Several reports have described that soil microbes are worthy to be used as the source of different antimicrobial substances including peptides for versatile applications . Most of the antimicrobial peptides produced by diverse bacteria inhibit Gram-positive strains like Listeria monocytogens but not Gram-negative . Among the various peptides, lipopeptides are well known to inhibit the growth of fungi and bacteria including opportunistic pathogens. Consequently, naturally produced antimicrobial lipopeptides have been receiving increased attention due to their anti-infective nature with wide antimicrobial spectrum. Besides the activity of natural peptides, any chemical modifications in structure of these lipopeptide are shown to improve their spectrum and activity. To this effect, daptomycin, an anionic lipopeptide has already been used for therapeutic applications . While antimicrobial lipopeptides are produced by different Gram-positive and Gram-negative bacteria, only lipopeptides produced by species of Pseudomonas and Bacillus have been studied in detail [13, 14, 27–29]. In the present study several antimicrobial substances producing bacterial strains were isolated from a fecal contaminated soil sample and characterization of these substances revealed them as antimicrobial lipopeptides.
The phenotypic features like Gram-negative staining, catalase positive, oxidase negative, facultative anaerobic growth and citrate utilization observed for all strains suggested that they belong to the Enterobacteriaceae family, usually observed in fecal matter. The 16S rRNA gene sequence blast analysis and subsequent phylogentic analysis assigned all strains to different species of the genera Citrobacter and Enterobacter. Interestingly, though strains S-5 and S-9 displayed high identity with E. hormaechei and E. mori respectively in 16S rRNA gene sequence, they only formed an out group to the cluster comprised of different Enterobacter and Citrobacter species (Figure 2). However, the overall topology of neighbour-joining tree revealed the phylogenetic complexity and discrepancies in 16S rRNA gene sequences of strains belonging to the family Enterobacteriaceae. It was also supported by the unusual inclusion of different species belonging to genera Citrobacter and Enterobacter in the same cluster suggesting the need to revisit the family Enterobacteriaceae.
The antimicrobial lipopeptides typically contain a cyclic or linear oligopeptide linked with a β-hydroxy fatty acid tail of varied lengths . Inhibition spectra of these lipopeptides are influenced by the composition of oligopeptide as well as fatty acid component [30, 31]. Antimicrobial lipopeptides are largely produced by Gram-positive bacteria like Bacillus sp. and are classified into different families based on the composition of oligopeptides and antibacterial or antifungal activities . Among the Gram-negative bacteria, Pseudomonas is the only genus reported to produce antimicrobial lipopeptides such as massetolide, viscosin , syringomycin , arthrofactin , pseudodesmins , orfamide  and putisolvin . In addition to these lipopeptides, species like P. fluorescens was reported to produce different massetolide analogues . However, lipopeptides produced by Gram-positive or Gram-negative bacteria had different composition and so far no bacterial strain has been reported to produce reciprocal combinations of lipopeptide composition. Therefore, in the present study we made an attempt to characterize lipopeptides produced by the strains of genera Citrobacter and Enterobacter.
The comprehensive mass spectral (MALDI-TOF MS and GC-MS) analysis of HPLC purified antimicrobial lipopeptides obtained from strains of Citrobacter and Enterobacter revealed the occurrence of different lipopeptide antibiotics belonging to groups like kurstakin, iturin, surfactin and fengycin, usually produced by Gram-positive bacteria. Further, individual lipopeptide belonging to a particular group shown to exhibit differences in their amino acids [13, 27], fatty acid chain length or isomers of fatty acids and thus generating various analogues with varied activity [13, 33]. Accordingly, lipopeptides of the present study showed differences in fatty acid composition and also differed in their antibacterial activity. Of the various lipopeptides, the lipopeptide fraction Fr-b produced by all strains had a molecular weight of 984/985 Da. Although amino acid composition of this peptide identified it as kurstakin, it differed in fatty acid composition (C15) when compared to other kurstakin members that contained fatty acids with chain length of C11-C14, suggesting the lipopeptide fraction (Fr-b) is an isoform of kurstakin. Further, differences in antimicrobial activity spectrum of these peptides attributed to the fatty acid composition differences .
A variety of lipopeptides produced by strains Citrobacter sp. strain S-3 and Enterobacter sp. strain S-11 were identified as lipopeptides belonging to iturin, kurstakin and fengycin with unusual broad spectrum antibacterial activity. It is pertinent to mention that the fraction Fr-e of strains S-3 and S-11, had an identical mass with the lipopeptide reported by Swart and Merwe , therefore, we have minimized further attempt to characterize the full sequence as reported [β-NC14NYNQPNS]. Additionally, identification of C14 fatty acid as the lipid content of the fraction Fr-e also confirmed their classification under iturins as they are known to contain a fatty acid chain length of C14 to C16 along with a cyclic peptide of seven amino acids. Cyclic lipopeptide biosurfactants like iturin, mycosubtilin, surfactin and kurstakin are largely produced by species of Bacillus exhibiting antimicrobial activity [12, 28]. In fact, iturin and fengycin produced by B. subtilis are recognized as potential biopharmaceutical agents due to their antimicrobial and biosurfactant properties . Although different types of lipopeptides varied in their amino acid and/or fatty acid composition, they all are usually thermostable, resistant to proteolytic enzymes and inhibits the growth by altering the membrane integrity. Similarly no reduction in antibacterial activity was observed for fraction Fr-e upon exposing it to 100°C temperature for 30 min suggesting it as heat stable. The amino acid composition analysis of highly active lipopeptide fraction (Fr-c) of strain S-3 and S-11 revealed the sequence as R(C17)EOrnYTEVPEYV which corresponds to linearized fengycin B’2, an isoform produced by a B. subtilis strain . Among the other lipopeptide fractions, Fr-f (m/z 607.21 Da) and Fr-d (m/z 637.23 Da), produced by strains S-3 and S-11, respectively, showed significant antimicrobial activity, but could not be assigned to any lipopeptide family as their molecular mass did not match with any reported antimicrobial lipopeptides. Other mass ions, except m/z 679 Da, produced by different strains did not show significant antimicrobial activity against any test strain. Although iturins, kurstakins, surfactins and fengycins differed in composition, they followed the same mechanisms such as involving pore formation on bacterial membrane  or by other non-specific interactions with the membrane  as a result of their antimicrobial activity. Findings of this study, together with the fact that the entire isolated strains belong to Citrobacter or Enterobacter and antimicrobial lipopeptide production ability, suggests that they are possibly produced by these bacteria as a part of defence mechanism to survive in complex environments.