Bacterial characterization by flow cytometry.

Bacteria were analyzed in a dual-beam flow cytometer after double staining with the fluorescent dyes chromomycin A3 and Hoechst 33258, which bind preferentially to DNA that is rich in guanine-cytosine and adenine-thymine, respectively. The measurements were indicative of the cellular DNA content and base composition, cell concentration, and proliferative state of the population. The ratio of the chromomycin A3 signal to the Hoechst 33258 signal increased with the guanine-cytosine content of the cellular DNA for the six cultured species measured, following expectation. Bacteria in urine from patients with urinary tract infections were characterized without interference from host cell DNA, debris, or other particulates.

The usefulness of diagnostic bone marrow examination in patients with human immunodeficiency virus (HIV) infection.

To determine the utility of bone marrow examination for the diagnosis of opportunistic infections and lymphoma in patients with known or suspected human immunodeficiency virus (HIV) infection, we retrospectively reviewed the medical and laboratory records of all patients undergoing diagnostic bone marrow examinations at San Francisco General Hospital between January 1, 1988 and December 31, 1989. All marrow examinations of patients with known or suspected HIV infection in which specimens were examined histopathologically and/or microbiologically for opportunistic pathogens or lymphoma were analyzed. Bone marrow examination resulted in the diagnosis of mycobacterial infection in 16% of the patients studied. Blood culture was 77% sensitive and bone marrow culture was 86% sensitive for detecting disseminated mycobacterial infection. This difference was not statistically significant (p greater than 0.05). Disseminated fungal infections occurred in less than 5% of the patients studied, and most were rapidly and accurately detected by examination of stained bone marrow samples. No case of lymphoma was diagnosed by bone marrow examination. Bone marrow examination may be useful for diagnosing opportunistic infections in patients with HIV infection. Mycobacterial blood cultures have a sensitivity comparable to bone marrow cultures in detecting disseminated mycobacterial infections, are less invasive, and may be less costly. Marrow examination is not useful for diagnosing lymphoma but can determine the extent of lymphoma that has been diagnosed by other means.

Predictive value of stool examination in acute diarrhea.

We prospectively evaluate the value of fecal blood and fecal leukocytes in predicting whether acute diarrhea in adults is associated with a stool culture positive for a bacterial pathogen. One hundred thirteen patients, aged 19 to 50 years, seen in a two-year period in an urban adult outpatient setting underwent stool culture for the presenting symptom of diarrhea. Heterosexual men represented 48% of the cohort, women represented 17%, and homosexual men represented 35%. Overall, 53 (47%) of the patients had positive stool cultures for enteric pathogens. Campylobacter jejuni was the most common organism in the entire cohort, but Shigella species were most common in homosexual men. The best predictive variables for a stool culture positive for a bacterial pathogen were the presence of both fecal leukocytes and fecal blood in the stool, compared with only one or neither. When both were present, the sensitivity was 81%, the specificity 74%, and the predictive values of a positive and negative test were 81% and 83%, respectively; the likelihood ratio was 4.87. When homosexual men and the rest of the cohort were analyzed separately, the combination of fecal leukocytes and fecal blood remained the best method of predicting a positive stool culture in both. Examination of stool for fecal leukocytes and fecal blood is a rapid, reliable, and inexpensive way to differentiate between bacterial and other causes of acute diarrhea in the adult acute care setting.

Lack of effect of prophylactic aerosolized pentamidine on the detection of Pneumocystis carinii in induced sputum or bronchoalveolar lavage specimens.

Two independent studies were undertaken to determine the effect of prophylactic treatment with aerosolized pentamidine on the laboratory diagnosis of Pneumocystis carinii pneumonia in individuals at risk for or with the acquired immunodeficiency syndrome. The first study was a retrospective analysis to determine the effect of prophylactic treatment with aerosolized pentamidine on the diagnostic yield and sensitivity of detection of P carinii in induced sputum specimens. The results of examinations of 110 induced sputum specimens from patients who had not received aerosolized pentamidine were compared with the findings in 57 specimens from patients who had. There was no statistically significant difference between the two groups for the diagnostic yield in induced sputum specimens (48% vs 47%) or in bronchoalveolar lavage fluid specimens subsequently obtained from patients with nondiagnostic induced sputum examinations (33% vs 37%). The sensitivity of induced sputum specimens for identifying P carinii was 76% to 78% for patients who had not received aerosolized pentamidine and 71% to 75% for patients who had received the drug. The second study was a prospective comparison of 118 bronchoalveolar lavage fluid specimens to determine the effect of prophylactic treatment with aerosolized pentamidine on the number of organisms present. One hundred eighteen bronchoalveolar lavage fluid specimens were quantitatively examined and scored according to the number of clumps of P carinii present. No statistically significant difference was seen in the number of clumps of P carinii found in specimens from patients who had received aerosolized pentamidine vs the number of clumps found in specimens from patients who had not. In conclusion, prophylactic treatment with aerosolized pentamidine had no effect on (1) the diagnostic yield and sensitivity of detection of P carinii in induced sputum specimens or (2) the number of organisms detected in bronchoalveolar lavage fluid specimens obtained from individuals at risk for or with the acquired immunodeficiency syndrome.

Tumor induction by Agrobacterium tumefaciens: specific transfer of bacterial deoxyribonucleic acid to plant tissue.

When Agrobacterium tumefaciens cells grown in the presence of tritiated thymidine to label specifically the bacterial deoxyribonucleic acid (DNA) are incubated with carrot root tissue for short periods of time, an appreciable fraction of the label becomes firmly associated with the root tissue. Such association is not observed in identical experiments when A. tumefaciens cell ribonucleic acid or protein are labeled. The extent of the retention of thymidine-derived label from bacterial cells by the root tissue in experiments with A. radiobacter and poorly tumorigenic strains of A. tumefaciens is significantly less than that afforded by tumorigenic strains of A. tumefaciens but greater than the level afforded by Escherichia coli. Transfer of DNA-specific label from A. tumefaciens to carrot root discs is not enhanced by treatments designed to provoke lysis of the bacterial cells, nor is it decreased by addition of deoxyribonuclease or excess unlabeled thymidine to the incubation medium. Bacterial cell-to-plant cell contact is necessary for transfer. Unlabeled A. radiobacter cells decrease in a competitive manner transfer of label when mixed with labeled A. tumefaciens cells. These findings suggest that transfer of DNA from A. tumefaciens to plant tissue after binding of the bacterial cells to specific plant tissue site(s) is a necessary feature of the mechanism by which A. tumefaciens provokes tumors in plants and provides an experimental technique of potentially great value in study of the early steps in the process of tumor induction by A. tumefaciens.

Mutations simultaneously affecting endonuclease II and exonuclease III in Escherichia coli.

We studied mutants of E. coli originally identified as being deficient in either endonuclease II (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.30) or exonuclease III [deoxyribonucleate (double-stranded) 5'-nucleotidohydrolase, EC 3.1.4.27] activity. Twelve independently derived mutants were tested, including three new endonuclease II mutants. Deficiency of one enzyme was always accompanied by deficiency of the other. Furthermore, temperature-sensitivity of one activity was always accompanied by temperature-sensitivity of the other, and the enzymes were co-purified. The results suggested a physical association between exonuclease III and endonuclease II, which may be of advantage in the excision-repair of DNA. A thermolabile endonuclease II was purified from one of the new mutants, indicating that it had an altered structural gene. This mutation, and all similar ones mapped by genetic transduction, was located between the pncA and aroD genes on the E. coli chromosome. One mutant had a prolonged generation time, an increased sensitivity to the alkylating agents methyl-methanesulfonate and mitomycin C, and a decreased plating efficiency for bacteriophage lambda, but no marked sensitivity to ultraviolet or gamma-irradiation. Its enzymatic and biological abnormalities were simultaneously revertible, suggesting they were caused by a single mutation. These results suggested a role for these enzymes in normal cell growth processes and in the repair of alkylation damage.

Changes in cyclic AMP levels during development in Myxococcus xanthus.

Cyclic AMP levels doubled in Myxococcus xanthus under conditions in which cells aggregate and form fruiting bodies. In liquid medium, glycerol- or dimethyl sulfoxide-induced sporulating cultures exhibited a sharp but transient rise in cyclic AMP concentration after 45 min.

Incidence and characterization of beta-hemolytic Streptococcus milleri and differentiation from S. pyogenes (group A), S. equisimilis (group C), and large-colony group G streptococci.

The biochemical characteristics of 172 clinical isolates of group A, C, F, or G or "nongroupable" beta-hemolytic streptococci were examined. Among these isolates, 91 were identified as beta-hemolytic strains of Streptococcus milleri. The remaining isolates included 20 Streptococcus pyogenes, 21 Streptococcus equisimilis, 37 large-colony group G streptococci, and 3 unidentified nongroupable isolates. A majority (84%) of the S. milleri strains possessed Lancefield group antigen (3 A, 27 C, 41 F, and 5 G), whereas 15 S. milleri strains (16%) were nongroupable. Serological tests did not differentiate S. milleri isolates with group A, C, or G antigen from S. pyogenes (group A), S. equisimilis (group C), or large-colony group G streptococci. Biochemical tests which were found useful for differentiation included the Voges-Proskauer test, hydrolysis of pyroglutamic acid and beta-D-glucuronide, bacitracin susceptibility, and acid production from ribose. S. milleri represented 56% of the group C, 100% of the group F, and 83% of the nongroupable beta-hemolytic streptococci isolated in our clinical laboratory, whereas the incidence of S. milleri among group A and group G streptococci was estimated to be low. The role of beta-hemolytic S. milleri as a cause of human infection remains obscured by the failure to routinely differentiate S. milleri from other beta-hemolytic streptococci.

Update on laboratory tests for the diagnosis of pulmonary disease in HIV-1-infected individuals.

Various diagnostic tests, both specific and nonspecific, are available in the clinical laboratories for diagnosing human immunodeficiency virus-1 (HIV-1) infection and associated respiratory pathogens. Pneumocystis carinii pneumonia remains the most common pulmonary disease in HIV-1-infected individuals and there have been no significant advances in the laboratory diagnosis of the pathogen beyond the traditional microscopic examination of specimens. In contrast, the greatest revolution in laboratory diagnostic testing has been for mycobacteria, with major advances resulting in significant reduction in the time necessary for isolation and identification to the species level. The application of the polymerase chain reaction for the identification of a variety of pulmonary pathogens observed in HIV-1 infected individuals is discussed.

Broth microdilution testing of susceptibilities to 30 antimicrobial agents of Mycobacterium avium strains from patients with acquired immune deficiency syndrome.

A total of 31 strains of Mycobacterium avium complex isolated from patients with acquired immune deficiency syndrome were tested for susceptibility to 30 antimicrobial agents by using microdilution trays containing dried antimicrobial agents. MICs were determined over a period of 7 days of growth in a broth medium (7HSF) that is equivalent to 7H11 agar. MICs obtained by this method showed good agreement with MICs determined by the agar dilution method. Strains could be divided into two groups by their antimicrobial susceptibility patterns. All group 1 strains (8 of the 31 strains tested) were at least moderately susceptible to inhibition by a variety of beta-lactam antimicrobial agents, including amoxicillin-clavulanic acid and cefmenoxime. Group 2 strains (23 of 31) were susceptible only to amikacin (22 of 23 strains). All 31 strains were resistant to oxacillin, clindamycin, erythromycin, tetracycline, chloramphenicol, vancomycin, nitrofurantoin, and aztreonam at the highest concentration of antimicrobial agent present in the microdilution trays. The addition of Tween 80 to 7HSF broth increased the susceptibility of M. avium complex to many of the antimicrobial agents tested. Killing of M. avium complex (i.e., less than or equal to 1% survival after 7 days) was found to vary for different strains and antimicrobial agents. Killing of some strains by amoxicillin-clavulanic acid, carbenicillin, azlocillin, cefmenoxime, cefotaxime, amikacin, and ampicillin occurred at concentrations of antimicrobial agent that are achievable in serum. Further studies are needed to determine whether any of these antimicrobial agents has activity against M. avium complex cells that have been ingested by macrophages.

Therapeutic implications of inhibition versus killing of Mycobacterium avium complex by antimicrobial agents.

Patients with the acquired immune deficiency syndrome (AIDS) with disseminated Mycobacterium avium infection have responded poorly to treatment with rifabutine (Ansamycin) and clofazimine, in spite of the good in vitro response of M. avium to these antimicrobial agents. We compared the ability of these and other antimicrobial agents to kill versus the ability to inhibit the growth of strains of the M. avium complex isolated from patients with AIDS. Killing curve experiments showed that the concentrations of rifabutine and clofazimine needed to kill two log units of M. avium are at least 32 times greater than the concentrations needed to inhibit growth. Little or no killing occurred at concentrations of these antimicrobial agents that are achievable in serum. In contrast, five of seven strains tested were killed by ciprofloxacin at concentrations that can be achieved in serum. Ciprofloxacin should be studied further for possible use in the treatment of M. avium infections.

Extrapulmonary pneumocystosis.

Extrapulmonary pneumocystosis is an exceedingly rare complication of Pneumocystis carinii pneumonia (PCP). Prior to the advent of the human immunodeficiency virus type 1 (HIV-1) epidemic, only 16 cases of extrapulmonary pneumocystosis in individuals who were immunocompromised by a variety of underlying diseases had been reported. Since the beginning of the HIV-1 and related PCP epidemic, at least 90 cases of extrapulmonary pneumocystosis have been reported. This review briefly presents a history of the discovery of P. carinii and its recognition as a human pathogen, the controversy regarding its taxonomy, and the epidemiology of this organism. A more detailed analysis of the incidence of extrapulmonary pneumocystosis in HIV-1-infected individuals and its occurrence despite widespread prophylaxis for PCP with either aerosolized pentamidine or systemic dapsone-trimethoprim is presented. The clinical features of published cases of extrapulmonary pneumocystosis in non-HIV-1-infected individuals are summarized and contrasted with those in HIV-1 infected individuals. The diagnosis of extrapulmonary pneumocystosis is discussed, and because clinical microbiologists and pathologists are the key individuals in establishing the diagnosis, the characteristic microscopic morphology of P. carinii as its appears when stained with a variety of stains is presented and reviewed. The review concludes with a brief discussion of treatments for extrapulmonary pneumocystosis.

Eradication of AIDS-related disseminated mycobacterium avium complex infection after 12 months of antimycobacterial therapy combined with highly active antiretroviral therapy.

To determine if microbiologic cure of AIDS-related disseminated Mycobacterium avium complex (MAC) is possible in patients receiving highly active antiretroviral therapy (HAART), 4 patients with a history of disseminated MAC received >/=12 months of macrolide-based antimycobacterial therapy. All were asymptomatic and had absolute CD4 cell count >100/microL (range, 137-301) and <10,000 copies/mL of human immunodeficiency virus RNA (range, <500-1250). A bone marrow aspirate and peripheral blood were obtained for mycobacterial culture. Follow-up blood cultures were obtained routinely at 4 weeks and every 8 weeks thereafter. All 4 patients had negative bone marrow and blood cultures and then discontinued antimycobacterial therapy. All patients' subsequent cultures remain sterile and all are clinically asymptomatic (range, 8-13 months follow-up). It appears that disseminated MAC infection can be cured by prolonged antimycobacterial therapy in some persons who experience sustained CD4 lymphocyte increases while receiving HAART.

Rapid confirmatory identification of Neisseria gonorrhoeae with lectins and chromogenic substrates.

A group of five tests utilizing wheat germ and soybean lectins and chromogenic substrates (orthonitrophenyl-beta-D-galactopyranoside, gamma-glutamyl-beta-naphthylamide, and prolyl-beta-naphthylamide derivatives) was used as a rapid (30-min) method for the identification of Neisseria gonorrhoeae. The rapid method agreed with Minitek test results for all 126 N. gonorrhoeae isolates and all 39 nongonococcal isolates tested. Soybean lectin was useful for the identification of rare strains (4 of 126) of N. gonorrhoeae which are not agglutinated by wheat germ lectin. The chromogenic substrates differentiate N. gonorrhoeae from Neisseria meningitidis, Neisseria lactamica, and other Neisseria species which may grow on Thayer-Martin or other selective media.

Antimicrobial synergism against Mycobacterium avium complex strains isolated from patients with acquired immune deficiency syndrome.

Pairs of 11 antimicrobial agents were tested in vitro for their ability to act synergistically against three strains of Mycobacterium avium complex isolated from patients with acquired immune deficiency syndrome. From the combinations tested, four drugs (ethambutol, rifampin, ciprofloxacin, and erythromycin) were selected for more extensive study against 20 strains of M. avium complex. The inhibitory and killing synergism obtained with combinations of two, three, or four drugs was assessed by determining the fractional inhibitory concentration index and fractional bactericidal concentration index. Inhibitory synergism occurred against 90 to 100% of the strains for all drug combinations in which ethambutol was included. Killing synergism occurred against 85 to 95% of the strains when ethambutol was used in combinations which included either rifampin or ciprofloxacin. However, killing synergism occurred against only 45% of the strains when drugs were tested at concentrations that can be obtained in patient serum. In other experiments, rifabutin (Ansamycin) gave results that were comparable to those obtained with rifampin. Clofazimine did not show synergistic killing activity at a concentration that is achievable in serum for any of the drugs tested. Our results indicate that there is considerable variability in the antimicrobial susceptibility of M. avium isolates obtained from patients with acquired immune deficiency syndrome. This variability could have significant impact on the clinical response to various therapies.

Clinical trial comparing bacitracin with Strep-A-Chek for accuracy and turnaround time in the presumptive identification of Streptococcus pyogenes.

In a clinical trial, Strep-A-Chek (a 10-min chromogenic test) was compared with the bacitracin disk susceptibility test for accuracy and turnaround time in the presumptive identification of Streptococcus pyogenes. Among 461 isolates of beta-hemolytic streptococci (344 throat isolates and 117 isolates from other sites), 303 group A S. pyogenes isolates were found. The sensitivities of the Strep-A-Chek and bacitracin tests were high (96.4 and 100%, respectively), but the bacitracin test had a lower specificity (84.2%) than the Strep-A-Chek test (98.7%). The predictive values for positive and negative test results were 99.3 and 93.4%, respectively, for Strep-A-Chek and 92.4 and 100%, respectively, for bacitracin. Strep-A-Chek correctly identified all isolates upon repeat testing. All bacitracin tests were performed on subcultures of isolates from the primary plate. Strep-A-Chek testing was performed on colonies from the primary plate when isolated colonies were available. This shortened the turnaround time for Strep-A-Chek compared with bacitracin by at least 24 h on nearly one-half (45%) of the isolates. A peripheral finding of this study was that sulfamethoxazole-trimethoprim blood agar offered no advantage over conventional blood agar with regard to the number of false-positive bacitracin tests obtained from each medium.

In vitro susceptibility of Mycobacterium avium complex to the new fluoroquinolone sparfloxacin (CI-978; AT-4140) and comparison with ciprofloxacin.

We tested the activity of the new fluoroquinolone sparfloxacin (CI-978; AT 4140) against 30 strains of Mycobacterium avium complex (MAC) isolated from patients with acquired immune deficiency syndrome. MICs of sparfloxacin (range, less than or equal to 0.06 to 4 micrograms/ml) were lower than MICs of ciprofloxacin for all 30 strains, and MBCs for acid-fast bacteria were lower for 28 of the 30 strains. In synergism experiments using 10 strains of MAC, fractional inhibitory concentration indices revealed that the combination of sparfloxacin plus ethambutol was synergistic against 9 strains, and the three-drug combination of sparfloxacin plus ethambutol plus rifampin was synergistic against all strains. In the absence of ethambutol, the combination of sparfloxacin plus rifampin appeared to be antagonistic against three of the MAC strains.

Killing by antimycobacterial agents of AIDS-derived strains of Mycobacterium avium complex inside cells of the mouse macrophage cell line J774.

The murine macrophage continuous cell line J774 was used to measure the ability of antimicrobial agents, either singly or in combination, to kill intracellular Mycobacterium avium complex. All of 14 strains of M. avium complex, isolated from patients with AIDS, grew inside J774 cells during an incubation period of 7 days. The susceptibility of macrophage-ingested M. avium complex to antimicrobial agents was determined by comparing the number of colony-forming units (cfu) of M. avium complex inside untreated macrophages at the time of drug addition with the number of cfu present in macrophages after treatment with drugs for 7 days. Simultaneous experiments were carried out in broth medium without macrophages in order to compare killing of free mycobacteria with killing of macrophage-ingested mycobacteria. Antimicrobial agents (rifampin, rifabutin [Ansamycin], ethambutol, ciprofloxacin, clofazimine, isoniazid, and amikacin) were tested using concentrations that are achievable in the serum of patients. Among drugs known to penetrate macrophages, there was 96.2% agreement in susceptibility test results between the broth experiments and the J774 experiments when single drugs were tested, but only 74% agreement when combinations of drugs were tested. Killing of M. avium complex inside J774 cells by any single drug was uncommon. However, killing in J774 cells occurred against 10 of 11 (91%) strains with the combination of rifabutin + ethambutol + ciprofloxacin and against all of seven strains tested with the combination of rifabutin + ethambutol + amikacin. Interpretive criteria of in vitro susceptibility data need to be developed so that these interpretations correlate with a predictable clinical response in patients.