Prevention of scrub typhus. Prophylactic administration of doxycycline in a randomized double blind trial.

We conducted a prospective randomized double blind study on the effects of doxycycline as a prophylactic antibiotic against scrub typhus. A total of 1,125 military subjects was followed for periods as long as 5 months of exposure in a hyperendemic focus in the Pescadores Islands of Taiwan. Oral 200 mg doses of doxycycline (Vibramycin) or placebo were given once each week throughout the trial. The incidence rate of scrub typhus in the placebo group was 2.5 times greater than that of the group taking doxycycline (P = 0.11). When subjects who failed to comply with scheduled administration of doxycycline were removed from the analysis, the incidence rate of scrub typhus in the control group was five times greater than that in the drug group (P = 0.04). The rates of infection with Rickettsia tsutsugamushi and of sick call reports were the same in experimental and control groups. The drug was well tolerated in pretrial tests and complaints were negligible during the conduct of the trial. Doxycycline appears to be an excellent antibiotic for the prevention of scrub typhus among personnel exposed to high risk of infection with R. tsutsugamushi.

Pathogenicity of encapsulated Bacteroides melaninogenicus group, B. oralis and B. ruminicola subsp. brevis in abscesses in mice.

The pathogenicity of 27 clinical isolates of the Bacteroides melaninogenicus (BM) group and four clinical isolates of B. oralis and B. ruminicola subsp. brevis were investigated by inoculating them into mice and subsequently determining their ability to cause subcutaneous (SC) or intraperitoneal abscesses. Only 11 isolates of BM group and one B. ruminicola induced abscesses in mice, and all were found to be heavily encapsulated on recovery from the abscesses (more than 50 per cent of the organisms were encapsulated). When the other 23 isolates, however, were injected SC in combination with either Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus or Streptococcus pyogenes, abscesses were formed in 16 of the 23 combinations. The Bacteroides spp. recovered from the mixed infection were heavily encapsulated. Capsules also formed in Bacteroides if the organisms were injected together with capsular material or formalin killed cells of K. pneumoniae or encapsulated Bacteroides sp. Once non-encapsulated or only slightly encapsulated strains acquired a capsule, they could induce abscesses on reinoculation into mice.

Pathogenicity of aeromonas.

The ability of 15 Aeromonas sobria and 9 Aeromonas hydrophila isolates to cause subcutaneous lesions was tested. An inoculum of 10(11) colony forming units/l was injected subcutaneously into mice. Surviving animals developed a subcutaneous abscess and/or localised skin sloughing and loss of hair (alopecia). An abscess was induced by all nine A. hydrophila isolates and by three of the 15 A. sobria isolates. The induction of local epidermal sloughing and loss of hair followed challenge with either A. hydrophila or A. sobria and correlated with the organisms' lethality for mice and their cytotoxicity in the Y-I adrenal cell assay. Local epidermal sloughing was not induced by the media used for growing the organisms or by sonicated cells of the isolates. The ability to cause epidermal sloughing was lost by incubating viable cells at 45 degrees C for 35 minutes. These yet unreported in vivo features of Aeromonas sp. may be useful in studies of the pathogenicity of the species as well as for rapid assay of toxicity of strains.

Changes in the bacterial flora of the external ear canal from the wearing of occlusive equipment.

External otitis has been associated with wearing obstructive gear covering the earlobe and external ear canal. In this study we investigated the changes in the bacterial flora of the external ear canal after wearing rubber hoods. The bacterial flora of external ear canals surfaces was investigated in a group of 19 volunteers who wore rubber hoods for a period of 25 to 30 minutes while doing manual work. The protective effect of 2% acetic acid was tested by instilling it in the left ear of all participants. Staphylococcus epidermidis, Propionibacterium acnes, and alpha-hemolytic streptococci were the predominant isolates from the ear samples. After hoods were worn, there was a substantial increase in the number of these organisms in 7 (36.8%) of the external ears. However, an increase in the bacterial counts in the external ear canals occurred in only 2 (10.5%) of the individuals treated prophylactically with acetic acid drops (P less than 0.02).

Production and characterization of two hemolysins of Bacillus cereus.

Bacillus cereus strain B-48 produced two hemolysins with molecular weights of 52,000 (H-I) and 31,000 (H-II). A mutant was isolated that produced only H-II but was identical with the wild type in all other respects. We exploited this mutant to produce H-II for study that was free of contamination by H-I. By manipulation of media composition, we produced H-I in the absence of H-II. The hemolysins were precipitated differently by ammonium sulfate, and both exhibited the Arrhenius effect when heated. Both hemolysins attached rapidly to erythrocytes; however, lysis by H-I was immediate, while lysis by H-II followed after a lag. Hemolysis by H-I and H-II increased in rate with increasing temperature and was absent at 0 degrees C. Only H-I was inhibited by cholesterol. The hemolysins of B. cereus appeared similar to the hemolysins of B. thuringiensis. H-I probably is identical with cereolysin.

Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation.

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

Microtitration of Bacillus cereus hemolysin.

A microtiter procedure for the quantitation of Bacillus cereus hemolysin is described.

Campylobacter fetus ss. Jejuni, a newly recognized enteric pathogen: morphology and intestinal colonization.

Campylobacter fetus ss. jejuni has recently been recognized as a frequent enteric pathogen of man. Although long known as a significant pathogen of domestic animals, little is known regarding mechanisms of colonization and pathogenesis. In an effort to study the colonization and morphology of this organism, we used scanning electron microscopy to examine ilea and colons from mice in which a human isolate of C. fetus ss. jejuni had been inoculated. When inoculated into the ileum of the mice, a transient colonization was observed by 24 hours and had disappeared by 48 hours. Colonization of the colon first appeared at 48 hours and persisted at least three weeks. When inoculated directly into the colon, colonization appeared within 24 hours in the colon and persisted at least 16 days. Severe colonic necrosis and mucosal degeneration were observed in colons only in those mice which were inoculated via the colon. In the infected colons, the C. fetus ss. jejuni organisms were enmeshed in a fibrous network which may play a role in the attachment of bacteria to the mucosal surface. Differences between the morphology of C. fetus ss. jejuni in the mouse model and that found in pure culture were observed and may represent distinct tissue and culture phases of growth.

Effect of diving and diving hoods on the bacterial flora of the external ear canal and skin.

The bacterial flora of the external ear canals and posterior auricular skin surfaces were investigated in a group of 26 divers after 25 dry-suit dives in harbor water and 20 dry-suit dives in clear test tank test. A control group of 16 divers wore rubber hoods 19 times for a similar period (25 to 30 min) but did not dive. The protective effect of 2% acetic acid was tested by instilling it in the left ear of 14 divers and 8 nondivers. Staphylococcus epidermidis, Propionibacterium acnes, alpha-hemolytic streptococci, and enteric gram-negative rods were the predominant isolates from skin and ear samples. After the divers dove or after they wore hoods without going in the water, there was a substantial increase in the number of these organisms on the skin (46.9%) or in the external ears (43.8%) of the divers. However, an increase in the bacterial counts in the external ear canals occurred in only 13.6% of the individuals treated prophylactically with acetic acid drops. Although no gram-negative rods were recovered from the skin or external ear canals of divers in clear tank water, 23 strains were isolated after the dives in harbor water. Identical gram-negative isolates also were recovered from the harbor water. Gram-negative organisms also were recovered from three newly acquired skin lacerations, where they persisted for at least 24 h. Our data show the acquisition of gram-negative rods when dives were made in polluted water. The data also demonstrate the increase in bacterial counts that occurs when rubber diving rods are worn (in or out of water) and that this increase can be controlled by pretreatment of ears with acetic acid.

Antibiotic and clavulanic acid treatment of subcutaneous abscesses caused by Bacteroides fragilis alone or in combination with aerobic bacteria.

A subcutaneous abscess model was utilized in investigating the usefulness of various antimicrobial combinations in the treatment of mixed aerobic-anaerobic infections in mice. The infected animals were treated for seven days with single or combined antimicrobial therapy. The single agents used were penicillin, clavulanic acid, oxacillin, metronidazole, and gentamicin. The antimicrobial combinations used were penicillin plus clavulanic acid and metronidazole plus penicillin, oxacillin, or gentamicin. Administration of a single agent was effective in treating abscesses caused by a single susceptible organism. The only effective therapies for mixed infections were the combinations of penicillin plus clavulanic acid or metronidazole plus gentamicin. This study demonstrated the efficacy of penicillin and clavulanic acid in the treatment of infections caused by Bacteroides fragilis alone or in combination with beta-lactamase-producing aerobic bacteria.

Enzymatic activities of cell-free extracts of Rickettsia typhi.

Cell-free extracts of Rickettsia typhi were tested for activities of enzymes of the tricarboxylic acid cycle, of glutamate catabolism, and of glycolysis. The organisms were grown in the yolk sacs of chicken embryos, harvested shortly before the time of embryo death, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The following enzymatic activities were demonstrated: high levels of malate dehydrogenase (MDH), moderate levels of glutamate-oxaloacetate transaminase, glutamate, succinate, and isocitrate dehydrogenases, and citrate synthase, and low levels of glutamate-pyruvate transaminase. The specific activities of some of these enzymes were higher when the rickettsiae were harvested at a time of active proliferation, 3 to 4 days prior to embryo death. Rickettsial MDH was differentiated from host MDH by its migration pattern on polyacrylamide gel electrophoresis. The activities of MDH and two other dehydrogenases, demonstrable after the cells had been disrupted, were absent from purified, intact rickettsial preparations. No activity was detected for glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, lactate dehydrogenases, phosphoglucose isomerase, fructoaldolase, or pyruvate kinase. Our results suggest that extracts of R. typhi that contain demonstrable enzymes involved in the catabolism of glutamate and tricarboxylic acid cycle intermediates, unlike Coxiella burnetti, lack detectable glycolytic activity.

Pathogenicity of piliated and encapsulated Bacteroides fragilis.

Of 20 isolates of the Bacteroides fragilis group, only three were encapsulated (C+) and these induced abscesses in mice. After coinoculation with live, formalized or capsular material from other bacteria seven other isolates formed abscesses. The Bacteroides recovered from these abscesses were C+; those that had been inoculated with Klebsiella had pili-like and bacteriophage-like structures. Once encapsulated, the Bacteroides isolates induced abscesses when injected alone.

Biphasic culture system for rapid Campylobacter cultivation.

We developed a biphasic culture system consisting of 4 ml of brucella agar (BA) and 6 ml of brucella broth (BB) in 25-cm2 tissue culture flasks, which were incubated in air (BB/BAa) or in a gas mixture of 5% O2, 10% CO2, and 85% N2 (BB/BAg). These media were also used with a supplement consisting of ferrous sulfate, sodium metabisulfite, and sodium pyruvate and incubated as above (FB/FAa and FB/FAg, respectively). Highly satisfactory growth of Campylobacter jejuni 301 was obtained with all medium-gas phase combinations provided that the number of viable cells in the inoculum was large (greater than or equal to 10(6)/ml). The use of FB/FAa permitted the inoculum to be reduced to 100 cells per ml. With an adjusted gas phase (BB/BAg and FB/FAg), near-optimal growth was obtained from an inoculum of 1 to 10 cells per ml. Under most of these conditions the generation time was approximately 90 min. During the logarithmic growth phase, the cells retained their typical spiral morphology and high motility. These media also proved to be highly satisfactory for the cultivation of fresh isolates as well as other stock strains of Campylobacter. When the broth phase of the cultures, after addition of 15% glycerol, was quickly frozen and maintained at -70 degrees C, all strains thus far examined were readily recoverable and satisfactorily cultivated without additional passage.

In-vivo protection of group A beta-haemolytic streptococci from penicillin by beta-lactamase-producing Bacteroides species.

The incidence of persistent group A beta-haemolytic streptococci in tonsils, despite penicillin therapy, has increased in recent years. beta-Lactamase-producing organisms have previously been recovered from 74% of patients with persisting group A streptococci. We investigated the possibility that beta-lactamase-producing strains of Bacteroides spp. can protect streptococci from penicillin. A mixed infection was induced in mice in the form of a subcutaneous abscess involving a penicillin-susceptible group A streptococcus, and a beta-lactamase-producing strain of either Bact. melaninogenicus or Bact. fragilis. The infected animals were treated for seven days with parenteral penicillin, penicillin and clavulanic acid or clindamycin. Penicillin treatment prevented the formation of abscesses in animals inoculated with group A streptococci alone, but not in those inoculated with group A streptococci and Bacteroides spp. We attributed this resistance to penicillin therapy, and the subsequent formation of abscesses in mice, to protection of the streptococci from penicillin by beta-lactamase-producing strains of Bacteroides spp. Effective therapy for mixed infections was achieved when clavulanic acid, a beta-lactamase inhibitor, was administered with penicillin. A similar effect was noted with clindamycin, which is active against both group A streptococci and Bacteroides spp.

In vivo protection of penicillin-susceptible Bacteroides melaninogenicus from penicillin by facultative bacteria which produce beta-lactamase.

We investigated the possibility that beta-lactamase producing strains of Klebsiella pneumoniae and Staphylococcus aureus can protect organisms of the Bacteroides melaninogenicus group from penicillin. A mixed infection was induced in mice in the form of a subcutaneous abscess involving a penicillin-susceptible encapsulated B. melaninogenicus, and a beta-lactamase producing strain of either K. pneumoniae or S. aureus. The infected animals were treated for 7 days with single or combined antimicrobial therapy. The single agents used were penicillin, clavulanic acid, metronidazole, and gentamicin. The antimicrobial combinations were penicillin and clavulanic acid, penicillin and gentamicin, and metronidazole and gentamicin. Administration of a single agent was effective in treating abscesses caused by susceptible organisms. The only effective therapy for mixed infections was by combination therapy of penicillin and clavulanic acid or metronidazole and gentamicin. This study supports the hypothesis that beta-lactamase producing facultative bacteria may shield their anaerobic counterparts from penicillin therapy, thereby contributing to the persistence of the infection.

Synergism between penicillin, clindamycin, or metronidazole and gentamicin against species of the Bacteroides melaninogenicus and Bacteroides fragilis groups.

Clinical isolates of the Bacteroides melaninogenicus and Bacteroides fragilis groups were tested for in vitro and in vivo susceptibility to penicillin, clindamycin, and metronidazole, used singly or in combination with gentamicin. The in vitro tests consisted of determinations of minimal inhibitory concentrations (MICs) carried out with or without constant amounts of gentamicin. When used alone, gentamicin had negligible effects on the bacteria but significantly reduced the MICs of penicillin, clindamycin, and metronidazole against 11, 10, and 3, of the 15 strains of the B. melaninogenicus group, respectively. The 15 strains of the B. fragilis group were all beta-lactamase producers and were highly resistant to penicillin or the combination of penicillin and gentamicin. However, gentamicin reduced the MICs of clindamycin and metronidazole against 1 and 7 strains of this group, respectively. The in vivo tests were carried out in mice and consisted of measurements of the effects of the antimicrobial agents on the sizes and bacterial content of abscesses induced by subcutaneous injection of bacterial suspensions. The results of the in vivo tests were generally consistent with those obtained in vitro with strains of the B. melaninogenicus group. Synergism between gentamicin and penicillin, clindamycin, or metronidazole was shown in 13, 10, and 3 strains of this group, respectively. In vivo synergism was not clearly demonstrated with the strains of the B. fragilis group, possibly because clindamycin and metronidazole used alone were highly efficacious. We suggest that the synergistic effect of gentamicin is due to its increased transport into the bacterial cell in the presence of penicillin and, possibly, other antimicrobial agents. The newly recognized in vitro and in vivo synergism between penicillin and other antimicrobial agents and an aminoglycoside in B. melaninogenicus may have clinical implications that deserve to be investigated.

Association of Aeromonas sobria with human infection.

Fifteen Aeromonas isolates from various human infections and nine isolates from polluted water were identified as either Aeromonas hydrophila or Aeromonas sobria and examined for cytotoxigenicity, enterotoxigenicity, adherence to epithelial cells, and other virulence-associated factors, including proteases, lipases, elastases, and hemolysins. Two groups of organisms (I and II) were distinguishable based on differences in median lethal doses in mice and cytotoxicity for Y-1 adrenal cells. Group I clinical and environmental strains had median lethal doses of less than 10(7) colony-forming units, were cytotoxic, frequently possessed several virulence-associated factors, and had lysine decarboxylase-positive or Voges-Proskauer-positive phenotypes or both. Piliation of Aeromonas was associated strongly with ability to adhere to human buccal cells, and these characteristics were associated with group I strains. Group II clinical and environmental strains had median lethal doses of greater than or equal to 10(7) colony-forming units, were not cytotoxic, and usually were lysine decarboxylase negative or Voges-Proskauer negative or both. Clinical strains in group II exhibited enterotoxigenicity, which was not detected in group II environmental strains. A sobria was more frequently associated with human infections; 13 of the 15 clinical strains were A. sobria, and 2 were A. hydrophila. On the other hand, the majority of the environmental strains (seven of nine) were A. hydrophila.