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.

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.

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.

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.

In vitro comparison of cefotetan with five other cephalosporins against nosocomial pathogens.

The in vitro activity of cefotetan against Staphylococcus aureus and gram-negative bacterial strains including nonfermenting species has been compared with that of cefoxitin, cefotaxim, moxalactam, cefoperazone and ceftazidime by use of an agar dilution method. Cefotetan was found to be inactive against Pseudomonas aeruginosa and Acinetobacter species and only moderately active against Staphylococcus aureus, Pseudomonas maltophilia, Pseudomonas cepacia, and Enterobacter cloacae. It was more active than cefoxitin and cefoperazone against Escherichia coli, Klebsiella pneumoniae, and indole-positive Proteus strains.