Remodeling of Cross-bridges Controls Peptidoglycan Cross-linking Levels in Bacterial Cell Walls.

Cell walls are barriers found in almost all known bacterial cells. These structures establish a controlled interface between the external environment and vital cellular components. A primary component of cell wall is a highly cross-linked matrix called peptidoglycan (PG). PG cross-linking, carried out by transglycosylases and transpeptidases, is necessary for proper cell wall assembly. Transpeptidases, targets of ß-lactam antibiotics, stitch together two neighboring PG stem peptides (acyl-donor and acyl-acceptor strands). We recently described a novel class of cellular PG probes that were processed exclusively as acyl-donor strands. Herein, we have accessed the other half of the transpeptidase reaction by developing probes that are processed exclusively as acyl-acceptor strands. The critical nature of the cross-bridge on the PG peptide was demonstrated in live bacterial cells, and surprising promiscuity in cross-bridge primary sequence was found in various bacterial species. Additionally, acyl-acceptor probes provided insight into how chemical remodeling of the PG cross-bridge (e.g., amidation) can modulate cross-linking levels, thus establishing a physiological role of PG structural variations. Together, the acyl-donor and -acceptor probes will provide a versatile platform to interrogate PG cross-linking in physiologically relevant settings.

Expression profiling in a mammalian host reveals the strong induction of genes encoding LysM domain-containing proteins in Enterococcus faecium.

Enterococcus faecium is an important health care-associated pathogen that is difficult to treat due to the high level of antibiotic resistance of clinical isolates. The identification of new potential therapeutic targets or vaccination strategies is therefore urgently needed. In this regard, we carried out a transcriptomic analysis of the E. faecium vancomycin-resistant strain AUS0004, comparing the gene expression of bacteria grown under laboratory conditions and bacteria isolated from an infection site. This analysis highlighted more than 360 genes potentially induced under infection conditions. Owing to their expression profiles, four LysM domain-containing proteins were characterized in more detail. The EFAU004_01059, 1150 and 494 proteins are highly homologous, whereas EFAU004_01209 has a unique domain-architecture and sequence. The analysis of corresponding mutants showed that all LysM proteins played relevant roles in the infection process of E. faecium in mice. The EFAU004_01209 mutant also displayed profound morphological modifications, suggesting it has a role in cell wall synthesis or cell division. Furthermore, the adhesion to kidney cells and growth of the mutant was affected in human urine. All these phenotypes and the surface exposure of EFAU004_01209 identify this protein as an interesting new drug target in E. faecium.

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

The cell wall architecture of Enterococcus faecium: from resistance to pathogenesis.

The cell wall of Gram-positive bacteria functions as a surface organelle that continuously interacts with its environment through a plethora of cell wall-associated molecules. Enterococcus faecium is a normal inhabitant of the GI tract of mammals, but has recently become an important etiological agent of hospital-acquired infections in debilitated patients. Insights into the assembly and function of enterococcal cell wall components and their interactions with the host during colonization and infection are essential to explain the worldwide emergence of E. faecium as an important multiantibiotic-resistant nosocomial pathogen. Understanding the biochemistry of cell wall biogenesis and principles of antibiotic resistance at the molecular level may open up new frontiers in research on enterococci, particularly for the development of novel antimicrobial strategies. In this article, we outline the current knowledge on the most important antimicrobial resistance mechanisms that involve peptidoglycan synthesis and the role of cell wall constituents, including lipoteichoic acid, wall teichoic acid, capsular polysaccharides, LPxTG cell wall-anchored surface proteins, WxL-type surface proteins and pili, in the pathogenesis of E. faecium.

Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content.

BACKGROUND: The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains. METHODOLOGY: Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs. PRINCIPAL FINDINGS: Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM. CONCLUSION: Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.

Reversible monensin adaptation in Enterococcus faecium, Enterococcus faecalis and Clostridium perfringens of cattle origin: potential impact on human food safety.

OBJECTIVES: To determine the stability/reversibility and mechanism of monensin adaptation in monensin-treated cattle isolates compared with reference bacterial isolates, exposed in vitro to high monensin concentrations. METHODS: We evaluated the potential for cattle-origin strains of Clostridium perfringens, Enterococcus faecium and Enterococcus faecalis exposed to monensin in vivo (in vivo monensin-exposed isolates) to maintain or achieve the ability to grow in the presence of high monensin concentrations (in vitro monensin-adapted isolates). Twenty-one consecutive subcultures of the in vitro monensin-adapted strains were performed, and monensin MICs were determined for the 3rd, 7th, 14th and 21st subcultures (subcultured isolates). SDS-PAGE and transmission electron microscopy (TEM) were used to determine protein expression and visualize extracellular morphology changes. RESULTS: Monensin-non-exposed isolates did not display monensin adaptation during in vitro monensin exposure. In contrast, in vivo monensin-exposed isolates displayed monensin adaptation enabling growth at 32× MIC. Upon consecutive subculturing, monensin MICs returned to baseline, or one dilution above, for the monensin-adapted strains. SDS-PAGE identified overexpression of a 14 kDa protein (C. perfringens) and a 20.5 kDa protein (E. faecium and E. faecalis) in the monensin-adapted isolates. TEM demonstrated that in vitro monensin-adapted strains had a significantly thicker cell wall or glycocalyx compared with in vivo monensin-exposed or subcultured isolates. CONCLUSIONS: In vivo monensin-exposed isolates of C. perfringens, E. faecium and E. faecalis have the ability to grow in the presence of high monensin concentrations in vitro. This is associated with an increased thickening of the cell wall or glycocalyx that is reversible upon serial passage, suggesting a phenotypically expressed, but not genetically stable, trait.

Characterization of an Enterococcus faecium small-colony variant isolated from blood culture.

Small-colony variants (SCVs) of bacteria are slow-growing subpopulations which can cause latent or recurrent infections due to better intracellular survival compared to their wild-type counterparts. Atypical colony morphology and altered biochemical profile may lead to failure in identification of SCV strains. We here report for the first time the isolation of an Enterococcus faecium SCV phenotype. The case of a 65-year-old woman with acute myeloid leukaemia who developed symptoms of sepsis during induction chemotherapy is presented. E. faecium with normal and SCV phenotype was isolated from blood cultures. At the same time urine culture was positive with E. faecium suggesting that bacteraemia originated from the urinary tract. The SCV phenotype was characterized by atypical growth behaviour. Electron microscopic analyses revealed perturbation of the separation of daughter cells and the accumulation of cell wall material. Accordingly, the SCV variant showed a dysfunction or lack of spontaneous autolysis whereas the normal phenotype did not. In contrast to conventional identification systems based on biochemical characteristics, the E. faecium SCV was precisely identified by MALDI-TOF MS analysis implemented in our laboratory. Hence, the increasing use of MALDI-TOF MS analysis for the identification of bacteria might be an appropriate tool for the detection of SCV variants, the diagnosis of which is of importance for the clinical outcome and the antibiotic treatment.

Purification and characterization of enterocin MC13 produced by a potential aquaculture probiont Enterococcus faecium MC13 isolated from the gut of Mugil cephalus.

A bacteriocin producer strain MC13 was isolated from the gut of Mugil cephalus (grey mullet) and identified as Enterococcus faecium. The bacteriocin of E. faecium MC13 was purified to homogeneity, as confirmed by Tricine sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE). Reverse-phase high-performance liquid chromatography (HPLC) analysis showed a single active fraction eluted at 26 min, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry analysis showed the molecular mass to be 2.148 kDa. The clear zone in native PAGE corresponding to enterocin MC13 band further substantiated its molecular mass. A dialyzed sample (semicrude preparation) of enterocin MC13 was broad spectrum in its action and inhibited important seafood-borne pathogens: Listeria monocytogenes , Vibrio parahaemolyticus, and Vibrio vulnificus. This antibacterial substance was sensitive to proteolytic enzymes: trypsin, protease, and chymotrypsin but insensitive to catalase and lipase, confirming that inhibition was due to the proteinaceous molecule, i.e., bacteriocin, and not due to hydrogen peroxide. Enterocin MC13 tolerated heat treatment (up to 90 °C for 20 min). Enterococcus faecium MC13 was effective in bile salt tolerance, acid tolerance, and adhesion to the HT-29 cell line. These properties reveal the potential of E. faecium MC13 to be a probiotic bacterium. Enterococcus faecium MC13 could be used as potential fish probiotic against pathogens such as V. parahaemolyticus, Vibrio harveyi, and Aeromonas hydrophila in fisheries. Also, this could be a valuable seafood biopreservative against L. monocytogenes.

Role of class A penicillin-binding proteins in the expression of beta-lactam resistance in Enterococcus faecium.

Peptidoglycan is polymerized by monofunctional d,d-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of beta-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the DeltapbpF DeltaponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the L,D-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the DeltapbpF DeltaponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different beta-lactam antibiotics differed as a function of its partner glycosyltransferase.

Expression of two distinct types of pili by a hospital-acquired Enterococcus faecium isolate.

Surface filamentous structures designated pili, and implicated in virulence, have been found on the surfaces of several Gram-positive pathogens. This work describes the conditional expression of two phenotypically distinct pilus-like structures, designated PilA and PilB, on the surface of a hospital-adapted Enterococcus faecium bloodstream isolate. E. faecium is an emerging Gram-positive opportunistic pathogen that can cause severe disease, particularly in immunocompromised patients. Expression of PilA- and PilB-type pili was analysed during different phases of growth in broth culture. During growth, PilA and PilB pilin subunits were expressed around the cross-wall in early-exponential-phase cells. Polymerization and migration of short PilB-type pili towards the poles occurred in cells from the exponential phase and long polymerized pili were expressed at the poles of cells grown to stationary phase. In contrast, PilA-type pili were not expressed in broth culture, but only when cells were grown on solid media. Furthermore, surface expression of the PilA- and PilB-type pili was regulated in a temperature-dependent manner, as polymerization of two distinct types of pili at the surface only occurred when cells were grown at 37 degrees C; no pili were observed on cells grown at 21 degrees C. Hospital-aquired E. faecium isolates were specifically enriched in pilin gene clusters, suggesting that conditional expression of pili may contribute to E. faecium pathogenesis.

Influence of peroxyacetic acid and nisin and coculture with Enterococcus faecium on Listeria monocytogenes biofilm formation.

Biofilm formation is a matter of concern in food industries because biofilms facilitate the survival of pathogenic bacteria such as Listeria monocytogenes, which may contaminate food-processing equipment and products. In this study, nisin and two Enterococcus faecium strains were evaluated for their effect on biofilm formation by L. monocytogenes cultured in brain heart infusion broth and on stainless steel coupons. Elimination of preformed L. monocytogenes biofilms by peroxyacetic acid also was tested. Adhesion control experiments were performed with pure cultures of L. monocytogenes after swab collection of adhered cells, which were then enumerated on PALCAM agar plates and visualized by scanning electron microscopy. Formation of a biofilm was recorded when the number of adhered cells was at least 10(3) CFU/cm2. When L. monocytogenes was cocultured with E. faecium bac-, the number of adhered L. monocytogenes cells was 2.5 log lower (P = 0.002) when initially compared with the control culture, but after 6 h of incubation a biofilm was again detected. However, in coculture on stainless steel coupons, E. faecium bac+ inhibited L. monocytogenes adherence and did not allow biofilm formation for up to 48 h (P < 0.001). In the presence of nisin or after treatment with peroxyacetic acid, bacterial growth was reduced (P < 0.001) up to 4.6 and 5.6 log CFU/cm2, respectively, when compared with L. monocytogenes cultures on untreated coupons. However, after these treatments, cells were still present, and after 24 h of incubation, a renewed biofilm was detected in L. monocytogenes cultures treated with nisin. Although all tested conditions reduced L. monocytogenes growth to some extent, only coculture with E. faecium bac+ efficiently reduced biofilm formation, suggesting a potential control strategy for this pathogen.

7-O-malonyl macrolactin A, a new macrolactin antibiotic from Bacillus subtilis active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and a small-colony variant of Burkholderia cepacia.

We report here the discovery, isolation, and chemical and preliminary biological characterization of a new antibiotic compound, 7-O-malonyl macrolactin A (MMA), produced by a Bacillus subtilis soil isolate. MMA is a bacteriostatic antibiotic that inhibits a number of multidrug-resistant gram-positive bacterial pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and a small-colony variant of Burkholderia cepacia. MMA-treated staphylococci and enterococci were pseudomulticellular and exhibited multiple asymmetric initiation points of septum formation, indicating that MMA may inhibit a cell division function.

Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii.

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.

Effect of quinupristin/dalfopristin alone or in combination with vancomycin on the structure of Enterococcus faecium.

Twenty strains of Enterococcus faecium susceptible to quinupristin/dalfopristin (< 2 mg/l) were DNA fingerprinted to exclude strain duplication. Ten strains were susceptible to vancomycin (minimal inhibitory concentration [MIC] < 2 mg/l) and 10 were resistant to vancomycin (MIC > 400 mg/l). Vancomycin at 1/2 MIC, quinupristin/dalfopristin at 1/4 MIC and their combination, except for a tube control, was added to 10 ml trypticase soy broth tubes which were planted with the respective 24-h trypticase soy broth cultures. The products of incubation were sampled periodically throughout 24 h for gram stain and electron microscopy. Cell size was measured on photographs at 20,000x final magnification and results were statistically analyzed. The cells of all strains of Enterococcus faecium exposed for 12 h to quinupristin/dalfopristin were comparable in size to the control, Most cells, however, showed areas of low density of ribosome in the center of the cells. The cells of Enterococcus faecium resistant to vancomycin exposed to vancomycin were larger than the controls with means of 1.96 micron -2.07 micron versus 1.16 micron (p < 0.001); these cells consisted of individual organisms connected by wide cross walls of abnormal fibrous structure. Enterococcus faecium sensitive to vancomycin exposed to vancomycin remained comparable to the control. The combination of quinupristin/dalfopristin plus vancomycin produced large cells with multiple abnormal cross walls in both vancomycin-resistant and vancomycin-sensitive Enterococcus faecium. The addition of quinupristin/dalfopristin to vancomycin appears to modify the vancomycin-susceptible strains to respond to vancomycin in the same manner as do the vancomycin-resistant organisms.

Resistance of Enterococcus faecium to neutrophil-mediated phagocytosis.

During a previous study of the opsonic requirements for neutrophil (polymorphonuclear leukocyte [PMN])-mediated killing of enterococci, we identified two strains of Enterococcus faecium (TX0015 and TX0016) that were resistant to PMN-mediated killing. To better define the mechanism of this resistance, we examined phagocytosis with a fluorescence assay and found that TX0016 was completely resistant to phagocytosis by PMNs; this finding was confirmed by electron microscopy. Examination of multiple strains of enterococci revealed that all 20 strains of Enterococcus faecalis tested were readily phagocytosed (mean, 18 intracellular organisms per PMN; range, 7 to 28). In contrast, only 13 (50%) of 26 strains of E. faecium tested were susceptible to phagocytosis (> or = 7 organisms per PMN); the other 13 strains showed < or = 3 organisms per PMN. Enterococcus casseliflavus ATCC 25788 and one strain of Enterococcus hirae were also resistant to phagocytosis, while two strains of Enterococcus durans, Enterococcus mundtii ATCC 43186, and one strain each of Enterococcus raffinosus and Enterococcus solitarius were readily phagocytosed. Exposure of E. faecium TX0016 to sodium periodate, but not to the protease trypsin or pronase or to phospholipase C, eliminated resistance to phagocytosis. Sialic acid, a common periodate-sensitive structure used by microorganisms to resist opsonization, could not be demonstrated in E. faecium TX0016 by the thiobarbituric acid method, nor was phagocytosis of TX0016 altered by neuraminidase treatment. This study suggests that there is a difference in susceptibility to phagocytosis by PMNs between different species of enterococci and that a carbohydrate-containing moiety which is not sialic acid may be involved in the resistance of E. faecium TX0016 to phagocytosis.

Growth and adhesion of Enterococcus faecium L-forms.

Comparisons of growth and surface colonisation of Enterococcus faecium L-forms and their cell-walled forms were undertaken to produce information about their ability to form sessile cells. The growth of L-forms in liquid culture was slower than that of the parent. This was reflected in their longer lag phase and slower specific growth rates: 0.16 h-1 for the L-form and 0.81 h-1 for the parent. Although E. faecium L-forms attached to a silastic rubber surface, the attached population density was 10-100-fold less than that of the parent. Confluent biofilms on the silastic surfaces were not observed for either bacterial form. Comparison of the attachment of E. faecium L-form and parent may provide important information on how bacteria overcome host defence mechanisms and antibiotic treatment.

Effect of extracellularly generated singlet oxygen on gram-positive and gram-negative bacteria.

In the separated surface-sensitizer system, a photosensitizer is physically separated from the substrate by a thin air layer under such conditions that only singlet oxygen can reach and oxidize the substrate, preventing the competition by type I photosensitized processes. This method has been used to study the reaction of singlet oxygen with Gram-positive (Streptococcus faecium) and Gram-negative (Escherichia coli) bacterial strains. Studies on cell samples exposed to singlet oxygen for different periods of time show a drastic decrease in survival for S. faecium, while E. coli becomes sensitive only when the integrity of the outer membrane is altered by treatment with CaCl2 or tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid (Tris-EDTA). Biochemical and ultrastructural analyses suggest that the cytoplasmic membrane and the genetic material are the main sites damaged by singlet oxygen.

Photosensitizing activity of water- and lipid-soluble phthalocyanines on prokaryotic and eukaryotic microbial cells.

The photosensitizing activity of lipophilic zinc-phthalocyanine (Zn-Pc) and its water-soluble sulphonated derivative (Zn-PcS) towards Streptococcus faecium and Candida albicans was studied and correlated with the amount of cell-bound photosensitizer. With both micro-organisms Zn-PcS was more tightly bound in larger amounts than Zn-Pc in the protoplasts of the cytoplasmic membrane. As a consequence, the photoinduced damage in S. faecium initially involved membrane proteins, while DNA was modified only upon prolonged irradiation. For C. albicans only Zn-PcS showed a preferential affinity for the spheroplasts and the decrease in cell survival was not accompanied by detectable modifications of the electrophoretic pattern of membrane proteins. The photoinduced ultrastructural alteration of both micro-organisms suggests damage at membrane level. This would indicate the involvement of different targets in bacteria and yeast for phthalocyanine photosensitization.