Biofilm formation by ST17 and ST19 strains of Streptococcus agalactiae.

Bacterial biofilms are an important virulence factor with a vital role in evasion from the host immune system, colonization and infection. The aim of the present study was to evaluate in vitro the effects of three environmental factors (H+, glucose and human plasma) in biofilm formation, by carrier and invasive Streptococcus agalactiae strains of ST17 and ST19 sequence types, including DNase producers and non-producers. Bacteria ability to assemble biofilms was classified based on crystal violet assay. Biofilm formation was also monitored by scanning electron microscopy. Depending on the growth medium used, each bacterial isolate could fit in different biofilm production categories. Our data showed that optimal conditions for S. agalactiae biofilm assembly were reached after 48 h incubation at pH 7.6 in the presence of glucose and inactivated human plasma. In the presence of inactivated human plasma, the biofilm biomass of ST19 strains experienced a higher increase than ST17 strains. The composition of the extracellular polymeric matrix of the three strongest biofilm producers (all from ST17) was accessed by enzymatic digestion of mature biofilms and proteins were shown to be the predominant component. The detailed identification of the extracellular protein components should contribute to the development of new therapeutic strategies to fight S. agalactiae infections.

Synthetic Ellagic Acid Glycosides Inhibit Early Stage Adhesion of Streptococcus agalactiae Biofilms as Observed by Scanning Electron Microscopy.

Ellagic acid derivatives possess antimicrobial and antibiofilm properties across a wide-range of microbial pathogens. Due to their poor solubility and ambident reactivity it is challenging to synthesize, purify, and characterize the activity of ellagic acid glycosides. In this study, we have synthesized three ellagic acid glycoconjugates and evaluated their antimicrobial and antibiofilm activity in Streptococcus agalactiae (Group B Streptococcus, GBS). Their significant impacts on biofilm formation were examined via SEM to reveal early-stage inhibition of cellular adhesion. Additionally, the synthetic glycosides were evaluated against five of the six ESKAPE pathogens and two fungal pathogens. These studies reveal that the ellagic acid glycosides possess inhibitory effects on the growth of gram-negative pathogens.

The Antibacterial Mechanism of Terpinen-4-ol Against Streptococcus agalactiae.

Streptococcus agalactiae, a highly contagious mastitis pathogen, caused huge economic losses; meanwhile, repeated use of antibiotics results in the emergence of serious antibiotic residues and drug resistance. Therefore, it is in great need to develop ecologically sustainable antimicrobial agents. In the study, the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and action mechanism of terpinen-4-ol against S. agalactiae was investigated to evaluate antibacterial activity of terpinen-4-ol. Results showed the MIC and MBC of terpinen-4-ol were 98 and 196 µg/mL, respectively. Time-kill curves displayed that the antibacterial activity of terpinen-4-ol was in a concentration-dependent manner. Transmission electron micrographs showed that the cell membrane and wall of S. agalactiae were damaged, and plasmolysis and chromatins were inconspicuous. Release of Ca2+ and Mg2+ proved that terpinen-4-ol could increase cell membrane permeability. And the release of lactate dehydrogenase (LDH) suggested that cell wall was destroyed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 4',6-diamidino-2-phenylindole (DAPI) staining results showed that terpinen-4-ol could affect the synthesis of protein and DNA. These results suggested that terpinen-4-ol might be used as candidate for treating S. agalactiae infection.

Human Milk Oligosaccharides Exhibit Antimicrobial and Antibiofilm Properties against Group B Streptococcus.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterial pathogen that causes invasive infections in both children and adults. During pregnancy, GBS is a significant cause of infection of the fetal membranes (chorioamnionitis), which can lead to intra-amniotic infection, preterm birth, stillbirth, and neonatal sepsis. Recently, breastfeeding has been thought to represent a potential mode of GBS transmission from mother to newborn, which might increase the risk for late-onset sepsis. Little is known, however, about the molecular components of breast milk that may support or prevent GBS colonization. In this study, we examine how human milk oligosaccharides (HMOs) affect the pathogenesis of GBS. HMOs from discrete donor samples were isolated and profiled by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Growth and biofilm assays show that HMOs from mothers of specific milk groups can modulate the growth and biofilm formation of GBS. High-resolution field-emission gun scanning electron microscopy (SEM) and confocal laser scanning microscopy confirmed the quantitative biofilm assays and demonstrated cell arrangement perturbations in bacterial cultures treated with specific oligosaccharides. These findings demonstrate that HMOs affect the growth and cell biology of GBS. Finally, this study provides the first example of HMOs functioning as antibiofilm agents against GBS.

Exploring the in vitro thrombolytic potential of streptokinase-producing ß-hemolytic Streptococci isolated from bovine milk.

The aim of this study was to isolate and characterize streptokinase-producing ß-hemolytic Streptococcus sp. from bovine milk. A total of 50 milk samples were collected randomly from different breeds of cow and goat (Vellore, Tamil Nadu, India). The samples were characterized and screened for streptokinase-producing isolates using microbial and biochemical analysis. About 97 colonies were isolated from milk samples showing hemolytic patterns of α (19.6%), ß (24.7%) and γ (55.6 %). Out of 20ß-hemolytic isolates, only 6 colonies (VB2, VB3, VB8, VB14, VB16, and VB17) were identified as ß-hemolytic Streptococci as potent producers of streptokinase. VB2 and VB14 showed the greatest streptokinase activities of 265 U mL(-1) and 225 U mL(-1), respectively. Based on biochemical and molecular characterization, the potent isolates VB2 and VB14 were identified and confirmed as S. equinus and S. agalactiae, respectively. The identified strains were named Streptococcus equinus VIT_VB2 (GenBank accession no. JX406835) and Streptococcus agalactiae VITVS5 (GenBank accession No. KF186620) The strains isolated from bovine milk provide a variance in the fibrinolytic activity on blood clots. The current study has demonstrated that the isolation of streptokinase producers from bovine milk, and the production of streptokinase from novel strain, enhanced the fibrinolytic activity. This study is the first to report that Streptococcus equinus produces streptokinase.

Antibacterial activity and mechanism of berberine against Streptococcus agalactiae.

The antibacterial activity and mechanism of berberine against Streptococcus agalactiae were investigated in this study by analyzing the growth, morphology and protein of the S. agalactiae cells treated with berberine. The antibacterial susceptibility test result indicated minimum inhibition concentration (MIC) of berberine against Streptococcus agalactiae was 78 µg/mL and the time-kill curves showed the correlation of concentration-time. After the bacteria was exposed to 78 µg/mL berberine, the fragmentary cell membrane and cells unequal division were observed by the transmission electron microscopy (TEM), indicating the bacterial cells were severely damaged. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) study demonstrated that berberine could damage bacterial cells through destroying cellular proteins. Meanwhile, Fluorescence microscope revealed that berberine could affect the synthesis of DNA. In conclusion, these results strongly suggested that berberine may damage the structure of bacterial cell membrane and inhibit synthesis of protein and DNA, which cause Streptococcus agalactiae bacteria to die eventually.

Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae.

The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide (CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the "pellicle") overlaid by a 15-45 nm thick layer of CPS (the "capsule"). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.

The enhancement of biofilm formation in Group B streptococcal isolates at vaginal pH.

Group B streptococcus (GBS) is a common asymptomatic colonizer in acidic vagina of pregnant women and can transmit to newborns, causing neonatal pneumonia and meningitis. Biofilm formation is often associated with bacterial colonization and pathogenesis. Little is known about GBS biofilm and the effect of environmental stimuli on their growth along with biofilm formation. The objective of this study was to investigate the survival and biofilm formation of GBS, isolated from pregnant women, in nutrient-limited medium under various pH conditions. Growth and survival experiments were determined by optical density and viable counts. Crystal violet staining, scanning electron microscopy, and atomic force microscopy (AFM) were used to analyze the capacity of biofilm production. Our results showed that GBS isolates proliferated with increasing pH with highest maximum specific growth rate (µmax) at pH 6.5, but survived at pH 4.5 for longer than 48 h. Biofilm formation of the 80 GBS isolates at pH 4.5 was significantly higher than at pH 7.0. This difference was confirmed by two other methods. The low elastic modulus obtained from samples at pH 4.5 by AFM revealed the softness of biofilm; in contrast, little or no biofilm was measured at pH 7.0. Under acidic pH, the capability of biofilm formation of serotypes III and V showed statistically significant difference from serotypes Ia and Ib. Our finding suggested that survival and enhanced biofilm formation at vaginal pH are potentially advantageous for GBS in colonizing vagina and increase the risk of vaginosis and neonatal infection.

A model for group B Streptococcus pilus type 1: the structure of a 35-kDa C-terminal fragment of the major pilin GBS80.

The Gram-positive pathogen Streptococcus agalactiae, known as group B Streptococcus (GBS), is the leading cause of bacterial septicemia, pneumonia, and meningitis among neonates. GBS assembles two types of pili-pilus islands (PIs) 1 and 2-on its surface to adhere to host cells and to initiate colonization for pathogenesis. The GBS PI-1 pilus is made of one major pilin, GBS80, which forms the pilus shaft, and two secondary pilins, GBS104 and GBS52, which are incorporated into the pilus at various places. We report here the crystal structure of the 35-kDa C-terminal fragment from GBS80, which is composed of two IgG-like domains (N2-N3). The structure was solved by single-wavelength anomalous dispersion using sodium-iodide-soaked crystals and diffraction data collected at the home source. The N2 domain exhibits a cnaA/DEv-IgG fold with two calcium-binding sites, while the N3 domain displays a cnaB/IgG-rev fold. We have built a model for full-length GBS80 (N1, N2, and N3) with the help of available homologous major pilin structures, and we propose a model for the GBS PI-1 pilus shaft. The N2 and N3 domains are arranged in tandem along the pilus shaft, whereas the respective N1 domain is tilted by approximately 20° away from the pilus axis. We have also identified a pilin-like motif in the minor pilin GBS52, which might aid its incorporation at the pilus base.

Specific involvement of pilus type 2a in biofilm formation in group B Streptococcus.

Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

Group B streptococcus exploits lipid rafts and phosphoinositide 3-kinase/Akt signaling pathway to invade human endometrial cells.

OBJECTIVE: We sought to determine the role lipid rafts and phosphoinositide 3-kinase (PI3K) in invasiveness of group B streptococci (GBS) to endometrial cells. STUDY DESIGN: Antibiotic protection assay and electron microscopy were used to evaluate the invasion of GBS to human endometrial Ishikawa cells cholesterol-depleted by using methyl-beta-cyclodextrin or treated with PI3K inhibitors: wortmannin or LY294002. Immunoblotting analysis of Akt phosphorylation and cellular imaging of GFP-Akt-PH probe were used to assess PI3Ks activation in infected cells. RESULTS: Infected Ishikawa cells streptococci are associated to membrane ruffles with morphological features of undergoing internalization. GBS remained attached but completely failed to invade to cholesterol-depleted human endometrial cells or displayed decreased invasiveness in the presence of PI3K inhibitors. Cholesterol depletion resulted in loss of membrane ruffling and dispersion of raft-associated molecules: monosialoganglioside GM1 and PI3K. CONCLUSION: This work provides the evidence that lipid rafts and raft-associated PI3K are implicated in GBS invasion to human endometrial cells.

[Comparison of antimicrobial and bactericidal activities and postantibiotic effects of macrolides antibiotics against clinical isolates, and examination of shape alteration by scanning electron microscope].

We examined antibacterial activities of 4 kinds of macrolides (MLs), erythromycin (EM), clarithromycin (CAM), azithromycin (AZM) and rokitamycin (RKM), against 4 bacterial species of clinical strains isolated in 2004. Bacterial isolates used were 51 strains of methicillin-susceptible Staphylococcus aureus (MSSA), 20 of Streptococcus pyogenes, 68 of Streptococcus agalactiae, and 120 of Streptococcus pneumoniae. Macrolide resistance genes, ermB and mefE, in macrolide-resistant S. pyogenes and S. agalactiae, and all of pneumococci were analyzed by PCR. Antimicrobial activities against macrolide-susceptible MSSA of EM and CAM, were more potent than those of RKM. By contrast, against S. pneumoniae, RKM was more effective than EM, CAM and AZM. Against S. pyogenes and S. agalactiae, 4 antibiotics showed similar antimicrobial activities. Twelve, 1 and 2 strains of MSSA, S. pyogenes and S. agalactiae, respectively, were resistant to EM, CAM and AZM, whereas RKM was active to almost, but not quite, of them. Among 120 strains of S. pneumoniae, 76 (63.3%) were resistant to EM (MIC; > or = 0.5 microg/mL), and 23, 15 and 28 strains were highly resistant (MIC; > 128 microg/mL) to EM, CAM and AZM, respectively. By contrast, for RKM, there were far fewer resistant strains, and there was no highly resistant strain. PCR analyses of macrolide-resistant genes revealed that 1 resistant strain of S. pyogenes and 2 of S. agalactiae carried mefE and ermB, respectively. In the case of S. pneumoniae, 59, 19 and 5 strains, respectively, carried ermB, mefE and both ermB and mefe. We also studied about bactericidal activities and postantibiotic effects (PAE) of MLs using macrolide-susceptible, and ermB- and mefE-carrying S. pneumoniae, and observed morphological alterations of the strains treated with the drugs by a scanning electron microscope. It was demonstrated that RKM had superior bactericidal activities and PAE than other 3 drugs, and potent destructive effects to all of 3 strains.

Identification of novel genomic islands coding for antigenic pilus-like structures in Streptococcus agalactiae.

We have recently reported the presence of covalently linked pilus-like structures in the human pathogen, Group B Streptococcus (GBS). The pilus operon codes for three proteins which contain the conserved amino acid motif, LPXTG, associated with cell wall-anchored proteins together with two genes coding for sortase enzymes. Analysis of the eight sequenced genomes of GBS has led to the identification of a second, related genomic island of which there are two variants, each containing genes coding for proteins with LPXTG motifs and sortases. Here we show that both variant islands also code for pilus-like structures. Furthermore, we provide a thorough description and characterization of the genomic organization of the islands and the role of each protein in the assembly of the pili. For each pilus, polymerization of one of the three component proteins is essential for incorporation of the other two proteins into the pilus structure. In addition, two sortases are required for complete pilus assembly, each with specificity for one of the pilus components. A component protein of one of the newly identified pili is also a previously identified protective antigen and a second component of this pilus is shown to confer protection against GBS challenge. We propose that pilus-like structures are important virulence factors and potential vaccine candidates.

Assembly and role of pili in group B streptococci.

Streptococcus agalactiae[group B streptococcus (GBS)] is the leading cause of neonatal pneumonia, sepsis and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes five putative sortases, including the major class A sortase enzyme and four class C sortases. The genes encoding the class C sortases are tandemly arranged in two different loci, srtC1-C2 and srtC3-C4, with a similar genetic organization and are thought to be involved in pilus biosynthesis. Each pair of sortase genes is flanked by LPXTG protein encoding genes, two upstream and one downstream, and a divergently transcribed regulatory gene located upstream from this locus. We demonstrated that strain NEM316 expresses only the srtC3-C4 locus, which encodes three surface proteins (Gbs1474, Gbs1477 and Gbs1478) that polymerize to form appendages resembling pili. Structural and functional analysis of this locus revealed that: (i) the transcriptional activator RogB is required for expression of the srtC3-C4 operon; (ii) Gbs1477, and either SrtC3 or SrtC4 are absolutely required for pilus biogenesis; and (iii) GBS NEM316 pili are composed of three surface proteins, Gbs1477, the bona fide pilin which is the major component, Gbs1474, a minor associated component, and Gbs1478, a pilus-associated adhesin. Surprisingly, pilus-like structures can be formed in the absence of the two minor components, i.e. the putative anchor Gbs1474 or the adhesin Gbs1478. Adherence assays showed that Gbs1478 confers adhesive capacity to the pilus. This study provides the first evidence that adhesive pili are also present in Gram-positive pathogens.

Anchors away: contribution of a glycolipid anchor to bacterial invasion of host cells.

Group B Streptococcus (GBS) is an important cause of infections, including meningitis. The molecular events underlying its pathogenesis are poorly understood. A study in this issue of the JCI reports that the GBS invasion-associated gene (iagA) contributes to meningeal infection and virulence by facilitating invasion of the cells that compose the blood-brain barrier and of other host cells. The mechanism involved most likely relates to the gene product's role in synthesis of a glycolipid anchor for a bacterial cell-surface entity that interacts directly with host cells.

Genome analysis reveals pili in Group B Streptococcus.

Pili are essential virulence factors in many Gram-negative bacteria; however, they have not been described in most important Gram-positive pathogens. While screening the sequence of multiple genomes of Group B Streptococcus, we identified protective antigens that formed high molecular weight polymers. Immunogold electron microscopy revealed that the structures have a pilus-like form. These large structures have gone unrecognized in decades of studies of Group B Streptococcus.

CovS/CovR of group B streptococcus: a two-component global regulatory system involved in virulence.

In this study, we carried out a detailed structural and functional analysis of a Streptococcus agalactiae (GBS) two-component system which is orthologous to the CovS/CovR (CsrS/CsrR) regulatory system of Streptococcus pyogenes. In GBS, covR and covS are part of a seven gene operon transcribed from two promoters that are not regulated by CovR. A DeltacovSR mutant was found to display dramatic phenotypic changes such as increased haemolytic activity and reduced CAMP activity on blood agar. Adherence of the DeltacovSR mutant to epithelial cells was greatly increased and analysis by transmission electron microscopy revealed the presence at its surface of a fibrous extracellular matrix that might be involved in these intercellular interactions. However, the DeltacovSR mutant was unable to initiate growth in RPMI and its viability in human normal serum was greatly impaired. A major finding of this phenotypic analysis was that the CovS/CovR system is important for GBS virulence, as a 3 log increase of the LD(50) of the mutant strain was observed in the neonate rat sepsis model. The pleiotropic phenotype of the DeltacovSR mutant is in full agreement with the large number of genes controlled by CovS/CovR as seen by expression profiling analysis, many of which encode potentially secreted or cell surface-associated proteins: 76 genes are repressed whereas 63 were positively regulated. CovR was shown to bind directly to the regulatory regions of several of these genes and a consensus CovR recognition sequence was proposed using both DNase I footprinting and computational analyses.

Septicaemia in emerald monitors (Varanus prasinus Schlegel 1839) caused by Streptococcus agalactiae acquired from mice.

The present study was performed to investigate both the identity and the source of the bacteria responsible for a fatal septicaemia observed in a group of three subadult emerald monitors (Varanus prasinus Schlegel 1839). The emerald monitors were necropsied and examined by light microscopy, including immunohistology, and by electron microscopy. Tissue samples were additionally submitted for bacteriological, virological and parasitological examinations. The virological and parasitological results were noncontributory, whereas the bacteriological investigation resulted in the isolation of gram-positive cocci which were characterized biochemically and serologically and by molecular analysis. The death of the emerald monitors was caused by a partially leukocyte-associated septicaemic infection with streptococci of serological group B of serotype V. Phenotypically and genotypically identical group B streptococci were isolated from the intestine of subadult mice, obtained from the feed used for the monitors. The genotypical characterization included an identical DNA fingerprint of strains of both origins, indicating the epidemiological relation between the feeding mice and the infections of the monitors.

Influence of proteins Bsp and FemH on cell shape and peptidoglycan composition in group B streptococcus.

Group B streptococcus (GBS) is surrounded by a capsule. However, little is known about peptidoglycan metabolism in these bacteria. In the present study, a 65 kDa protein was isolated from the culture supernatant of GBS and N-terminally sequenced, permitting isolation of the corresponding gene, termed bsp. The bsp gene was located close to another gene, designated femH, and reverse transcription-PCR revealed a bicistronic transcriptional organization for both genes. The Bsp protein was detected in the culture supernatant from 31 tested clinical isolates of GBS, suggesting a wide distribution of Bsp in these bacteria. Overexpression of bsp resulted in lens-shaped GBS cells, indicating a role for bsp in controlling cell morphology. Insertional disruption of femH resulted in a reduction of the L-alanine content of the peptidoglycan, suggesting that femH is involved in the incorporation of L-alanine residues in the interpeptide chain of the peptidoglycan of GBS.

Regulation of D-alanyl-lipoteichoic acid biosynthesis in Streptococcus agalactiae involves a novel two-component regulatory system.

The dlt operon of gram-positive bacteria comprises four genes (dltA, dltB, dltC, and dltD) that catalyze the incorporation of D-alanine residues into the lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Streptococcus agalactiae, which, in addition to the dltA to dltD genes, included two regulatory genes, designated dltR and dltS, located upstream of dltA. The dltR gene encodes a 224-amino-acid putative response regulator belonging to the OmpR family of regulatory proteins. The dltS gene codes for a 395-amino-acid putative histidine kinase thought to be involved in the sensing of environmental signals. The dlt operon of S. agalactiae is mainly transcribed from the P(dltR) promoter, which directs synthesis of a 6.5-kb transcript encompassing dltR, dltS, dltA, dltB, dltC, and dltD, and from a weaker promoter, P(dltA), which is located in the 3' extremity of dltS. We demonstrate that P(dltR), but not P(dlA), is activated by DltR in the presence of DltS in D-Ala-deficient LTA mutants resulting from insertional inactivation of the dltA gene, which encodes the cytoplasmic D-alanine-D-alanyl carrier ligase DltA. Expression of the dlt operon does not require DltR and DltS, since the basal activity of P(dltR) is high, being 20-fold that of the constitutive promoter P(aphA-3) which directs synthesis of the kanamycin resistance gene aphA-3 in various gram-positive bacteria. We hypothesize that the role of DltR and DltS in the control of expression of the dlt operon is to maintain the level of D-Ala esters in LTAs at a constant and appropriate value whatever the environmental conditions. The DltA(-) mutant displayed the ability to form clumps in standing culture and exhibited an increased susceptibility to the cationic antimicrobial polypeptide colistin.