Volker Brade

Molecular analysis of fusidic acid resistance in Staphylococcus aureus

Fusidic acid is a potent antibiotic against severe Gram‐positive infections that interferes with the function of elongation factor G (EF‐G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF‐G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid‐resistant clinical S. aureus strains as well as in 10 fusidic acid‐resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF‐G. To confirm the importance of observed amino acid exchanges in EF‐G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF‐G derivatives with the exchanges P406L, H457Y and L461K were constructed by site‐directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid‐susceptible S. aureus strain RN4220 caused a fusidic acid‐resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid‐resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF‐G for the generation of fusidic acid resistance in S. aureus.

Is Serological Testing a Reliable Tool in Laboratory Diagnosis of Syphilis? Meta-Analysis of Eight External Quality Control Surveys Performed by the German Infection Serology Proficiency Testing Program

ABSTRACT The accuracy of diagnostic tests is critical for successful control of epidemic outbreaks of syphilis. The reliability of syphilis serology in the nonspecialist laboratory has always been questioned, but actual data dealing with this issue are sparse. Here, the results of eight proficiency testing sentinel surveys for diagnostic laboratories in Germany between 2000 and 2003 were analyzed. Screening tests such as Treponema pallidum hemagglutination assay (mean accuracy, 91.4% [qualitative], 75.4% [quantitative]), Treponema pallidum particle agglutination assay (mean accuracy, 98.1% [qualitative], 82.9% [quantitative]), and enzyme-linked immunosorbent assays (ELISAs) (mean qualitative accuracy, 95%) were more reliable than Venereal Disease Research Laboratory (VDRL) testing (mean accuracy, 89.6% [qualitative], 71.1% [quantitative]), the fluorescent treponemal antibody absorption test (FTA-ABS) (mean accuracy, 88% [qualitative], 65.8% [quantitative]), and immunoblot assays (mean qualitative accuracy, 87.3%). Clearly, immunoglobulin M (IgM) tests were more difficult to manage than IgG tests. False-negative results for samples that have been unambiguously determined to be IgM and anti-lipoid antibody positive accounted for 4.7% of results in the IgM ELISA, 6.9% in the VDRL test, 18.5% in the IgM FTA-ABS, and 23.0% in the IgM immunoblot assay. For negative samples, the mean percentage of false-positive results was 4.1% in the VDRL test, 5.4% in the IgM ELISA, 0.7% in the IgM FTA-ABS, and 1.4% in the IgM immunoblot assay. On average, 18.3% of participants misclassified samples from patients with active syphilis as past infection without indicating the need for further treatment. Moreover, 10.2% of laboratories wrongly reported serological evidence for active infection in samples from patients with past syphilis or in sera from seronegative blood donors. Consequently, the continuous participation of laboratories in proficiency testing and further standardization of tests is strongly recommended to achieve better quality of syphilis serology.

Immune evasion of Borrelia burgdorferi: Insufficient killing of the pathogens by complement and antibody

The innate immune system and, in particular, the complement system play a key role in the elimination of micro-organisms after entrance in the human host. Like other pathogens, borreliae must develop strategies to inactivate host defence mechanisms. By investigating serum (NHS)-susceptibility of borreliae, we found that mainly B. afzelii isolates are serum-resistant, whereas the majority of B. burgdorferi s. s. isolates display an intermediate serum-sensitive phenotype. In contrast, B. garinii isolates are killed effectively by complement and therefore are classified as serum-sensitive. Up to now, we have identified two distinct proteins of 27.5 kDa and 20.7 kDa expressed on the outer surface of borreliae, which interact directly with FHL-1/reconectin and factor H, the two major regulators of the alternative complement pathway. These borrelial proteins are termed CRASPs (complement regulator-acquiring surface proteins). CRASPs are detectable only on serum-resistant borreliae and, accordingly, binding of FHL-1/reconectin and factor H only occur with serum-resistant borrelial isolates. We conclude from these results that the control of complement activation on the borrelial surface is due to the interaction of borrelial CRASPs with host complement regulatory proteins. Thus, CRASPs represent an important mechanism of immune evasion on the part of borrelial isolates belonging mostly to the genospecies B. afzelii. By analysing the humoral adaptive immune response of patients, we detected sera that killed NHS-resistant borreliae. Borreliacidal activity is observed most frequently with sera of patients at stage III of the disease. The killing of NHS-resistant isolates by these immune sera always requires the combination of antibodies and complement. Bactericidal activity, however, is not detected in all immune sera at the different disease stages, although specific anti-Borrelia antibodies are present according to serological test results. This observation suggests that not all borrelial antigens are able to induce a borreliacidal immune response. In an extensive analysis of 24 immune sera, we identified up to 12 borrelial antigens, including OspC, which possess the greatest potential for the induction of borreliacidal antibody. The borreliacidal potential of anti-OspC antibodies was tested directly on an OspC-expressing borrelial wild-type isolate and a corresponding variant lacking OspC. In these studies, only the wild-type isolate expressing OspC on its surface proved positive for the lytic complement complex, thereby indicating the great importance of this antigen for the control of the infection. Additional studies are required to identify further protective antigens among these 12 proteins, all of which are candidates for infection control according to our studies involving patient immune sera. These antigens may include the recently detected CRASPs.

Two cases of chronic arthritis of the forearm due to Mycobacterium tuberculosis.

In the absence of coexisting active pulmonary disease, tuberculosis is frequently not considered in the differential diagnosis of chronic inflammation of the joints. The cases of two immigrant patients with tuberculous arthritis involving the forearm are reported. In both cases non-specific arthritis or trauma was suspected, resulting in a delay between the onset of symptoms and institution of specific therapy of 21 and 24 months, respectively. Diagnosis was achieved by histological and microbiological examination of synovial biopsy material. Polymerase chain reaction forMycobacterium tuberculosis complex was positive in only one patient. Treatment consisted of antituberculosis chemotherapy, surgical synovectomy, and debridement of the affected joints. These cases serve as a reminder that, although rare, tuberculosis can cause chronic arthritis.

Prevalence of Antibodies against the Human Granulocytic Ehrlichiosis Agent in Lyme Borreliosis Patients from Germany

Abstractu2002To contribute to the discussion of whether or not human granulocytic ehrlichiosis (HGE) occurs in midwestern Germany, sera from individuals with different risk categories for tick exposure were retrospectively examined by means of an immunofluorescence assay. The seroreprevalence for the HGE agent accounted for 5.5% of the 270 patients tested. Specific antibodies were detected more often in patients with early Lyme infection than in patients with stage III disease or in asymptomatic individuals seropositive for Lyme disease. Investigation of 50 patients with an active or recent syphilis infection revealed no cross-reactivity between Treponema pallidum antibodies and the HGE agent. The prevalence of HGE antibodies (13.1%) among 76 Lyme borreliosis patients from this urban area was significantly higher (P<0.05) than that in the control groups (2.6%). The findings indicate that concomitant or serial infections with Borrelia burgdorferi and the HGE agent or closely related organisms may be a common occurrence in tick-exposed patients from Germany.

New colorimetric microdilution method for in vitro susceptibility testing of Borrelia burgdorferi against antimicrobial substances.

Abstractu2002A newly developed colorimetric microdilution method was used to analyze the activity of 12 antimicrobial agents against nine Borrelia burgdorferi isolates, including all three genospecies pathogenic for humans. In addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and Borrelia bissettii tick isolates were investigated. The applied test system is based upon color changes that occur in the presence of phenol red and result from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes. After 72u2009h of incubation, minimal inhibitory concentrations (MICs) were determined from the decrease of absorbance by software-assisted calculation of growth curves. MIC values were lowest for azlocillin (MIC, ≤0.125u2009μg/ml), ceftriaxone (MIC range, ≤0.015–0.06u2009μg/ml), and azithromycin (MIC range, ≤0.015–0.06u2009μg/ml). Whereas tobramycin (MIC range, 8–64u2009μg/ml) exhibited little activity, spectinomycin (MIC range, 0.25–2u2009μg/ml) showed in vitro antimicrobial activity against Borrelia burgdorferi. The MICs of penicillin G for Borrelia afzelii isolates were ten times higher than those for Borrelia burgdorferi, Borrelia valaisiana, and Borrelia bissettii isolates (P<0.05) and 100 times higher than those for isolates belonging to the genospecies Borrelia garinii (P<0.05). Further significant differences with respect to the MIC values of the other antimicrobial agents tested were not noted. The colorimetric microdilution method offered the advantages of reliability, reproducibility, and convenience and could handle large numbers of isolates and antibiotics.

Clonal heterogeneity, distribution, and pathogenicity of methicillin-resistantStaphylococcus aureus

Four thousand eighty-eightStaphylococcus aureus isolates obtained from patients hospitalised in a university clinic and four community hospitals over a period of one year were screened for methicillin resistance. A resistance rate of 5% was detected among initial isolates. Distribution of methicillin-resistantStaphylococcus aureus (MRSA) and methicillin-sensitiveStaphylococcus aureus showed an increased prevalence of MRSA in clinically significant specimens such as blood, central venous catheter tips, bronchial secretions, and wound secretions. Typing of 110 MRSA strains (initial isolates) by macrorestriction analysis of chromosomal DNA revealed 26 different genotypes that could be divided into five epidemic and 21 sporadic strains. More than 50% of all isolates belonged to one type that was confirmed to be closely related to the “southern-German” epidemic strain. Production of virulence factors such as enterotoxin A-D and toxic shock syndrome-toxin 1 among MRSA strains (initial isolates) occurred in ten of 26 different MRSA types. A strong correlation between genotype and toxin production was demonstrated.

Disseminated Cutaneous Mycobacterium marinum Infection in a Patient with Non-Hodgkin's Lymphoma

Abstract.A 60-year-old woman with non-Hodgkins lymphoma was admitted to the hospital because of extensive subcutaneous abscesses developing on all limbs. The patient had an aquarium and kept tropical fish as pets. After repeated investigations, the diagnosis of Mycobacterium marinum was established from skin biopsy by PCR and culture. Long-term therapy with several drugs regimens had only a limited efficacy and was accompanied by severe adverse reactions. This report highlights the therapeutic problems posed by disseminated cutaneous M. marinum infection in the immunosuppressed host.

Systematic analysis highlights the key role of TLR2/NF-κB/MAP kinase signaling for IL-8 induction by macrophage-like THP-1 cells under influence of Borrelia burgdorferi lysates

Lyme borreliosis is a spirochetal infection caused by the Borrelia burgdorferi sensu lato complex that can proceed towards an inflammatory joint manifestation known as Lyme arthritis. Production of chemokines orchestrating neutrophil infiltration is supposed to be key to early arthritic pathogenesis. Using PMA-differentiated macrophage-like THP-1 (mTHP-1) cells we identified by antibody array methodology or mRNA analysis IL-8, GRO-alpha, NAP-2, and SDF-1alpha as being among those chemokines that are upregulated by bacterial lysates obtained from B. burgdorferi. Based on these observations, we set out to characterize in detail mechanisms mediating IL-8 release in this cellular model. TLR2 blocking antibodies, analysis of p65 translocation, and electromobility-shift analysis revealed activation of the TLR2/NF-kappaB axis by B. burgdorferi. The functional importance of this pathway was substantiated by suppression of IL-8 after inhibition of IkappaB kinase. Notably, MAP kinases, specifically the MEK1/2-ERK1/2 pathway, were essential for IL-8 secretion. Those data were confirmed by using freshly isolated adherent peripheral blood mononuclear cells. On the contrary, B. burgdorferi-induced IL-8 in mTHP-1 was unlikely related to flagellin, alpha3beta1-integrin signaling, lipopolysaccharide, bacterial DNA, NOD1/NOD2 agonists, or to intermediate production of IL-1beta and TNF-alpha. Induction of IL-8 by B. burgdorferi was not due to amplification of constitutive AP-1 DNA-binding activity detectable in mTHP-1 cells. Data presented herein validate that TLR2, particularly on mTHP-1 cells, holds a central position in mediating IL-8 secretion associated with extracellular B. burgdorferi and beyond that suggest inhibition of IkappaB kinase and MEK1/2 kinases as promising pharmacological strategies aiming at IL-8 in early Lyme arthritis.

Evaluation of modern agglutination tests for identification of methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

effect, which appears to be mediated by inhibition of Candida aspartate proteinase activity [A. Cassone et al., 12th World AIDS Conference, 1998, Abstract no. 42358]. In our study, however, indinavir did not show any direct antifungal effect, even when used at concentrations tenfold those of HIV-1 ID95 (0.07 mg/l). Under experimental conditions (using an assay based on resorufin-labelled casein substrate), a dose-dependent inhibition of adherence of Candida albicans caused by HIV-1 protease inhibitors has been demonstrated, but these antiretroviral agents had no effect on the fungal vitality [Borg-von Zepelin et al., 9th European Congress of Clinical Microbiology and Infectious Diseases, 1999, Abstract no. P1164].