Correction: Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0060143.].

Redefining Peptide 14D: Substitutional Analysis for Accelerated TB Diagnosis and Enhanced Activity against Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a predominant cause of mortality, especially in low- and middle-income nations. Recently, antimicrobial peptides have been discovered that at low concentrations could stimulate the growth of M. tuberculosis (hormetic response). In this study, such a peptide was used to investigate the effects on the time to positivity (TTP). A systematic substitution analysis of peptide 14D was synthesized using Spot synthesis technology, resulting in 171 novel peptides. Our findings revealed a spectrum of interactions, with some peptides accelerating M. tuberculosis growth, potentially aiding in faster diagnostics, while others exhibited inhibitory effects. Notably, peptide NH2-wkivfiwrr-CONH2 significantly reduced the TTP by 25 h compared to the wild-type peptide 14D, highlighting its potential in improving TB diagnostics by culture. Several peptides demonstrated potent antimycobacterial activity, with a minimum inhibitory concentration (MIC) of 20 µg/mL against H37Rv and a multidrug-resistant M. tuberculosis strain. Additionally, for two peptides, a strongly diminished formation of cord-like structures was observed, which is indicative of reduced virulence and transmission potential. This study underscores the multifaceted roles of antimicrobial peptides in TB management, from enhancing diagnostic efficiency to offering therapeutic avenues against M. tuberculosis.

Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.

Discovery of Antimicrobial Peptides That Can Accelerate Culture Diagnostics of Slow-Growing Mycobacteria Including Mycobacterium tuberculosis.

Antimicrobial peptides (AMPs) can directly kill Gram-positive bacteria, Gram-negative bacteria, mycobacteria, fungi, enveloped viruses, and parasites. At sublethal concentrations, some AMPs and also conventional antibiotics can stimulate bacterial response increasing their resilience, also called the hormetic response. This includes stimulation of growth, mobility, and biofilm production. Here, we describe the discovery of AMPs that stimulate the growth of certain mycobacteria. Peptide 14 showed a growth stimulating effect on Mycobacteria tuberculosis (MTB), M. bovis, M. avium subsp. paratuberculosis (MAP), M. marinum, M. avium-intracellulare, M. celatum, and M. abscessus. The effect was more pronounced at low bacterial inocula. The peptides induce a faster transition from the lag phase to the log phase and keep the bacteria longer in the log phase before entering stationary phase when compared to nontreated controls. In some cases, an increase in the division rate was observed. An initial screen using MAP and a collection of 75 peptides revealed 13 peptides with a hormetic effect. For MTB, a collection of 25 artificial peptides were screened and 13 were found to reduce the time to positivity (TTP) by at least 5%, improving growth. A screen of 43 naturally occurring peptides, 11 fragments of naturally occurring peptides and 5 designed peptides, all taken from the database APD3, identified a further 44 peptides that also lowered TTP by at least 5%. Lasioglossin LL-III (Bee) and Ranacyclin E (Frog) were the most active natural peptides, and the human cathelicidin LL37 fragment GF-17 and a porcine cathelicidin protegrin-1 fragment were the most active fragments of naturally occurring peptides. Peptide 14 showed growth-stimulating activity between 10 ng/mL and 10 µg/mL, whereas the stability-optimised Peptide 14D had a narrow activity range of 0.1-1 µg/mL. Peptides identified in this study are currently in commercial use to improve recovery and culture for the diagnostics of mycobacteria in humans and animals.

Antimicrobial Peptides Designed against the Ω-Loop of Class A ß-Lactamases to Potentiate the Efficacy of ß-Lactam Antibiotics.

Class A serine ß-lactamases (SBLs) have a conserved non-active site structural domain called the omega loop (Ω-loop), in which a glutamic acid residue is believed to be directly involved in the hydrolysis of ß-lactam antibiotics by providing a water molecule during catalysis. We aimed to design and characterise potential pentapeptides to mask the function of the Ω-loop of ß-lactamases and reduce their efficacy, along with potentiating the ß-lactam antibiotics and eventually decreasing ß-lactam resistance. Considering the Ω-loop sequence as a template, a group of pentapeptide models were designed, validated through docking, and synthesised using solid-phase peptide synthesis (SPPS). To check whether the ß-lactamases (BLAs) were inhibited, we expressed specific BLAs (TEM-1 and SHV-14) and evaluated the trans-expression through a broth dilution method and an agar dilution method (HT-SPOTi). To further support our claim, we conducted a kinetic analysis of BLAs with the peptides and employed molecular dynamics (MD) simulations of peptides. The individual presence of six histidine-based peptides (TSHLH, ETHIH, ESRLH, ESHIH, ESRIH, and TYHLH) reduced ß-lactam resistance in the strains harbouring BLAs. Subsequently, we found that the combinational effect of these peptides and ß-lactams sensitised the bacteria towards the ß-lactam drugs. We hypothesize that the antimicrobial peptides obtained might be considered among the novel inhibitors that can be used specifically against the Ω-loop of the ß-lactamases.

Four-Month High-Dose Rifampicin Regimens for Pulmonary Tuberculosis.

BACKGROUND: Shorter but effective tuberculosis treatment regimens would be of value to the tuberculosis treatment community. High-dose rifampicin has been associated with more rapid and secure lung sterilization and may enable shorter tuberculosis treatment regimens. METHODS: We randomly assigned adults who were given a diagnosis of rifampicin-susceptible pulmonary tuberculosis to a 6-month control regimen, a similar 4-month regimen of rifampicin at 1200 mg/d (study regimen 1 [SR1]), or a 4-month regimen of rifampicin at 1800 mg/d (study regimen 2 [SR2]). Sputum specimens were collected at regular intervals. The primary end point was a composite of treatment failure and relapse in participants who were sputum smear positive at baseline. The noninferiority margin was 8 percentage points. Using a sequence of ordered hypotheses, noninferiority of SR2 was tested first. RESULTS: Between January 2017 and December 2020, 672 patients were enrolled in six countries, including 191 in the control group, 192 in the SR1 group, and 195 in the SR2 group. Noninferiority was not shown. Favorable responses rates were 93, 90, and 87% in the control, SR1, and SR2 groups, respectively, for a country-adjusted absolute risk difference of 6.3 percentage points (90% confidence interval, 1.1 to 11.5) comparing SR2 with the control group. The proportions of participants experiencing a grade 3 or 4 adverse event were 4.0, 4.5, and 4.4% in the control, SR1, and SR2 groups, respectively. CONCLUSIONS: Four-month high-dose rifampicin regimens did not have dose-limiting toxicities or side effects but failed to meet noninferiority criteria compared with the standard 6-month control regimen for treatment of pulmonary tuberculosis. (Funded by the MRC/Wellcome Trust/DFID Joint Global Health Trials Scheme; ClinicalTrials.gov number, NCT02581527.)

Characterization of the MurT/GatD complex in Mycobacterium tuberculosis towards validating a novel anti-tubercular drug target.

OBJECTIVES: Identification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study. METHODS: Biochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome. RESULTS: Rv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likely to be the mycobacterial PG precursor-modifying enzymes MurT and GatD, respectively. An in vivo network involving Mtb-MurT, regulatory proteins and cell division proteins was also identified. CONCLUSIONS: Understanding the role of the enzyme complex in the context of PG metabolism and cell division, and the implications for antimicrobial resistance and host immune responses will facilitate the design of therapeutics that are targeted specifically to M. tuberculosis.

The Antimicrobial Peptide, Bactenecin 5, Supports Cell-Mediated but Not Humoral Immunity in the Context of a Mycobacterial Antigen Vaccine Model.

Bactenecin (Bac) 5 is a bovine antimicrobial peptide (AMP) capable of killing some species of bacteria through the inhibition of protein synthesis. Bac5 and other AMPs have also been shown to have chemotactic properties and can induce inflammatory cytokine expression by innate immune cells. Recently, AMPs have begun to be investigated for their potential use as novel vaccine adjuvants. In the current work, we characterise the functionality of Bac5 in vitro using murine macrophage-like cells, ex vivo using human tonsil tissue and in vivo using a murine model of vaccination. We report the effects of the peptide in isolation and in the context of co-presentation with mycobacterial antigen and whole, inert Bacillus subtilis spore antigens. We find that Bac5 can trigger the release of nitric oxide from murine macrophages and upregulate surface marker expression including CD86, MHC-I and MHC-II, in the absence of additional agonists. When coupled with mycobacterial Ag85 and B. subtilis spores, Bac5 also enhanced IFNγ secretion. We provide evidence that B. subtilis spores, but not the Bac5 peptide, act as strong adjuvants in promoting antigen-specific immunoglobulin production in Ag85B-vaccinated mice. Our findings suggest that Bac5 is an important regulator of the early cell-mediated host immune response.

3-(5-Nitrofuran-2-yl)prop-2-en-1-one Derivatives, with Potent Antituberculosis Activity, Inhibit A Novel Therapeutic Target, Arylamine N-acetyltransferase, in Mycobacteria.

In this study, the inhibitory potential of 3-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives was evaluated against a panel of bacteria, as well as mammalian cell lines to determine their therapeutic index. In addition, we investigated the mechanism of antibiotic action of the derivatives to identify their therapeutic target. We discovered compound 2 to be an extremely potent inhibitor of Mycobacterium tuberculosis H37Rv growth (MIC: 0.031 mg/L) in vitro, performing better than the currently used first-line antituberculosis drugs such as isoniazid, rifampicin, ethambutol, and pretomanid in vitro. Furthermore, compound 3 was equipotent to pretomanid against a multidrug-resistant M. tuberculosis clinical isolate. The derivatives were selective and bactericidal towards slow-growing mycobacteria. They showed low cytotoxicity towards murine RAW 264.7 and human THP-1 cell lines, with high selectivity indices. Compound 1 effectively eliminated the intracellular mycobacteria in a mycobacteria-infected macrophage model. The derivatives were assessed for their potential to inhibit mycobacterial arylamine N-acetyltransferase (NAT) and were identified as good inhibitors of recombinant mycobacterial NAT, a novel target essential for the intracellular survival of M. tuberculosis. This study provided hits for designing new potent and selective antituberculosis leads, having mycobacterial NAT inhibition as their possible endogenous mechanisms of action.

Exploration of 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazoles as selective, multitargeted antimycobacterial agents.

We report the biological evaluation of 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazole derivatives against bacteria, eukaryotic cell lines and the assessment of their mechanisms of action to determine their prospects of being developed into potent antituberculosis agents. The compounds were evaluated for their antibacterial property against Mycobacterium tuberculosis H37Rv, multidrug-resistant M. tuberculosis, Mycobacterium bovis BCG, Mycobacterium aurum, Escherichia coli, and Staphylococcus aureus using high-throughput spot-culture growth inhibition assay. They were found to be selective toward slow-growing mycobacteria and Gram-positive bacteria. In M. bovis BCG, they exhibited a bactericidal mode of action. Cytotoxicity was assessed in human THP-1 and murine RAW 264.7 cell lines, and the compounds showed a lower cytotoxicity potential when compared with their antibacterial activity. They were found to be excellent whole-cell efflux pump inhibitors of the mycobacterial surrogate M. aurum, performing better than known efflux pump inhibitor verapamil. The 5-nitrothiophene moiety was identified for the first time as a prospective inhibitor scaffold of mycobacterial arylamine N-acetyltransferase enzyme, which is the key enzyme in metabolizing isoniazid, a first-line antituberculosis drug. The two aforementioned findings make the compounds potential hits in the development of adjunctive tuberculosis therapy.

Cell wall peptidoglycan in Mycobacterium tuberculosis: An Achilles' heel for the TB-causing pathogen.

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.

DNA sequence-selective C8-linked pyrrolobenzodiazepine-heterocyclic polyamide conjugates show anti-tubercular-specific activities.

New chemotherapeutic agents with novel mechanisms of action are in urgent need to combat the tuberculosis pandemic. A library of 12 C8-linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD)-heterocyclic polyamide conjugates (1-12) was evaluated for anti-tubercular activity and DNA sequence selectivity. The PBD conjugates were screened against slow-growing Mycobacterium bovis Bacillus Calmette-Guérin and M. tuberculosis H37Rv, and fast-growing Escherichia coli, Pseudomonas putida and Rhodococcus sp. RHA1 bacteria. DNase I footprinting and DNA thermal denaturation experiments were used to determine the molecules' DNA recognition properties. The PBD conjugates were highly selective for the mycobacterial strains and exhibited significant growth inhibitory activity against the pathogenic M. tuberculosis H37Rv, with compound 4 showing MIC values (MIC=0.08 mg l-1) similar to those of rifampin and isoniazid. DNase I footprinting results showed that the PBD conjugates with three heterocyclic moieties had enhanced sequence selectivity and produced larger footprints, with distinct cleavage patterns compared with the two-heterocyclic chain PBD conjugates. DNA melting experiments indicated a covalent binding of the PBD conjugates to two AT-rich DNA-duplexes containing either a central GGATCC or GTATAC sequence, and showed that the polyamide chains affect the interactions of the molecules with DNA. The PBD-C8 conjugates tested in this study have a remarkable anti-mycobacterial activity and can be further developed as DNA-targeted anti-tubercular drugs.

Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals.

The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase in recoverability and an improved sensitivity of up to three logs when compared with conventional culture. Using TiKa culture, MAP clumping was minimal and produced visible colonies in half the time required by standard culture methods. Parallel quantitative evaluation of the TiKa culture approach and qPCR on MAP loads in tissue and gut mucosal samples from a MAP vaccine-challenge study, showed good correlations between colony counts (cfu) and qPCR derived genome equivalents (Geq) over a large range of loads with a 30% greater sensitivity for TiKa culture approach at low loads (two logs). Furthermore, the relative fold changes in Geq and cfu from the TiKa culture approach suggests that non-mucosal tissue loads from MAP infected animals contained a reduced proportion of non-viable MAP (mean 19-fold) which was reduced significantly further (mean 190-fold) in vaccinated "reactor" calves. This study shows TiKa culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin.

Tetrahydroisoquinolines affect the whole-cell phenotype of Mycobacterium tuberculosis by inhibiting the ATP-dependent MurE ligase.

OBJECTIVES: (S)-Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñan, was found to inhibit the growth of Mycobacterium tuberculosis H37Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action. METHODS: Biomimetic Pictet-Spengler or Bischler-Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds. RESULTS: In this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H37Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis. CONCLUSIONS: As the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis.

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis.

The emergence of total drug-resistant tuberculosis (TDRTB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new anti-mycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 N-terminal residues, the other mutated residues are essential for catalysis. Variations in K(m) values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.

ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation.

New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0A resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.

Bacterial population structure of the jute-retting environment.

Jute is one of the most versatile bast fibers obtained through the process of retting, which is a result of decomposition of stalks by the indigenous microflora. However, bacterial communities associated with the retting of jute are not well characterized. To investigate the presence of microorganisms during the process of jute retting, full-cycle rRNA approach was followed, and two 16S rRNA gene libraries, from jute-retting locations of Krishnanagar and Barrackpore, were constructed. Phylotypes affiliating to seven bacterial divisions were identified in both libraries. The bulk of clones came from Proteobacteria ( approximately 37, 41%) and a comparatively smaller proportion of clones from the divisions-Firmicutes ( approximately 11, 12%), Cytophaga-Flexibacter-Bacteroidetes group (CFB; approximately 9, 7%), Verrucomicrobia ( approximately 6, 5%), Acidobacteria ( approximately 4, 5%), Chlorobiales ( approximately 5, 5%), and Actinobacteria ( approximately 4, 2%) were identified. Percent coverage value and diversity estimations of phylotype richness, Shannon-Weiner index, and evenness confirmed the diverse nature of both the libraries. Evaluation of the retting waters by whole cell rRNA-targeted flourescent in situ hybridization, as detected by domain- and group-specific probes, we observed a considerable dominance of the beta-Proteobacteria (25.9%) along with the CFB group (24.4%). In addition, 32 bacterial species were isolated on culture media from the two retting environments and identified by 16S rDNA analysis, confirming the presence of phyla, Proteobacteria ( approximately 47%), Firmicutes ( approximately 22%), CFB group ( approximately 19%), and Actinobacteria ( approximately 13%) in the retting niche. Thus, our study presents the first quantification of the dominant and diverse bacterial phylotypes in the retting ponds, which will further help in improving the retting efficiency, and hence the fiber quality.