Christiane Szekat

Presence of Staphylococcus aureus with Reduced Susceptibility to Vancomycin in Germany

Abstract A total of 457 Staphylococcus aureus strains from the culture collection of the National Reference Center for Staphylococci in Bonn, Germany, were screened for susceptibility to vancomycin because some Staphylococcus aureus strains are able to form subpopulations that show intermediate resistance to vancomycin. Two methicillin-resistant Staphylococcus aureus strains (isolated in 1993) exhibited intermediate resistance. One of these, Staphylococcus aureus 137-93, which displayed the genomic DNA fragment pattern of the northern German epidemic strain, appeared homogeneously resistant. Neither of these strains had been identified by routine susceptibility testing. The resistance of the German isolates was lower than that of the Japanese isolate Mu50. To determine whether a similar mechanism confers vancomycin resistance in Staphylococcus aureus Mu50 and 137-93, the intracellular cell wall precursor concentration was measured and was not found to be comparably increased in Staphylococcus aureus 137-93. In conclusion, strains showing intermediate resistance have been present in Germany for some time (at least since 1993), but the subpopulations with decreased sensitivity were overlooked during antibiotic susceptibility testing.

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.

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

ABSTRACT 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.

Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin

ABSTRACT The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.

The lantibiotic mersacidin is an autoinducing peptide.

ABSTRACT The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.

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

BackgroundThe 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.ResultsHere, 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 (0.15 × MIC) 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.ConclusionIn 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.

Pulsed-Field Gel Electrophoresis of Staphylococcus aureus Isolates from Atopic Patients Revealing Presence of Similar Strains in Isolates from Children and Their Parents

ABSTRACT 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.

Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42

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.

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 strains 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.

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.