In vitro evaluation of combined usage of fosfomycin and 5-fluorouracil for selective isolation of Leptospira species.

The combined usage of fosfomycin (FOM) and 5-fluorouracil (5-FU) as selective agents for the isolation of leptospires from contaminated materials was investigated. Additive or synergistic antibacterial activity was apparent with the combination compared with each agent used separately. Of 54 bacterial strains tested, 52 were inhibited, while all 5 Leptospira strains tested were unaffected by the combined addition of FOM (400 micrograms/ml) and 5-FU (100 micrograms/ml) to Korthof medium. Furthermore, this combination successfully supported the selective growth of Leptospira interrogans serovar copenhageni in experimentally contaminated specimens. This FOM-5-FU combination is surmised to be useful for the selective isolation of leptospires from contaminated clinical, pathological, or environmental materials.     

Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells.

In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled borreliae. Significant (P < 0.00005) differences in the liver uptake of the agents of Lyme borreliosis, Borrelia burgdorferi IRS, Borrelia afzelii VS461, and Borrelia garinii PBi, and that of the agents of relapsing fever, Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae, were observed. The liver uptakes ranged between 65.9% for B. burgdorferi IRS and 40.5% for B. turicatae. Neither relapsing fever nor Lyme disease borreliae were recovered from infected livers when the livers were cultured in Barbour-Stoenner-Kelly II medium. The in vitro uptake of B. burgdorferi IRS by isolated rat Kupffer cells was rapid, and within 30 min of the infection, large intracellular aggregates of amorphous material were detectable by immunofluorescence with specific anti-B. burgdorferi antibody. The reculturing of B. burgdorferi IRS from Kupffer cells incubated for 24 h in RPMI medium before inoculation with bacteria was negative. The results obtained in this study indicated that borreliae are efficiently taken up and killed by rat hepatic macrophages in the absence of serum factors.     

Interpretive criteria and quality control parameters for determining bacterial susceptibility to fosfomycin tromethamine

Studies with fosfomycin tromethamine disks containing 200 µg of fosfomycin and 50 µg of glucose-6-phosphate confirmed the following zone diameter criteria for the NCCLS method: 12 mm for resistant (MIC256 µg/ml), 13–15 mm for intermediate (MIC 128 µg/ml) and 16 mm for susceptible (MIC64 µg/ml). Additional studies defined acceptable MIC and zone diameter ranges for the following quality control strains:Escherichia coli ATCC 25922, MIC 0.5 to 4.0 µg/ml, zone diameter 23 to 29 mm;Staphylococcus aureus ATCC 25923, zone diameter 26 to 32 mm;Pseudomonas aeruginosa ATCC 27813, MIC 2.0 to 8.0 µg/ml; andEnterococcus faecalis, ATCC 29212, MIC 16 to 64 µg/ml.     

Culture ofBorrelia burgdorferi on six solid media

Six solid substrates were compared for their support of growth of ten isolates ofBorrelia burgdorferi, the causative agent of Lyme borreliosis. The following substrates with or without rabbit serum and bovine serum albumin were tested: BSK agar, BHIAM agar, TAROM agar, MEM agar, MKP agar and PMR agar. After incubation in a candle jar and a GasPak for two to four weeks,Borrelia colonies were counted and characterized. Colony morphology was related more to the growth substrate than to the characteristics of the variousBorrelia burgdorferi isolates. Culture on PMR agar resulted in the highest recovery rate and the best colony formation, with a size variation of 0.3–3.0 mm. Culture ofBorrelia burgdorferi on solid media may facilitate the diagnosis of Lyme borreliosis by enabling easier biological and genetical analysis of isolates and more accurate differentiation of strains.     

Glycerophosphodiester phosphodiesterase gene (glpQ) of Borrelia lonestari identified as a target for differentiating Borrelia species associated with hard ticks (Acari:Ixodidae)

A glpQ ortholog was identified in DNA from Borrelia lonestari-positive Amblyomma americanum, providing further evidence that B. lonestari is more closely related to the relapsing fever group spirochetes than to borreliae that cause Lyme disease. This finding provides a basis for developing diagnostic assays to differentiate species of borrelia transmitted by hard ticks.     

Selective medium for isolation of Clostridium botulinum from human feces.

A selective medium, Clostridium botulinum isolation (CBI) agar, was developed for the isolation of C. botulinum from human feces. This medium contains cycloserine (250 microgram/ml), sulfamethoxazole (76 microgram/ml), and trimethoprim (4 microgram/ml) as selective inhibitory agents. Qualitative tests indicated complete recovery of C. botulinum types A, B, F, and G on CBI medium. It was more difficult to recognize type G colonies on the medium because of their lack of lipase activity. Except for a few species of Clostridium, the growth of other obligate anaerobes and of the facultative anaerobes tested on CBI medium was suppressed. Quantitative studies of C. botulinum on the selective medium yielded counts comparable to those obtained on egg yolk agar control plates. Isolation of C. botulinum types A, B, and F from seeded fecal specimens was easily achieved with CBI medium. The use of CBI agar should aid the rapid isolation of C. botulinum from fecal specimens associated with foodborne and infant botulism.     

The relapsing fever spirochete Borrelia hermsii contains multiple, antigen-encoding circular plasmids that are homologous to the cp32 plasmids of Lyme disease spirochetes

Borrelia hermsii, an agent of tick-borne relapsing fever, was found to contain multiple circular plasmids approximately 30 kb in size. Sequencing of a DNA library constructed from circular plasmid fragments enabled assembly of a composite DNA sequence that is homologous to the cp32 plasmid family of the Lyme disease spirochete, B. burgdorferi. Analysis of another relapsing fever bacterium, B. parkeri, indicated that it contains linear homologs of the B. hermsii and B. burgdorferi cp32 plasmids. The B. hermsii cp32 plasmids encode homologs of the B. burgdorferi Mlp and Bdr antigenic proteins and BlyA/BlyB putative hemolysins, but homologs of B. burgdorferi erp genes were absent. Immunoblot analyses demonstrated that relapsing fever patients produced antibodies to Mlp proteins, indicating that those proteins are synthesized by the spirochetes during human infection. Conservation of cp32-encoded genes in different Borrelia species suggests that their protein products serve functions essential to both relapsing fever and Lyme disease spirochetes. Relapsing fever borreliae replicate to high levels in the blood of infected animals, permitting direct detection and possible functional studies of Mlp, Bdr, BlyA/BlyB, and other cp32-encoded proteins in vivo.     

Salivary gland extract from engorged Ixodes ricinus (Acari: Ixodidae) stimulates in vitro growth of Borrelia burgdorferi sensu lato

In vitro effect of salivary gland extract from fed Ixodes ricinus, the competent vector of Lyme borreliosis in Europe, on the growth of Borrelia burgdorferi sensu lato (B. garinii, B. afzelii and B. burgdorferi sensu stricto) was examined in BSK‐H medium. Motility rate, concentration of motile spirochetes and their morphology were estimated at intervals of 0, 2, 4, 6 and 8 days using darkfield microscopy. Salivary gland extract derived from I. ricinus stimulated markedly the growth of three genomic species of borreliae. The results confirm a substantial role of salivary glands in the mechanism of pathogen transmission to vertebrate host. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

GlpQ: an antigen for serological discrimination between relapsing fever and Lyme borreliosis.

Tick-borne relapsing fever is caused by numerous Borrelia species maintained in nature by Ornithodoros tick-mammal cycles. Serological confirmation is based on either an immunofluorescence assay or an enzyme-linked immunosorbent assay using whole cells or sonicated Borrelia hermsii as the antigen. However, antigenic variability of this bacterium's outer surface proteins and antigens shared with the Lyme disease spirochete (B. burgdorferi), may cause both false-negative and false-positive results when testing sera of patients suspected to have either relapsing fever or Lyme disease. To develop a specific serological test for relapsing fever, we created a genomic DNA library of B. hermsii, screened transformed Escherichia coli cells for immunoreactivity with high-titered (> or = 1:2,048) human anti-B. hermsii antiserum, and selected an immunoreactive clone (pSPR75) expressing a 39-kDa protein. DNA sequencing, subcloning, and serum adsorption experiments identified the immunoreactive protein as a homolog of GlpQ, a glycerophosphodiester phosphodiesterase identified previously in E. coli, Haemophilus influenzae, and Bacillus subtilis. Serum samples from humans and mice infected with B. hermsii or other species of relapsing fever spirochetes contained antibodies recognizing GlpQ, whereas serum samples from Lyme disease and syphilis patients were nonreactive. Serologic tests based on this antigen will identify people exposed previously to relapsing fever spirochetes and help clarify the distribution of relapsing fever and Lyme disease in situations in which the occurrence of their causative agents is uncertain.     

In vitro activity of cefpodoxime against bacterial isolates obtained from patients with cancer

The in vitro activity of cefpodoxime, an oral cephalosporin ester, against 792 bacterial isolates representing 36 species was evaluated in comparison to that of ciprofloxacin and trimethoprim/sulfamethoxazole (TMP/SMX). Cefpodoxime inhibited the majority ofStreptococcus spp.,Haemophilus influenzae andProteus mirabilis at a concentration of 0.12 µg/ml. It was also active againstCitrobacter diversus, Escherichia coli, Klebsiella spp.,Proteus vulgaris, Serratia marcescens and methicillin-susceptibleStaphylococcus aureus isolates. Overall, cefpodoxime appeared to be less active than ciprofloxacin and TMP/SMX against many pathogens common in cancer patients.     

Fibronectin-Binding Protein of Borrelia hermsii Expressed in the Blood of Mice with Relapsing Fever

To identify and characterize surface proteins expressed by the relapsing fever (RF) agent Borrelia hermsii in the blood of infected mice, we used a cell-free filtrate of their blood to immunize congenic naive mice. The resultant antiserum was used for Western blotting of cell lysates, and gel slices corresponding to reactive bands were subjected to liquid chromatography-tandem mass spectrometry, followed by a search of the proteome database with the peptides. One of the immunogens was identified as the BHA007 protein, which is encoded by a 174-kb linear plasmid. BHA007 had sequence features of lipoproteins, was surface exposed by the criteria of in situ protease susceptibility and agglutination of Vtp⁻ cells by anti-BHA007 antibodies, and was not essential for in vitro growth. BHA007 elicited antibodies during experimental infection of mice, but immunization with recombinant protein did not confer protection against needle-delivered infection. Open reading frames (ORFs) orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of Borrelia duttonii and B. recurrentis, but not in Lyme disease Borrelia species. Recombinant BHA007 bound both human and bovine fibronectin with K_d (dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is a member of a large family of multifunctional proteins in Borrelia species that bind to fibronectin as well as other host proteins.     

Use of fosfomycin disks for isolation of diphtheroids

A fosfomycin disk (200 µg) placed on blood agar was used to select diphtheroids from clinical specimens. AllCorynebacterium type strains tested, representing ten different groups of diphtheroids, and 150 toxigenic strains ofCorynebacterium diphtheriae from different outbreaks, overgrew the fosfomycin disk, while other aerobic gram-positive rods had a large inhibition zone. The method increased the rate of isolation of diphtheroids in 1267 clinical specimens fivefold, from 1.4 % to 7.6 %, especially when the diphtheroids were found concomitantly with other bacteria. Overgrowth of the disk by other microorganisms occurred in 11 % of the samples tested, but such microorganisms could easily be distinguished by culture morphology or Gram stain. Fosfomycin disks provide a simple, highly sensitive but not very specific aid for isolation ofCorynebacterium diphtheriae and diphtheroids from clinical specimens.     

In vitro activity of fosfomycin alone and in combination with amoxicillin, clarithromycin and metronidazole against Helicobacter pylori compared with combined clarithromycin and metronidazole

In order to evaluate the suitability of fosfomycin in combination with other agents for the treatment of Helicobacter pylori infections, the susceptibility profiles of 65 H. pylori strains were determined against multiple antimicrobial agents and combinations thereof using the agar dilution method. For fosfomycin alone, the range of minimum inhibitory concentration (MIC) results and the MICs at which 50% and 90% of strains were inhibited were 0.5–32 g/ml and 2 and 4 g/ml, respectively. For the combination of fosfomycin with amoxicillin, clarithromycin or metronidazole, the means calculated for the minimum and maximum fractional inhibitory concentration index were 0.70–1.17 and 1.15–2.03, respectively, suggesting partial synergy or indifference in the majority of strains. The combination of clarithromycin and metronidazole showed synergistic activity against 14 of 28 H. pylori strains tested. The in vitro activity results suggest the combination of fosfomycin with either amoxicillin or clarithromycin may be a promising alternative for the treatment of H. pylori infection. However, the clinical efficacy of these regimens remains to be investigated.     

Comparison of growth and morphology of Borrelia burgdorferi sensu lato in BSK-H and BSK-II media stored for prolonged periods

Barbour-Stoenner-Kelly II (BSK-II) and BSK-H media were used for cultivation and isolation of fastidious Borrelia species - the causative agents of Lyme borreliosis. Culture media have a limited shelf life and require adequate storage. Our goal was to assess how the growth of Borrelia would be affected by prolonged storage of media and inadequate storage conditions (BSK-H stored at +4 °C for 2.5 years and BSK-II stored at −20 °C for 11 years). Growth of different Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana strains was assessed during 2 weeks of incubation at 33 °C. Monitored parameters included cell count per mL, morphology and motility. The results of this study have shown weaker growth of borrelia strains in BSK-H at +4 °C (median final cell number of 1.5 × 10⁶/mL) than in BSK-II at −20 °C (median final cell number of 7.75 × 10⁶/mL) and in fresh BSK-H media (median final cell number of 8.95 × 10⁶/mL). Duration of storage of media had no impact on Borrelia morphology and motility. Our results indicate that temperature of −20 °C is optimal for long-term storage of medium, BSK-II stored for 11 years provided effective support to growth of Borrelia and may be employed for cultivation.     

Two cases of multidrug-resistant Nocardia farcinica infection in immunosuppressed patients and implications for empiric therapy

Presented here are two cases of multidrug-resistant Nocardia farcinica infection that occurred in immunocompromised patients. One of the patients developed the infection despite being on trimethoprim/sulfamethoxazole prophylaxis for Pneumocystis jiroveci. These cases demonstrate the propensity of Nocardia spp. to cause disseminated disease and to develop resistance to multiple antimicrobial agents used in the initial treatment of serious Nocardia infection. These factors lead to the conclusion that empiric monotherapy with trimethoprim/sulfamethoxazole may not be sufficient. Treatment with a combination regimen of imipenem and amikacin may be a more promising initial therapy.     

Efficacy of fosfomycin and its combination with linezolid, vancomycin and imipenem in an experimental peritonitis model caused by a Staphylococcus aureus strain with reduced susceptibility to vancomycin

The objective of this study was to evaluate the in vitro and in vivo efficacies of therapies including fosfomycin against clinical Staphylococcus aureus isolates with reduced susceptibility to vancomycin (hGISA). Time–kill curves were performed over 24 h. Peritonitis in C57BL/6 mice was induced by intraperitoneal inoculation of 10⁸ CFU/ml. Four hours later (0 h), therapy was started and the treatment groups were: control (not treated), fosfomycin (100 mg/kg/5 h), vancomycin (60 mg/kg/5 h), imipenem (30 mg/kg/5 h), fosfomycin plus linezolid, fosfomycin plus vancomycin and fosfomycin plus imipenem, receiving subcutaneous therapy over 25 h. Bacterial counts in peritoneal fluid, bacteraemia and mortality rates were determined. In vitro, fosfomycin showed a synergistic effect when combined with the other antimicrobials tested. In the animal model, fosfomycin combinations were effective and significantly reduced the bacteraemia rates achieved in the control, imipenem and vancomycin groups (p < 0.05). The best combination in vivo was fosfomycin plus imipenem. Also, fosfomycin plus linezolid was significantly better than vancomycin alone, reducing the bacterial concentration in the peritoneal fluid. In conclusion, in vitro and in vivo, fosfomycin in combination with linezolid, vancomycin or imipenem exerted a good activity. Fosfomycin plus imipenem was the most active combination, decreasing 3 log CFU/ml, and appears to be a promising combination for clinical practice.     

Beneficial effect of sulfamethoxazole and trimethoprim on accelerated coagulation

We have shown that the drug combination of sulfamethoxazole and trimethoprim has a radio-protective effect in mice. Since radiation injury is due in part to microvascular thrombosis produced by the release or action of thromboplastin, we evaluated the effect of these drugs individually on thromboplastin-accelerated coagulation. Sulfamethoxazole at a final concentration of 0.05 g/ml and trimethoprim at concentrations of 0.005 and 0.05 g/ml significantly prolonged the recalcification times of human plasma. Whether these drugs have an anticoagulant effect when employed clinically remains to be determined.     

In vitro susceptibility of“Campylobacter upsaliensis” to twenty-four antimicrobial agents

The in vitro susceptibility of 41 strains ofCampylobacter upsaliensis to 24 antimicrobial agents was determined using a broth microdilution procedure. Most isolates were susceptible to the fluoroquinolones and -lactam antibiotics tested, but all strains were resistant to trimethoprim (MBCs 128 µg/ml) and teicoplanin (MBCs 32 µg/ml). These agents may be useful in a selective isolation medium forCampylobacter upsaliensis.     

Naegleria fowleri andN. lovaniensis: Differences in sensitivity to trimethoprim and other antifolates

The growth ofNaegleria fowleri cultures in a BCS medium was not affected either by trimethoprim at 400 g/ml or by aminopterine, 3,5-diaminopterine and methotrexate at 500 g/ml.N. lovaniensis propagation in the same medium was inhibited with 10 g/ml of trimethoprim, 50 g/ml methotrexate and 100 g/ml 3,5-diaminopteridine. Aminopterine was ineffective at a concentration of 500 g/ml. The inhibitory effect of trimethoprim onN. lovaniensis cultures depended on the medium composition and could be neutralized by an addition of folic or tetrahydrofolic acids and a suspension of heat-killedEnterobacter aerogenes. Thymine, thymidine, hypoxantine and 2-amino-4-hydroxy-6-(tatrahydroxybutyl)-pteridine did not have an adverse effect. Trimethoprim activity inN. fowleri cultures could not be enhanced by the addition of Triton X-100 and Polymyxine B. Cryolyzate ofN. fowleri amoebae did not influence the trimethoprim inhibition ofN. lovaniensis cultures. Deviation in dihydrofolatereductase chemical structure or thymine dependency seems to be the probable explanation forN. fowleri antifolate resistance.     

Improved selective medium for the isolation of lipase-positive Clostridium botulinum from feces of human infants.

Isolation of lipase-positive Clostridium botulinum from fecal specimens establishes the diagnosis of infant botulism, contributes to the diagnosis of food-borne botulism, and is most easily accomplished by use of selective media. Modification of an available selective medium, C. botulinum inhibitory medium (CBI), enabled more rapid isolation of C. botulinum. The modified medium (botulinum selective medium [BSM] contained (per liter) 25 g of dehydrated heart infusion broth, 20 g of agar, 30 ml of egg yolk suspension, 250 mg of cycloserine, 76 mg of sulfamethoxazole, 4 mg of trimethoprim, and 100 IU of thymidine phosphorylase at pH 7.4. The two media were compared by using 15 fresh fecal specimens from infant botulism patients (10 type A and 5 type B) and a C. botulinum isolate that had been obtained from an infant botulism patient and that was mixed into a fresh stool specimen from a healthy human infant. In comparison to CBI, BSM always provided better suppression of the nonbotulinum fecal flora and earlier emergence of lipase-positive colonies. Diagnosis of infant botulism was accomplished sooner with BSM than with CBI because isolation of lipase-positive C. botulinum was easier.