Successful management of an MRSA outbreak in a neonatal intensive care unit.

We report an MRSA outbreak in our 25-bed tertiary neonatal intensive care unit (NICU), which was successfully contained. Methods include a retrospective review of patient files, microbiology records and meeting protocols. During the seven months of outbreak, 27 patients and seven health care workers (HCWs) had positive cultures for MRSA. The outbreak was caused by the epidemic Rhine-Hessen strain; cultured isolates were monoclonal. After a sharp increase of the number of new MRSA-cases the installation of an outbreak management team (OMT) and implementation of comprehensive measures (extensive screening and decolonization strategy including orally applied vancomycin, isolation wards, intensive disinfection regimen) successfully terminated the outbreak within one month. Ten (53%) of 19 patients with completed follow-up and all of the HCWs were decolonized successfully. Gastrointestinal colonization was present in 15 of 27 (56%) neonates, and was associated with poor decolonization success (30% vs. 78% in absence of gastrointestinal colonization). A comprehensive outbreak management can terminate an outbreak in a NICU setting within a short time. Thorough screening of nares, throat and especially stool is necessary for correct cohorting. Gastrointestinal decolonization in neonates seems difficult.

Association between antibiotic residues, antibiotic resistant bacteria and antibiotic resistance genes in anthropogenic wastewater - An evaluation of clinical influences.

The high use of antibiotics in human and veterinary medicine has led to a wide spread of antibiotics and antimicrobial resistance into the environment. In recent years, various studies have shown that antibiotic residues, resistant bacteria and resistance genes, occur in aquatic environments and that clinical wastewater seems to be a hot spot for the environmental spread of antibiotic resistance. Here a representative statistical analysis of various sampling points is presented, containing different proportions of clinically influenced wastewater. The statistical analysis contains the calculation of the odds ratios for any combination of antibiotics with resistant bacteria or resistance genes, respectively. The results were screened for an increased probability of detecting resistant bacteria, or resistance genes, with the simultaneous presence of antibiotic residues. Positive associated sets were then compared, with regards to the detected median concentration, at the investigated sampling points. All results show that the sampling points with the highest proportion of clinical wastewater always form a distinct cluster concerning resistance. The results shown in this study lead to the assumption that ciprofloxacin is a good indicator of the presence of multidrug resistant P. aeruginosa and extended spectrum ß-lactamase (ESBL)-producing Klebsiella spec., Enterobacter spec. and Citrobacter spec., as it positively relates with both parameters. Furthermore, a precise relationship between carbapenemase genes and meropenem, regarding the respective sampling sites, could be obtained. These results highlight the role of clinical wastewater for the dissemination and development of multidrug resistance.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and reliable identification of clinical yeast isolates.

The clinical impact of severe infections with yeasts and yeast-like fungi has increased, especially in immunocompromised hosts. In recent years, new antifungal agents with different and partially species-specific activity patterns have become available. Therefore, rapid and reliable species identification is essential for antifungal treatment; however, conventional biochemical methods are time-consuming and require considerable expertise. We evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid routine identification of clinical yeast isolates. A total of 18 type collection strains and 267 recent clinical isolates of Candida (n = 250), Cryptococcus, Saccharomyces, Trichosporon, Geotrichum, Pichia, and Blastoschizomyces spp. were identified by MALDI-TOF MS. The results were compared with those obtained by conventional phenotyping and biochemical tests, including the API ID 32C system (bioMérieux, Nürtingen, Germany). Starting with cells from single colonies, accurate species identification by MALDI-TOF MS was achieved for 247 of the clinical isolates (92.5%). The remaining 20 isolates required complementation of the reference database with spectra for the appropriate reference strains which were obtained from type culture collections or identified by 26S rRNA gene sequencing. The absence of a suitable reference strain from the MALDI-TOF MS database was clearly indicated by log(score) values too low for the respective clinical isolates; i.e., no false-positive identifications occurred. After complementation of the database, all isolates were unambiguously identified. The established API ID 32C biochemical diagnostic system identified 244 isolates in the first round. Overall, MALDI-TOF MS proved a most rapid and reliable tool for the identification of yeasts and yeast-like fungi, with the method providing a combination of the lowest expenditure of consumables, easy interpretation of results, and a fast turnaround time.

Antibiotic resistant bacteria and resistance genes in biofilms in clinical wastewater networks.

Increasing isolation rates of resistant bacteria in the last years require identification of potential infection reservoirs in healthcare facilities. Especially the clinical wastewater network represents a potential source of antibiotic resistant bacteria. In this work, the siphons of the sanitary installations from 18 hospital rooms of two German hospitals were examined for antibiotic resistant bacteria and antibiotic residues including siphons of showers and washbasins and toilets in sanitary units of psychosomatic, haemato-oncological, and rehabilitation wards. In addition, in seven rooms of the haemato-oncological ward, the effect of 24 h of stagnation on the antibiotic concentrations and MDR (multi-drug-resistant) bacteria in biofilms was evaluated. Whereas no antibiotic residues were found in the psychosomatic ward, potential selective concentrations of piperacillin, meropenem and ciprofloxacin were detected at a rehabilitation ward and ciprofloxacin and trimethoprim were present at a haemato-oncology ward. Antibiotic resistant bacteria were isolated from the siphons of all wards, however in the psychosomatic ward, only one MDR strain with resistance to piperacillin, third generation cephalosporins and quinolones (3MRGN) was detected. In contrast, the other two wards yielded 11 carbapenemase producing MDR isolates and 15 3MRGN strains. The isolates from the haemato-oncological ward belonged mostly to two specific rare sequence types (ST) (P. aeruginosa ST823 and Enterobacter cloacae complex ST167). In conclusion, clinical wastewater systems represent a reservoir for multi-drug-resistant bacteria. Consequently, preventive and intervention measures should not start at the wastewater treatment in the treatment plant, but already in the immediate surroundings of the patient, in order to minimize the infection potential.

Rapid monitoring of vancomycin-resistant Enterococcus faecium in hospital departments by repetitive element palindromic polymerase chain reaction.

BACKGROUND: The current increase in nosocomial infections caused by vancomycin-resistant enterococci (VRE) warrants improvement of detection methods and hygiene measures. Knowledge of the local epidemiology is important for monitoring compliance of medical personnel with hygiene measures. AIM: To evaluate semi-automated repetitive element palindromic polymerase chain reaction (rep-PCR) for rapid molecular typing of VRE. METHODS: Primary VRE isolates were collected during an observation period of one year and retrospectively typed by rep-PCR. Molecular typing was performed on isolates from two departments with elevated VRE rates and patients with increased risk for systemic VRE infections. Typing results were correlated with temporal and spatial information on patient moves, VRE laboratory results and multi-locus sequence typing (MLST). FINDINGS: Approximately 70% of VRE isolates within a department could be assigned to similarity clusters. Spread of VRE was limited to the individual departments. There was no evidence for spread of endemic VRE strains within the geographical catchment area of the hospital. Our results demonstrate the utility of rep-PCR typing on a department level. However, a Diversilab® threshold of ≥98% had to be applied to claim similarity, and suspected transmissions needed to be confirmed by vanA/B genotyping and compiled information on spatial and temporal patient contact. MLST verified the findings. CONCLUSION: Spread of predominantly detected vancomycin-resistant Enterococcus faecium was limited to the department level with no evidence for wider dissemination within the hospital. Well-standardized and validated (semi-)automated rep-PCR systems are useful for rapid detection of possible VRE transmission. However, suspected transmissions need to be confirmed by clinical and microbiological parameters.

Influence of cationic peptides on the activity of the autolytic endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22.

The peptidoglycan hydrolyzing endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22 is not able to attack intact cell walls of S. simulans 22, but hydrolyzes cell walls of Micrococcus luteus and soluble peptidoglycan chains of S. simulans 22. Hydrolysis of cell walls of M. luteus is activated in presence of organic cations such as poly-L-lysine (n = 17) and the peptide antibiotics Pep 5 and nisin, whereas hydrolysis of soluble peptidoglycan chains is not influenced. High concentrations of inorganic cations inhibit enzyme activity. These effects are discussed with respect to the cationic nature of the enzyme (pI greater than 9.5) and the regulation of the concerted action of the N-acetylmuramoyl-L-alanine amidase and the glucosaminidase during S. simulans 22 autolysis in vivo.

Cloning, sequencing and production of the lantibiotic mersacidin.

Mersacidin is a lanthionine-containing peptide antibiotic that shows a good in vivo efficiency against methicillin-resistant Staphylococcus aureus. It is excreted during early stationary phase and could be purified from culture supernatant in a one-step procedure by reversed phase HPLC. Its structural gene was cloned from chromosomal DNA of the producer strain Bacillus subtilis HIL Y-85,54728. Sequencing revealed that pre-mersacidin consists of an unusually long 48 amino acid leader sequence and a 20 amino acid propeptide part which is modified during biosynthesis to the mature lantibiotic. The comparison of the mersacidin prepeptide with those of hitherto known lantibiotics demonstrates that mersacidin is more closely related to type B lantibiotic cinnamycin than to type A lantibiotics.

Identification and analysis of a gene encoding a Fur-like protein of Staphylococcus epidermidis.

A gene (fur) for a Fur-like protein was identified on a 1.1 kb chromosomal DNA fragment of Staphylococcus epidermidis BN 280; the fur gene is followed by an open reading frame coding for the N-terminus of a putative superoxide dismutase. Within the -35 promoter region of both genes, as sequence motif was detected with low similarity to Fur-binding regulatory DNA segments, the so-called Fur boxes. Fur titration in Escherichia coli strain H1717 demonstrated that the E. coli Fur protein binds to the Fur box of the promoter region of the S. epidermidis fur gene. The S. epidermidis Fur protein was expressed in E. coli as indicated by the formation of inactive dimers with the chimeric repressor CI(N)-Fur(C) (Stojiljkovic, I. and Hantke. K. (1995) Mol. Gen. Genet. 247, 199-205), but was not able to complement the Fur mutation in E. coli H1681.

Effect of leader peptide mutations on biosynthesis of the lantibiotic Pep5.

Lantibiotics are lanthionine-containing antibiotic peptides which are synthesized from ribosomal prepeptides by post-translational modification. In order to elucidate the function of a conserved motif in the N-terminal leader sequence of lantibiotic prepeptides, three amino acids were exchanged in the leader peptide sequence of the lantibiotic Pep5. Exchanging Phe-19 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging Asp-6 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging ASp-6 for Lys, the production was decreased only to 82%. Proteolytic fragments of Pep5 or incorrectly modified Pep5 molecules, indicative of incorrect modifications, were not found in the culture supernatant. Thus, in contrast to the biosynthesis of the lantibiotic nisin, the FDLEI-motif is not essential for biosynthesis of Pep5 and has no influence on correct ring formation or processing, but seems to be important for optimal biosynthesis rates.

Human-associated Staphylococcus aureus strains within great ape populations in Central Africa (Gabon).

The risk of serious infections caused by Staphylococcus aureus is well-known. However, most studies regarding the distribution of (clinically relevant) S. aureus among humans and animals took place in the western hemisphere and only limited data are available from (Central) Africa. In this context, recent studies focused on S. aureus strains in humans and primates, but the question of whether humans and monkeys share related S. aureus strains or may interchange strains remained largely unsolved. In this study we aimed to evaluate the distribution and spread of human-like S. aureus strains among great apes living in captivity. Therefore, a primate facility at the International Centre for Medical Research of Franceville (Gabon) was screened. We detected among the primates a common human S. aureus strain, belonging to the spa-type t148. It was isolated from three different individuals of the western lowland gorilla (Gorilla gorilla gorilla), of which one individual showed a large necrotizing wound. This animal died, most probably of a staphylococcal sepsis. Additionally, we discovered the t148 type among chimpanzees (Pan troglodytes) that were settled in the immediate neighbourhood of the infected gorillas. A detailed analysis by pulsed field gel electrophoresis showed that the gorilla and chimpanzee isolates represented two closely related strains. To our knowledge, this is the first report of a human-associated S. aureus strain causing disease in great apes. The simultaneous detection in gorillas and chimpanzees indicated an interspecies transmission of this S. aureus strain. Our results recommend that protection of wild animals must not only be based on habitat conservation, but also on the assessment of the risk of contact with human pathogens.

Lantibiotics: mode of action, biosynthesis and bioengineering.

Lantibiotics are gene-encoded peptides that contain intramolecular ring structures, introduced through the thioether containing lanthionine and methyllanthionine residues. The overwhelming majority of the lantibiotics shows antibacterial activity. Some lantibiotics, e.g. nisin, are characterized by a dual mode of action. These peptides form a complex with the ultimate cell wall precursor lipid II, thereby inhibiting cell wall biosynthesis. The complexes then aggregate, incorporate further peptides and form a pore in the bacterial membrane. Recent results show that complexing of lipid II is widespread among lantibiotics; however, pore formation depends on the overall length of the peptide and the lipid composition of the test strain membrane. In the two-component system of lacticin 3147, the two functions are performed by the two different peptides. The genetic information for production of lantibiotics is organized in gene clusters which contain a structural gene (lanA) for the lantibiotic prepeptide. The modifications are introduced by one biosynthetic enzyme (LanM) or a combination of a dehydratase (LanB) and a cyclase (LanC). These enzymes have been in the focus of recent bioengineering studies: The structure of NisC has been resolved, the reaction mechanism of LctM was elucidated and the active site residues were characterized by mutagenesis studies. In vitro modification systems have successfully been used to introduce thioether rings into other biologically active peptides. Furthermore, variant lantibiotics with enhanced properties have been engineered and at least one promising new lantibiotic with strong activity against multiresistant pathogens has been described.

Autolytic system of Staphylococcus simulans 22: influence of cationic peptides on activity of N-acetylmuramoyl-L-alanine amidase.

Pep 5 and nisin are cationic peptide antibiotics which in addition to their membrane-disruptive action induce autolysis in staphylococci. To investigate the mechanism of lysis induction, the influence of the peptides on the activity of the N-acetylmuramoyl-L-alanine amidase of Staphylococcus simulans 22 was studied. In experiments with isolated cell walls at low ionic strength, the amidase activity was stimulated by the addition of Pep 5 and nisin, as well as by polylysine, streptomycin, and mono- and divalent cations. The concentrations necessary for activation depended on the nature of the cation and ranged from 5 microM for poly-L-lysine (n = 17) to 150 mM for Na+ at a cell wall concentration of 100 micrograms of cell walls per ml. No effect was observed if the cell walls were devoid of polyanionic constituents. Kinetic data suggested that the amidase bound to the teichoic and teichuronic acids of the cell wall and was thereby inhibited. Cationic molecules reversed this inhibition, most likely by displacing the enzyme from the polyanions. If the concentrations of the larger peptides were high in relation to cell wall concentration, the activation turned into inhibition, presumably by interfering with the access of the enzyme to its substrate. These experiments demonstrate that the activity of the amidase is modulated by basic peptides in vitro and help to explain how Pep 5 and nisin may cause lysis of treated cells.

Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria.

A plethora of novel gene-encoded antimicrobial peptides from animals, plants and bacteria has been described during the last decade. Many of the bacterial peptides possess modified building blocks such as thioethers and thiazoles or unsaturated and stereoinverted amino acids, which are unique among ribosomally made peptides. Genetic and biochemical studies of many of these peptides, mostly the so-called lantibiotics, have revealed the degree to which cells are capable of transforming peptides by posttranslational modification. The biosynthesis follows a general scheme: Precursor peptides are first modified and then proteolytically activated; the latter may occur prior to, concomitantly with or after export from the cell. The genes for the biosynthetic machinery are organized in clusters and include information for the antibiotic prepeptide, the modification enzymes and accessory functions such as dedicated proteases and ABC transporters as well as immunity factors and regulatory proteins. These fundamental aspects are discussed along with the biotechnological potential of the peptides and of the biosynthesis enzymes, which could be used for construction of novel, peptide-based biomedical effector molecules.

Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes.

Pep 5 and nisin are cationic bactericidal peptides which were shown to induce autolysis in Staphylococcus cohnii 22. In contrast to nisin, Pep 5 induced lysis could be stimulated in the presence of glucose. Addition of lipoteichoic acids (LTA) (D-alanine:phosphorus = 0.475:1) inhibited all effects of Pep 5 on susceptible cells in a molar ratio LTA:Pep 5 of 10:1. Treatment of S. cohnii 22 with Pep 5 or nisin for 20 min and subsequent washing with 2.5 M NaCl released autolysin activity. Crude preparations of the hydrolyzing enzymes produced free amino groups as well as polysaccharide fragments from the murein backbone, suggesting the presence of a muramidase or glucosamidase, and endopeptidase or amidase. Both enzyme activities were inhibited by lipoteichoic acid; they could be fully reactivated by addition of Pep 5 in sufficient concentrations. The velocity of hydrolysis was not influenced by nisin, whereas it was doubled in presence of Pep 5. The results are discussed in view of a possible mechanism of induction of lysis by Pep 5 and nisin.

Lantibiotics--unusually modified bacteriocin-like peptides from gram-positive bacteria.

Lantibiotics are antibacterial peptides frequently produced by Gram-positive bacteria. They are distinguished by unique structural properties unprecedented so far in peptide chemistry. The most striking feature is the occurrence of intramolecular rings introduced by the thioether amino acids lanthionine and 3-methyllantionine. Additional usual amino acids such as didehydroalanine and didehydrobutyrine are found. Lantibiotics are produced from ribosomally synthesized prepeptides and the unusual amino acids are formed by post-translational modifications. This review summarizes the current knowledge on the biosynthetic mechanisms and enzymes taking part in biosynthesis, on the primary and spatial structures of the active peptides and the correlation between structural aspects and the antibacterial activity. Furthermore, the mode of action of type-A lantibiotics and the immunity phenomenon are described, and an outlook for future research and potential applications is given.

Molecular analysis of expression of the lantibiotic pep5 immunity phenotype.

The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Within its biosynthetic gene cluster, the immunity gene pepI, providing producer self-protection, is localized upstream of the structural gene pepA. Pep5 production and the immunity phenotype have been found to be tightly coupled (M. Reis, M. Eschbach-Bludau, M. I. Iglesias-Wind, T. Kupke, and H.-G. Sahl, Appl. Environ. Microbiol. 60:2876-2883, 1994). To study this phenomenon, we analyzed pepA and pepI transcription and translation and constructed a number of strains containing various fragments of the gene cluster and expressing different levels of immunity. Complementation of a pepA-expressing strain with pepI in trans did not result in phenotypic immunity or production of PepI. On the other hand, neither pepA nor its product was found to be involved in immunity, since suppression of the translation of the pepA mRNA by mutation of the ATG start codon did not reduce the level of immunity. Moreover, homologous and heterologous expression of pepI from a xylose-inducible promoter resulted in significant Pep5 insensitivity. Most important for expression of the immunity phenotype was the stability of pepI transcripts, which in the wild-type strain, is achieved by an inverted repeat with a free energy of -56.9 kJ/mol, localized downstream of pepA. We performed site-directed mutagenesis to study the functional role of PepI and constructed F13D PepI, I17R PepI, and PepI 1-65; all mutants showed reduced levels of immunity. Western blot analysis indicated that F13D PepI and PepI 1-65 were not produced correctly or were partially degraded, while I17R PepI apparently was less efficient in providing self-protection than the wild-type PepI.

Biosynthesis of the lantibiotic mersacidin: organization of a type B lantibiotic gene cluster.

The biosynthetic gene cluster (12.3 kb) of mersacidin, a lanthionine-containing antimicrobial peptide, is located on the chromosome of the producer, Bacillus sp. strain HIL Y-85,54728 in a region that corresponds to 348 degrees on the chromosome of Bacillus subtilis 168. It consists of 10 open reading frames and contains, in addition to the previously described mersacidin structural gene mrsA (G. Bierbaum, H. Brötz, K.-P. Koller, and H.-G. Sahl, FEMS Microbiol. Lett. 127:121-126, 1995), two genes, mrsM and mrsD, coding for enzymes involved in posttranslational modification of the prepeptide; one gene, mrsT, coding for a transporter with an associated protease domain; and three genes, mrsF, mrsG, and mrsE, encoding a group B ABC transporter that could be involved in producer self-protection. Additionally, three regulatory genes are part of the gene cluster, i.e., mrsR2 and mrsK2, which encode a two-component regulatory system which seems to be necessary for the transcription of the mrsFGE operon, and mrsR1, which encodes a protein with similarity to response regulators. Transcription of mrsA sets in at early stationary phase (between 8 and 16 h of culture).

Construction of an expression system for engineering of the lantibiotic Pep5.

Pep5 is a lanthionine-containing antimicrobial peptide which is produced by Staphylococcus epidermidis 5. Its structural gene, pepA, is located on the 20-kb plasmid pED503. A 6.2-kb fragment of pED503 containing pepA, the immunity gene pepI, and 5.4 kb of downstream sequence was able to direct biosynthesis of biologically active Pep5 in a nonproducing variant of the producer strain which is devoid of pED503. In addition to producing wild-type Pep5 with a molecular mass of 3,488 Da, the clone produced a peptide with an eightfold-lower bactericidal activity and a mass of 3,506 Da, indicative of incomplete dehydration of one hydroxyamino acid. For construction of the expression system, this 6.2-kb fragment was cut into a 1.39-kb fragment containing pepA and pepI and a 4.8-kb fragment covering the remaining downstream region. This 4.8-kb fragment was directly cloned into an Escherichia coli-Staphylococcus shuttle vector, yielding a new plasmid (pGB9) into which mutated pepA genes generated on the 1.39-kb fragment can be reinserted to yield a functional Pep5 biosynthesis gene cluster. To test the expression system, two mutants were constructed. Lys-18-Pro Pep5 was produced in its dehydrated form and a partially hydrated form in amounts comparable to those of the wild-type peptide. In contrast, only small amounts of Phe-23-Asp Pep5 were excreted, indicating that some residues in the propeptide part of the prelantibiotic may be crucial for certain steps in the biosynthetic pathway of lantibiotics.

Biosynthesis and biological activities of lantibiotics with unique post-translational modifications.

Lantibiotics are biologically active peptides which contain the thioether amino acid lanthionine as well as several other modified amino acids. They can be broadly divided into two groups on the basis of their structures: type-A lantibiotics are elongated, amphiphilic peptides, while type-B lantibiotics are compact and globular. In the last decade there has been a marked increase in research interest in these peptides due both to the novel biosynthetic mechanisms by which they are produced, as well as to their potential applications. Lantibiotics are synthesised on the ribosome as a prepeptide which undergoes several post-translational modification events, including dehydration of specific hydroxyl amino acids to form dehydroamino acids, addition of neighbouring sulfhydryl groups to form thioethers and, in specific cases, other modifications such as introduction of D-alanine residues from L-serine, formation of lysinoalanine bridges, formation of novel N-terminal blocking groups and oxidative decarboxylation of a C-terminal cysteine. The genetic elements responsible for these specific modification reactions encode unique enzymes with hitherto unknown reaction mechanisms. Production of these peptides also requires accessory proteins including processing proteases, translocators of the ATP-binding cassette transporter family, regulatory proteins and dedicated producer self-protection mechanisms. While the principle biological activity of most type-B lantibiotics appears to be directed at the inhibition of enzyme functions, the type-A lantibiotics kill bacterial cells by forming pores in the cytoplasmic membrane.

The genetics of lantibiotic biosynthesis.

The lantibiotics are a rapidly expanding group of biologically active peptides produced by a variety of Gram-positive bacteria, and are so-called because of their content of the thioether amino acids lanthionine and beta-methyllanthionine. These amino acids, and indeed a number of other unusual amino acids found in the lantibiotics, arise following post-translational modification of a ribosomally synthesised precursor peptide. A number of genes involved in the biosynthesis of these highly modified peptides have been identified, including genes encoding the precursor peptide, enzymes responsible for specific amino acid modifications, proteases able to remove the leader peptide, ABC-superfamily transport proteins involved in lantibiotic translocation, regulatory proteins controlling lantibiotic biosynthesis and proteins that protect the producing strain from the action of its own lantibiotic. Analysis of these genes and their products is allowing greater understanding of the complex mechanism(s) of the biosynthesis of these unique peptides.