not Correlate Directly with Known Biofilm-Related Mutations.

Non-typhoidal Salmonella (NTS) serovars have a broad host range and cause gastroenteritis in humans. However, invasive NTS (iNTS) bloodstream infections have increased in the last decade, causing 60,000 deaths annually. Human-specific typhoidal Salmonella colonizes and forms biofilms on gallstones, resulting in chronic, asymptomatic infection. iNTS lineages are undergoing genomic reduction and may have adapted to person-to-person transmission via mutations in virulence, bile resistance, and biofilm formation. As such, we sought to determine the capacity of iNTS lineages for biofilm formation and the development of chronic infections in the gallbladder in our mouse model. Of the lineages tested (L1, L2, L3 and UK), only L2 and UK were defective for the rough, dry and red (RDAR) morphotype, correlating with the known bcsG (cellulose) mutation but not with csgD (curli) gene mutations. Biofilm-forming ability was assessed in vitro, which revealed a biofilm formation hierarchy of L3 > ST19 > UK > L1 = L2, which did not correlate directly with either the bcsG or the csgD mutation. By confocal microscopy, biofilms of L2 and UK had significantly less curli and cellulose, while L1 biofilms had significantly lower cellulose. All iNTS strains were able to colonize the mouse gallbladder, liver, and spleen in a similar manner, while L3 had a significantly higher bacterial load in the gallbladder and increased lethality. While there was iNTS lineage variability in biofilm formation, gallbladder colonization, and virulence in a chronic mouse model, all tested lineages were capable of colonization despite possessing biofilm-related mutations. Thus, iNTS strains may be unrecognized chronic pathogens in endemic settings.

The synergetic effect of sitafloxacin–arbekacin combination in the Mycobacterium abscessus species

Mycobacterium abscessus species (MABS) is the most commonly isolated rapidly growing mycobacteria (RGM) and is one of the most antibiotic-resistant RGM with rapid progression, therefore, treatment of MABS is still challenging. We here presented a new combination treatment with sitafloxacin that targeted rough morphotypes of MABS, causing aggressive infections. Thirty-four clinical strains of MABS were isolated from various clinical samples at the Juntendo university hospital from 2011 to 2020. The susceptibility to a combination of sitafloxacin and antimicrobial agents was compared to that of the antimicrobial agents alone. Out of 34 MABS, 8 strains treated with sitafloxacin–amikacin combination, 9 of sitafloxacin–imipenem combination, 19 of sitafloxacin–arbekacin combination, and 9 of sitafloxacin–clarithromycin combination showed synergistic effects, respectively. Sitafloxacin–arbekacin combination also exhibited the synergistic effects against 10 of 22 Mycobacterium abscessus subspecies massiliense (Mma) strains and 8 of 11 Mycobacterium abscessus subspecies abscessus (Mab) strains, a highly resistant subspecies of MABS. The sitafloxacin–arbekacin combination revealed more synergistic effects in rough morphotypes of MABS (p = 0.008). We demonstrated the synergistic effect of the sitafloxacin–arbekacin combination against MABS. Further, this combination regimen might be more effective against Mab or rough morphotypes of MABS.