Biological properties of experimental dental alginate modified for self-disinfection using green nanotechnology.

OBJECTIVES: Disinfection of alginate impression materials is a mandatory step to prevent cross-infection in dental clinics. However, alginate disinfection methods are time-consuming and exert a negative impact on accuracy and mechanical properties. Thus, this study aimed to prepare disinfecting agents (CHX and AgNO3) and silver nanoparticles reduced by a natural plant extract to produce a self-disinfecting dental alginate. METHODS: Conventional alginate impression material was used in this study. Silver nitrate (0.2% AgNO3 group) and chlorohexidine (0.2% CHX group) solutions were prepared using distilled water, and these solutions were later employed for alginate preparation. Moreover, a 90% aqueous plant extract was prepared from Boswellia sacra (BS) oleoresin and used to reduce silver nitrate to form silver nanoparticles that were incorporated in the dental alginate preparation (BS+AgNPs group). The plant extract was characterized by gas chromatography/mass spectrometry (GC/MS) analysis while green-synthesized silver nanoparticles (AgNPs) were characterized by UV-visible (UV-vis) spectroscopy and scanning electron microscopy (SEM). An agar disc diffusion assay was used to test the antimicrobial activity against Candida albicans, Streptococcus mutans, Escherichia coli, methicillin-resistant and susceptible Staphylococcus aureus strains, and Micrococcus luteus. Agar plates were incubated at 37 ± 1 °C for 24 h to allow microbial growth. Diameters of the circular inhibition zones formed around each specimen were measured digitally by using ImageJ software. RESULTS: Chemical analysis of the plant extract revealed the presence of 41 volatile and semi-volatile active compounds. UV-Vis spectrophotometry, SEM, and EDX confirmed the formation of spherical silver nanoparticles using the BS extract. CHX, AgNO3, and the BS+AgNPs modified groups showed significantly larger inhibition zones than the control group against all tested strains. BS+AgNPs and CHX groups showed comparable efficacy against all tested strains except for Staphylococcus aureus, where the CHX-modified alginate had a significantly higher effect. CONCLUSIONS AND CLINICAL RELEVANCE: CHX, silver nitrate, and biosynthesized silver nanoparticles could be promising inexpensive potential candidates for the preparation of a self-disinfecting alginate impression material without affecting its performance. Green synthesis of metal nanoparticles using Boswellia sacra extract could be a very safe, efficient, and nontoxic way with the additional advantage of a synergistic action between metal ions and the phytotherapeutic agents of the plant extract.

Antibiotic concentrations in raw hospital wastewater surpass minimal selective and minimum inhibitory concentrations of resistant Acinetobacter baylyi strains.

Antibiotics are essential for modern medicine, they are employed frequently in hospitals and, therefore, present in hospital wastewater. Even in concentrations, that are lower than the minimum inhibitory concentrations (MICs) of susceptible bacteria, antibiotics may exert an influence and select resistant bacteria, if they exceed the MSCs (minimal selective concentrations) of resistant strains. Here, we compare the MSCs of fluorescently labelled Acinetobacter baylyi strains harboring spontaneous resistance mutations or a resistance plasmid with antibiotic concentrations determined in hospital wastewater. Low MSCs in the µg/L range were measured for the quinolone ciprofloxacin (17 µg/L) and for the carbapenem meropenem (30 µg/L). A 24 h continuous analysis of hospital wastewater showed daily fluctuations of the concentrations of these antibiotics with distinctive peaks at 7-8 p.m. and 5-6 a.m. The meropenem concentrations were always above the MSC and MIC values of A. baylyi. In addition, the ciprofloxacin concentrations were in the range of the lowest MSC for about half the time. These results explain the abundance of strains with meropenem and ciprofloxacin resistance in hospital wastewater and drains.

Detection of methicillin-resistant coagulase-negative staphylococci harboring the class A mec complex by MALDI-TOF mass spectrometry.

The aim of this study was to test the identification of methicillin resistance in coagulase-negative staphylococci by routine matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). SCCmec cassettes of type II, III and VIII encode a small peptide called PSM-mec in the vicinity of mecA. It is visible at m/z 2415 during MALDI-TOF MS of whole cells of Staphylococcus aureus. In view of the fact that psm-mec has been identified in methicillin-resistant coagulase-negative staphylococci, we evaluated a collection of clinical coagulase-negative staphylococci, that contained 77.03% of methicillin-resistant isolates, for the presence of the structural gene encoding PSM-mec and the appearance of the corresponding signal during mass spectroscopy. In MALDI-TOF MS spectra, 89.65% of the strains that harbored the gene yielded the correct signal, corresponding to a sensitivity of 0.897 and a specificity of 1.0. However, regarding detection of methicillin resistance, i. e. considering all resistant strains as positive regardless of the presence of the gene, the overall sensitivity of the test decreased to 0.285, due to the fact that only 29.43% of all resistant isolates contained psm-mec. In conclusion, the presence of the signal in MALDI-TOF MS quickly indicates methicillin-resistance in coagulase-negative staphylococci but its absence does not indicate susceptibility to methicillin.

Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus.

Staphylococcus aureus has acquired resistance to nearly all antibiotics used in clinical practice. Whereas some resistance mechanisms are conferred by uptake of resistance genes, others evolve by mutation. In this study, IS256 has been shown to play a role, e.g., in S. aureus strains displaying intermediate resistance to vancomycin (VISA). To characterize the IS256 insertion sites in the genomes of two closely related sequence type 247 (ST247) VISA strains, all insertions were mapped in both VISA and a susceptible control strain. The results showed that the three ST247 strains contained the highest number so far of IS256 insertions for all sequenced S. aureus strains. Furthermore, in contrast to the case with the other IS elements in these genomes, the IS256 insertion sites were not identical in the closely related strains, indicating a high transposition frequency of IS256 When IS256 was introduced into a laboratory strain which was then cultured in the presence of antibiotics, it was possible to isolate small-colony variants (SCVs) that possessed IS256 insertions in guaA and hemY that displayed increased resistance to vancomycin and aminoglycosides, respectively. For these clones, a very rapid reversion to the wild type that resembled the fast reversion of clinical SCVs was observed. The reversion was caused by excision of IS256 in a small number of fast-growing clones that quickly outcompeted the SCVs in broth cultures. In conclusion, the presence of IS256 confers a strong genomic plasticity that is useful for adaptation to antibiotic stress.

Differentiation of Staphylococcus argenteus (formerly: Staphylococcus aureus clonal complex 75) by mass spectrometry from S. aureus using the first strain isolated from a wild African great ape.

The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132T. Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice.

Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides.

Lantibiotics are ribosomally synthesized antimicrobial peptides with substantial posttranslational modifications. They are characterized by the unique amino acids lanthionine and methyllanthionine, which are introduced by dehydration of Ser/Thr residues and linkage of the resulting dehydrated amino acids with Cys residues. BLAST searches using the mersacidin biosynthetic enzyme (MrsM) in the NCBI database revealed a new class II lantibiotic gene cluster in Bacillus pseudomycoides DSM 12442. Production of an antimicrobial substance with activity against Gram-positive bacteria was detectable in a cell wash extract of this strain. The substance was partially purified, and mass spectrometric analysis predicted a peptide of 2,786 Da in the active fraction. In order to characterize the putative lantibiotic further, heterologous expression of the predicted biosynthetic genes was performed in Escherichia coli. Coexpression of the prepeptide (PseA) along with the corresponding modification enzyme (PseM) resulted in the production of a modified peptide with the corresponding mass, carrying four out of eight possible dehydrations and supporting the presence of four thioether and one disulfide bridge. After the proteolytic removal of the leader, the core peptide exhibited antimicrobial activity. In conclusion, pseudomycoicidin is a novel lantibiotic with antimicrobial activity that was heterologously produced in E. coli.

Identification of agr-positive methicillin-resistant Staphylococcus aureus harbouring the class A mec complex by MALDI-TOF mass spectrometry.

A small peptide called PSM-mec is encoded on the type II, III and VIII SCCmec cassettes present in the genomes of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains. This peptide is excreted by agr-positive strains, which represent about 89% of the strains of our collection and can be identified by the presence of delta toxin in mass spectrometry. The presence of the peptide in the MALDI-TOF MS spectra of whole cells was proved by a knock-down experiment employing a clone that expressed antisense RNA to psm-mec. Furthermore, evaluation of a collection of clinical agr-positive MRSA and MSSA isolates and type strains showed that, using a detection window of m/z 2411-2419, the PSM-mec is detected by mass spectrometry of whole cells with a sensitivity of 0.95 and a specificity of 1, thereby enabling rapid identification of a subgroup of MRSA with a method that is used during routine identification procedures.

Antibiotic-induced autoactivation of IS256 in Staphylococcus aureus.

The 3' end of rsbU, encoding the positive regulator of the stress factor sigma B, was identified as a hot spot for spontaneous IS256 insertion in Staphylococcus aureus SA137/93G. Interestingly, subinhibitory concentrations of chloramphenicol in combination with heat stress, as well as linezolid and spectinomycin at physiological temperatures, selected for such rsbU::IS256 insertion mutants. In consequence of the inactivation of rsbU, the IS256 transposition frequency was increased 4-fold in S. aureus HG001.

Analysis of the matrix-assisted laser desorption ionization-time of flight mass spectrum of Staphylococcus aureus identifies mutations that allow differentiation of the main clonal lineages.

Nosocomial infections involving epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains are a serious problem in many countries. In order to analyze outbreaks, the infectious isolates have to be typed; however, most molecular methods are expensive or labor-intensive. Here, we evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of cell extracts for the molecular characterization of S. aureus strains. The peak patterns of 401 MRSA and methicillin-susceptible S. aureus (MSSA) strains, including clinical and laboratory strains, were analyzed. Database searches indicated the peptides that were represented by the corresponding peaks in the spectra. The identities of the peptides were confirmed by the sequencing of mutants, the expression of antisense RNA fragments that resulted in the knockdown of the peptide of interest and the concomitant loss of the signal, or tandem MALDI-TOF MS (MALDI-TOF/TOF MS). It was shown that the signals derive mainly from stress proteins and ribosomal proteins. Peak shifts that differentiate the main S. aureus clonal complexes CC5, CC22, CC8, CC45, CC30, and CC1 correlate to point mutations in the respective genes. Retrospective typing of an MRSA outbreak showed that it is possible to differentiate unrelated MSSA, MRSA, and borderline resistant S. aureus (BORSA) strains isolated from health care workers. In conclusion, this method allows for the detection of the epidemic lineages of S. aureus during species identification by MALDI-TOF MS analysis.

Production of capsular polysaccharide does not influence Staphylococcus aureus vancomycin susceptibility.

BACKGROUND: Diverse mechanisms (increased cell wall thickness, low cross linking, decreased autolysis, etc.) have been reported for Staphylococcus aureus strains with intermediate vancomycin susceptibility (VISA). This study was conducted to identify common mechanisms responsible for decreased vancomycin susceptibility in a VISA strain pair. RESULTS: Transcriptional profiling of the clinical heterogeneous VISA isolate SA137/93A and its spontaneous homogeneous mutant strain SA137/93G pointed to an increased capsule production in the strain pair compared to a susceptible control. Furthermore, transcript quantification of the gene cap5E, which is essential for capsule biosynthesis, revealed elevated levels in the VISA strains SA137/93A, SA137/93G and Mu50 in comparison with susceptible strains Reynolds, Newman and SA1450/94. The increased expression was observed in bacteria from exponential as well as stationary growth phase. However, suppression of type 5 capsule formation by expression of antisense RNA did not increase vancomycin susceptibility in the VISA strain SA137/93G. Likewise, construction of inducible mutants of S. aureus Newman or repair of capsule biosynthesis of S. aureus HG001 and S. aureus 1450/94 did not influence resistance to vancomycin. Furthermore, purified type 5 polysaccharide did not protect indicator strains from the action of vancomycin. CONCLUSIONS: The VISA strain tested in this study displayed an increased production of type 5 capsular polysaccharide. However, the production of capsule material did not protect strain SA137/93G and three vancomycin sensitive strains in the presence of vancomycin and thus is not part of the resistance mechanism; however it may represent a by-product of VISA life style that is often characterized by a high sigma factor B activity.

A Staphylococcus capitis strain with unusual bacteriocin production.

Staphylococcus capitis is a member of the human and mammal skin microbiomes and is considered less harmful than Staphylococcus aureus. S. capitis subsp. urealyticus BN2 was isolated from a cat and expressed strong antibacterial activity against a range of Gram-positive species, most notably including S. aureus strains with resistance to methicillin (MRSA) and strains with intermediate resistance to vancomycin (VISA). These latter strains are normally relatively resistant to bacteriocins, due to cell wall and cell membrane modifications. Genomic sequencing showed that the strain harboured at least two complete gene clusters for biosynthesis of antagonistic substances. The complete biosynthetic gene cluster of the well-known lantibiotic gallidermin was encoded on a large plasmid and the mature peptide was present in isopropanol cell extracts. In addition, a chromosomal island contained a novel non-ribosomal peptide synthetase (NRPS) gene cluster. Accidental deletion of two NRPS modules and partial purification of the anti-VISA activity showed that this novel bacteriocin represents a complex of differently decorated, non-ribosomal peptides. Additionally, a number of phenol-soluble modulins (PSMs) was detected by mass spectrometry of whole cells. Producing these compounds, the strain was able to outcompete several S. aureus strains, including MRSA and VISA, in tube cultures.

Genome sequence of Staphylococcus aureus VC40, a vancomycin- and daptomycin-resistant strain, to study the genetics of development of resistance to currently applied last-resort antibiotics.

The increasing emergence of multidrug-resistant Staphylococcus aureus is a problem of global importance. Here, we report the genome of S. aureus VC40, which is resistant to the last-resort antibiotics vancomycin and daptomycin. Its genome sequence will allow insights into the mechanisms that convey full resistance to these compounds.

Dissemination of carbapenem resistant bacteria from hospital wastewater into the environment.

Infections with antibiotic resistant pathogens threaten lives and cause substantial costs. For effective interventions, knowledge of the transmission paths of resistant bacteria to humans is essential. In this study, carbapenem resistant bacteria were isolated from the wastewater of a maximum care hospital during a period of two years, starting in the patient rooms and following the sewer system to the effluent of the wastewater treatment plant (WWTP). The bacteria belonged to six different species and 44 different sequence types (STs). The most frequent STs, ST147 K. pneumoniae (blaNDM/blaOXA-48) and ST235 P. aeruginosa (blaVIM) strains, were present at nearly all sampling sites from the hospital to the WWTP effluent. After core genome multi-locus sequence typing (cgMLST), all ST147 K. pneumoniae strains presented a single epidemiological cluster. In contrast, ST235 P. aeruginosa formed five cgMLST clusters and the largest cluster contained the strain from the WWTP effluent, indicating without doubt, a direct dissemination of both high-risk clones into the environment. Thus, there are - at least two - possible transmission pathways to humans, (i) within the hospital by contact with the drains of the sanitary installations and (ii) by recreational or irrigation use of surface waters that have received WWTP effluent. In conclusion, remediation measures must be installed at both ends of the wastewater system, targeting the drains of the hospital as well as at the effluent of the WWTP.

The RNA Polymerase Inhibitor Corallopyronin A Has a Lower Frequency of Resistance Than Rifampicin in Staphylococcus aureus.

Corallopyronin A (CorA) is active against Gram-positive bacteria and targets the switch region of RNA polymerase. Because of the high frequency of mutation (FoM) leading to rifampicin resistance, we determined the CorA FoM in S. aureus using fluctuation analysis at 4 × minimum inhibitory concentration (MIC). Resistant mutants were characterized. S. aureus strains HG001, Mu50, N315, and USA300 had an MIC of 0.25 mg/L. The median FoM for CorA resistance was 1.5 × 10−8, 4.5-fold lower than the median FoM of 6.7 × 10−8 for rifampicin, and was reflected in a 4-fold lower mutation rate for CorA than rifampicin (6 × 10−9 for CorA vs. 2.5 × 10−8 for rifampicin). In CorA-resistant/rifampicin-sensitive strains, the majority of amino acid exchanges were S1127L in RpoB or K334N in RpoC. S. aureus Mu50, a rifampicin-resistant clinical isolate, yielded two further exchanges targeting amino acids L1131 and E1048 of the RpoB subunit. The plating of >1011 cells on agar containing a combination of 4 × MIC of rifampicin and 4 × MIC of CorA did not yield any growth. In conclusion, with proper usage, e.g., in combination therapy and good antibiotic stewardship, CorA is a potential antibiotic for treating S. aureus infections.

Cervimycin-Resistant Staphylococcus aureus Strains Display Vancomycin-Intermediate Resistant Phenotypes.

Resistance to antibiotics is an increasing problem and necessitates novel antibacterial therapies. The polyketide antibiotics cervimycin A to D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug-resistant staphylococci and vancomycin-resistant enterococci. To initiate mode of action studies, we selected cervimycin C- and D-resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized CmR mutants harbored a combination of mutations in walK and clpP or clpC. In vitro and in vivo analyses showed that the mutations in the Clp proteins abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not all Bacillus subtilis strains cervimycin-resistant. The essential gene walK was the second mutational hotspot in the CmR S. aureus strains, which decreased WalK activity in vitro and generated a vancomycin-intermediate resistant phenotype, with a thickened cell wall, a lower growth rate, and reduced cell lysis. Transcriptomic and proteomic analyses revealed massive alterations in the CmR strains compared to the parent strain S. aureus SG511, with major shifts in the heat shock regulon, the metal ion homeostasis, and the carbohydrate metabolism. Taken together, mutations in the heat shock genes clpP, clpC, and dnaK, and the walK kinase gene in CmR mutants induced a vancomycin-intermediate resistant phenotype in S. aureus, suggesting cell wall metabolism or the Clp protease system as primary target of cervimycin. IMPORTANCE Staphylococcus aureus is a frequent cause of infections in both the community and hospital setting. Resistance development of S. aureus to various antibiotics is a severe problem for the treatment of this pathogen worldwide. New powerful antimicrobial agents against Gram-positives are needed, since antibiotics like vancomycin fail to cure vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant enterococci (VRE) infections. One candidate substance with promising activity against these organisms is cervimycin, which is an antibiotic complex with a yet unknown mode of action. In our study, we provide first insights into the mode of action of cervimycins. By characterizing cervimycin-resistant S. aureus strains, we revealed the Clp system and the essential kinase WalK as mutational hotspots for cervimycin resistance in S. aureus. It further emerged that cervimycin-resistant S. aureus strains show a VISA phenotype, indicating a role of cervimycin in perturbing the bacterial cell envelope.

Influence of ciprofloxacin and vancomycin on mutation rate and transposition of IS256 in Staphylococcus aureus.

In Staphylococcus aureus, the development of intermediate resistance to vancomycin is due to an accumulation of mutations. To elucidate the mechanisms involved here, a standard laboratory strain (S. aureus HG001) and a clinical MRSA mutator strain (S. aureus SA1450/94, which is characterized by a spontaneous insertion of IS256 into the gene of the mismatch repair enzyme MutS) were incubated at subinhibitory concentrations of ciprofloxacin and vancomycin. Ciprofloxacin increased the mutation rates of both strains, but this effect was inhibited when the SOS response was blocked by the presence of a non-cleavable variant of the LexA repressor. In the presence of vancomycin, the mutation rate was slightly elevated in the mutator strain, and this increase also depended on the strain's ability to induce the SOS response. Furthermore, treatment with subinhibitory concentrations of both antibiotics resulted in an activation of transposition frequency of the insertion element IS256 in S. aureus HG001. Transposition was dependent on the presence of a functional transposase, and the activation of transposition depended on the presence of the functional phosphatase RsbU, which activates SigB transcription activity. An in silico analysis indicated a putative antisense sigma B promoter sequence within the transposase gene. Scrambling of this promoter resulted in an about 20-fold activation of transposition activity of IS256. These data indicate that sigma B is involved in the regulation of IS256 activity by generation of an antisense RNA.

Pulsed-field gel electrophoresis of Staphylococcus aureus isolates from atopic patients revealing presence of similar strains in isolates from children and their parents.

Skin colonization with Staphylococcus aureus is often associated with atopic dermatitis, and staphylococcal enterotoxins have been implicated in the etiology of atopic disease. In this study, the colonization of patients with atopic dermatitis and their parents was investigated in order to evaluate the possibility of intrafamiliar transmission. S. aureus strains were isolated from 30 of 45 patients (66%). In 19 of 29 families (65%), at least one parent carried S. aureus, and the overall colonization rate of the parents was 48%. All strains were typed by pulsed-field gel electrophoresis (PFGE), and the presence of enterotoxin genes in the strains was assayed by multiplex PCR. A high percentage (84%) of the isolates present on the children and on at least one of their parents displayed identical PFGE and enterotoxin patterns as well as identical antibiotic resistance profiles, indicating intrafamiliar transmission. Forty-five percent of the strains did not carry any enterotoxin gene. The most frequently found enterotoxin genes were seg and sei, which were present in 36% of the strains, and seb, which was found in 24% of the strains. The other toxin genes occurred only in low frequencies. Most strains were resistant to penicillin (82%), and 15% showed resistance to more than one antibiotic. Intermediately-vancomycin-resistant S. aureus or methicillin-resistant S. aureus strains were not detected. In conclusion, this study indicates that the colonization rate of parents of atopic children is rather high and may increase the risk of recolonization of the child.

The lantibiotic mersacidin is a strong inducer of the cell wall stress response of Staphylococcus aureus.

BACKGROUND: The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids that is ribosomally produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin acts by complexing the sugar phosphate head group of the peptidoglycan precursor lipid II, thereby inhibiting the transglycosylation reaction of peptidoglycan biosynthesis. RESULTS: Here, we studied the growth of Staphylococcus aureus in the presence of subinhibitory concentrations of mersacidin. Transcriptional data revealed an extensive induction of the cell wall stress response, which is partly controlled by the two-component regulatory system VraSR. In contrast to other cell wall-active antibiotics such as vancomycin, very low concentrations of mersacidin (0.15xMIC) were sufficient for induction. Interestingly, the cell wall stress response was equally induced in vancomycin intermediately resistant S. aureus (VISA) and in a highly susceptible strain. Since the transcription of the VraDE ABC transporter genes was induced up to 1700-fold in our experiments, we analyzed the role of VraDE in the response to mersacidin. However, the deletion of the vraE gene did not result in an increased susceptibility to mersacidin compared to the wild type strain. Moreover, the efficacy of mersacidin was not affected by an increased cell wall thickness, which is part of the VISA-type resistance mechanism and functions by trapping the vancomycin molecules in the cell wall before they reach lipid II. Therefore, the relatively higher concentration of mersacidin at the membrane might explain why mersacidin is such a strong inducer of VraSR compared to vancomycin. CONCLUSION: In conclusion, mersacidin appears to be a strong inducer of the cell wall stress response of S. aureus at very low concentrations, which reflects its general mode of action as a cell wall-active peptide as well as its use of a unique target site on lipid II. Additionally, mersacidin does not seem to be a substrate for the resistance transporter VraDE.

Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42.

UNLABELLED: Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent. METHODOLOGY/PRINCIPAL FINDINGS: The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens. CONCLUSIONS/SIGNIFICANCE: The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.

Production of the novel two-peptide lantibiotic lichenicidin by Bacillus licheniformis DSM 13.

BACKGROUND: Lantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. The structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG) are organized in biosynthetic gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: Sequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides. CONCLUSIONS/SIGNIFICANCE: In conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains.