274 Emerging Infectious Diseases • Vol. 9, No. 2, February 2003 and no reports of disease transmission were received from any other recipients of organs from this donor. In addition, the patient had no history of receiving infusions of holistic or alternative medicines. The organism was initially detected in the aerobic Bact/Alert blood culture system (bioMérieux, Inc., Durham, NC) after 72 h incubation at 35°C. Presumptive identification of the pleomorphic gram-positive bacillus as Streptomyces sp. was based on phenotypic characterization by using standard conventional tests and cellular fatty acid analysis. Species identification was determined by DNA sequencing of the 16S rRNA gene. DNA sequencing reactions were performed with the Tag Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA), and data were generated with an ABI 377 automated instrument. The sequence data were assembled, edited, and compared with published sequences for the 16S rRNA gene of S. bikiniensis (2). The genus Streptomyces belongs to the order Actinomycetales, which includes Mycobacterium, Nocardia, and Actinomyces. Streptomyces are gram-positive, extensively branched, filamentous bacteria that form aerial hyphae with chains of spores. Their natural habitat is soil, and each species has a defined geographic distribution. None are common in the United States. With the exception of specimens from actinomycotic mycetoma, the isolation of Streptomyces from clinical specimens frequently is considered laboratory contamination (3). Rare cases of clinical disease attributed to Streptomyces have been published, including bloodstream infection (1,4) and focal invasive infections (5–9). Streptomyces was not the only potential pathogen isolated from some of the clinical specimens in these studies. Scant data are available on effective treatment of Streptomyces infection. Mycetoma caused by Streptomyces is often treated with penicillin, sulfonamides, or tetracycline; however, the cure rate is low. The recommended duration of therapy is lengthy (up to 10 months). Isolates of S. griseus referred to the Centers for Disease Control and Prevention were frequently resistant to ampicillin (80%), sulfamethoxazole (43%), cotrimoxazole (29%), and ciprofloxacin (57%) (10). Resistance to doxycycline (19%) and minocycline (10%) was lower. Vancomycin susceptibility was not tested. Resistance patterns must be interpreted cautiously because Streptomyces can synthesize antibiotics, potentially confounding results of invitro susceptibility testing. The patient described in this report had no signs or symptoms of infection. The transient fever that prompted the first blood culture was probably due to the methotrexate infusion and not infection with S. bikiniensis. That the fever was of short duration despite persistently positive blood cultures supports this conclusion. The potential for causing minimal symptoms may contribute to assignment of Streptomyces as a contaminant. Clinical correlation is difficult if the infection is silent. Streptomyces isolated from blood cultures should not be dismissed as contaminants without careful consideration of the clinical situation; the isolation of Streptomyces from repeat blood cultures strongly suggests a pathogenic role.

BACKGROUND:Mycobacterium avium subspecies paratuberculosis (MAP) has been suspected of involvement in Crohn's disease (CD). We investigated this potential association by testing whole blood from CD patients and healthy controls for the presence of MAP by culture and molecular methods. In addition, each blood sample was analyzed for polymorphisms in the NOD2/CARD15 gene previously associated with CD. METHODS:Four 4-mL K(2)-EDTA tubes of whole blood were drawn from each subject (n = 260, 130 CD patients and 130 healthy controls). Two tubes of blood were cultured for MAP by the following methods: Mycobacterial Growth Indicator Tube, Herrold's Egg Yolk Agar, BACTEC 460, and Hungate. The remaining 2 tubes of blood were tested for MAP DNA and polymorphisms in the NOD2/CARD15 gene by polymerase chain reaction (PCR). RESULTS:One healthy control patient was positive for MAP via PCR; however, no viable MAP was cultured from this individual. All blood cultures were negative for MAP. One CD patient's blood was culture-positive for M. tuberculosis complex. CD patients exhibited a higher rate of polymorphism in the NOD2/CARD15 gene than healthy control patients. CONCLUSIONS:In this study MAP was not recovered from the blood of CD patients or healthy controls. However, CD patients showed higher mutation rates in the NOD2/CARD15 gene, compared with healthy controls, supporting the findings of other investigators. No correlation between these polymorphisms and MAP bacteremia in CD patients could be identified in this study.

The extended prophylactic use of the antiretroviral drug nevirapine in breastfeeding infants of human immunodeficiency virus (HIV)-infected mothers in developing countries has been shown to reduce HIV transmission ([2][1], [5][2]). Nevirapine's use in this setting was also associated with a

DSA is a beta-sulfonylacetamide with in vitro activity against pathogenic mycobacteria. Although the enzymatic target(s) of DSA has not been identified, studies to date suggest that this class of compounds may interfere directly or indirectly with ATP synthase and other components of the mycobacterial respiratory chain. In this study we further evaluated the in vitro activity of DSA against anaerobically adapted BCG using two established models. DSA killed BCG in the anaerobic Wayne model. Bactericidal activity ranged from >99% to 60%. DSA killed rifampin-tolerant persisters with a reduction in viable counts of 1.5log(10) versus controls. Conclusive identification of the DSA-specific target(s) will permit a better understanding of the unique mechanism of action of this class of compounds against both aerobically growing and anaerobically adapted bacilli in vitro.

BACKGROUND. Inflammation, both acute and chronic, is a common feature of Prostate histology. While inflammation has been proposed to play an important role in both benign and malignant growth of the prostate, the stimuli for this inflammation remain poorly characterized. Infectious pathogens are potential stimuli for prostatic inflammation.METHODS. Universal eubacterial PCR was used to test 170 prostate tissue core samples from 30 cancer patients for 16S rDNA gene sequences. Positive PCR products (n = 64, 37%) were cloned and sequenced. For comparison, tissue samples from 30 patients were cultured using standard clinical microbiological techniques. DNA samples from 200 additional patients were tested by organism-specific PCR for the presence of Chlamydia trachomatis, Propionibacterium acnes, Trichomonas vaginalis, BK virus, Epstein-Barr virus, human cytomegalovirus, human papillomavirus, and xenotropic murine leukemia-related virus.RESULTS. 16S sequencing results indicated the presence of 83 distinct microorganisms. Microbiological culture isolated markedly fewer species. In general, organism-specific PCR failed to detect multiple organisms previously reported as common in the prostate. There was no significant association between the presence of particular species of bacteria and histologic evidence of acute or chronic inflammation.CONCLUSIONS. Most prostates from men undergoing prostatectomy (87%) contain bacterial DNA from one or more species. However, the majority of individual tissue core samples were negative, suggesting regional heterogeneity in the presence of bacteria and a lack of a generalized or ubiquitous prostatic flora. Culture results suggest either the "unculturable" nature of species present in the prostate or that 16S rDNA sequences were derived from nonviable bacteria.

ABSTRACT Multidrug-resistant (MDR) Mycobacterium tuberculosis and extrensively drug-resistant (XDR) M. tuberculosis are emerging public health threats whose threats are compounded by the fact that current techniques for testing the susceptibility of M. tuberculosis require several days to weeks to complete. We investigated the use of high-performance liquid chromatography (HPLC)-based quantitation of mycolic acids as a means of rapidly determining drug resistance and susceptibility in M. tuberculosis . Standard susceptibility testing and determination of the MICs of drug-susceptible ( n = 26) and drug-resistant M. tuberculosis strains, including MDR M. tuberculosis strains ( n = 34), were performed by using the Bactec radiometric growth system as the reference method. The HPLC-based susceptibilities of the current first-line drugs, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), were determined. The vials were incubated for 72 h, and aliquots were removed for HPLC analysis by using the Sherlock mycobacterial identification system. HPLC quantitation of total mycolic acid peaks (TMAPs) was performed with treated and untreated cultures. At 72 h, the levels of agreement of the HPLC method with the reference method were 99.5% for INH, EMB, and PZA and 98.7% for RIF. The inter- and intra-assay reproducibilities varied by drug, with an average precision of 13.4%. In summary, quantitation of TMAPs is a rapid, sensitive, and accurate method for antibiotic susceptibility testing of all first-line drugs currently used against M. tuberculosis and offers the potential of providing susceptibility testing results within hours, rather than days or weeks, for clinical M. tuberculosis isolates.

We report a 4-year-old girl with a complicated Enterococcus faecalis ventriculoperitoneal shunt infection who failed vancomycin therapy. We demonstrate linezolid's high CSF penetration and its CSF bacteriostatic activity against E. faecalis. Linezolid may be a good alternative for treatment of ventriculoperitoneal shunt infections in cases of vancomycin-resistant organisms or apparent treatment failures.

ABSTRACT Blood culture bottles with antimicrobial removal systems are recommended for patients who develop fever while on antibiotics. This study compared the ability of Becton Dickinson (Sparks, MD) BACTEC PLUS bottles and bioMerieux (Durham, NC) BacT/Alert FA bottles to effectively remove vancomycin, cefoxitin, ceftriaxone, cefepime, piperacillin-tazobactam, ampicillin, oxacillin, gentamicin, and a combination of gentamicin/penicillin, thus allowing bacterial pathogens to grow. Each bottle was spiked with 10 ml of human blood, antibiotic, and strains of organisms susceptible to the antibiotic evaluated. The organisms used were type strains and clinical isolates of Staphylococcus aureus (methicillin susceptible and resistant), Streptococcus pneumoniae , a viridans streptococcus, Enterococcus faecalis , Enterococcus faecium , Streptococcus agalactiae , Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa. Testing was completed in triplicate, using 10 to 100 CFU/ml of organisms with various concentrations of each antibiotic. Two rounds of testing were completed per antibiotic/organism combination. Bottles were mixed and loaded onto their respective instruments as per the manufacturer's instructions. Antimicrobial removal was evaluated on the basis of time to detection of organism growth, for up to 5 days of incubation. Overall, the BacT/Alert FA system recovered 25.1% of strains from test bottles and 96.9% of strains from growth control bottles (no antibiotic added), and the BACTEC PLUS system recovered 95.1% of strains from test bottles and 100% of strains from growth control bottles. Both systems performed well in the detection of Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa in the presence of gentamicin. In the presence of ceftriaxone, neither system was able to recover Streptococcus pneumoniae . The ability to remove vancomycin and cefoxitin was also determined by measuring antibiotic levels remaining in bottles after 1 h of incubation. The results demonstrated remaining levels of 72 to 90% of vancomycin and 71 to 72% of cefoxitin in the BacT/Alert system. For the BACTEC system, remaining levels were 0 to 30% of vancomycin and 0% of cefoxitin. Under these simulated conditions, the BACTEC PLUS system was superior to the BacT/Alert FA system in recovering gram-positive and gram-negative bacterial pathogens in the presence of β-lactam antibiotics, gentamicin/penicillin, and vancomycin.

Background: Antimicrobial-resistant Shigella sonnei is a growing problem in the United States and poses treatment challenges particularly among children. Azithromycin is recommended as an alternative oral agent for shigellosis. Methods: All isolates of Shigella submitted to Johns Hopkins clinical laboratory during the outbreak year (2002) were compared with a historical comparison group (1996–2000). Isolates were considered multiresistant if they were resistant to ampicillin and trimethoprim-sulfamethoxazole (TS). Selected outbreak and reference isolates were tested for azithromycin susceptibility by E-test, disk diffusion and broth dilution methods. Results: Between 1996–2000, among the 111 isolates submitted, 63% were from pediatric patients; 63% of isolates were resistant to ampicillin and 12% to TS. In 2002, among the 205 isolates submitted, 82% were from pediatric patients; 91% isolates were resistant to ampicillin and 67% to TS. The proportion of multiresistant isolates increased from 6% in 1996 to 65% in 2002 (P < 0.05). Azithromycin susceptibility by E-test and disk diffusion demonstrated 2 zones of inhibition for S. sonnei. Interpretation using the inner zone resulted in higher MICs (minimal inhibitory concentration) compared with the outer zones by E-test (P < 0.0001) and disk diffusion (P < 0.0001). Conclusions: With increasing interest in using azithromycin for shigellosis, clinical laboratories should be aware of the interpretation difficulty caused by the dual-zone phenomenon seen with E-test and disk diffusion methods for S. sonnei.

OBJECTIVE:To determine the comparative efficacy of topical gatifloxacin with ciprofloxacin, fortified amikacin, and clarithromycin against Mycobacterium chelonae keratitis in an animal model.METHODS:Experimental M chelonae keratitis was induced via intrastromal inoculation in a rabbit model. Thirty-five rabbits were randomly divided into 5 groups and each group was treated hourly for 12 hours with topical 0.9% balanced salt solution, 0.3% gatifloxacin, 0.3% ciprofloxacin hydrochloride, a combination of topical fortified amikacin sulfate (50 mg/mL) and clarithromycin (10 mg/mL), or a triple combination of topical 0.3% gatifloxacin, fortified amikacin sulfate (50 mg/mL), and clarithromycin (10 mg/mL). Antibacterial efficacy of each regimen was determined by quantitative bacteriologic analysis.RESULTS:Treatment with 0.3% gatifloxacin or the triple combination of 0.3% gatifloxacin, topical fortified amikacin sulfate (50 mg/mL), and clarithromycin (10 mg/mL) reduced the number of mycobacterial organisms more significantly than the controls that were treated with a topical balanced salt solution (both P<.001). Therapy with 0.3% gatifloxacin was more effective than 0.3% ciprofloxacin alone (P<.001) and demonstrated synergy by enhancing the efficacy of the combination of fortified amikacin (50 mg/mL) and clarithromycin (10 mg/mL) (P<.001). Neither 0.3% ciprofloxacin nor the combination of fortified amikacin (50 mg/mL) and clarithromycin (10 mg/mL) demonstrated a significant difference in activity against mycobacteria compared with the topical balanced salt solution.CONCLUSION:These results suggest that topical 0.3% gatifloxacin ophthalmic solution can be a new initial treatment agent against M chelonae keratitis.Clinical Relevance Topical gatifloxacin 0.3% may provide an initial alternative in therapy of M chelonae keratitis.

OBJECTIVE:To determine the effect on BCG of n-octanesulphonylacetamide (OSA), a novel compound of the class beta-sulphonylcarboxamides, which has potent in vitro activity against pathogenic mycobacteria.METHODS AND RESULTS:The effect of OSA in BCG was examined using two-dimensional protein electrophoresis. Treatment of BCG with OSA resulted in overexpression of two proteins identified as the b-subunit of ATP synthase (Rv1306) and a 17 kDa heat shock protein (Rv0251c). [35S]Methionine pulse-labelling revealed that overexpression occurred within as little as 3.5 h post-exposure. These results were confirmed by RT-PCR. ATP levels decreased in OSA-treated BCG at 5 min, and 1, 3 and 24 h, with a 64%, 45%, 54% and 73% reduction in ATP, respectively. Only dicyclohexylcarbodiimide (DCCD), a known ATP synthase inhibitor, had a similar effect. No appreciable difference in ATP level was observed in BCG treated with standard antimycobacterial drugs, additional respiratory chain inhibitors or a fatty acid synthase inhibitor at a comparable time-point. Protein synthesis decreased within 5 min of exposure to OSA (56%), DCCD (74%) and thenoyltrifluoroacetone (TTFA) (77%). Ethanol (2.3%) potentiated the activity of OSA. In contrast, no synergic effect was observed with streptomycin and ethanol. Mycolic acid levels decreased 79% with DCCD, 46% with TTFA, a complex II inhibitor, and 43% with OSA compared with untreated controls.CONCLUSIONS:Our results suggest that OSA may interfere directly or indirectly with ATP synthase and possibly other components of the mycobacterial respiratory chain. These effects may hinder energy production, leading to interruption in the synthesis of large macromolecules including proteins and mycolic acids.

OBJECTIVE:Mycobacterium avium subspecies (subsp.) paratuberculosis (MAP) is the causative agent of Johne's disease in ruminants and has been associated with Crohn's disease in humans. We sought to test growth rates and susceptibilities of various strains of MAP in two available growth media.DESIGN:Paired comparison design.METHODS:Using the BACTEC macrobroth radiometric growth system and Congo Red-staining agar media, we determined inherent differences in growth characteristics of three bovine and two human strains of MAP and compared susceptibility results obtained in each growth system.RESULTS:Significant differences were observed in growth rate as well as mycobactin J dependence between strains and between a laboratory-adapted isolate of the same strain in the macrobroth system. Similarly, colonial morphology and Congo Red staining on agar media were observed. Two strains, one human and one bovine, demonstrated a 100% rough transparent colony with white coloration on Congo Red agar, while one bovine isolate exclusively grew as a smooth opaque colony with red coloration on Congo Red agar. The remaining strains exhibited mixtures of these two colonial morphotypes on agar media. Comparative susceptibility results between the BACTEC radiometric macrobroth method and the agar proportionality method showed good correlation for most antibiotics/inhibitors tested. However, erratic or poor growth in the macrobroth system prevented minimal inhibitory concentration determinations for two bovine strains by this method.CONCLUSION:This study demonstrates the variability in the colonial morphology of MAP on Congo Red agar as well as the correlation of antibiotic susceptibility results between the BACTEC macro broth method and the agar proportionality method. This study also emphasizes the need for the development of improved, standardized culture and susceptibility test methods for MAP.

We report a case of a surgical site infection caused by clindamycin-susceptible, erythromycin-resistant methicillin-resistant Staphylococcus aureus (MRSA) that did not respond to treatment with clindamycin. The MRSA isolate obtained after treatment was resistant to clindamycin but was found to be identical by pulsed-field gel electrophoresis to the clindamycin-susceptible isolate obtained before treatment. A post hoc erythromycin-induction test (D test) confirmed the presence of in vitro inducible macrolide-lincosamide-streptogramin B resistance (iMLS) in the pretreatment isolate. Erythromycin induction testing confirmed in vitro iMLS in 90 (56%) of 161 erythromycin-resistant, clindamycin-susceptible clinical S. aureus isolates overall and in a significantly higher proportion (78%) of methicillin-susceptible S. aureus isolates from pediatric patients. Our clinical laboratory currently tests all S. aureus isolates for iMLS before reporting clindamycin susceptibility.