Small colony variants (SCVs) represent minority sub-populations of Staphylococcus aureus that grow slowly (>72 h) on routine media, yielding small, non-pigmented, non-hemolytic colonies. Although S. aureus SCVs have been recognized for many years [1Barbour RGH Small colony variants (‘G’ forms) produced by Staphylococcus pyogenes during the development of resistance to streptomycin.Aust J Exp Biol Med Sci. 1950; 28: 415-420Crossref PubMed Scopus (1) Google Scholar, 2Wise RI Spink WW The influence of antibiotics on the origin of small colonies (G variants) of Micrococcus pyogenes var. aureus.J Clin Invest. 1954; 33: 1611-1612Crossref PubMed Scopus (34) Google Scholar, 3Acar JF Goldstein FW Lagrange P Human infections caused by thiamine or menadione requiring Staphylococcus aureus.J Clin Microbiol. 1978; 8: 142-147PubMed Google Scholar], the connection between this phenotype and persistent recurrent infections has only recently been appreciated. Clinical and laboratory-generated S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds required in the biosynthesis of menaquinone and cytochromes, components of the electron transport chain. The subsequent decrease in electron transport activity in SCVs may account for their resistance to a variety of antibiotics and other antistaphylococcal compounds, such as protamine, some antibiotics, and platelet microbiocidal proteins, and also provide a mechanism for their persistence within host tissues [4Proctor RA van Langevelde P Kristjansson M Maslow JN Arbeit RD Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus.Clin Infect Dis. 1995; 20: 95-102Crossref PubMed Scopus (322) Google Scholar, 5Proctor RA Vesga O Otten MF et al.Staphylococcus aureus small colony variants cause persistent and resistant infections.Chemotherapy. 1996; 42: 47-52Crossref PubMed Scopus (14) Google Scholar, 6von Eiff C Heilmann C Proctor R Woltz C Peters G Götz F A site-directed Staphylococcus aureus hemB mutant is a small colony variant which persists intracellularly.J Bacteriol. 1997; 179: 4706-4712Crossref PubMed Scopus (230) Google Scholar]. It is well known that S. aureus SCVs can be derived from clinical isolates both in vitro and in vivo following exposure to aminoglycosides and β-lactam antibiotics [1Barbour RGH Small colony variants (‘G’ forms) produced by Staphylococcus pyogenes during the development of resistance to streptomycin.Aust J Exp Biol Med Sci. 1950; 28: 415-420Crossref PubMed Scopus (1) Google Scholar, 2Wise RI Spink WW The influence of antibiotics on the origin of small colonies (G variants) of Micrococcus pyogenes var. aureus.J Clin Invest. 1954; 33: 1611-1612Crossref PubMed Scopus (34) Google Scholar, 4Proctor RA van Langevelde P Kristjansson M Maslow JN Arbeit RD Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus.Clin Infect Dis. 1995; 20: 95-102Crossref PubMed Scopus (322) Google Scholar, 5Proctor RA Vesga O Otten MF et al.Staphylococcus aureus small colony variants cause persistent and resistant infections.Chemotherapy. 1996; 42: 47-52Crossref PubMed Scopus (14) Google Scholar]. Mitsuyama et al. recently described the emergence of SCVs after exposure of wild-type parent strains to an MIC concentration of the fluoroquinolone pazufloxacin [7Mitsujama J Yamada H Maehana J et al.Characteristics of quinolone-induced small colony variants in Staphylococcus aureus.J Antimicrob Chemother. 1997; 39: 697-705Crossref PubMed Scopus (17) Google Scholar]. Emergent SCVs were half as susceptible to pazufloxacin and ciprofloxacin as wild-type S. aureus. Reduced susceptibilities of SCVs to these compounds were not a result of mutations in gyrA, gyrB, grlA or grlB, or efflux via NorA, but rather were mediated by decreased fluoroquinolone uptake, perhaps as a result of permeability changes in the cell wall. Little is known about the ability of different quinolone compounds to select for SCVs in S. aureus or the MICs of these antibiotics for the SCV isolates subsequently selected. In this study we investigated the spontaneous mutation rate and frequency of SCV emergence in S. aureus isolates exposed to five different fluoroquinolones (ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin). The in vitro activities of these quinolone compounds were also measured against SCVs and sibling strains with a normal phenotype. Two clinical isolates of S. aureus (i57, methicillin susceptible, and S78, methicillin resistant) were used to investigate spontaneous mutation rates and the frequencies of SCV emergence when they were exposed to each of the five quinolone compounds. Two control isolates were also used in this study, I10 (a stable SCV generated by interrupting hemB, the hemin biosynthetic gene in S. aureus, by inserting an ermB cassette), and A2 (a stable strain of I10 complemented with an intact hemB [6von Eiff C Heilmann C Proctor R Woltz C Peters G Götz F A site-directed Staphylococcus aureus hemB mutant is a small colony variant which persists intracellularly.J Bacteriol. 1997; 179: 4706-4712Crossref PubMed Scopus (230) Google Scholar], resulting in a wild-type phenotype). All four isolates were ciprofloxacin susceptible (Table 1).Table 1Quinolone susceptibility of randomly selected mutants of S. aureus strains i57, S78, A2 and I10 after exposure to four times the MIC of ciprofloxacin and moxifloxacinMIC of mutants (mg/L)MIC of parent strain (mg/L)CiprofloxacinMoxifloxacinParent strainCiprofloxacinMoxifloxacinNo. of mutantsColony identificationRangeMIC50MIC90RangeMIC50MIC90i570.25≤0.0325SCV1–2120.125–0.50.250.525Non‐SCV1–2120.125–0.50.250.5S780.25≤0.0325SCV1–2120.125–0.50.250.525Non‐SCV1–2120.125–0.50.250.5110a110, a stable SCV; a hemB mutant.0.1250.0625SCV0.5–2120.25–10.51A2bA2, a normal phenotype; hemB‐complemented 110 isolate.0.1250.0625SCV0.5–2120.25–10.5125Non‐SCV0.5–2120.25–10.51a 110, a stable SCV; a hemB mutant.b A2, a normal phenotype; hemB‐complemented 110 isolate. Open table in a new tab Mutant colonies with higher MIC values than those of the parent strains were obtained by spreading 109–1010 colony-forming units (CFUs) per mL of each of the four test isolates over agar plates containing, in parallel, two-fold and four-fold MICs of each of the compounds tested. IsoSensitest agar plates were used for i57 and S78, while trypticase soy (TSA) agar plates supplemented with 2.5 mg/L of erythromycin (and 10 mg/L of chloramphenicol to select for the complementing plasmid in strain A2) were used for the control strains. Following overnight incubation at 37°C, the number of colonies growing was determined. The frequency of appearance of mutants with raised MICs was calculated as the ratio of mutants arising after overnight incubation to the number of CFUs originally inoculated. This experiment was repeated four times for each of the five fluoroquinolones tested. SCVs selected from isolates i57, S78 and A2 were recognized and characterized as previously described [4Proctor RA van Langevelde P Kristjansson M Maslow JN Arbeit RD Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus.Clin Infect Dis. 1995; 20: 95-102Crossref PubMed Scopus (322) Google Scholar, 5Proctor RA Vesga O Otten MF et al.Staphylococcus aureus small colony variants cause persistent and resistant infections.Chemotherapy. 1996; 42: 47-52Crossref PubMed Scopus (14) Google Scholar]. In brief, isolates considered to be stable SCVs were grown on TSA agar with disks containing 2.0 μg of menadione or 20 μg of hemin, in the presence of 5% CO2, to test for auxotrophism for these compounds. Gram stain characteristics, hemolytic activity, coagulase production and susceptibility tests for lysostaphin were evaluated by standard procedures. From one agar plate, for each strain, and for each of the fluoroquinolones tested, 25 isolates demonstrated to be SCVs and 25 isolates with normal phenotypes were randomly selected for MIC testing. In addition, we paralleled our MIC studies using four S. aureus SCV isolates shown to fulfil the microbiological definition of SCV, derived from patients each of whom had previously been treated for osteomyelitis with gentamicin-impregnated beads. In each case, clinical SCV isolates were isolated concomitantly with normal methicillin-susceptible wild-type organisms shown to be clonally identical as determined by pulsed-field gel electrophoresis [8von Eiff C Bettin D Proctor RA et al.Recovery of small colony variants of Staphylococcus aureus following gentamicin bead placement for osteomyelitis.Clin Infect Dis. 1997; 25: 1250-1251Crossref PubMed Scopus (195) Google Scholar]. The MIC values for ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin were determined by both broth microdilution and agar dilution methods, using an inoculum of 105 CFU/mL, with results being analyzed after 48 h of incubation at 35°C. Following exposure of isolates i57, S78, A2 and I10 to two and four times the MIC of each test drug, the spontaneous mutation rates for the appearance of colonies with higher MICs were in the range of 10–5 to 10–7 for ciprofloxacin, 10–7 to 10–8 for ofloxacin and levofloxacin, and 10–8 to 10–9 for sparfloxacin and moxifloxacin. Thus, resistance against the newer fluoroquinolones such as sparfloxacin and moxifloxacin develops less frequently compared to other quinolones tested. Selection with two and four times the MIC led to the emergence of SCVs in the wild-type parent strains i57 and S78 as well as in the plasmid-complemented mutant A2, with each of the five fluoroquinolones tested. Mean percentage values of the frequency of SCV isolation from four independent experiments were as follows, for ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin respectively; for strain i57, 37%, 33%, 43%, 38% and 40%; for strain S78, 65%, 72%, 49%, 50% and 35%; and for strain A2, 41%, 38%, 43%, 35% and 33%. Thus, the frequency of SCV isolation from these three strains ranged between 33% and 72%, with no significant difference between any of the antibiotics tested (p > 0.1). In contrast to Mitsujama et al [7Mitsujama J Yamada H Maehana J et al.Characteristics of quinolone-induced small colony variants in Staphylococcus aureus.J Antimicrob Chemother. 1997; 39: 697-705Crossref PubMed Scopus (17) Google Scholar] (who used 0.25, 1.0 and 4.0 times the MIC to select for mutants), we were unable to find any differences in MIC values of any of the quinolones tested between randomly selected resistant SCVs and sibling strains with a normal phenotype, after exposure to two and four times the MIC, independent of the susceptibility test method used. None of the SCVs or normal wild-type organisms had MIC values above 2 mg/L for any of the drugs tested. Table 1 shows the range and MIC50 and MIC90 values for ciprofloxacin and moxifloxacin, the least active and most active of the compounds tested, against mutants selected with four times the MIC. Comparable data were obtained for mutants selected with two times the MIC. In line with this observation, there were also no differences in quinolone MIC values between the four clinically derived SCVs (data not shown). The differences between our study and that reported by Mitsujama et al with regard to the ability of quinolones to select for stable SCVs may be due to the phenomenon being strain and/or quinolone dependent, as pazufloxacin was used as a study compound [7Mitsujama J Yamada H Maehana J et al.Characteristics of quinolone-induced small colony variants in Staphylococcus aureus.J Antimicrob Chemother. 1997; 39: 697-705Crossref PubMed Scopus (17) Google Scholar]. Ciprofloxacin showed the highest MIC values, followed by ofloxacin, levofloxacin, sparfloxacin and moxifloxacin as the most active with the lowest MIC values (Table 1). This is in accordance with previous investigations, in which moxifloxacin was the most active compound, in response to all characterized mutations in grlA, grlB, gyrA and gyrB in 116 unrelated clinical S. aureus isolates [9Schmitz FJ Hofmann B Hansen B et al.Relationship between ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin MICs and with mutations in grlA, grlB, gyrA and gyrB in 116 unrelated clinical isolates of Staphylococcus aureus.J Antimicrob Chemother. 1998; 41: 481-484Crossref PubMed Scopus (95) Google Scholar]. The development of quinolone resistance in S. aureus requires multiple and stepwise mutations, with mutations in grlA preceding mutations in gyrA [10Ferrero L Cameron B Crouzet J Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus.Antimicrob Agents Chemother. 1995; 39: 1554-1558Crossref PubMed Scopus (239) Google Scholar]. Therefore, we were not able to select fluoroquinolone-resistant SCVs or strains with normal phenotype using only one overnight incubation at two or four times the MIC. By comparing SCVs to strains with a wild-type phenotype, our study offers the possibility to investigate the effect that a lower electrochemical gradient across the cell membrane has on susceptibility to both old and new fluoroquinolones. In contrast to susceptibility to aminoglycoside compounds, our data suggest that the lower electrochemical gradient across the cell membrane has no effect on fluoroquinolone susceptibility. While quinolone monotherapies are not generally used in the treatment of infections caused by S. aureus, they are often used as empirical therapies for respiratory tract infections, of which a proportion will be caused by S. aureus or present as part of a mixed infection, particularly for those that are hospital acquired. In such instances, exposure to quinolone compounds clearly provides an opportunity for the selection of SCVs which may play a role in recurrent infections. In summary, moxifloxacin is the most active compound tested, with the lowest spontaneous mutation rate. Selection with two and four times the MIC led to the emergence of SCVs with all five fluoroquinolones tested. While the selection of SCVs seems to be strain dependent, the lower electrochemical gradient across the cell membrane in SCVs has no effect on fluoroquinolone susceptibility.

In murine cells the most important effector mechanism directed against the intracellular pathogen Toxoplasma gondii is the production of toxic nitrogen oxides. In contrast the induction of the tryptophan degrading enzyme indolamine 2,3-dioxygenase (IDO) has been described to be the most effective anti-parasitic mechanism in most human cells. In this report we analysed IDO induction and NO production in the human uroepithelial carcinoma cell line RT4. We found that after stimulation with IFN-γ these cells were able to restrict toxoplasma growth. This was due to an activation of IDO, and the anti-parasitic effect mediated by RT4 cells was abrogated by the addition of l-tryptophan. In addition we found that the costimulation of RT4 cells with IL-1 and IFN-γ results in the production of nitric oxide, and that in RT4 cells stimulated with both these cytokines, IDO activity and toxoplasmostasis was lower than in cells stimulated with IFN-γ alone. This IL-1-mediated inhibition of IFN-γ-induced IDO activity and toxoplasmostasis could be blocked by monomethyl l-arginine, an inhibitor of NO production. We therefore conclude that the induction of indolamine 2,3-dioxygenase activity in human cells is a very important effector mechanism directed against Toxoplasma gondii, and that in human cells the production of NO might be involved in the regulation of IDO activity

Over a period of three years the incidence of methicillin resistant Staphylococcus aureus (MRSA) isolates in 11 hospitals in the greater Düsseldorf area was observed. From a total of 7,814 S. aureus isolates, 489 (6.3%) were methicillin resistant. From 198 different patients, MRSA first isolates and 291 second isolates could be cultured. Methicillin resistance among all S. aureus isolates from 11 hospitals in the greater Düsseldorf area, ranged from 0.5 to 7.8% dependant on the size of the hospital. The highest incidence (7.8%) was found in a 1,500 bed hospital and the lowest incidence in a smaller 200 bed hospital (0.5%). With respect to the distribution among clinical departments the highest incidence of MRSA isolates was found on intensive care units and surgical wards, 25.5% and 13.0% respectively. The commonest specimen from which the MRSA isolates were cultured were respiratory secretions (17.6%) followed by central venous catheter tips (12.8%). In terms of the drug resistance pattern: all isolates were resistant to the aminoglycosides and gyrase inhibitors, whereas between 80% and 90% were sensitive to fusidic acid, chloramphenicol and pyrimethamine-sulfamethoxazole. All the strains were sensitive to the glycopeptide antibiotics, vancomycin and teicoplanin. Strain typing of 181 available first isolates (from a total of 198 first isolates) by PFGE and phage lysotyping produced identical results in more than 90% of all cases. Twenty-eight different MRSA strain types were identified by PFGE and in total 23 lysotypes could be determined. During the period of investigation an increased incidence of MRSA on an intensive care unit was observed, in which a total of 204 MRSA (42% of the total number) were isolated. The strain typing using both methods showed that on that ICU eight different MRSA types were involved in this outbreak. A hygiene plan was implemented on the unit with considerable success in reducing the incidence and spread of MRSA.

In this study the production of enterotoxin A-D and toxic shock syndrome toxin-1 (TSST-1) of 181 methicillin resistant (MRSA) and 100 methicillin sensitive (MSSA) Staphylococcus aureus first isolates from different patients was investigated. All the MRSA- and MSSA isolates in the study were collected in a period between 1993 and 1995 from specimens sent from 11 different acute care hospitals in the greater Düsseldorf area. As far as possible the isolates were matched according to ward and hospital. The isolates were collected in the same time period and matched for specimen from which isolated. Furthermore, only first isolates were analysed in both groups. No significant difference in the production of toxin of any type between MRSA and MSSA could be detected (51 and 40% respectively). When the individual toxins were analysed, again no significant difference between MRSA and MSSA was demonstrable (enterotoxin production by MRSA 40% and MSSA 36%, and TSST-1 16% and 8% respectively). Despite this, a slight tendency for MRSA to produce enterotoxin A and B and for MSSA to produce enterotoxin C was observed. In addition, generation of TSST-1 by both groups was independent of enterotoxin A-D production. Interestingly, no increase in the proportion of TSST-1- or enterotoxin-producing MRSA and MSSA isolates was observed in strains isolated from blood cultures from patients with a clinical diagnosis of sepsis. Genotypical pulsed-field-gel-electrophoresis (PFGE) and phenotypical (bacteriophage typing, lysotyping) characterization of the 181 MRSA isolates resulted in 28 different PFGE patterns (of which 19 were toxin producers) and 22 lysotyping groups (18 of which produced toxin). In summary, the investigated clinical S. aureus isolates showed no difference in their ability to produce toxin and this was independent of their sensitivity to methicillin.

Toxoplasma gondii is an obligate intracellular parasite which, after primary infection of humans, is maintained in a dormant state by the host cellular immune system. In the event of an acquired immunosuppression, those parasites surviving as dormant cysts in the host may undergo a change in status, proliferate and cause a life-threatening toxoplasmic encephalitis. Over the last decade much knowlege has accumulated concerning the immune response against T. gondii. This review focuses attention particularly on the anti-parasitic effector mechanisms and the cellular immune reactions in the central nervous system during the course of reactivated toxoplasmic encephalitis.

In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2+ neurons, GFAP+ astrocytes, F4/80+ microglia, and O1+ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection.

Microglia were isolated from a murine neonatal brain cell culture in which their development had been stimulated by supplementation with the macrophage/microglial growth factor macrophage colony-stimulating factor (M-CSF). Using the whole-cell configuration of the patch-clamp technique, voltage-gated membrane currents were recorded from these microglial cells. Hyperpolarization induced inward rectifying K+ currents, as described for microglia from untreated cultures. These currents activated negative to the K+ equilibrium potential and, with a strong hyperpolarization, displayed time-dependent inactivation. The inactivation was abolished when extracellular NaCl was replaced by N-methyl-D-glucamine (NMG), thereby indicating a partial block of this K+ conductance by Na+. Inward rectifying currents were also blocked by extracellularly applied Cs+ or Ba2+. They were slightly diminished following treatment with extracellular tetraethylammonium chloride (TEA) but were not affected by 4-aminopyridine (4-AP). Upon long lasting depolarizing voltage pulses to potentials positive to 0 mV, the cells exhibited a slowly activating H+ current which could be reduced by application of inorganic polyvalent cations (Ba2+, Cd2+, Co2+, La3+, Ni2+, Zn2+) as well as by 4-AP or TEA. Based on their kinetics and pharmacological characteristics, both currents detected on M-CSF-grown microglia are suggested to correspond to the inward rectifier and the H+ current of macrophages.

Interferon-gamma (IFN-gamma) is a potent immune regulatory cytokine and is, in addition, involved in the induction of antiparasitic effector mechanisms in different cell types. The first step of IFN-gamma action is its binding to a specific receptor. Furthermore, it has been shown that IFN-gamma binds with a great affinity to the heparin-like structure of heparan sulfate, which is localized in basement membranes and on cell surfaces. In this study, we analyze the effect of heparin and heparan sulfate on three different IFN-gamma-mediated activities inducible in human glioblastoma cells (87HG31 and 86HG39). We find firstly that heparin is able to inhibit IFN-gamma-mediated induction of major histocompatibility complex (MHC) class II antigen expression on 87HG31 cells, an effect which can be abrogated by protamine. Secondly, we show that heparin inhibits the IFN-gamma-induced toxoplasmostasis within 86HG39 cells in a dose-dependent fashion, and thirdly that heparin inhibits the IFN-gamma-mediated induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase. In contrast to IFN-gamma-induced effects, the activity of other cytokines, such as interleukin (IL)-1, IL-2 and IL-6, is not influenced by heparin. The possible mechanism of heparin-induced inhibition of IFN-gamma is discussed.