Rough Morphotype Research Articles

Biofilm Formation by and Multicellular Behavior of Escherichia coli O55:H7, an Atypical Enteropathogenic Strain

Enteropathogenic Escherichia coli (EPEC) is an important causal agent of diarrheal illness throughout the world. Nevertheless, researchers have only recently begun to explore its capacity to form biofilms. Strain O55:H7 (DMS9) is a clinical isolate belonging to the atypical EPEC (aEPEC) group, which displays a high degree of genetic relatedness to enterohemorrhagic E. coli. Strain DMS9 formed a robust biofilm on an abiotic surface at 26 degrees C, but not at 37 degrees C. It also formed a dense pellicle at the air-liquid interface and developed a red, rough, and dry (RDAR) morphotype on Congo red agar. Unlike a previously described E. coli O157:H7 strain, the aEPEC strain seems to express cellulose. Transposon mutagenesis was used to identify biofilm-deficient mutants. One of the mutants was inactivated in the csgFG genes, required for assembly and secretion of curli fimbriae, while a second mutant had a mutation in crl, a thermosensitive global regulator that modulates sigma(S) activity and downstream expression of curli and cellulose. The two mutants were deficient in their biofilm formation capabilities and did not form a pellicle at the air-liquid interface. Unlike in Salmonella, the csgFG mutant in aEPEC completely lost the RDAR phenotype, while the crl mutant displayed a unique RDAR "pizza"-like morphotype. Genetic complementation of the two mutants resulted in restoration of the wild-type phenotype. This report is the first to describe and analyze a multicellular behavior in aEPEC and support a major role for curli and the crl regulator in biofilm development at low temperatures corresponding to the nonmammalian host environment.

Bistable Expression of CsgD in Biofilm Development ofSalmonella entericaSerovar Typhimurium

Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.

The cell surface-exposed glycopeptidolipids confer a selective advantage to the smooth variants ofMycobacterium smegmatis in vitro

The cell surface of mycobacteria is quite rich in lipids. Glycopeptidolipids, surface-exposed lipids that typify some mycobacterial species, have been associated with a phenotypic switch between rough and smooth colony morphotypes. This conversion in Mycobacterium smegmatis is correlated with the absence/presence of glycopeptidolipids on the cell surface and is due to insertion sequence mobility. Here, we show that the occurrence of a high amount of glycopeptidolipids in the smooth variant leads to lower invasion abilities and lower internalization by macrophages. We further show that the high production of glycopeptidolipids on the cell surface can confer a selective advantage to the smooth variant when grown in vitro. This higher fitness under the laboratory condition might explain the selection of smooth variants in several independent laboratories. The implications of these findings are discussed.

Genotyping of Different Pseudomonas Aeruginosa Morphotypes Arising from the Lower Respiratory Tract of a Patient Taken to an Intensive Care Unit

Pseudomonas aeruginosa is an opportunistic pathogen and an ubiquitous environmental bacterium. Fifty-seven days after hospitalization, we isolated three distinct P. aeruginosa morphotypes (smooth, rough and mucoid) from the lower respiratory tract of a patient admitted to a Cardiology Intensive Care Unit (ICU). Moreover, a group of nine colony variants, arising from the three P. aeruginosa isolates growing in laboratory growth media, were also isolated. The resulting 12 isolates were characterised for antibiotic resistance profile and subjected to genotypic analysis by fluorescent-Amplified Fragment Length Polymorphism (f-AFLP) and automated repetitive extragenic palindromic-PCR (rep-PCR) fingerprinting. The three smooth, rough and mucoid morphotypes presented different antibiotic resistance profiles and genotyping analysis showed that they belonged to distinct clones, indicating that at day 57 after the admission the patient was simultaneously colonized by three distinct P. aeruginosa isolates. On the other hand, the nine colony variants presented heterogeneous antibiotic resistance profiles and clustered together with the three parental isolates. The understanding of the link between genotype plasticity and antibiotic resistance may contribute to improving our knowledge of this life-threatening pathogen.

A LysR-Type Transcriptional Regulator in Burkholderia cenocepacia Influences Colony Morphology and Virulence

Burkholderia cenocepacia strain K56-2 typically has rough colony morphology on agar medium; however, shiny colony variants (shv) can appear spontaneously. These shv all had a minimum of 50% reduction in biomass formation and were generally avirulent in an alfalfa seedling infection model. Three shv-K56-2 S15, K56-2 S76, and K56-2 S86-were analyzed for virulence in a chronic agar bead model of respiratory infection and, although all shv were able to establish chronic infection, they produced significantly less lung histopathology than the rough K56-2. Transmission electron microscopy revealed that an extracellular matrix surrounding bacterial cells was absent or reduced in the shv compared to the rough wild type. Transposon mutagenesis was performed on the rough wild-type strain and a mutant with an insertion upstream of ORF BCAS0225, coding for a putative LysR-type regulator, exhibited shiny colony morphology, reduced biofilm production, increased N-acyl homoserine lactone production, and avirulence in alfalfa. The rough parental colony morphotype, biofilm formation, and virulence in alfalfa were restored by providing BCAS0225 in trans in the BCAS0225::pGSVTp-luxCDABF mutant. Introduction of BCAS0225 restored the rough morphotype in several shv which were determined to have spontaneous mutations in this gene. In the present study, we show that the conversion from rough wild type to shv in B. cenocepacia correlates with reduced biofilm formation and virulence, and we determined that BCAS0225 is one gene involved in the regulation of these phenotypes.

Colony morphology of Xylella fastidiosa almond leaf scorch strains

Xylella fastidiosa is the causal agent of almond leaf scorch disease (ALSD), which is currently reemerging in California as a potential threat to almond (Prunus dulcis) production. We previously reported the presence of different colony morphotypes of X. fastidiosa ALSD strains on periwinkle wilt medium solidified with Gelrite and their association with genotypes or pathotypes after a low number of serial transfers. The morphotypes could be differentiated by single nucleotide polymorphisms and were classified as A- and G-genotypes. The stability of colony morphology was not studied. Yet, it is an important issue in the bacterial characterization. In this project, we evaluated the variations of colony morphology through 14 subculture passages. The G-genotype strains were consistently (>99%) associated with smooth colony morphotypes. Similarly, A-genotype strains were consistently associated with rough colony morphotypes. Rough A-genotype strains reversed to smooth colony variants at a low frequency suggesting phase variation in X. fastidiosa. In an ALSD survey, we successfully used colony morphology to discern X. fastidiosa genotypes with >95% accuracy. The phenotypic traits described in this study are of value for further biological and genetic studies of X. fastidiosa.

Contribution of proteases and plasmin‐acquired activity in migration of Peptostreptococcus micros through a reconstituted basement membrane

Peptostreptococcus micros is a gram-positive bacterium that has been associated with chronic periodontitis and endodontic infections. The aims of this study were to investigate the production of proteases and the acquisition of plasmin activity by rough and smooth morphotypes of P. micros. The contribution of these properties in the migration of bacteria through a reconstituted basement membrane was also evaluated. Protease activities were determined using chromogenic and fluorogenic substrates as well as by zymography. Plasminogen binding activity was studied using an enzyme-linked immunosorbent assay. The role of proteases and plasmin-acquired activity in tissue penetration was investigated using Matrigel. The rough morphotype strains of P. micros, but not the smooth morphotype strains, were found to possess chymotrypsin-like and gelatinase activities, both of which were inhibited by a serine protease inhibitor. By zymography, three gelatinase bands (165, 129, and 115 kDa) were identified. Both morphotypes of P. micros can bind human plasminogen on their cell surface. Once bound to P. micros, plasminogen activators of bacterial (streptokinase) and human (urokinase) origins were found to activate plasminogen into plasmin. Our results also showed that plasmin activity can be acquired by P. micros following co-incubation with human brain microvascular endothelial cells in culture. When non-coated cells were used, the rough morphotype strain (HG1262), which possesses chymotrypsin-like and gelatinase activities, showed a better capacity to penetrate a reconstituted basement membrane (Matrigel) than the smooth morphotype strain (HG1251). Penetration of the Matrigel by P. micros HG1262 was inhibited by the presence of a serine protease inhibitor. In addition, cells of P. micros with plasmin activity showed a significantly greater tissue penetration capacity. Our study suggests that endogenous proteolytic activities of P. micros as well as plasmin-acquired activity, may facilitate dissemination of bacterial cells to surrounding periodontal tissues and blood vessels.

Spontaneous reversion of Mycobacterium abscessus from a smooth to a rough morphotype is associated with reduced expression of glycopeptidolipid and reacquisition of an invasive phenotype

Mycobacterium abscessus is an increasingly important cause of human disease; however, virulence determinants are largely uncharacterized. Previously, it was demonstrated that a rough, wild-type human clinical isolate (390R) causes persistent, invasive infection, while a smooth isogenic mutant (390S) has lost this capability. During serial passage of 390S, a spontaneous rough revertant was obtained, which was named 390V. This revertant regained the ability to cause persistent, invasive infection in human monocytes and the lungs of mice. Glycopeptidolipid (GPL), which plays a role in environmental colonization, was present in abundance in the cell wall of 390S, and was associated with sliding motility and biofilm formation. In contrast, a marked reduction in the amount of GPL in the cell wall of 390R and 390V was correlated with cord formation, a property associated with mycobacterial virulence. These results indicate that the ability to switch between smooth and rough morphologies may allow M. abscessus to transition between a colonizing phenotype and a more virulent, invasive form.

The production of a new extracellular putative long-chain saturated polyester by smooth variants of Mycobacterium vaccae interferes with Th1-cytokine production

Mycobacterium vaccae is of major pharmaceutical interest as an immunotherapeutic agent. Although M. vaccae 15483 ATCC(T) strain displays smooth and rough colonial morphologies on solid culture media, it is not known in which conditions M. vaccae switches from one colonial morphotype to the other or whether there are biological differences, especially immunological, between them. We have found that the change from a smooth to rough stable variant occurs spontaneously at 30 degrees C. The analysis of the composition of the cell wall in both variants showed that the smooth morphotype presents an extracellular material that has never previously been described and was identified as a long-chain saturated polyester that, interestingly, is not produced by the rough morphotype. Our results also indicate that this compound could be implicated in the spreading ability of smooth colonies. Proliferation, IFN-gamma and IL-12(p40) production by splenocyte cultures was significantly higher in mice immunised with the rough variant compared with those immunised with the smooth one. This latter finding suggests that the different colonial morphology of M. vaccae may affect the immunomodulatory effects induced from M. vaccae preparations.

Morphotypic conversion in Listeria monocytogenes biofilm formation: biological significance of rough colony isolates.

Adherence to a stainless steel surface selected isolates of Listeria monocytogenes with enhanced surface colonization abilities and a change in phenotype from the common smooth colony morphology to a succession of rough colony morphotypes. Growth in broth culture of the best-adapted, surface-colonizing rough colony morphotype gave a smooth colony revertant. Comparative analysis revealed that the smooth and rough variants had similar phenotypic and biochemical characteristics (e.g., identical growth rates and tolerances to antibiotics and environmental stressors). Rough colony isolates, however, failed to coordinate motility or induce autolysis. The defect in autolysis of rough colony isolates, which involved impaired cellular localization of several peptidoglycan-degrading enzymes, including cell wall hydrolase A (CwhA), suggested a link to a secretory pathway defect. The genetic basis for the impairment was studied at the level of the accessory secretory pathway component SecA2. DNA sequencing of the secA2 gene in smooth and rough colony isolates found no mutations in the coding or promoter regions. Analysis of SecA2 expression with an integrated secA2-FLAG tag construct found the protein to be upregulated in the rough and revertant backgrounds compared to the parental smooth colony isolate. A compensatory mechanism involving the SecA2 secretion pathway components is postulated to control smooth to rough interconversion of L. monocytogenes. Such phenotypic variation may enhance the ability of this opportunistic pathogen to colonize environments as diverse as processing surfaces, food products, and animal hosts.

Characterization of virulence, colony morphotype and the glycopeptidolipid of Mycobacterium avium strain 104

Setting: Members of the Mycobacterium avium complex (MAC) are responsible for mycobacterial disease in children, the aged and in immunocompromised individuals. The complex consists of different species, serovars and morphologic forms that vary in virulence. One isolate of the MAC is currently being sequenced (MAC 104) and was chosen based on its derivation from an AIDS patient and the fact that it could be genetically manipulated. Objective: MAC 104 was therefore analyzed for virulence, colony morphotype and expression of the glycopeptidolipid (GPL) responsible for serotying differences and the rough to smooth morphological switch. Results: The isolate was found to be virulent in the murine model of low-dose aerosol infection in that it could colonize the lung, proliferate within the tissue and disseminate to other organs. MAC 104 expressed a variety of colony morphotypes, the most prevalent of which were smooth opaque, smooth transparent and rough. All three morphotypes could persist in the lung; however, the transparent and rough morphotypes grew more rapidly in vivo . The rough morphotype was unusual in that it expressed an atypical form of the GPL usually absent from rough morphotypes. Conclusion: This characterization complements the genome data and confirms that MAC 104 behaves similarly to other MAC isolates.

Rapid differentiation of "Mycobacterium canettii" from other Mycobacterium tuberculosis complex organisms by PCR-restriction analysis of the hsp65 gene.

A total of 102 isolates of the Mycobacterium tuberculosis complex, including available "M. canettii" isolates, were studied by PCR-restriction analysis of a 441-bp fragment of the hsp65 gene. PRA upon HhaI enzyme digestion (GCGC) allowed easy differentiation of "M. canettii" from other members of the M. tuberculosis complex (three bands of 260, 105, and 60 bp for "M. canetti," compared to four bands of 185, 105, 75, and 60 bp for other members of the M. tuberculosis complex). Sequencing of the 441-bp hsp65 fragment of "M. canettii" isolates showed the disappearance of an HhaI site at position 235 due to a C-to-T transition that corresponded to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv. Considering that "M. canettii" may also exist as a stable rough morphotype, we suggest that the true number of "M. canettii" isolates may be underestimated in clinical microbiology laboratories.

5] Genetic and phenotypic analysis of multicellular behavior in salmonella typhimurium

Publisher Summary The genetic and phenotypic analysis of muilticellular behavior in salmonella typhimurium. According to Costerton et al. , biofilms can be defined in a narrower sense as “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface,” or in a broader sense as “matrix-enclosed bacterial populations adherent to each other and/or to surfaces or interfaces.” In Salmonella typhimurium, a morphotype in which cells are acting as a community is identified. This morphotype displayed various modes of multicellular behavior in stationary phase, such as cell clumping in aerated, liquid culture, pellicle formation in standing liquid culture, formation of a rigid cell network accompanied by surface translocation on solid medium, adhesion to biological macromolecules, and biofilm formation on various abiotic surfaces such as glass, polystyrene (PS), and stainless steel. On solid medium, the colony bound the dye Congo red (CR) included in the agar plates, thereby leading to the pronounced display of a peculiar morphotype that is an irregularly shaped, flat, wrinkled colony with undulate margin. Referring to the morphotype on plates, the complete multicellular behavior has been called rdar—red, dry, and rough—morphotype. Genetic and electron microscopy analyses revealed that the extracellular matrix consists of at least two components; thin aggregative fimbriae (agf); and an unknown substance.

Colony morphotypes on Congo red agar segregate along species and drug susceptibility lines in the Mycobacterium avium-intracellulare complex.

Isolates of the Mycobacterium avium-intracellulare complex (MAC) have long been known to segregate into transparent opaque and rough colony morphotypes that differ from each other in clinically important parameters including drug susceptibility and virulence. Here the authors report additional morphotypic variation that occurs on two levels: interspecific (between M. avium and M. intracellulare) and intraspecific (within individual M. avium isolates). Clinical isolates of M. avium grown on Congo red (CR) plates formed red, pink or mixed (red and white) opaque colonies, while M. intracellulare isolates formed purely white opaque colonies. A quantitative CR binding assay showed that this interspecific differential applies to transparent as well as opaque colony variants; however, it was less pronounced among laboratory reference strains than among recent clinical isolates. Opaque colonies of M. avium isolates with 'mixed' phenotypes segregated into stable opaque red and white variants with shared IS1245 banding patterns (intraspecific segregation). White segregants of M. avium were more flocculent and significantly more resistant to ciprofloxacin and rifamycin drugs than were red segregants. Thus, cultivation on CR agar revealed a previously unknown multidrug resistant colony morphotype of M. avium.

Pathogenicity of Peptostreptococcus micros morphotypes and Prevotella species in pure and mixed culture.

Recently, an atypical rough colony morphotype of Peptostreptococcus micros, a species which is found in ulcerating infections, including periodontitis, was isolated. The virulence of morphotypes alone and in combination with Prevotella intermedia and P. nigrescens was investigated both in vivo and in vitro. All strains tested induced abscesses containing fluid pus in a mouse skin model, and lesions caused by monocultures of the rough morphotype strains of P. micros were statistically significantly larger than those induced by the smooth morphotype strains. Inocula containing both morphotypes produced similar sized abscesses compared to mono-inocula containing the same bacterial load. Both Prevotella species induced small abscesses when inoculated alone, and when Pr. nigrescens was inoculated with one of the other strains, the abscesses were not significantly different from the abscesses induced by the mono-infections of this strain. Synergy, in terms of higher numbers of colony forming units (cfu) in the mixed inocula, was found for all combinations of the rough morphotypes of P. micros and both Prevotella spp. Pus from abscesses caused by combinations of Peptostreptococcus and Prevotella spp. transmitted the infection to other mice, but no abscesses were formed in mice inoculated with pus induced by mono-inocula. These results demonstrated synergic activity between both rough and smooth P. micros strains and oral Prevotella strains. The in-vitro co-culture experiments produced no evidence of growth stimulation. The effect of P. micros strains on the immune system was investigated by testing their ability to initiate luminol-dependent chemiluminescence of polymorphonuclear leucocytes in the presence and absence of human serum. In the latter, the rough morphotype strains initiated higher counts than the smooth morphotype strains. Further work is needed to elucidate the difference in virulence between the smooth and the rough morphotype cells of P. micros and the nature of the interaction with the Prevotella spp.

Prevalence of Peptostreptococcus micros morphotypes in patients with adult periodontitis.

The prevalence of the smooth and rough colonial morphotypes of Peptostreptococcus micros was examined with culture technique in 123 patients with adult periodontitis (age 24-68 years). Of all subgingival samples, 91% contained the smooth morphotype of P. micros. The smooth morphotype constituted a mean percentage of the total anaerobic viable biota of 6.0%, with a range of 0.02-35.7%. Of these samples, 49% contained colonies of the rough morphotype as well, with a mean percentage of the total anaerobic viable biota of 2.3% (range 0.01-16.2%). None of the samples contained only the rough morphotype. The total percentage of P. micros varied from 0.02-35.71% with a mean of 7.2%. No correlation was found between the prevalence of both morphotypes of P. micros and the age of the subjects or with loss of attachment or pocket depth.

Description of Two Morphotypes of Peptostreptococcus micros

Peptostreptococcus micros is often isolated from abscesses in several parts of the human body. The oral cavity is considered the natural habitat for the species, which has been implicated as a periodontal pathogen. In plaque samples from periodontitis patients we observed the presence of a rough morphotype of P. micros in addition to the previously recognized smooth morphotype. The rough morphotype has not been described previously. Both morphotypes are frequently isolated simultaneously from the same patient. In this paper strains of both morphotypes are described. The smooth morphotype, represented by the type strain, grew as small, dome-shaped, bright white, nonhemolytic colonies. The rough morphotype grew as equally white dry colonies which were hemolytic and had wrinkled edges. DNA-DNA reassociation studies revealed homology at the species level between the two morphotypes; in addition, no differences in physiological characteristics were observed when the organisms were tested with API-32A and API-ZYM kits. The rough cells had long, thin fibrillar structures outside the cell envelope when they were stained negatively for electron microscopy. In the smooth morphotype these structures were not present. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole-cell extracts were different for the two morphotypes. In xylene-water phase partition studies, the smooth morphotype was found to be hydrophobic, whereas the rough morphotype was found to be relatively hydrophilic. The distinct morphotypes were stable on blood agar; however, the rough morphotype changed to a nonfibrillar type with a smooth colony morphology after repeated subculturing in broth.

Properties of Wine Lactic Acid Bacteria: Their Potential Enological Significance

Lactic acid bacteria isolated from Australian red wines were examined for properties of potential enological significance. Some 166 strains were tested. Strains of <i>Leuconostoc oenos</i> were divided into rough and smooth morphotypes on the basis of colony appearance. Rough morphotypes produced cells arranged in long chains and readily grew in wine. Smooth morphotypes produced cells arranged in pairs or short chains, but most of these strains exhibited poor growth in wine. As tested in API 50CH galleries (pH 6.0), strains of <i>L. oenos, Pediococcus parvulus</i>, and several <i>Lactobacillus</i> species exhibited considerable heterogeneity in their abilities to ferment carbohydrates. The fermentation profile of individual strains was altered when the pH was reduced to 4.0. Strains of <i>L. oenos</i> were more tolerant of low pH than those of <i>P. parvulus</i> or <i>Lactobacillus</i> spp. Strains of <i>P. parvulus</i> and <i>Lactobacillus</i> spp. were more tolerant of high concentrations of ethanol and total sulfur dioxide. None of the strains produced extracellular protease. Lipases were produced by several strains of <i>L. oenos</i> and one strain of <i>Lactobacillus</i>. The majority of strains produced esterases.

Pseudomonas aeruginosa: changes in antibiotic susceptibility, enzymatic activity, and antigenicity among colonial morphotypes.

Colonial variants of Pseudomonas aeruginosa have received renewed interest because of their occurrence in sputum cultures of patients with cystic fibrosis. We encountered 11 strains of P. aeruginosa from various body sites of non-cystic fibrosis patients. The strains showed two to three colonial variants, including smooth, rough, and iridescent morphotypes that arose from subculture of a single colony of P. aeruginosa originating from a primary source. The colonial segregants differed in antibiotic susceptibility (resistance to gentamicin, carbenicillin, chloramphenicol, and tetracycline), presence or absence of exoenzymes (gelatinase and elastase), degree of proteolytic activity (caseinase), pigmentation, and antigenicity. These observations suggest that in vivo dissociation with concomitant changes in enzymatic and surface properties might greatly enhance invasiveness. Concurrent differences in antimicrobial susceptibility among the colonial variants could account in some instances for the failure of antibiotic treatment in P. aeruginosa infections in which one would anticipate a positive therapeutic response.