Analysis of Six tonB Gene Homologs in Bacteroides fragilis Revealed That tonB3 is Essential for Survival in Experimental Intestinal Colonization and Intra-Abdominal Infection.

The opportunistic, anaerobic pathogen and commensal of the human large intestinal tract, Bacteroides fragilis strain 638R, contains six predicted TonB proteins, termed TonB1-6, four ExbBs orthologs, ExbB1-4, and five ExbDs orthologs, ExbD1-5. The inner membrane TonB/ExbB/ExbD complex harvests energy from the proton motive force (Δp), and the TonB C-terminal domain interacts with and transduces energy to outer membrane TonB-dependent transporters (TBDTs). However, TonB's role in activating nearly one hundred TBDTs for nutrient acquisition in B. fragilis during intestinal colonization and extraintestinal infection has not been established. In this study, we show that growth was abolished in the ΔtonB3 mutant when heme, vitamin B12, Fe(III)-ferrichrome, starch, mucin-glycans, or N-linked glycans were used as a substrate for growth in vitro. Genetic complementation of the ΔtonB3 mutant with the tonB3 gene restored growth on these substrates. The ΔtonB1, ΔtonB2, ΔtonB4, ΔtonB5, and ΔtonB6 single mutants did not show a growth defect. This indicates that there was no functional compensation for the lack of TonB3, and it demonstrates that TonB3, alone, drives the TBDTs involved in the transport of essential nutrients. The ΔtonB3 mutant had a severe growth defect in a mouse model of intestinal colonization compared to the parent strain. This intestinal growth defect was enhanced in the ΔtonB3 ΔtonB6 double mutant strain, which completely lost its ability to colonize the mouse intestinal tract compared to the parent strain. The ΔtonB1, ΔtonB2, ΔtonB4, and ΔtonB5 mutants did not significantly affect intestinal colonization. Moreover, the survival of the ΔtonB3 mutant strain was completely eradicated in a rat model of intra-abdominal infection. Taken together, these findings show that TonB3 was essential for survival in vivo. The genetic organization of tonB1, tonB2, tonB4, tonB5, and tonB6 gene orthologs indicates that they may interact with periplasmic and nonreceptor outer membrane proteins, but the physiological relevance of this has not been defined. Because anaerobic fermentation metabolism yields a lower Δp than aerobic respiration and B. fragilis has a reduced redox state in its periplasmic space-in contrast to an oxidative environment in aerobes-it remains to be determined if the diverse system of TonB/ExbB/ExbD orthologs encoded by B. fragilis have an increased sensitivity to PMF (relative to aerobic bacteria) to allow for the harvesting of energy under anaerobic conditions.

First case of abdominal infection caused by bacteroides fluxus.

Bacteroides fluxus is a Gram-negative anaerobic bacillus isolated from human faeces in healthy individuals. Until now, this bacterium had not been involved in human diseases. We report the first case of abdominal infection due to this microorganism in an elderly patient. A 76-year-old man with a history of chronic pulmonary obstructive disease presented with dyspnea, orthopnea and cough. The clinical evolution worsened with both a colonic ischemia and further diffuse peritonitis of pancreatic origin. Peritoneal fluid was obtained and the culture yielded B. fluxus in pure culture. Resistance to penicillin, amoxicillin-clavulanate, clindamycin and moxifloxacin was documented. Treatment with meropenem + linezolid was started, but the patient finally died due to a multiorganic failure.

Anaerobic bacteraemia: a 10-year retrospective epidemiological survey.

In order to identify current trends in anaerobic bacteraemia, a 10-year retrospective study was performed in the University Hospital Brussel, Belgium. All clinically relevant bacteraemia detected from 2004 until 2013 were included. Medical records were reviewed in an attempt to define clinical parameters that might be associated with the occurrence of anaerobic bacteraemia. 437 of the isolated organisms causing anaerobic bacteraemia were thawed, subcultured and reanalyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). There were an average of 33 cases of anaerobic bacteraemia per year during 2004-2008 compared to an average of 27 cases per year during 2009-2013 (P = 0.017), corresponding to a decrease by 19% between the first and the latter period. Also, the total number of cases of anaerobic bacteraemia per 100,000 patient days decreased from 17.3 in the period from 2004 to 2008 to 13.7 in the period 2009 to 2013 (P = 0.023). Additionally, the mean incidence of anaerobic bacteraemia decreased during the study period (1.27/1000 patients in 2004 vs. 0.94/1000 patients in 2013; P = 0.008). In contrast, the proportion of isolated anaerobic bacteraemia compared to the number of all bacteraemia remained stable at 5%. Bacteroides spp. and Parabacteroides spp. accounted for 47.1% of the anaerobes, followed by 14.4% Clostridium spp., 12.6% non-spore-forming Gram-positive rods, 10.5% anaerobic cocci, 8.2% Prevotella spp. and other Gram-negative rods and 7.1% Fusobacterium spp. The lower gastrointestinal tract (47%) and wound infections (10%) were the two most frequent sources for bacteraemia, with the origin remaining unknown in 62 cases (21%). The overall mortality rate was 14%. Further studies focusing on the antimicrobial susceptibility and demographic background of patients are needed to further objectify the currently observed trends.

Clinical characteristics associated with mortality of patients with anaerobic bacteremia.

The presence of anaerobes in the blood stream is known to be associated with a higher rate of mortality. However, few prognostic risk factor analyses examining whether a patient's background characteristics are associated with the prognosis have been reported. We performed a retrospective case-controlled study to assess the prognostic factors associated with death from anaerobic bacteremia. Seventy-four patients with anaerobic bacteremia were treated between January 2005 and December 2014 at Aichi Medical University Hospital. The clinical information included drug susceptibility was used for analysis of prognostic factors for 30-day mortality. Multivariate logistic analyses revealed an association between the 30-day mortality rate and malignancy (OR: 3.64, 95% CI: 1.08-12.31) and clindamycin resistance (OR: 7.93, 95% CI: 2.33-27.94). The result of Kaplan-Meier analysis of mortality showed that the 30-day survival rate was 83% in clindamycin susceptible and 38.1% in clindamycin resistant anaerobes causing bacteremia. The result of log-rank test also showed that susceptibility to clindamycin affected mortality (P < 0.001). Our results indicated that malignancy and clindamycin susceptibility could be used to identify subgroups of patients with anaerobic bacteremia with a higher risk of 30-day mortality. The results of this study are important for the early and appropriate management of patients with anaerobic bacteremia.

Antagonic effect of the inhibition of inducible nitric oxide on the mortality of mice acutely infected with Escherichia coli and Bacteroides fragilis.

Sepsis, the leading cause of death in intensive care units, is associated with overproduction of nitric oxide (NO) due to inducible NO synthase (iNOS), responsible for some of the pathologic changes. Aminoguanidine (AG) is a selective iNOS inhibitor with reported inconsistent actions in sepsis. To investigate the influence of iNOS, we studied models of acute bacterial sepsis using acute challenges with aerobic (Escherichia coli) and anaerobic (Bacteroides fragilis) bacteria in the presence of AG. Six-week-old, 23 g, male and female BALB/c and C57Bl/6j mice, in equal proportions, were inoculated (ip) with bacteria in groups of 4 animals for each dose and each experiment in the absence or presence of AG (50 mg/kg, ip, starting 24 h before challenge and daily until day 6) and serum nitrate was measured by chemiluminescence. Both types of bacteria were lethal to mice, with an LD50 of 6 nephelometric units (U) for E. coli and 8 U for B. fragilis. Nitrate production peaked on the second day after E. coli inoculation with 8 and 6 U (P < 0.05), but was absent after non-lethal lower doses. After challenge with B. fragilis this early peak occurred at all tested doses after 24 h, including non-lethal ones (P < 0.05). AG-treated mice challenged with E. coli presented higher survival (P < 0.05) and increased LD50. AG-treated mice challenged with B. fragilis had lower LD50 and higher mortality. Control AG-treated animals presented no toxic effects. The opposite effect of iNOS blockade by AG in these models could be explained by restriction of oxygen for immune cells or an efficient action of NO in anaerobic localized infections. The antagonic role of NO production observed in our bacterial models could explain the reported discrepancy of NO action in sepsis.

Antagonic effect of the inhibition of inducible nitric oxide on the mortality of mice acutely infected with Escherichia coli and Bacteroides fragilis

Sepsis, the leading cause of death in intensive care units, is associated with overproduction of nitric oxide (NO) due to inducible NO synthase (iNOS), responsible for some of the pathologic changes. Aminoguanidine (AG) is a selective iNOS inhibitor with reported inconsistent actions in sepsis. To investigate the influence of iNOS, we studied models of acute bacterial sepsis using acute challenges with aerobic (Escherichia coli) and anaerobic (Bacteroides fragilis) bacteria in the presence of AG. Six-week-old, 23 g, male and female BALB/c and C57Bl/6j mice, in equal proportions, were inoculated (ip) with bacteria in groups of 4 animals for each dose and each experiment in the absence or presence of AG (50 mg/kg, ip, starting 24 h before challenge and daily until day 6) and serum nitrate was measured by chemiluminescence. Both types of bacteria were lethal to mice, with an LD50 of 6 nephelometric units (U) for E. coli and 8 U for B. fragilis. Nitrate production peaked on the second day after E. coli inoculation with 8 and 6 U (P < 0.05), but was absent after non-lethal lower doses. After challenge with B. fragilis this early peak occurred at all tested doses after 24 h, including non-lethal ones (P < 0.05). AG-treated mice challenged with E. coli presented higher survival (P < 0.05) and increased LD50. AG-treated mice challenged with B. fragilis had lower LD50 and higher mortality. Control AG-treated animals presented no toxic effects. The opposite effect of iNOS blockade by AG in these models could be explained by restriction of oxygen for immune cells or an efficient action of NO in anaerobic localized infections. The antagonic role of NO production observed in our bacterial models could explain the reported discrepancy of NO action in sepsis.

Effect of moxifloxacin versus imipenem/cilastatin treatment on the mortality of mice infected intravenously with different strains of Bacteroides fragilis and Escherichia coli.

OBJECTIVES: To study the effect of moxifloxacin versus imipenem/cilastatin (hereafter referred to as imipenem) treatment on the mortality of mice infected intravenously with different strains of Bacteroides fragilis and Escherichia coli. METHODS: Groups of 20 mice each were infected intravenously with different strains of B. fragilis [moxifloxacin and imipenem susceptible or resistant, and enterotoxin (ET) positive or negative] and E. coli (moxifloxacin and imipenem susceptible). Twenty-four hours post-infection, intravenous therapy with either moxifloxacin (2.0 mg twice a day) or imipenem (2.4 mg three times a day) was started and continued for 3 days. Control groups were left untreated. Survival rates were recorded at day 7 post-infection. At that time, surviving mice were killed and numbers of bacteria in the liver and kidneys were determined. RESULTS: If compared with untreated animals, mice treated with either moxifloxacin or imipenem showed significantly improved survival (P < 0.001). There was no significant difference (P = 0.97) in the survival rates comparing the two treatment regimens irrespective of the ET positivity or the susceptibility to moxifloxacin or imipenem of the infective B. fragilis strain. However, there was a tendency that B. fragilis was recovered more often from the liver and kidneys of mice infected with ET positive strains. CONCLUSIONS: The data show that moxifloxacin was as efficacious as imipenem in reducing the mortality rate of mice suffering from a severe systemic aerobic/anaerobic infection.

T-cell proliferative responses following sepsis in neonatal rats.

Both experimental and clinical evidence suggest a suppression of T-cell function in burn and sepsis. The objective of the present study was to evaluate splenocyte and purified T-cell proliferative response and IL-2 production in septic neonatal rats. We also examined if alterations in T-cell proliferation and IL-2 production in neonatal sepsis is due to elevation in PGE2. PGE2 is known to play a significant role in T-cell suppression during sepsis in adults. Sepsis was induced in 15-day-old neonatal Sprague-Dawley rats by implanting 0.1 cm3 of fecal pellet impregnated with Escherichia coli (50 CFU) and Bacteroides fragilis (10(3) CFU). Animals receiving fecal pellets without the bacteria were designated as sterile. A group of septic and sterile rats were treated with PGE2 synthesis inhibitors, NS398 and resveratrol. These treatments of animals allowed us to evaluate the role of PGE2 in T-cell suppression during neonatal sepsis. Splenocytes as well as purified T cells were prepared and then proliferative response and IL-2 productive capacities were measured. A significant suppression of splenocyte proliferation and IL-2 production was noticed in both sterile and septic animals compared to the T cells from unoperated control rats. In contrast, the proliferation and IL-2 production by nylon wool purified T cells in sterile rats was not significantly different from control rats, whereas, a significant suppression in Con A-mediated T-cell proliferation and IL-2 production noticed in septic rat T cells compared to the sterile and control rat T cells. Such decrease in T-cell proliferation and IL-2 production was accompanied with 20-25% deaths in neonates implanted with septic pellets. No mortality was noted in sterile-implanted neonates. Treatment of animals with COX-1 inhibitor had no effect on T-cell proliferation response in both septic and sterile groups, whereas COX-2 inhibitor abrogated the decrease in T-cell proliferative response in the septic group. The treatment of animals with COX-2 inhibitor also significantly prevented the sepsis-associated mortality in neonates. In conclusion, the present study demonstrated T-cell suppression during neonatal sepsis is accompanied by a decrease in IL-2 production. Such suppressions were ameliorated with COX-2 inhibitor suggesting a role for PGE2 in the suppressed T-cell-mediated immune function in neonatal sepsis.

[Anaerobic paraproctitis]. / Anaérobnyi paraproktit.

Anaerobic paraproctitis (AP) was seen in 8.6% of 1057 cases with acute paraproctitis. Severe general condition of the patient, vast damage, fetid wound secretion, extensive detachment and necrosis of tissues were the main AP symptoms. Radical excision of affected tissues with daily revision and sanatation in combination with intensive general therapy permits to reduce mortality to 4.3%. In early postoperative period reversible insufficiency of anal sphincter developed in 16.1% AP patients. It is necessary to close wounds as early as possible. Perforated cutaneous pedicle flap from posterior wall of the scrotum may be successfully used for plastic repair of perineum wound.

Antimicrobial resistance and clinical outcome of Bacteroides bacteremia: findings of a multicenter prospective observational trial.

There is debate regarding the correlation between in vitro susceptibility testing and clinical response to therapy for Bacteroides bacteremia. We conducted a prospective multicenter observational study of 128 patients with bacteroides bacteremia. Outcome was correlated with results of in vitro susceptibility testing of Bacteroides isolates recovered from blood and/or nonblood sites, determined with use of 3 end points: mortality at 30 days, clinical response (cure vs. failure), and microbiological response (eradication vs. persistence). The mortality rate among patients who received inactive therapy (45%) was higher than among patients who received active therapy (16%; P=.04). Clinical failure (82%) and microbiological persistence (42%) were higher for patients who received inactive therapy than for patients who received active therapy (22% and 12%, respectively; P=.0002 and.06, respectively). In vitro activity of agents directed at Bacteroides species reliably predicts outcome: the specificity was 97%, and positive predictive value was 82%. Antimicrobial susceptibility testing may be indicated for patients whose blood specimens yield Bacteroides species.

A rat model of non-lethal bacterial infection.

The purpose of the study was to develop a small animal model of intraperitoneal infection without mortality and with a catabolic response to the infection, viz., to mimic the clinical situation in man. Intraperitoneal infection was induced in female Wistar rats by deposition of a gelatin capsule containing a mixture of Escherichia coli and Bacteroides fragilis and adjuvant substances. Seven groups of animals were infected with different bacterial inocula (0.2-4.3 x 10(6) CFU) to establish reproducible and dose-dependent changes in mortality, body weight in relation to food intake, blood cultures, peripheral blood leukocyte counts, and abscess formation on autopsy. No mortality was observed in animals with an inoculum below 2.2 x 10(6) CFU in spite of positive blood cultures. Initial weight loss was followed by weight gain in all animals except the group infected with the low inoculum (0.2 x 10(6) CFU). This group had no mortality, was in a catabolic state for three days, indicated by weight loss in spite of nearly normal food intake, and the infectious state was supported by intraperitoneal dissemination of small abscesses. The low-grade character of the infection was reflected by changes in peripheral blood lymphocyte and neutrophil granulocyte concentrations. In conclusion, this study presents a small animal model with a reproducible dose response to the bacterial challenge, allowing prolonged studies of metabolic changes following infection.

Attributable mortality of bacteremia associated with the Bacteroides fragilis group.

Uncontrolled studies have suggested that Bacteroides fragilis group bacteremia has an attributable mortality rate between 13% and 33%. To determine the true attributable mortality rate and the mortality risk ratio associated with bacteremia due to the B. fragilis group, we conducted a matched-pair study in which cases were matched to controls for age, gender, year of admission, principal discharge diagnosis, and types of major surgery by an investigator blinded to survival status. Cases and controls were comparable in demographic and clinical characteristics. Cases had a significantly higher mortality rate (28% vs. 8.7%, P = .002, McNemar's test), with an attributable mortality rate of 19.3% (95% CI, 8%-30%; P = .003) and a mortality risk ratio of 3.2. In a multivariate analysis, three clinical factors were independently correlated with mortality: the presence of B. fragilis group bacteremia (RR: 4.9; 95% CI: 3.7-6.0; P = .009), congestive heart failure (RR: 8.0; 95% CI: 6.6-9.3; P = .003) or chronic liver disease (RR: 6.3; 95% CI: 4.8-7.7; P = .01). Cases also had a 16-day-longer stay in the hospital (P = .0007, Wilcoxon's signed rank test) compared with controls. Thus, B. fragilis group bacteremia contributes significantly to morbidity and mortality.

Effectiveness of fluconazole in murine Candida albicans and bacterial C. albicans peritonitis and abscess formation.

The role of fluconazole in the treatment of many forms of focal mycoses remains unclear. We studied the effectiveness of three different oral doses of fluconazole in three murine models of Candida albicans peritonitis leading to intra-abdominal abscess formation. During monomicrobial Candida infection, fluconazole decreased mortality and the number of C. albicans cultured per abscess; prolonged treatment also eliminated Escherichia coli translocation. In mixed C. albicans/E. coli/Bacteroides fragilis infection, prolonged treatment with higher doses of fluconazole decreased mortality, the number of abscesses formed, and the number C. albicans per abscess. In animals with a similar polymicrobial infection but with concurrent cefoxitin treatment, fluconazole decreased mortality and the number of C. albicans per abscess; in addition, prolonged treatment reduced the number of abscesses. Amphotericin B gave similar results in all three models. These data indicate that the clinical use of fluconazole in peritonitis should be investigated.

Anti-tumor necrosis factor antibody reduces mortality in the presence of antibiotic-induced tumor necrosis factor release.

The systemic tumor necrosis factor (TNF) response has been extensively studied during infection. In addition, antibiotics that cause cell-wall lysis have been associated with endotoxinemia and, therefore, could trigger TNF release. We studied the effects of pretreatment with cefoxitin and/or anti-TNF antibody on mortality and early (90 minutes) and delayed (6 hours) serum TNF levels in a murine model of mixed Escherichia coli/Bacteroides fragilis peritonitis. At low and intermediate inocula levels, cefoxitin, but not anti-TNF antibody, prevented death, and low serum TNF levels were noted in all groups. At the highest inoculum level, mortality was uniform in control, cefoxitin, and anti-TNF antibody groups, and a significant elevation in serum TNF levels was seen only at the 6-hour point in animals receiving cefoxitin. The addition of anti-TNF antibody to cefoxitin at this inoculum level abrogated the 6-hour rise in serum TNF levels and reduced mortality to 40%. These results emphasize that the cytokine response in disease is dependent on both the nature of the insult and other forms of therapeutic interventions.

Anaerobic bacteremia: incidence, patient characteristics, and clinical significance.

PURPOSE: In the 1970s, blood culture for obligate anaerobic bacteria became routine in most United States hospitals. Since then, various authorities have reported isolation of obligate anaerobes in 5% to 25% of blood cultures. Our experience suggests a much lower frequency; therefore, we retrospectively assessed the occurrence and significance of these cultures at our institutions. PATIENTS AND METHODS: Sixty-six patients at the University of Michigan Hospitals (UMH) and nine patients at the Ann Arbor Veteran's Administration Medical Center (AAVAMC) had one or more blood cultures positive for an obligate anaerobe between July 1, 1987, and December 31, 1988. Their medical records were reviewed retrospectively. RESULTS: The proportion of positive blood cultures yielding obligate anaerobes was 3.2% at the UMH and 1.8% at the AAVAMC. The incidences of clinically significant anaerobic bacteremia at the two hospitals were 0.68 and 0.54 cases per 1,000 patient admissions. Among the 40 patients from whom significant isolates were obtained, 15 (38%) had a fatal outcome. Bacteroides and Clostridium species accounted for 90% of the isolates and all of the fatal cases. The source for anaerobic bacteremia was usually obvious; 30 of the 40 patients were given empiric antibiotic therapy for anaerobes. The gastrointestinal tract was the source in two thirds of the cases and was clearly implicated as the source of 80% of the fatal bacteremias. CONCLUSIONS: The frequency of anaerobic bacteremia in our hospitals is much lower than was suggested in several large studies during the 1970s, probably reflecting a real decline in the incidence. The clinical features of our cases are similar to those of previous studies, and the mortality is still high despite the use of antibiotics effective against anaerobes. Since most patients were thought to have anaerobic infections at the time that cultures were obtained, they were usually treated empirically. Subsequent blood cultures positive for anaerobes infrequently influenced clinical management.

The acute phase of experimental infection with Trypanosoma cruzi is more severe in mice monoassociated with strict anaerobic bacteria.

1. The influence of some components of the normal human intestinal flora on the acute phase of experimental infection with strain CL of Trypanosoma cruzi was studied in 30-day-old germ-free or gnotobiotic CFW (LOB) mice monoassociated with Bacteroides fragilis, Peptostreptococcus sp or Clostridium sp by intragastric inoculation of 10(6) bacteria 10 days before the intraperitoneal infection with 5 x 10(3) trypomastigotes/g body weight. 2. Significantly earlier parasitemia peak and mortality were observed in Bacteroides fragilis- and Clostridium-associated mice (16.75 +/- 0.96 and 15.00 +/- 1.15 days, respectively) when compared with germfree animals (18.83 +/- 1.17 days). More precocious mortality (10.40 +/- 2.06 days) and, curiously, much lower blood parasitemia were observed in Peptostreptococcus-associated mice than in other gnotobiotic mice. 3. The extent of cardiac tissue parasitism decreased in the following order: germfree, B. fragilis-associated, Clostridium-associated, and Peptostreptococcus-associated animals. The levels of inflammatory reaction decreased in the following order: germfree, Peptostreptococcus-associated, Clostridium-associated, and B. fragilis-associated mice. 4. These results show that the acute phase of experimental infection with T. cruzi was more severe in mice associated with strict anaerobic bacteria when compared with germfree animals. This suggests that a normal intestinal flora may be another factor, in addition to nutritional and genetic factors, responsible for the different susceptibility of organisms of the same species infected with T. cruzi.

[Bacteremia caused by Bacteroides. An analysis of 50 episodes]. / Bacteriemia por Bacteroides. Un análisis de 50 episodios.

During the 32 months between October 1984 and May 1987 there were 72,800 admissions in our center; 31,523 blood cultures were undertaken and in 3,831 of them (11.5%) at least one significant organism was recovered; 6.7% of blood cultures yielded anaerobic bacteria, and 3.1% Bacteroides spp. In the 1,478 instances of bacteremia recorded during that period, the relative frequencies of bacteremia by anaerobic bacteria and by Bacteroides were 8.6% and 3.9%, respectively. In overall 50 Bacteroides bacteremias (47 due to Bacteroides fragilis and 3 to Bacteroides spp), 32 (64%) were hospital acquired and 14 (28%) postsurgical. Their most common source was the abdominal cavity (25, 50%), followed by the skin and soft tissues (7, 14%), feminine pelvis (4, 8%), miscellaneous (8, 16%), and unknown (7, 14%). Fifteen bacteremias (30%) were polymicrobial, 20 (40%) were associated with septic shock, and 15 (30%) with renal failure. Overall mortality rate was 52%, and that directly attributable to bacteremia was 32%. Poor prognostic predictors included the source from a perforated viscus, septic shock, renal failure or gastrointestinal hemorrhage, and the absence of adequate antimicrobial therapy.