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1.
Arch Intern Med ; 161(9): 1217-20, 2001 May 14.
Article in English | MEDLINE | ID: mdl-11343444

ABSTRACT

BACKGROUND: Therapy for Helicobacter pylori is generally empiric despite the fact that resistance to metronidazole and clarithromycin compromise therapeutic efficacy. The aim of this study was to aid clinicians in choosing a course of therapy for H pylori infection in the United States. METHODS: The frequency of primary clarithromycin and metronidazole resistance among H pylori isolated from patients enrolled in US-based clinical trials between 1993 and 1999 was reviewed in relation to patient age, sex, region of the United States, and test method (Etest and 2 agar dilution procedures). RESULTS: Clarithromycin and metronidazole resistance rates were based on the results of 3439 pretreatment Etest determinations and 3193 agar dilution determinations. Sex and age were available on 900 and 823 individuals, respectively. Metronidazole resistance was 39% by Etest and 21.6% by agar dilution (P<.001). Clarithromycin resistance was 12% by Etest and 10.6% by agar dilution. Amoxicillin or tetracycline resistance was rare. Metronidazole and clarithromycin resistance was more common in women than men (eg, 34.7% vs 22.6% for metronidazole and 14.1% vs 9.7% for clarithromycin (P =.01 and P =.06, respectively). Antibiotic resistance increased gradually up to age 70 years, then declined significantly (P<.05) regardless of test method. Regional differences in antimicrobial resistance did not occur. CONCLUSIONS: While age and sex had significant effects on resistance rates, regional differences were not present. The high prevalence of resistance to metronidazole and clarithromycin may soon require the performance of antimicrobial susceptibility testing of H pylori isolates prior to initiating treatment.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Antitrichomonal Agents/therapeutic use , Clarithromycin/therapeutic use , Helicobacter Infections/drug therapy , Helicobacter pylori/drug effects , Metronidazole/therapeutic use , Adult , Age Factors , Aged , Drug Resistance, Microbial , Helicobacter Infections/epidemiology , Helicobacter Infections/microbiology , Helicobacter pylori/pathogenicity , Humans , Middle Aged , Prevalence , Retrospective Studies , Sex Factors , United States/epidemiology
2.
FEMS Microbiol Lett ; 196(2): 93-8, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11267762

ABSTRACT

D-Alanine is a necessary precursor in the biosynthesis of the bacterial peptidoglycan. The naturally occurring L-alanine isomer is racemized to its D-form through the action of a class of enzymes called alanine racemases. These enzymes are ubiquitous among prokaryotes, and with very few exceptions are absent in eukaryotes, making them a logical target for the development of novel antibiotics. The alanine racemase gene from both Mycobacterium tuberculosis and M. avium was amplified by PCR and cloned in Escherichia coli. Overexpression of the proteins in the E. coli BL21 system, both as native and as His-tagged recombinant products, has been achieved. The proteins have been purified to electrophoretic homogeneity and analyzed biochemically. A D-alanine requiring double knock-out mutant of E. coli (alr, dadX) was constructed and the cloned genes were able to complement its deficiencies.


Subject(s)
Alanine Racemase/metabolism , Mycobacterium avium/enzymology , Mycobacterium tuberculosis/enzymology , Alanine Racemase/genetics , Alanine Racemase/isolation & purification , Amino Acid Sequence , Cloning, Molecular , DNA Primers , Escherichia coli , Genes, Bacterial , Molecular Sequence Data , Mycobacterium avium/genetics , Mycobacterium tuberculosis/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment
3.
Int J Antimicrob Agents ; 17(1): 39-44, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11137647

ABSTRACT

Although the NCCLS has approved the agar dilution method as the test of choice for antimicrobial susceptibility testing of Helicobacter pylori, a critical evaluation of this method in clinical trials to detect antibiotic resistance has not been performed. This study compares the Etest and agar dilution methods for detection of metronidazole and clarithromycin resistance in clinical isolates of H. pylori. MIC data were gathered from US-based clinical trials. The Etest was performed on Mueller-Hinton sheep blood agar plates following incubation for 4 days under 12% CO(2). The agar dilution test was performed according to the recently approved NCCLS methodology using aged sheep blood in a Mueller-Hinton agar base. Metronidazole resistance as determined by Etest was significantly higher than that determined by agar dilution (39%; 690/1768 vs. 25. 1%; 367/1465)(P<0.01). Clarithromycin resistance as determined by Etest was higher than that determined by agar dilution, but was not significantly different (12.5%; 209/1671 vs. 10.6%; 150/1414)(P>0.5). Inter-patient metronidazole resistance showed that the MIC values for identical isolates tested by both methods were equivalent in 58% (109/188). Of the 42% with a >2log(2) difference in MIC values, 17. 6% had a change in susceptibility pattern. For clarithromycin, 71.4% (237/332) of the MIC values for identical isolates tested by both methods had equivalent MIC values. Of the MIC values with a >2log(2) difference in MIC values, only 3% showed a change in susceptibility pattern. Intra-patient variability, i.e. paired isolates from the same patient, was assessed only for metronidazole. Of the 1393 paired isolates tested by Etest, 38.8% were shown to be resistant. Almost 69% of the Etest MIC determinations were deemed equivalent and 16.7% had a change in susceptibility pattern. Of the 639 paired isolates tested by agar dilution, 23.9% were resistant to metronidazole. Almost 72% of the agar dilution MIC values were equivalent and 11.3% of the determinations had a change in susceptibility pattern. Clarithromycin resistance rates are similar, when determined by either test method. The Etest yields a significantly higher prevalence of metronidazole resistance among H. pylori compared with the agar dilution method and both methods yield discordant results, when isolates from different parts of the same stomach are compared. Neither method is reliable in determining metronidazole resistance in H. pylori.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clarithromycin/pharmacology , Helicobacter pylori/drug effects , Metronidazole/pharmacology , Microbial Sensitivity Tests , Colony Count, Microbial/methods , Drug Resistance, Microbial , Evaluation Studies as Topic , Humans , Microbial Sensitivity Tests/methods , Quality Control , Reproducibility of Results
4.
Cornea ; 19(4): 517-20, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10928770

ABSTRACT

PURPOSE: To compare acid-base and oxidation-reduction indicators and to investigate the effect of buffer and temperature on the colorimetric detection of microbial growth in corneal preservation media. METHODS: Corneal preservation media containing gentamicin, without or with HEPES buffer, were prepared with either phenol red or AlamarBlue indicators (AccuMed International, Westlake, OH, U.S.A.). Both media were inoculated with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, Serratia marcescens, or Candida albicans and then incubated at 4 degrees C, 22 degrees C, or 35 degrees C. The pH or percent reduction were determined hourly for eight hours, then daily for one week. RESULTS: The length of time before a confirmed change in pH or reduction occurred varied by microorganism, storage temperature, and buffering capacity. At 4 degrees C, none of the microorganisms caused a detectable pH change in buffered medium within one day after inoculation, although two bacterial species reduced AlamarBlue within four hours. At 22 degrees C and 35 degrees C, all bacteria except P. aeruginosa produced a pH shift within a few hours, and all tested bacterial species reduced AlamarBlue. For bacteria producing detectable pH changes, HEPES-buffered medium took longer to change than medium without HEPES. C. albicans was not detectable in HEPES-buffered medium at any temperature by phenol red and was only detectable by AlamarBlue after 2-3 days at 22 degrees C and 35 degrees C. CONCLUSION: Acidic shifts in refrigerated corneal preservation medium do not occur during contamination by several microorganisms. AlamarBlue, a redox indicator, is more sensitive than phenol red in detecting some bacteria. C. albicans is not reliably detected by pH or redox indicators.


Subject(s)
Bacteria/growth & development , Colorimetry/methods , Cornea , Drug Contamination , Fungi/growth & development , Organ Preservation Solutions/chemistry , Organ Preservation , Oxazines , Xanthenes , Coloring Agents , Corneal Transplantation , Humans , Hydrogen-Ion Concentration , Indicators and Reagents , Phenolsulfonphthalein , Quality Control
5.
Cornea ; 19(1): 26-9, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10632004

ABSTRACT

PURPOSE: To describe the epidemiology of Vibrio eye infections. METHOD: We reviewed the records of a patient from our institution with V. vulnificus keratitis and conducted a literature search for other cases of ocular infections with Vibrio species. RESULTS: A 39-year-old fisherman was struck in his left eye with an oyster shell fragment, developed suppurative V. vulnificus keratitis, and was successfully treated with combined cefazolin and gentamicin. Including our patient, 17 cases of eye infections with Vibrio spp. have been reported, and 11 (65%) involved exposure to seawater or shellfish. Of the seven cases due to V. vulnificus (six keratitis and one endophthalmitis), six had known exposure to shellfish or seawater along the U.S. coast of the Gulf of Mexico. Of five cases of V. alginolyticus conjunctivitis, three had been exposed to fish or shellfish. Three infections with V. parahaemolyticus (one keratitis and two endophthalmitis) were reported; two of these occurred in people exposed to brackish water on or near the Gulf Coast. Two cases of postsurgical endophthalmitis, one with V. albensis and one with V. fluvialis, also were reported. CONCLUSIONS: In addition to septicemia, gastroenteritis, and wound infections, halophilic noncholera Vibrio species can cause sight-threatening ocular infections. Ocular trauma by shellfish from contaminated water is the most common risk factor for Vibrio conjunctivitis and keratitis. Nearly one half of reported Vibrio infections of the eye occurred along the U.S. coast of the Gulf of Mexico.


Subject(s)
Corneal Ulcer/epidemiology , Eye Infections, Bacterial/epidemiology , Vibrio Infections/epidemiology , Vibrio/isolation & purification , Adult , Animals , Cefazolin/therapeutic use , Cornea/microbiology , Corneal Injuries , Corneal Ulcer/drug therapy , Corneal Ulcer/microbiology , Drug Therapy, Combination , Eye Infections, Bacterial/drug therapy , Eye Infections, Bacterial/etiology , Eye Injuries/drug therapy , Eye Injuries/epidemiology , Eye Injuries/microbiology , Gentamicins/therapeutic use , Humans , Male , Risk Factors , Southeastern United States , Vibrio Infections/drug therapy , Vibrio Infections/etiology , Visual Acuity
6.
Ophthalmology ; 106(8): 1500-3, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10442894

ABSTRACT

OBJECTIVE: To analyze commercially available bottled water as a possible source of microbial contamination of contact lenses. METHODS: Two different lots of 23 brands of noncarbonated bottled water were tested for coliforms, total bacteria, fungi, and free-living amebae. A sample consisted of three separate 100-ml aliquots from one lot of each brand (46 samples). Aliquots were vacuum-filtered using a 0.45-microm Nalgene analytical filter unit, and the membrane filter was placed on a filter pad in a Petri dish containing test medium. Plates were examined under a stereomicroscope, and the number of colony-forming units (CFUs) was calculated for each sample. To test for the presence of free-living amebae, three aliquots totaling approximately 3800 ml were concentrated using 8-microm filters, and the filters were placed on non-nutrient agar with live Enterobacter aerogenes. To assess the possibility of contaminating contact lenses, etafilcon lenses were rinsed in 2-ml aliquots of four brands of bottled water and then cultured. RESULTS: Seventeen (37%) of 46 samples, representing 11 (48%) of 23 brands, contained viable micro-organisms. Bacteria, including coliforms, were recovered from 12 samples of 8 brands. Yeasts or molds were recovered from seven samples of five brands. Free-living amebae were isolated from two samples, and fresh-water algae were found in both samples of one brand. Nine (20%) of 46 samples, representing 7 (30%) of the 23 brands, had more than 500 CFUs per ml or contained coliforms. Sterile contact lenses became contaminated when exposed for 1 minute to two of four brands of water from which micro-organisms were recovered. CONCLUSION: Some bottled waters contain high numbers of potential ocular pathogens. Bottled water is not safe for routine use with contact lenses.


Subject(s)
Amoeba/isolation & purification , Bacteria/isolation & purification , Contact Lenses , Fungi/isolation & purification , Water Microbiology , Water/parasitology , Amoeba/growth & development , Animals , Bacteria/growth & development , Colony Count, Microbial , Contact Lens Solutions , Contact Lenses/microbiology , Fungi/growth & development , Safety
7.
Am J Ophthalmol ; 126(4): 590-2, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9780107

ABSTRACT

PURPOSE: To evaluate the use of buffered charcoal-yeast extract agar for the isolation of Acanthamoeba from clinical specimens. METHODS: We retrospectively reviewed laboratory records of patients with ocular acanthamebic infection from October 1993 to September 1997 to compare the recovery of Acanthamoeba from clinical specimens inoculated onto various media. We then compared the experimental recovery of 10 corneal isolates of Acanthamoeba on buffered charcoal-yeast extract and blood agars. RESULTS: Paired data for buffered charcoal-yeast extract and blood agars were available from 24 cultures performed in 13 cases of ocular acanthamebic infection. Acanthamebic trails were detected on both buffered charcoal-yeast extract and blood agars in nine cultures, only on buffered charcoal-yeast extract agar in nine cultures, and only on blood agar in one culture (P = .027). In the experimental study, all 10 clinical isolates produced trails on buffered charcoal-yeast extract agar, and the mean recovery after 10 days of incubation ranged from 38% to 95% of the original inoculum number. For seven of the 10 isolates, more than 70% of the original inoculum was recovered on buffered charcoal-yeast extract agar. Only two of the 10 strains produced persistent trails on the blood agar, and the mean recoveries after 10 days of incubation were 0.67% and 1.17%. Recovery was significantly better on buffered charcoal-yeast extract agar than blood agar (P < or = .0003). CONCLUSION: Buffered charcoal-yeast extract agar is an excellent commercially available culture medium for the recovery of Acanthamoeba.


Subject(s)
Acanthamoeba Keratitis/parasitology , Acanthamoeba/isolation & purification , Cornea/parasitology , Parasitology/methods , Acanthamoeba Keratitis/diagnosis , Agar , Animals , Buffers , Charcoal , Culture Media , Humans , Retrospective Studies , Yeasts
8.
Retina ; 18(2): 169-73, 1998.
Article in English | MEDLINE | ID: mdl-9564700

ABSTRACT

PURPOSE: This study sought to determine whether the use of an antimicrobial removal device (ARD) to process intraocular fluids increases microbial detection compared with conventional cultures. METHODS: The authors retrospectively reviewed all cases of endophthalmitis submitted to their laboratory from January 1982 through December 1996. Aqueous or vitreous specimens or both that were cultured on conventional media (blood agar, chocolate agar, anaerobic blood agar, and thiol broth) and by ARD processing were included in the study. Specimens were inoculated into tubes with ARD for 5 to 10 minutes. The fluid was then withdrawn and cultured using conventional media; thioglycolate broth was added to the tube containing the resin beads. The conventional and ARD-processed cultures were incubated at 35 degrees C for at least 7 days. RESULTS: Of the 338 endophthalmitis cultures processed using both conventional cultures and a parallel ARD, 166 (49.1%) yielded positive microbial growth on one or more media. Of the 166 culture-confirmed cases, 127 (76.5%) were positive in both the ARD-processed and direct cultures, 17 (10.2%) were positive by conventional culture only, and 22 (13.3%) were positive by the ARD-processed sample alone (P = 0.52). The spectrum of microorganisms was similar among all culture groups. The detection of coagulase-negative staphylococci and micrococci by ARD alone was slightly better than detection by conventional culture only (P = 0.06). Of 93 positive cultures from 179 patients in whom prior antibiotic use was documented, 75 (80.6%) were positive by both methods, 8 (8.6%) by conventional cultures only, and 10 (10.8%) only by the ARD-processed specimen (P = 0.81). CONCLUSION: Use of an antimicrobial removal device does not significantly increase the microbial yield of endophthalmitis cultures compared with conventional culture techniques, whether or not antimicrobial therapy is being used.


Subject(s)
Endophthalmitis/microbiology , Microbiological Techniques/instrumentation , Anti-Bacterial Agents/therapeutic use , Aqueous Humor/microbiology , Drainage , Endophthalmitis/drug therapy , Evaluation Studies as Topic , Humans , Retrospective Studies , Vitrectomy , Vitreous Body/microbiology
9.
Cornea ; 17(2): 135-40, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9520187

ABSTRACT

BACKGROUND: Approximately 15-20% of Streptococcus pneumoniae clinical isolates in the United States are not susceptible to penicillin. Isolates with a minimum inhibitory concentration (MIC) of 0.12-1.0 mg/ml are intermediately resistant, and those with an MIC > 2.0 microg/ml are classified as having high-level penicillin resistance. PURPOSE: We determined the proportion of penicillin-resistant S. pneumoniae recovered from ocular and periocular infections from 1976 through 1995, compared these cases with penicillin-susceptible controls, and evaluated the susceptibility of penicillin-resistant isolates to selected cephalosporins and fluoroquinolones. METHODS: MICs for cephalothin, ceftazidime, ciprofloxacin, and ofloxacin were determined for available isolates by the E test. We performed a case-comparison study to evaluate differences between patients with penicillin-susceptible and -nonsusceptible ocular pneumococcal infections. RESULTS: Of 173 ocular isolates of S. pneumoniae isolated in the 20-year period, 12 (7%) were not susceptible to penicillin, including eight (5%) intermediate isolates and four (2%) highly resistant isolates. Penicillin-intermediate and -resistant pneumococci were recovered at a rate of none of 46 isolates from 1976 through 1980, one (4%) of 25 from 1981 through 1985, one (2%) of 51 from 1986 through 1990, and 10 (20%) of 51 from 1991 through 1995 (p < 0.0004). We found no significant differences in presenting characteristics between patients with ocular infections due to penicillin-susceptible pneumococci and those caused by nonsusceptible strains. All retested isolates with intermediate susceptibility to penicillin had a cephalothin MIC < or = 1.5 microg/ml, and all retested isolates with high-level penicillin resistance had a cephalothin MIC > or = 4 microg/ml. The ceftazidime MIC for all penicillin-resistant isolates was > or = 24 microg/ml. All penicillin-nonsusceptible isolates had MICs < or = 1.5 microg/ml for ciprofloxacin and < or = 3 microg/ml for ofloxacin. CONCLUSION: Penicillin resistance has recently emerged among ocular S. pneumoniae. Cephalothin, ciprofloxacin, and ofloxacin have good activity against some ocular isolates with intermediate penicillin susceptibility, although another agent such as vancomycin may be needed for pneumococci with high-level penicillin-resistance.


Subject(s)
Eye Infections, Bacterial/microbiology , Penicillin Resistance , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , Anti-Infective Agents/pharmacology , Cephalosporins/pharmacology , Child , Child, Preschool , Eye Infections, Bacterial/diagnosis , Eye Infections, Bacterial/epidemiology , Female , Fluoroquinolones , Humans , Infant, Newborn , Male , Microbial Sensitivity Tests , Middle Aged , Penicillins/pharmacology , Pneumococcal Infections/diagnosis , Pneumococcal Infections/epidemiology , Retrospective Studies , Risk Factors , Streptococcus pneumoniae/drug effects
10.
J Clin Microbiol ; 35(4): 915-22, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9157153

ABSTRACT

Acanthamoeba is a genus of ubiquitous, free-living amebae that can be difficult to isolate by standard microbiologic techniques. We retrospectively reviewed the laboratory records of patients with ocular acanthamoebic infection for the period from January 1973 to June 1996 and found that Acanthamoeba isolates were recovered from 73, 71, and 70% of clinical specimens inoculated onto buffered charcoal-yeast extract agar (BCYE), nonnutrient agar with live or dead Escherichia coli, and tryptic soy agar (TSA) with horse or sheep blood, respectively. We then prospectively compared the recovery of a corneal isolate of Acanthamoeba on commercial media from Remel and BBL (TSA with 5% sheep blood, TSA with 5% horse blood, TSA with 5% rabbit blood, V agar, chocolate agar, BCYE, and selective BCYE with polymyxin B, anisomycin, and vancomycin) and on axenic and monoxenic media prepared with live or dead bacteria (Enterobacter aerogenes, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, and Stenotrophomonas maltophilia). Good recovery of trophozoites was obtained on BCYE, TSA with rabbit blood, TSA with horse blood, and Remel TSA with sheep blood. BBL TSA with horse blood or rabbit blood provided good recovery of cysts. All species of live or dead bacteria yielded good recovery of trophozoites; however, only nonnutrient agar with live P. aeruginosa, live E. aerogenes, or live S. maltophilia gave good recovery of cysts. TSA with either rabbit blood or horse blood, BCYE, and nonnutrient agar prepared with live P. aeruginosa, E. aerogenes, or S. maltophilia offer optimal recovery of Acanthamoeba.


Subject(s)
Acanthamoeba/isolation & purification , Parasitology/methods , Animals , Culture Media , Rabbits
11.
Arch Ophthalmol ; 114(4): 433-6, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8602781

ABSTRACT

OBJECTIVES: To determine if the number of ocular infections associated with Stenotrophomonas maltophilia is increasing, to identify predisposing factors, and to evaluate antimicrobial susceptibility. METHODS: Retrospective review of ocular microbiology laboratory records from January 1, 1972, through December 31, 1995. RESULTS: Stenotrophomonas maltophilia was recovered from 15 cases of ocular infection, at a rate of one in every 1339 ocular specimens in the 1970s, one in 413 in the 1980s, and one in 363 in the 1990s through 1995. The organism was the predominant isolate in five cases and was part of a polybacterial infection in the remaining 10 cases. Eight of the 15 cases had bacterial keratitis, including one with infectious crystalline keratopathy. Of the remaining seven infections, S maltophilia was recovered from two cases of acute conjunctivitis, two infected scleral buckles (one with orbital cellulitis), two cases of infantile dacryocystitis, and one case of preseptal cellulitis. Ocular isolates of S maltophilia were resistant to the aminoglycosides and most beta-lactams, and showed variable susceptibility to the fluoroquinolones. CONCLUSIONS: Stenotrophomonas maltophilia is emerging as an important opportunistic ocular pathogen. Most infections by this organism occur in patients with ocular compromise, and the characteristically resistant antibiogram of S maltophilia limits the therapeutic options.


Subject(s)
Eye Infections, Bacterial/microbiology , Gram-Negative Bacterial Infections/microbiology , Xanthomonas/isolation & purification , Adult , Aged , Aged, 80 and over , Anti-Bacterial Agents/pharmacology , Cellulitis/microbiology , Conjunctivitis/microbiology , Dacryocystitis/microbiology , Female , Humans , Infant , Keratitis/microbiology , Male , Microbial Sensitivity Tests , Middle Aged , Opportunistic Infections/etiology , Retrospective Studies , Xanthomonas/drug effects
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