RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus.

In Staphylococcus aureus, metabolism is intimately linked with virulence determinant biosynthesis, and several metabolite-responsive regulators have been reported to mediate this linkage. S. aureus possesses at least three members of the RpiR family of transcriptional regulators. Of the three RpiR homologs, RpiRc is a potential regulator of the pentose phosphate pathway, which also regulates RNAIII levels. RNAIII is the regulatory RNA of the agr quorum-sensing system that controls virulence determinant synthesis. The effect of RpiRc on RNAIII likely involves other regulators, as the regulators that bind the RNAIII promoter have been intensely studied. To determine which regulators might bridge the gap between RpiRc and RNAIII, sarA, sigB, mgrA, and acnA mutations were introduced into an rpiRc mutant background, and the effects on RNAIII were determined. Additionally, phenotypic and genotypic differences were examined in the single and double mutant strains, and the virulence of select strains was examined using two different murine infection models. The data suggest that RpiRc affects RNAIII transcription and the synthesis of virulence determinants in concert with σ(B), SarA, and the bacterial metabolic status to negatively affect virulence.

Emergence of Citrobacter freundii carrying IMP-8 metallo-ß-lactamase in Germany.

Metallo-ß-lactamases (MBLs) in Enterobacteriaceae are an increasing problem worldwide. This report describes the isolation of Citrobacter freundii carrying IMP-8 MBL from three patients during the period from March 2012 until March 2013 in Germany. The bla IMP-8 enzyme is predominantly found in Asia, where IMP-8 has spread to various enterobacterial species causing serious infections. To our best knowledge, this is the first report of bla IMP-8 habouring Enterobacteriaceae in Europe.

Expression of staphylococcal superantigens during nasal colonization is not sufficient to induce a systemic neutralizing antibody response in humans.

Staphylococcus aureus carriers have high-titer serum antibodies against non-enterotoxin gene cluster (egc) superantigens, whereas they lack anti-egc antibodies, suggesting different superantigen expression profiles in vivo. We measured the superantigen transcripts in S. aureus directly isolated from the nose of persistent carriers and correlated them with the superantigen-neutralizing antibody response. While neutralizing serum antibodies against the staphylococcal enterotoxins A and C (SEA and SEC) were found in carriers, antibodies against the egc-encoded staphylococcal enterotoxin-like toxin O (SElO) were rare. Surprisingly, the transcription of selo was comparable to sea and sec during nasal colonization. Thus, egc superantigens are transcribed during nasal colonization, but this is not sufficient to induce a serum antibody response.

Resistance to dermcidin-derived peptides is independent of bacterial protease activity.

Dermcidin (DCD) is an antimicrobial peptide constitutively expressed in eccrine sweat glands in human skin. By post-secretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides that are able to kill several Gram-positive and Gram-negative bacteria but are only weakly active against Pseudomonas aeruginosa. Here, we questioned whether bacterial resistance to DCD peptides is mediated by proteolytic degradation. It was shown that DCD-derived peptides are degraded by purified bacterial proteases and by extracellular proteases secreted by P. aeruginosa in a concentration-dependent manner. However, protease-deficient mutants of P. aeruginosa PAO1 lacking either lasA, lasB (elastase) or both showed a similar sensitivity towards DCD-derived peptides as the wild-type strain. Finally, inhibition of total protease activity indicated that proteases secreted by P. aeruginosa are not responsible for the poor activity of DCD-derived peptides against P. aeruginosa. These data suggest that the decreased sensitivity of P. aeruginosa to DCD-derived peptides is not mediated by proteolytic degradation under physiological conditions.

Emergence of increasing linezolid-resistance in enterococci in a post-outbreak situation with vancomycin-resistant Enterococcus faecium.

During 2004 and at the start of 2005 a university hospital in Southwest Germany was affected by an extensive outbreak of vancomycin-resistant Enterococcus faecium (VRE). Although the outbreak was contained, linezolid-resistant enterococci emerged during and after the outbreak as the usage of linezolid became more common. Linezolid resistance was no longer limited to VRE. Nosocomial spread of linezolid-resistant but vancomycin-susceptible E. faecium was detected and these strains also emerged in patients without prior drug exposure. Linezolid should therefore be used with caution and the susceptibility of isolates monitored over time. Isolation precautions and screening of contacts should be considered to avoid spread of resistant isolates.

Discrimination between epidemic and non-epidemic glycopeptide-resistant E. faecium in a post-outbreak situation.

Vancomycin-resistant enterococci (VRE) have been isolated in increasing numbers. Hospital-adapted VRE exhibit relatively high pathogenicity by expressing factors like enterococcal surface protein (Esp), which facilitates epidemic spread. By contrast, 'community-acquired' VRE show low pathogenicity and non-epidemic features. In 2004 and 2005 an extended outbreak of VRE occurred at a university hospital in Southwestern Germany and an infection control programme was implemented to confine the outbreak. Pulsed-field gel electrophoresis (PFGE), esp PCR, multiple-locus variable number of tandem repeat analysis (MLVA), purK1 typing and multiple-locus sequence typing (MLST) were performed on representative VRE isolates. Twenty-six non-epidemic and two epidemic VRE types (MLST203, MLST280) were identified by PFGE. Seven of the non-outbreak VRE types were esp gene negative, whereas 19 non-outbreak and both epidemic VRE types were esp positive. Eight MLVA types were identified. MLVA type 1 included five PFGE types and MLVA type 159 included 16 PFGE types. Currently there is no efficient method available to identify non-epidemic VRE and avoid unnecessary isolation of patients. More than 50% non-epidemic clones were esp positive; nevertheless, esp PCR appears to be the most promising approach to identify non-epidemic VRE.

Metallo-beta-lactamase expressing multi-resistant Acinetobacter baumannii transmitted in the operation area.

Outbreaks of Acinetobacter baumannii demonstrating multiple antibiotic resistance, including meropenem resistance, have been described as severe therapeutic problems. Here we describe a monoclonal outbreak of infection and colonization with multidrug-resistant A. baumannii over a two-month period. Resistance to meropenem was mediated by expression of a metallo-beta-lactamase enzyme. Four of 14 patients showed clinical signs of infection and two died. Contamination of the environment, water, or instruments were excluded as causes of the outbreak. All patients, except one, underwent surgery in a specific operation theatre where surgery of contamination class IV (infected, dirty) was performed. Although individual surgeon error was eliminated, analyses of the patients' histories suggested that bacterial transmission had occurred during surgery. Five patients showed signs of A. baumannii infection and two of these patients suffered from large abdominal wounds infected with a high density of A. baumannii requiring repeated revisions. Presumably, these revisions favoured the transmission of A. baumannii, which is remarkably resistant to various environmental stresses including soaps, disinfectants and dry conditions. No case of meropenem-resistant A. baumannii had been observed in the hospital before the outbreak. Interestingly, the resistant bacteria appear to have been imported by a patient returning from West Africa. This indicates that, similar to MRSA, multiresistant A. baumannii may be introduced by patients from foreign hospitals. The outbreak was stopped in the following months by reinforcing standard procedures and by taking all necessary precautions such as patient isolation, and finally only one new case was detected.

Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of alpha-toxin during device-related infection resolved by direct quantitative transcript analysis.

The cytotoxic alpha-toxin (encoded by hla) of Staphylococcus aureus is regulated by three loci, agr, sarA and sae, in vitro. Here, we assess the regulation of hla in a guinea pig model of device-related infection by quantifying RNAIII (the effector molecule of agr) and hla directly in exudates accumulating in infected devices without subculturing of the bacteria. LightCycler reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the transcripts. Strains RN6390 and Newman expressed considerably smaller amounts of RNAIII in the guinea pig than during in vitro growth. The residual RNAIII expression decreased during the course of infection and was negatively correlated with bacterial densities. As with RNAIII, the highest hla expression was detected in both strains early in infection. Even in strain Newman, a weak hla producer in vitro, a pronounced expression of hla was observed during infection. Likewise, four S. aureus isolates from cystic fibrosis (CF) patients expressed Q1hla despite an inactive agr during device-related infection as in the CF lung. Mutation of agr and sarA in strain Newman and RN6390 had no consequence for hla expression in vivo. In contrast, the mutation in sae resulted in severe downregulation of hla in vitro as well as in vivo. In conclusion, S. aureus seems to be provided with regulatory circuits different from those characterized in vitro to ensure alpha-toxin synthesis during infections.

Regulation of Staphylococcus aureus type 5 and type 8 capsular polysaccharides by CO(2).

Staphylococcus aureus expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO(2). Here we show that CO(2) reduces CP5 expression at the transcriptional level and that CO(2) regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO(2) caused a significant decrease in CP8 expression in four S. aureus strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine S. aureus strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of cap5 and cap8. To test whether these sequence variations are responsible for the different responses to CO(2), promoter regions from selected strains were fused to the reporter gene xylE in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO(2) in all derivatives of strain Newman and was always positively regulated by CO(2) in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO(2) response. trans-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive S. aureus strains.

Quantification of bacterial transcripts during infection using competitive reverse transcription-PCR (RT-PCR) and LightCycler RT-PCR.

Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)-competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated Staphylococcus aureus RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic S. aureus lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (gyr) and a highly regulated alpha-toxin gene (hla) of S. aureus. Reproducible results were obtained with both methods. A sensitivity of 10(4) (gyr) and 10(3) (hla) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples.

Adherence of Staphylococcus aureus to endothelial cells: influence of capsular polysaccharide, global regulator agr, and bacterial growth phase.

The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase on S. aureus adherence to EC. Whereas S. aureus Newman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in the agr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC. S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P<0.005). Complementation of the cap5O mutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant and cap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

Agr-independent regulation of fibronectin-binding protein(s) by the regulatory locus sar in Staphylococcus aureus.

Fibronectin-binding proteins (FnBPs) are thought to be important for the attachment of Staphylococcus aureus during infection. The regulation of the genes fnbA and fnbB by the global regulatory loci sar and agr was examined using site-specific regulatory mutants of S. aureus strain Newman. The results from binding assays using both aqueous and solid-phase fibronectin as well as ligand blotting with biotinylated fibronectin showed that the expression of FnBPA is enhanced in the agr mutant but inhibited in the sar mutant and the sar-agr double mutant. The same regulatory pattern was observed in Northern blot analysis using fnbA-specific probes. The introduction of sar on a multicopy plasmid increased the already enhanced fnbA transcription of the agr mutant. FnBPB was not detectable by ligand blotting and the fnbB promoter activity in promoter fusion assays was not affected by either sar or agr. The sequence encompassing ORF3 located upstream of sarA was found to be essential for the activation of fnbA transcription. We hypothesize that this sequence may modulate SarA expression and/or activity on the post-transcriptional level. Gel shift assays demonstrated that SarA binds to the fnbA promoter fragments, probably as a dimer. DNase I footprinting assays with SarA revealed a protected area of 102 bp upstream of fnbA.

Molecular epidemiology of community-acquired Staphylococcus aureus in families with and without cystic fibrosis patients.

The molecular epidemiology of Staphylococcus aureus nasal commensal strains and community-acquired infecting strains was assessed by comparison of prevalence, persistence, transmission rate, and clonal distribution of S. aureus in families with and without cystic fibrosis (CF) patients. Isolates were typed by pulsed-field gel electrophoresis. CF patients without antibiotic treatment had a significantly higher nasal prevalence (66%) of S. aureus than did treated patients (29%; P<.001) or healthy controls (32%; P<.001), suggesting that persons with CF have a higher susceptibility to this organism. Strain transmission was frequent within both CF (55%) and non-CF (62%) families. After 3 and 19 months, 57% and 21%, respectively, of all persons still harbored the same S. aureus strain. Most of the isolates (78%) belonged to 8 of 38 genome types common in CF patients and in healthy persons. The predominant occurrence of a limited number of S. aureus clones within the community suggests evolutionary mechanisms for the selection of certain strains without an obvious association with disease.

Direct quantitative transcript analysis of the agr regulon of Staphylococcus aureus during human infection in comparison to the expression profile in vitro.

Bacteria possess a repertoire of distinct regulatory systems promoting survival in disparate environments. Under in vitro conditions it was demonstrated for the human pathogen Staphylococcus aureus that the expression of most virulence factors is coordinated by the global regulator agr. To monitor bacterial gene regulation in the host, we developed a method for direct transcript analysis from clinical specimens. Quantification of specific transcripts was performed by competitive reverse transcription-PCR, and results were normalized against the constitutively expressed gene for gyrase (gyr). Using sputum from cystic fibrosis (CF) patients infected with S. aureus we examined the transcription of the effector molecule RNAIII of agr, of spa (protein A), generally repressed by agr, and of hla (alpha-toxin), generally activated by agr. In the CF lung RNAIII was expressed poorly, indicating an inactive agr in vivo. Despite the low level of RNAIII expression, spa was detectable only in minute amounts and an irregular transcription of hla was observed in all sputum samples. After subculturing of patient strains agr-deficient isolates and isolates with unusual expression profiles, i.e., not consistent with those obtained from prototypic strains, were observed. In conclusion, the agr activity seems to be nonessential in CF, and from the described expression pattern of spa and hla, other regulatory circuits aside from agr are postulated in vivo.

Characterization of a sar homolog of Staphylococcus epidermidis.

Coagulase-negative staphylococci are common nosocomial pathogens. A regulatory element, designated sar, partially controls exoprotein synthesis in coagulase-positive Staphylococcus aureus by modulating the expression of another regulatory locus, called agr. We report here the cloning of a sar homolog in S. epidermidis. The major open reading frame within sar in S. epidermidis is highly homologous (84%) to the S. aureus SarA protein. Primer extension studies revealed three sar transcripts (0.64, 0.76, and 0.85 kb) initiated from three distinct promoters. The interpromoter region in S. epidermidis differs from its S. aureus counterpart, possibly suggesting target gene differences and a disparate pattern for sar activation. Remarkably, the S. epidermidis sar homolog interacts with an agr promoter fragment of S. aureus in gel shift assays. Additionally, S. epidermidis sar fragments could restore hemolysin production in an S. aureus sar mutant. As typical virulence determinants controlled by sar in S. aureus are not present in S. epidermidis, an examination of functional and structural similarities and divergence of sar in staphylococci will be of major interest.

Influence of agr on fibrinogen binding in Staphylococcus aureus Newman.

The ability of Staphylococcus aureus to bind fibrinogen is believed to be important in promoting bacterial adherence to both intravascular catheters and host tissues during infection. We investigated the influence of the global regulator agr on the fibrinogen binding capacity and its relationship to the expression of coagulase (encoded by coa) and clumping factor (encoded by clfA) in strain Newman. Strains were obtained by transducing site-specific mutations of clfA, coa, and agr into strain Newman to obtain single, double, and triple mutants of the respective genes. As expected, the clfA mutant bound less soluble 125I-labeled fibrinogen than the corresponding coa mutant in agr+ strains; however, with agr mutant strains, the upregulation in fibrinogen binding capacity correlated mostly with the increased expression and transcription of coagulase as shown by Western (immunoblot) and Northern (RNA) blot analysis. In particular, the coa agr double mutant resulted in a significant reduction in fibrinogen binding compared with that of the agr mutant. The contribution of clfA to fibrinogen binding in agr-negative strains was less than that of coa (32,740 +/- 1,189 versus 18,141 +/- 334 and 38,919 +/- 1,021 cpm for clfA agr, coa agr, and the single agr mutant, respectively). Thus, coagulase is a major binding protein for soluble fibrinogen in the agr-negative background. In in vitro microtiter and catheter adherence assays with solid-phase fibrinogen, clumping factor, but not coagulase, plays a major role in binding to immobilized fibrinogen. coa transcription was negatively modulated by agr and occurred mainly during the exponential growth phase. In contrast, clfA transcription was agr independent and was strongest during the postexponential phase. Although an agr coa clfA triple mutant bound less soluble fibrinogen than the agr coa double mutant (8,504 +/- 831 versus 18,141 +/- 334 cpm), significant residual fibrinogen binding capacity remained in the triple mutant, thus suggesting an additional fibrinogen binding component. By using direct ligand affinity blotting with 125I-fibrinogen, we could identify coagulase and an additional unidentified 52-kDa protein as a fibrinogen binding component in cell extracts. This band was absent in the extract of the coa clfA double mutant.

Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus.

A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this locus is distinct from agr, xpr, and sar, three previously described global regulatory loci. Transduction experiments demonstrated that the genotype associated with mutant 1E3 could be transferred back into the parental strain ISP479C. The transductant 1E3-2 displayed a phenotypic profile similar to that of the original mutant. Northern (RNA) blot studies showed that this locus may be involved in modulating target genes at the mRNA level. In the rabbit endocarditis model, there was a significant decrease in both the infectivity rate and intravegetation bacterial density with mutant 1E3 compared with the parent at an inoculum of 10(3) CFU. Since protein A and the fibrinogen-binding protein(s) are major surface proteins that may mediate bacterial adhesion to host tissues, this locus may be an important genetic element involved in the expression of virulence determinants in S. aureus.

Iron release from transferrin by pyoverdin and elastase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces the siderophores pyoverdin and pyochelin as well as receptors for siderophores in response to iron deprivation. Previously, it has been shown in vitro that at neutral pH purified pyoverdin acquires iron from transferrin only in the presence of P. aeruginosa elastase (LasB), which proteolytically degrades transferrin. We constructed a LasB-negative mutant, PAO1E, by insertional mutagenesis to investigate whether this mutant differs in growth from the parental strain PAO1 in an iron-depleted medium supplemented with transferrin or human serum. PAO1 and PAO1E did not differ in growth with 1.25 microM Fe2-transferrin as the only iron source. Urea gel electrophoresis indicated iron release from intact transferrin during the logarithmic growth phase of PAO1 and PAO1E. A total of 333 microM LasB was synthesized from PAO1 after onset of stationary-phase growth. Quantification of pyoverdin by spectroscopy revealed that up to 900 microM pyroverdin was produced during growth of the strains in medium supplemented with Fe2-transferrin or 10% human serum. Incubation of Fe2-transferrin and purified pyoverdin in concentrations similar to those found in the culture supernatant resulted in release iron from transferrin after 10 h at 37 degrees C. However, LasB significantly enhanced the rate constant for iron acquisition of pyoverdin from transferrin. We conclude that P. aeruginosa can use transferrin as an iron source without further need of LasB or pH changes. This is further supported by experiments with P. aeruginosa K437, which has a defective iron uptake system, and its LasB-negative mutant, K437E. Though K437 and K437E did not differ in growth with Fe2-transferrin as the only iron source, their growth was significantly reduced relative to that of PAO1 and PAO1E.

A new extracellular protein of Pseudomonas aeruginosa PA103 regulated by regA.

The expression of exotoxin A (ExoA) from Pseudomonas aeruginosa is influenced by iron and is under the control of the regulatory gene regA. To test whether regA plays a role in the expression of other iron-regulated proteins a RegA- mutant was constructed by insertional mutagenesis. The polypeptide pattern of this mutant (PA103R) was compared with the parental strain (PA103) and a trans-complemented strain PA103R(pREX18) after growth of the strains in conditions containing low or high concentrations of iron. An iron-regulated 42 kDa protein (RRP) was identified and purified from the culture supernatant of PA103 and PA103R(pREX18) which was missing in PA103R. Database analysis of the N-terminal sequence of this regA-regulated protein (RRP) revealed no similarity to other proteins. Preliminary investigations into the function of RRP revealed that it has no proteolytic or cytotoxic activity. Using two-dimensional electrophoretic analysis of whole cells, a technique which allowed separation of over 600 polypeptides, we were unable to identify any other iron-regulated protein whose expression was regulated by regA.