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PubMed1999

In vitro activities of 13 fluoroquinolones against Staphylococcus aureus isolates with characterized mutations in gyrA, gyrB, grlA, and norA and against wild-type isolates.

The in vitro activities of 13 fluoroquinolones (FQs) were tested against 90 Staphylococcus aureus clinical isolates: 30 wild type for gyrA, gyrB, grlA and norA and 60 with mutations in these genes. Clinafloxacin (CI-960), sparfloxacin, and grepafloxacin were the most active FQs against wild-type isolates (MICs at which 90% of isolates were inhibited, 0.06 to 0.1 microgram/ml). Mutations in grlA did not affect the MICs of newer FQs. grlA-gyrA double mutations led to higher MICs for all the FQs tested. Efflux mechanisms affected the newer FQs to a much lesser extent than the less recently developed FQs.

Anti-Infective Agents↗
PubMed2000

Isolation and characterization of tetracycline-resistant clinical isolates of Helicobacter pylori.

Tetracycline is an important component of combination therapies for Helicobacter pylori eradication. Twenty-nine tetracycline-resistant isolates requiring MICs ranging from 4 to 16 microgram/ml were isolated from Korean (22 of 460) and Japanese (7 of 105) patients. Interestingly, all of the 29 tetracycline-resistant isolates exhibited cross-resistance to metronidazole, and the cross-resistance was transferred to tetracycline-sensitive H. pylori strains.

Drug Resistance, Multiple↗
PubMed2000

A Canadian national surveillance study of urinary tract isolates from outpatients: comparison of the activities of trimethoprim-sulfamethoxazole, ampicillin, mecillinam, nitrofurantoin, and ciprofloxacin. The Canadian Urinary Isolate Study Group.

Ampicillin, trimethoprim-sulfamethoxazole, mecillinam, nitrofurantoin, and ciprofloxacin mean resistance rates for 2,000 urinary tract isolates collected from outpatients across Canada in 1998 were 41.1, 19.2, 14.7, 5.0, and 1.8%, respectively. For Escherichia coli isolates alone (n = 1,681) comparable rates were 41. 0, 18.9, 7.4, 0.1, and 1.2%, respectively. The majority of E. coli isolates resistant to ampicillin, trimethoprim-sulfamethoxazole, or ciprofloxacin were susceptible (MIC, <16 microg/ml) to mecillinam.

Amdinocillin↗
PubMed2003

In vitro activities of caspofungin compared with those of fluconazole and itraconazole against 3,959 clinical isolates of Candida spp., including 157 fluconazole-resistant isolates.

Caspofungin is an echinocandin antifungal agent with broad-spectrum activity against Candida and Aspergillus spp. The in vitro activities of caspofungin against 3,959 isolates of Candida spp. obtained from over 95 different medical centers worldwide were compared with those of fluconazole and itraconazole. The MICs of the antifungal drugs were determined by broth microdilution tests performed according to the NCCLS method using RPMI 1640 as the test medium. Caspofungin was very active against Candida spp. (MIC at which 90% of the isolates were inhibited [MIC(90)], 1 micro g/ml; 96% of MICs were < or =2 micro g/ml). Candida albicans, C. dubliniensis, C. tropicalis, and C. glabrata were the most susceptible species of Candida (MIC(90), 0.25 to 0.5 micro g/ml), and C. guilliermondii was the least susceptible (MIC(90), >8 micro g/ml). Caspofungin was very active against Candida spp., exhibiting high-level resistance to fluconazole and itraconazole (99% of MICs were < or =1 micro g/ml). These results provide further evidence for the spectrum and potency of caspofungin activity against a large and geographically diverse collection of clinically important isolates of Candida spp.

Anti-Bacterial Agents↗
PubMed2006

In vivo transfer of the vanA resistance gene from an Enterococcus faecium isolate of animal origin to an E. faecium isolate of human origin in the intestines of human volunteers.

Transient colonization by vancomycin-resistant enterococci of animal origin has been documented in the intestines of humans. However, little is known about whether transfer of the vanA gene occurs in the human intestine. Six volunteers ingested a vancomycin-resistant Enterococcus faecium isolate of chicken origin, together with a vancomycin-susceptible E. faecium recipient of human origin. Transconjugants were recovered in three of six volunteers. In one volunteer, not only was vancomycin resistance transferred, but also quinupristin-dalfopristin resistance. This study shows that transfer of the vanA gene from an E. faecium isolate of animal origin to an E. faecium isolate of human origin can occur in the intestines of humans. It suggests that transient intestinal colonization by enterococci carrying mobile elements with resistance genes represents a risk for spread of resistance genes to other enterococci that are part of the human indigenous flora, which can be responsible for infections in certain groups of patients, e.g., immunocompromised patients.

Animals↗
PubMed1993

Isolation of Typical Marine Bacteria by Dilution Culture: Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions.

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 10 to 1.07 x 10 cells per liter. The mean cell volume varied between 0.042 and 0.074 mum, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 x 10 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 mum and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a degrees (A), 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a degrees (A), +/- 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.

Journal Article↗
PubMed1998

Study of toxin production by isolates of Stachybotrys chartarum and Memnoniella echinata isolated during a study of pulmonary hemosiderosis in infants.

A cluster of cases of pulmonary hemosiderosis among infants was reported in Cleveland, Ohio, during 1993 and 1994. These unusual cases appeared only in infants ranging in age from 1 to 8 months and were characterized by pulmonary hemorrhage, which caused the babies to cough up blood. A case-control study identified major home water damage (from plumbing leaks, roof leaks, or flooding) as a risk factor for development of pulmonary hemorrhage in these infants. Because of an interest in the possibility that trichothecene mycotoxins might be involved in this illness, a number of isolates of Stachybotrys chartarum were grown in the laboratory on rice, and extracts were prepared and analyzed both for cytotoxicity and for specific toxins. Two isolates of Memnoniella echinata, a fungus closely related to S. chartarum, were also included in these studies. S. chartarum isolates collected from the homes were shown to produce a number of highly toxic compounds, and the profiles of toxic compounds from M. echinata were similar; the most notable difference was the fact that the principal metabolites produced by M. echinata were griseofulvins.

Animals↗
PubMed1999

Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding bacteriophage phi3538 isolated from Escherichia coli O157:H7.

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx(2) gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage phi3538(Deltastx(2)::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with phi3538(Deltastx(2)::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for phi3538(Deltastx(2)::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of phi3538(Deltastx(2)::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that phi3538(Deltastx(2)::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.

Bacterial Toxins↗
PubMed2001

In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I.

We previously showed that very thin filamentous bacteria affiliated with the division green non-sulfur bacteria were abundant in the outermost layer of thermophilic methanogenic sludge granules fed with sucrose and several low-molecular-weight fatty acids (Y. Sekiguchi, Y. Kamagata, K. Nakamura, A. Ohashi, H. Harada, Appl. Environ. Microbiol. 65:1280-1288, 1999). Further 16S ribosomal DNA (rDNA) cloning-based analysis revealed that the microbes were classified within a unique clade, green non-sulfur bacteria (GNSB) subdivision I, which contains a number of 16S rDNA clone sequences from various environmental samples but no cultured representatives. To investigate their function in the community and physiological traits, we attempted to isolate the yet-to-be-cultured microbes from the original granular sludge. The first attempt at isolation from the granules was, however, not successful. In the other thermophilic reactor that had been treating fried soybean curd-manufacturing wastewater, we found filamentous microorganisms to outgrow, resulting in the formation of projection-like structures on the surface of granules, making the granules look like sea urchins. 16S rDNA-cloning analysis combined with fluorescent in situ hybridization revealed that the projections were comprised of the uncultured filamentous cells affiliated with the GNSB subdivision I and Methanothermobacter-like cells and the very ends of the projections were comprised solely of the filamentous cells. By using the tip of the projection as the inoculum for primary enrichment, a thermophilic, strictly anaerobic, filamentous bacterium, designated strain UNI-1, was successfully isolated with a medium supplemented with sucrose and yeast extract. The strain was a very slow growing bacterium which is capable of utilizing only a limited range of carbohydrates in the presence of yeast extract and produced hydrogen from these substrates. The growth was found to be significantly stimulated when the strain was cocultured with a hydrogen-utilizing methanogen, Methanothermobacter thermautotrophicus, suggesting that the strain is a sugar-fermenting bacterium, the growth of which is dependent on hydrogen consumers in the granules.

Animals↗
PubMed2004

Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis.

Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).

Bacterial Adhesion↗
PubMed1991

Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure.

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates but not deoxyribonucleotide triphosphates. Ribonucleotide reductase is the only enzyme known to catalyze the direct conversion of a ribonucleotide to a deoxyribonucleotide. Hydroxyurea inhibits ribonucleotide reductase by inactivating the tyrosine free radical present in the small subunit of the enzyme. In this report, we show that Chlamydia trachomatis growth is inhibited by hydroxyurea in both wild-type mouse L cells and hydroxyurea-resistant mouse L cells. Hydroxyurea was used as a selective agent in culture to isolate, by a stepwise procedure, a series of C. trachomatis isolates with increasing levels of resistance to the cytotoxic effects of the drug. One of the drug-resistant C. trachomatis isolates (L2HR-10.0) was studied in more detail. L2HR-10.0 retained its drug resistance phenotype even after passage in the absence of hydroxyurea for 10 growth cycles. In addition, L2HR-10.0 was cross resistant to guanazole, another inhibitor of ribonucleotide reductase. Results obtained from hydroxyurea inhibition studies using various host cell-parasite combinations indicated that inhibition of host cell and C. trachomatis DNA synthesis by hydroxyurea can occur but need not occur simultaneously. Crude extract prepared from highly purified C. trachomatis reticulate bodies was capable of reducing CDP to dCDP. The CDP reductase activity was not inhibited by monoclonal antibodies to the large and small subunits of mammalian ribonucleotide reductase, suggesting that the activity is chlamydia specific. The CDP reductase activity was inhibited by hydroxyurea. Crude extract prepared from drug-resistant L2HR-10.0 reticulate bodies contained an elevation in ribonucleotide reductase activity. In total, our results indicate that C. trachomatis obtains the precursors for DNA synthesis as ribonucleotides with subsequent conversion to deoxyribonucleotides catalyzed by a chlamydia-specific ribonucleotide reductase.

Adenine↗
PubMed2006

Molecular epidemiology of multidrug-resistant Mycobacterium bovis isolates with the same spoligotyping profile as isolates from animals.

PCR-based characterization techniques have been adopted in most laboratories for Mycobacterium bovis typing. We report a molecular characterization of human multidrug-resistant M. bovis isolates and three bovine isolates that share the spoligotyping profile. The analysis of the direct repeat region showed that both groups differed in the presence of spacers not included in the current membrane. They were also distinguished by two out of the nine mycobacterial interspersed repetitive unit variable-number tandem repeat loci tested, indicating that the human infection was not acquired from the cattle from which isolates were obtained. These results highlight that a combination of techniques is required for appropriate discrimination, even for those spoligotypes that have a low frequency.

Animals↗
PubMed1985

Isolation and enumeration of Clostridium botulinum by direct inoculation of infant fecal specimens on egg yolk agar and Clostridium botulinum isolation media.

Direct plating of stool specimens on selective (Clostridium botulinum isolation) and nonselective (egg yolk agar) media was evaluated as an aid in confirming infant botulism. C. botulinum was isolated from 13 of 14 culture-positive specimens with C. botulinum isolation and from 8 of 14 egg yolk agar. No lipase reaction was seen on plates of 31 culture-negative specimens.

Agar↗
PubMed2000

Frequency of isolation of Staphylococcus lugdunensis among staphylococcal isolates causing endocarditis: a 20-year experience.

Eighty-nine staphylococcal isolates recovered from patients with bacterial endocarditis at the Mayo Clinic from 1980 to 1999 were studied to determine the prevalence of Staphylococcus lugdunensis among clinical isolates of staphylococci causing endocarditis. Four isolates, all from patients with native mitral valve endocarditis, were identified as S. lugdunensis.

Adult↗
PubMed2002

Human cytomegalovirus and human umbilical vein endothelial cells: restriction of primary isolation to blood samples and susceptibilities of clinical isolates from other sources to adaptation.

In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 examined. Analysis of other clinical samples inoculated as controls revealed, in the presence of highly infected HELF monolayers, either the presence of very few infected HUVEC with urine specimens (n = 10 samples) or the lack of infected HUVEC with throat washes (n = 3) or amniotic fluid samples (n = 2). Thus, peripheral blood leukocytes (PBL) appear essential for primary isolation of HCMV in HUVEC. In this respect, HCMV strains, recovered from clinical samples other than buffy coats in HELF only, could be readily adapted to growth in HUVEC by coculturing PBL from healthy blood donors with infected HELF and then inoculating infected PBL onto HUVEC. Recently elucidated mechanisms of interaction of leukocytes and HUVEC with bidirectional transfer of virus seem to provide the basis for the restriction of HCMV primary isolation in HUVEC to blood samples. However, virus strains recovered from only HELF could be adapted to growth in HUVEC when inoculated with HELF-derived (either cell-associated or cell-free) HCMV strains upon primary isolation. In conclusion, due to the in vitro selection of virus variants provided with both PBL tropism and HUVEC tropism, HCMV recovery in HUVEC is PBL mediated and substantially restricted to blood samples. Lack of HCMV recovery in HUVEC from clinical samples other than blood leads to the assumption that epithelial cells, such as urinary, amniotic, or pharyngeal cells, do not possess adequate adhesion molecules to establish close contacts with HUVEC.

Cells, Cultured↗
PubMed2004

Trends in antimicrobial resistance in 1,968 invasive Streptococcus pneumoniae strains isolated in Spanish hospitals (2001 to 2003): decreasing penicillin resistance in children's isolates.

To address the public health problem of antibiotic resistance, the European Union (EU) founded the European Antimicrobial Resistance Surveillance System. A network of 40 hospitals that serve approximately 30% of the Spanish population (about 12 million) participated. Each laboratory reported data on antimicrobial susceptibility testing using standard laboratory procedures that were evaluated by an external quality control program. The antibiotic consumption data were obtained from the National Health System. We compared the antibiotic susceptibility of Spanish isolates of invasive Streptococcus pneumoniae (2001 to 2003) with antibiotic consumption. Invasive S. pneumoniae was isolated from 1,968 patients, 20% of whom were children at or below the age of 14 years. Of non-penicillin-susceptible strains (35.6%; 95% confidence interval, 34 to 37.2), 26.4% were considered intermediate and 9.2% were considered resistant. Between 2001 and 2003, penicillin resistance decreased from 39.5 to 33% overall and from 60.4 to 41.2% in children at or below the age of 14 years (P = 0.002). Resistance to erythromycin was at 26.6%, and coresistance with penicillin was at 19.1%. Of total isolates, the ciprofloxacin MIC was >2 mug/ml for 2.1%, with numbers increasing from 0.4% (2001) to 3.9% (2003). Total antibiotic use decreased from 21.66 to 19.71 defined daily doses/1,000 inhabitants/day between 1998 and 2002. While consumption of broad-spectrum penicillins, cephalosporins, and erythromycin declined, use of amoxicillin-clavulanate and quinolones increased by 17.5 and 27%, respectively. The frequency of antibiotic resistance in invasive S. pneumoniae in Spain was among the highest in the EU. However, a significant decrease in penicillin resistance was observed in children. This decrease coincided with the introduction of a heptavalent conjugate pneumoccocal vaccine (June 2001) and with a global reduction in antibiotic consumption levels.

Adolescent↗
PubMed2005

In vitro activities of anidulafungin against more than 2,500 clinical isolates of Candida spp., including 315 isolates resistant to fluconazole.

Anidulafungin is an echinocandin antifungal agent with potent activity against Candida spp. We assessed the in vitro activity of anidulafungin against 2,235 clinical isolates of Candida spp. using the CLSI broth microdilution method. Anidulafungin was very active against Candida spp. (the MIC at which 90% of strains are inhibited [MIC(90)] was 2 microg/ml when MIC endpoint criteria of partial inhibition [MIC-2] were used). Candida albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr were the most susceptible species of Candida (MIC(90), 0.06 to 0.12 microg/ml), and C. parapsilosis, C. lusitaniae, and C. guilliermondii were the least susceptible (MIC(90), 0.5 to 2 microg/ml). In addition, 315 fluconazole-resistant isolates were tested, and 99% were inhibited by < or =1 microg/ml of anidulafungin. These results provide further evidence for the spectrum and potency of anidulafungin activity against a large and geographically diverse collection of clinically important isolates of Candida spp.

Anidulafungin↗