Contribution of the enterococcal surface protein Esp to pathogenesis of Enterococcus faecium endocarditis

The enterococcal surface protein Esp, specifically linked to nosocomial Enterococcus faecium, is involved in biofilm formation. To assess the role of Esp in endocarditis, a biofilm-associated infection, an Esp-expressing E. faecium strain (E1162) or its Esp-deficient mutant (E1162Δesp) were inoculated through a catheter into the left ventricle of rats. After 24 h, less E1162Δesp than E1162 were recovered from heart valve vegetations. In addition, anti-Esp antibodies were detected in Esp-positive E. faecium bacteremia and endocarditis patient sera. In conclusion, Esp contributes to colonization of E. faecium at the heart valves. Furthermore, systemic infection elicits an Esp-specific antibody response in humans.     

The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis

Enterococci have emerged as one of the leading causes of nosocomial bloodstream, surgical site, and urinary tract infections. More recently, enterococci have been associated with biofilms, which are bacterial communities attached to a surface and encased in an extracellular polymeric matrix. The enterococcal cell surface-associated protein, Esp, enhances biofilm formation by Enterococcus faecalis in a glucose-dependent manner. Mature Esp consists of a nonrepeat N-terminal domain and a central region made up of two types of tandem repeats followed by a C-terminal membrane-spanning and anchor domain. This study was undertaken to localize the specific domain(s) of Esp that plays a role in Esp-mediated biofilm enhancement. To achieve this objective, we constructed in-frame deletion mutants expressing truncated forms of Esp in an isogenic background. By comparing strains expressing the mutant forms of Esp to those expressing wild-type Esp, we found that the strain expressing Esp lacking the N-terminal domain formed biofilms that were quantitatively less in biovolume than the strain expressing wild-type Esp. Furthermore, an E. faecalis strain expressing only the N-terminal domain of Esp fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. This suggested that the minimal region contributing to Esp-mediated biofilm enhancement in E. faecalis was confined to the nonrepeat N-terminal domain. Expression of full-length E. faecalis Esp in heterologous host systems of esp-deficient Lactococcus lactis and Enterococcus faecium did not enhance biofilm formation as was observed for E. faecalis. These results suggest that Esp may require interaction with an additional E. faecalis-specific factor(s) to result in biofilm enhancement.     

The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.     

Enterococcal surface protein Esp is not essential for cell adhesion and intestinal colonization of Enterococcus faecium in mice

Background  

Enterococcus faecium has globally emerged as a cause of hospital-acquired infections with high colonization rates in hospitalized patients. The enterococcal surface protein Esp, identified as a potential virulence factor, is specifically linked to nosocomial clonal lineages that are genetically distinct from indigenous E. faecium strains. To investigate whether Esp facilitates bacterial adherence and intestinal colonization of E. faecium, we used human colorectal adenocarcinoma cells (Caco-2 cells) and an experimental colonization model in mice.     

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.     

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.     

Five genes encoding surface-exposed LPXTG proteins are enriched in hospital-adapted Enterococcus faecium clonal complex 17 isolates

Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.     

Frequent occurrence of multidrug‐resistant CC17 Enterococcus faecium among clinical isolates in Sweden

Aims: To screen for the globally spread cluster of Enterococcus faecium, clonal complex 17 (CC17) and characterize the genetic profile of Swedish clinical Ent. faecium isolates. Methods: A total of 203 consecutive isolates collected from 2004 to 2007 from patients with bacteraemia in Sweden. All isolates were genotyped using multiple‐locus variable‐number tandem repeat analysis (MLVA) and 20 isolates representing different MLVA types (MT) were chosen for multilocus sequence typing (MLST). Minimal inhibitory concentrations against clinically relevant antibiotics were determined with agar dilution. Presence of the virulence genes esp and hyl was investigated using PCR. Results: A total of 65% (n = 109) of all isolates belonged to MT‐1, and the second most common MLVA type was MT‐159 (13%, n = 21). MLST analysis confirmed the presence of CC17 during the entire study period. The number of isolates resistant to gentamicin and vancomycin, as well as the presence of hyl, increased significantly during the investigation period. Conclusions: The present study demonstrates that nosocomial infections caused by Ent. faecium CC17 are commonly occurring in Sweden. Significance and Impact of the Study: This is the first report of CC17 Ent. faecium in Sweden. The increase of antibiotic resistance and virulence indicates that these strains are further adapting to the hospital environment.     

Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis

Aims: Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression–secretion system. Methods and Results: The esp gene was fused with the N‐terminal Sec‐dependent signal sequence of the B. choshinensis cell wall protein and a C‐terminal hexa‐histidine‐tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. Conclusions: The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20‐ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1‐l culture). Significance and Impact of the Study: Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications.     

Modulation of eDNA Release and Degradation Affects Staphylococcus aureus Biofilm Maturation

Recent studies have demonstrated a role for Staphylococcus aureus cidA-mediated cell lysis and genomic DNA release in biofilm adherence. The current study extends these findings by examining both temporal and additional genetic factors involved in the control of genomic DNA release and degradation during biofilm maturation. Cell lysis and DNA release were found to be critical for biofilm attachment during the initial stages of development and the released DNA (eDNA) remained an important matrix component during biofilm maturation. This study also revealed that an lrgAB mutant exhibits increased biofilm adherence and matrix-associated eDNA consistent with its proposed role as an inhibitor of cidA-mediated lysis. In flow-cell assays, both cid and lrg mutations had dramatic effects on biofilm maturation and tower formation. Finally, staphylococcal thermonuclease was shown to be involved in biofilm development as a nuc mutant formed a thicker biofilm containing increased levels of matrix-associated eDNA. Together, these findings suggest a model in which the opposing activities of the cid and lrg gene products control cell lysis and genomic DNA release during biofilm development, while staphylococcal thermonuclease functions to degrade the eDNA, possibly as a means to promote biofilm dispersal.     

Genetic Variability of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates from Humans,Chickens, and Pigs in Malaysia

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.     

Dogs Are a Reservoir of Ampicillin-Resistant Enterococcus faecium Lineages Associated with Human Infections

Ampicillin resistance is a marker for hospital-associated Enterococcus faecium. Feces from 208 dogs were selectively screened for the occurrence of ampicillin-resistant E. faecium (AREF). AREF was detected in 42 (23%) of 183 dogs screened in a cross-sectional study in the United Kingdom and in 19 (76%) of 25 dogs studied longitudinally in Denmark. AREF carriage was intermittent in all dogs studied longitudinally. Multilocus sequence typing of 63 canine AREF isolates revealed the presence of 13 distinct sequence types. Approximately 76% of the isolates belonged to hospital-adapted clonal complex 17 (CC17), including those of sequence types ST-78 and ST-192, which are widespread in European and Asian hospitals. Longitudinal screening of 18 healthy humans living in contact with 13 of the dogs under study resulted in the identification of a single, intermittent CC17 carrier. This person carried one of the sequence types (ST-78) recovered from his dog. Based on PCR and Southern hybridization analyses, the putative virulence gene cluster from orf903 to orf907 was widespread in canine AREF isolates (present in 97%), whereas orf2351 (present in 26% of isolates) and orf2430 (present in 31%) were strongly associated with CC17-related sequence types (P < 0.05). Surprisingly, esp and hyl were not detected in any of the isolates. The antimicrobial resistance profiles of canine AREF isolates generally differed from those previously described for clinical human isolates. The results indicate that dogs are frequent carriers of CC17-related lineages and may play a role in the spread of this nosocomial pathogen. The distinctive virulence and antimicrobial resistance profiles observed among canine AREF isolates raise interesting questions about the origin and evolution of the strains causing human infections.Enterococci are opportunistic pathogens and form part of the normal gastrointestinal flora in humans and animals. Over the last two decades, nosocomial infections caused by enterococci have emerged and their incidence has increased rapidly, first in the United States and recently in Europe (25, 26, 29). Although Enterococcus faecalis is the causative agent in most enterococcal infections, a shift toward infections caused by multidrug-resistant E. faecium has been noted in the last years, and presently, up to one-third of enterococcal infections in some countries are attributed to this species (17). This shift may be explained by changes in the patterns of antimicrobial usage, which may have resulted in the emergence of a distinct genogroup of hospital-associated ampicillin-resistant E. faecium (AREF) strains, currently labeled clonal complex 17 (CC17) (33). CC17 isolates are characterized by resistance to ampicillin and fluoroquinolones, as well as by the presence in most isolates of putative virulence genes encoding the enterococcal surface protein (esp) and hyaluronidase (hyl) and five recently described open reading frames (ORFs; orf903, orf904.5, orf906.7, orf2351, and orf2430) encoding LPXTG surface proteins, which are found less frequently among other E. faecium lineages (15, 20, 27).Based on the results of multilocus sequence typing (MLST) (28) and amplified fragment length polymorphism analysis (34), E. faecium isolates of animal origin seem to be host specific and generally unrelated to human lineages of clinical importance. Prior to this study, AREF CC17 strains have been isolated only sporadically from animals, including pigs (2, 10) and more recently dogs (8). Following these unexpected findings, the present study was designed to investigate the prevalence and shedding patterns of AREF in dogs. A cross-sectional study and two longitudinal studies involving a total of 208 dogs and 479 canine fecal samples were conducted in the United Kingdom and in Denmark, respectively. Canine isolates were characterized by MLST, antimicrobial susceptibility testing, and putative virulence gene profiling to assess the genetic relationship between human and canine AREF strains.     

Virulence determinants in vancomycin-resistant <Emphasis Type="Italic">Enterococcus faecium van</Emphasis>A isolated from different sources at University Hospital of Londrina,Paraná, Brazil

Enterococcus faecium, especially those showing multidrug resistance, has emerged as a significant cause of healthcare-associated infections worldwide. However, relatively little is known about the virulence and pathogenesis of this species. The aim of this study was to determine the occurrence of four putative virulence determinants of E. faecium and to correlate them with phenotypic traits. Using forty E. faecium vanA-type isolates from hospitalized patients and their environmental vicinity, we determined the following: the antimicrobial susceptibility profile, occurrence of the genes cylA, efaA, esp, and gelE, hemolytic and gelatinase activities, capacity to form biofilm and in vitro adhesion to epithelial cells. All isolates were shown to be resistant to vancomycin and teicoplanin, as well as to two or more other antimicrobials. All isolates harbored at least one putative virulence marker, and the prevalence was as follows: esp, 87.5%; efaA, 82.5%; gelE, 70%; and cylA, 65%. The presence of 4 genes was observed in 32.5% isolates. The presence of the efaA was associated with the presence of esp, regardless of the source of the isolates. A positive association with the presence of cylA and hemolytic activity in the sheep blood agar assay was observed. No association was found for gelE and gelatinase production in the agar plate assay, for efaA and LLC-MK2 cell adhesion, and for esp and biofilm formation on polystyrene surface. These results show the presence of putative virulence genes in multiple antimicrobial resistant E. faecium isolates from different sources in a hospital setting.     

Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

Identification of a Novel Genomic Island Specific to Hospital-Acquired Clonal Complex 17 Enterococcus faecium Isolates

Hospital-acquired clonal complex 17 (CC17) Enterococcus faecium strains are genetically distinct from indigenous strains and are enriched with resistance genes and virulence genes. We identified a genomic island in CC17 E. faecium tentatively encoding a metabolic pathway involved in carbohydrate transport and metabolism, which may provide a competitive advantage over the indigenous E. faecium microbiota.     

Role of ClpP in biofilm formation and virulence of Staphylococcus epidermidis

Infections caused by the leading nosocomial pathogen Staphylococcus epidermidis are characterized by biofilm formation on implanted medical devices. However, the molecular basis of biofilm formation and its regulation are not completely understood. Here, we describe an important role of the ClpP protease in biofilm development and virulence of S. epidermidis. We constructed an isogenic clpP mutant strain of a biofilm-forming clinical isolate of S. epidermidis. The mutant strain showed decreased biofilm formation in vitro and reduced virulence in a rat model of biofilm-associated infection. Biofilm forming ability of the mutant strain could be restored by expressing clpP on a plasmid, but not when a catalytically inactive allele of clpP gene was introduced. These observations indicate that the peptidase function of ClpP determines its role in biofilm formation. Experimental data in this work also suggested that clpP influenced initial attachment of bacteria on the plastic surface, the first step of biofilm formation. Furthermore, clpP was found to be regulated by the quorum-sensing agr, suggesting that part of the previously described influence of agr on the initial attachment to plastic surfaces may be mediated by clpP.     

Phosphoglycerate mutase affects Stenotrophomonas maltophilia attachment to biotic and abiotic surfaces

Stenotrophomonas maltophilia biofilm formation is of increasing medical concern, particularly for lung infections. However, the molecular mechanisms facilitating the biofilm lifestyle in S. maltophilia are poorly understood. We generated and screened a transposon mutant library for mutations that lead to altered biofilm formation compared to wild type. One of these mutations, in the gene for glycolytic enzyme phosphoglycerate mutase (gpmA), resulted in impaired attachment on abiotic and biotic surfaces. As adherence to a surface is the initial step in biofilm developmental processes, our results reveal a unique factor that could affect S. maltophilia biofilm initiation and, possibly, subsequent development.     

Cross‐transmission of clinical Enterococcus faecium in relation to esp and antibiotic resistance

Aims: To investigate clonality among clinical Enterococcus faecium isolates and normal intestinal microflora isolates as well as cross‐transmission between patients in relation to the presence of the esp gene and antibiotic resistance. Methods and Results: Blood‐culture isolates (n = 101) deriving from tertiary, secondary and primary hospitals were analysed. Antibiotic susceptibility was investigated. Polymerase chain reaction and pulsed‐field gel electrophoresis were used for detection of esp and genotyping, respectively. Nearly half (43%) of the patients included were involved in a cross‐transmission event with Ent. faecium. These strains disseminated both within and between all hospitals. The antibiotic resistance and presence of esp were highest in isolates from the tertiary hospital. Isolates harbouring esp showed less genetic diversity compared with esp negative ones. Conclusions: Cross‐transmission with Ent. faecium between patients was readily detected, indicating that hospital‐adapted clones circulate within and between hospitals. Acquired characteristics, such as antibiotic resistance and esp, seem to accumulate in the isolates disseminating in the tertiary hospital. Significance and Impact of the Study: It is important to characterize Ent. faecium isolates causing infections and to determine the extent of dissemination in order to prevent further spread of these pathogens.     

Type I fimbriae mediate in vitro adherence of porcine F18ac+ enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (ETEC)

Type I fimbriae commonly expressed by Escherichia coli mediate initial attachment of bacteria to host epithelial cells. However, the role of type I fimbriae in the adherence of porcine enterotoxigenic E. coli (ETEC) to host receptors is unclear. In this study, we examined the role of type I fimbriae in the adherence and biofilm formation of F18ac+ ETEC by constructing mutant strains with deletion of type I fimbrial major subunit (fimA) or minor subunit (fimH). The data indicated that the isogenic ΔfimA and ΔfimH mutants showed significantly lower adherence to porcine epithelial IPEC-1 and IPEC-J2 cells as compared to the F18ac+ ETEC parent strain. In addition, the adherence of F18ac+ ETEC to both cell lines was blocked by the presence of 0.5% D-mannose in the cell culture medium. In addition, both mutant strains impaired their ability to form biofilm in vitro. Interestingly, the deletion of fimA or fimH genes resulted in remarkable up-regulation of the expression of adhesin involved in diffuse adherence (AIDA-I). These results indicated that type I fimbriae may be required for efficient adherence of F18ac+ ETEC to pig epithelial cells and, perhaps, biofilm formation.     

Random insertion transposon mutagenesis of Mycobacterium fortuitum identified mutant defective in biofilm formation

Mycobacterium fortuitum has emerged as a nosocomial infectious agent and biofilm formation attributed for the presence of this bacterium in hospital environment. Transposon random mutagenesis was used to identify membrane-proteins for biofilm formation in M. fortuitum. Ten mutants were shortlisted from a library of 450 mutants for examine their biofilm forming ability. Comparative biofilm ability with respect to wild type M. fortuitum ATCC 6841 showed an altered and delayed biofilm formation in one mutant namely, MT721. Sequence analysis revealed mutation in anthranilate phosphoribosyl transferase (MftrpD), which is associated with tryptophan operon. Functional interaction study of TrpD protein through STRING showed its interaction with chorismate utilizing proteins, majorly involved in synthesis of aromatic amino acid and folic acid, suggesting that biofilm establishment and maintenance requires components of central metabolism. Our study indicates important role of MftrpD in establishment and maintenance of biofilm by M. fortuitum, which may further be explored for drug discovery studies against mycobacterial infections.