Cefdinir-induced modification of the susceptibility of bacteria to the antibacterial activity of human serum and polymorphonuclear neutrophils

The effect of serum on the bactericidal activity of cefdinir, and the ability of the antibiotic to modify the interaction of bacteria with human polymorphonuclear neutrophils were assessed. In the presence of antibiotic, serum-resistantEscherichia coli were sensitised to the bactericidal activity of normal human serum. Cefdinir enhanced opsonophagocytic killing ofEscherichia coli andStaphylococcus aureus at suprainhibitory concentrations. Significant potentiation of killing occurred with the combination of inhibitory concentrations of cefdinir, neutrophils and sub-optimal levels of serum opsonins. Pre-exposure ofEscherichia coli, but notStaphylococcus aureus, to cefdinir enhanced phagocytic uptake and killing of the antibiotic-damaged bacteria. These results indicate that cefdinir-mediated phenotypic modification ofEscherichia coli renders the bacteria susceptible to serum antibacterial activity and phagocytic uptake and intracellular killing.     

Funktionen der körpereigenen Abwehr gegen Anaerobier bei Gesunden und Patienten mit chronisch-septischer Granulomatose

Summary Different strains ofBacteroides fragilis exhibit great differences in sensitivity towards serum from healthy volunteers. In the presence of 10% autologous serum, neutrophilic granulocytes and monocytes (macrophages) caused significant killing ofB. fragilis. The measured phagocytic and killing activity of the cells is comparable to their activity against aerobic bacteria (S. aureus). In four patients with chronic granulomatous disease of childhood, phagocytosis was normal but killing ofB. fragilis andS. aureus in granulocytes or monocytes (macrophages) was appreciably lowered. This malfunction of the cells was accompanied by a disturbance in oxidative metabolism and inadequate iodination after phagocytosis ofB. fragilis. The results suggest that granulocytes and monocytes play an important role in host defense against endogenous infections with anaerobes.

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Abkürzungen NBT Nitroblautetrazolium - B. fragilis Bacteroides fragilis Herrn Prof. Dr. A.K. Kleinschmidt zum 70. Geburtstag gewidmet     

In vitro stimulation ofBacteroides fragilis growth byEscherichia coli

The interaction betweenBacteroides fragilis andEscherichia coli was investigated by following their growth kinetics in an in vitro mixed culture system. In Bacto-peptone or in a semisynthetic medium (SSM-M)Bacteroides fragilis could initiate growth only when a small number of viableEscherichia coli initially coexisted for a short period of time. Both Bacto-peptone and SSM-M previously spent byEscherichia coli also allowedBacteroides fragilis growth to occur but the supplementation of heat killedEscherichia coli cells or viable cell homogenates to fresh media failed to stimulateBacteroides fragilis growth. The in vitro stimulation ofBacteroides fragilis growth byEscherichia coli observed in this study may be best explained by the combination of (a) removal of oxygen and other toxic substances and (b) de novo production ofBacteroides fragilis growth stimulating factor(s) by coexistentEscherichia coli.     

Role of intestinal anaerobic bacteria in colonization resistance

The purpose of this study was to clarify the role of the intestinal anaerobe bacteria in colonization resistance. Germfree mice were associated withEscherichia coli C25 and either (a) no other species; (b) enterococcus; (c)Escherichia coli M14 andProteus mirabilis, or (d)Bacteroides fragilis andBacteroides vulgatus. All species colonized the cecum in high numbers, but only enterococcus significantly limited the translocation ofEscherichia coli C25 to mesenteric lymph nodes. However, the overall translocation rates were similar in all groups and ranged from 60% to 100%, due to translocation of other intestinal flora in addition toEscherichia coli C25. Conventionally reared mice were given either streptomycin, bacitracin/streptomycin or metronidazole which selectively eliminated facultative gramnegative bacteria, nearly all bacterial species or strictly anaerobic bacteria respectively. Only metronidazole significantly increased the rates of translocation of normal intestinal bacteria into mesenteric lymph nodes. Cohort groups of mice were then orally inoculated with drug resistantEscherichia coli C25, which actively colonized the cecum of all drug treated mice and translocated to the mesenteric lymph nodes of approximately half the streptomycin and metronidazole treated mice and nearly all the bacitracin/streptomycin treated mice. These results indicate that anaerobic bacteria play a pivotal role in limiting the translocation of normal intestinal bacteria, but that other bacterial groups also have a role in preventing the intestinal colonization and translocation of potential pathogens.     

German multicentre study on the in vitro susceptibility ofBacteroides species

In 1990 the first German multicentre study on the in vitro susceptibility ofBacteroides species was completed. Employing a commercially prepared microbroth dilution assay, nine participating institutions evaluated approximately 100 consecutive isolates ofBacteroides species from relevant clinical specimens. A total of 911 strains (449Bacteroides fragilis, 201Bacteroides thetaiotaomicron, 79Bacteroides ovatus, 78Bacteroides vulgatus, 77Bacteroides distasonis, 25Bacteroides uniformis, 2 others) were tested. Most of the isolates came from surgical patients (72 %); other sources included gynaecological patients (9 %) and medical patients (5 %). Seventy-eight percent of the anaerobes were found in mixed culture together with at least one aerobic organism (Escherichia coli 36 %, streptococci 15 %, or enterococci 13 %), while in 22 % of the cases, the anaerobes were the only bacteria grown from the specimens. The results showed that many of the strains were potent -lactamase producers (as judged by resistance to amoxicillin). However, all but one of the isolates demonstrated susceptibility in vitro when clavulanic acid was added to amoxicillin or ticarcillin. At the same time, 13 % of the organisms were resistant to mezlocillin, 5 % to cefoxitin and 4 % to clindamycin. Three strains were reported resistant to imipenem and one strain to metronidazole.Collaborating investigators: U. Höffler, Düsseldorf; W. Klietmann, Moers; K. Pelz, Freiburg; G. Peters, Köln; A. Rodloff, Berlin; G. Ruckdeschel, München; K.P. Schaal, Bonn, U. Ullmann, Kiel; H. Werner, Tübingen.     

Experimental animal model of surgical wound infection applicable to antibiotic prophylaxis

Wound infection, defined as accumulation of pus draining spontaneously or after opening of the Wound, developed in 19 out of 22 guinea pigs (86 %) after intraincisional contamination with 10⁷ Escherichia coli plus 10⁸ Bacteroides fragilis before wound closure. Antibiotic prophylaxis with gentamicin plus clindamycin significantly reduced the wound sepsis rate from 86 % to 29 % (p<0,001). Pus for bacteriological studies was available in all cases of wound infection but one, and culture always revealed bothEscherichia coli andBacteroides fragilis. The animal model Presented employs widely accepted criteria for wound infection, and avoids culture of tissue homogenates.     

Antibiotic-induced modification ofBacteroides fragilis and its susceptibility to phagocytosis by human polymorphonuclear leukocytes

Bacteroides fragilis grown in the presence of sub-inhibitory concentrations of clindamycin was shown to be altered its degree of encapsulation and susceptibility to phagocytosis by human polymorphonuclear leukocytes. Little polysaccharide capsule could be demonstrated either by light or transmission electron microscopy when the bacteria were grown anaerobically for four hours in the presence of 1/2 MIC of clindamycin. Such clindamycin-grown cells could be opsonized by normal human serum, and although less complement was consumed in the process, were more effectively taken up by the leukocytes than bacteria grown in the absence of the drug (45 % versus 24 %). It was also shown that drug treatment caused significant cellular leakage in the presence of serum, the³H-label appearing extracellularly. In addition there was greater loss of viability of the bacterial cells grown in the presence of the drug and subsequently exposed to the leukocytes for 60 min.     

Experimental intraabdominal abscess formation byEscherichia coli andBacteroides fragilis

An improved animal model of intraperitoneal abscess formation was developed in order to study the role ofBacteroides fragilis andEscherichia coli. Both microorganisms were found to independently cause abscesses, althoughBacteroides fragilis-induced abscesses were invariably minute. The combined inoculation of these microorganisms resulted in the induction of intraabdominal abscesses larger than those formed byBacteroides fragilis orEscherichia coli alone. However, the size difference between abscesses produced byEscherichia coli alone and by the combination of both organisms was not statistically significant. The animal model defined in this study differed from earlier models in that use of the complex sterile cecal contents can be avoided. This allows for a more precise evaluation of the contribution of each organism to the pathogenic process of intraabdominal abscess formation.     

Virulence factors in anaerobic bacteri

Various surface structures can be expressed inBacteroides fragilis, but little is known about capsular structures in other non-spore-forming anaerobes. Fimbriae have been isolated fromBacteroides fragilis andPorphyromonas gingivalis. The importance of iron-repressible outer membrane proteins as virulence factors inBacteroides fragilis is under study. The low endotoxic activity ofBacteroides fragilis lipopolysaccharide can be attributed to the chemical composition of this organism's lipid A. A tissue culture system for the demonstration ofBacteroides fragilis enterotoxin has recently been described. The toxins A and B ofClostridium difficile are immunologically distinct. The importance of IgA proteases and other enzymes as virulence factors in anaerobic bacteria remains unclear.     

In vitro susceptibility of clinical isolates ofBacteroides fragilis andBacteroides thetaiotaomicron in Japan

A nationwide survey of the susceptibility of 433 isolates ofBacteroides fragilis and 149 isolates ofBacteroides thetaiotaomicron was conducted from December 1986 through November 1989 in Japan. These strains were collected from 16 university hospitals and one metropolitan hospital. Metronidazole was the most active drug against both species, with no resistant isolates found. The activity of imipenem and sulbactam-cefoperazone was good, with very low resistance rates determined inBacteroides fragilis (1.4 % and 1.6 %, respectively) and inBacteroides thetaiotaomicron (3.4 % for both drugs), and was comparable to that of metronidazole. Cefoxitin, cefmetazole, cefotetan, cefbuperazone, latamoxef and ceftizoxime were found to be more active againstBacteroides fragilis, for which resistance rates were 3.2 to 9.5 %, than againstBacteroides thetaiotaomicron, for which resistance rates were 18.1 to 21.8 %. Rates of piperacillin resistance in the two species were 12.9 % and 26.8 %, respectively. Clindamycin was very active at a low concentration (MIC50 of 0.39 to 1.56 mg/l), but 24 % and 27.5 % ofBacteroides fragilis andBacteroides thetaiotaomicron isolates, respectively, were resistant to this agent.     

Changes in the susceptibility ofBacteroides fragilis group organisms to various antimicrobial agents 1979–1989

The in vitro activity of metronidazole, chloramphenicol, clindamycin and 11 -lactam antibiotics against 135 clinical isolates of theBacteroides fragilis group was compared. In addition, changes in the resistance patterns of members of theBacteroides fragilis group isolated at the Hospital Universitario San Carlos in Madrid, Spain, between 1979 and 1989 were documented. The most active -lactam drugs were imipenem and -lactamase inhibitor combinations. In 1989, however, two strains were found to be resistant to imipenem and to all other -lactam agents tested. There was no emergence of resistance to metronidazole. Chloramphenicol was very effective: only one resistant strain was detected in 1979 and no chloramphenicol-resistant isolates were found during the rest of the study period. An outbreak of clindamycin resistance was noted in 1982, and the first cefoxitin resistant strains were recovered in 1985. The changing patterns of susceptibility of anaerobic bacteria to antimicrobial agents and the emergence ofBacteroides fragilis strains resistant to new -lactam agents suggest that periodic antimicrobial susceptibility tests should be performed in order to guide the selection of antimicrobial agents for therapy.     

Effect of the presence of black pigmentedBacteroides of differing pathogenicity on the phagocytosis ofEscherichia coli by rat polymorphonuclear leucocytes

An investigation was made of the ability of rat polymorphonuclear leucocytes (PMNLs) to phagocytoseEscherichia coli in the presence of two species of black pigmentedBacteroides (B. melaninogenicus andB. intermedius). When both the bacteria were opsonized together in the presence of normal rat serum,B. melaninogenicus andB. intermedius impaired the phagocytosis ofE. coli significantly. However, the phagocytosis of these black pigmentedBacteroides remained unaffected in the presence ofE. coli. The inhibition of phagocytosis was seen only after the initial first hour of incubation. The inhibition of phagocytosis ofE. coli in the presence ofB. intermedius was more than in the presence ofB. melaninogenicus. The above observation confirms the important role played by black pigmentedBacteroides in experimental mixed infections withE. coli as observed by us earlier.     

Survey of the susceptibility patterns ofBacteroides fragilis group strains in France from 1977 to 1992

Rates of antibiotic resistance within theBacteroides fragilis group were monitored over a 15-year period in France by examining studies that employed the same methodology to test susceptibility of anaerobic bacteria. Chloramphenicol, metronidazole, beta-lactam/beta-lactamase inhibitor combinations and imipenem remained very active againstBacteroides fragilis. There was little or no change in rates of resistance to these antibiotics. Resistance to clindamycin increased from 1 % in 1977 to a peak of 19 % in 1987, and since then has remained at 8 to 12 %. There was some evidence that resistance to most beta-lactam agents increased during the same period. These results emphasize the need for periodic surveys of resistance patterns of theBacteroides fragilis group in each country.     

Evaluation of microdilution trays (Sensititre) for antimicrobial susceptibility testing ofBacteroides fragilis

Microdilution trays (Sensititre) for antimicrobial susceptibility testing ofBacteroides fragilis has been compared for accuracy with the standard agar dilution method. The microtrays were found to be reliable for all antimicrobials tested (benzylpenicillin, clindamycin, doxycycline, chloramphenicol, fusidic acid, cefoxitin, and cephalotin) but not for metronidazole.     

In vitro activity of the new quinolone BAY y 3118 against anaerobic bacteria

The in vitro activity of BAY y 3118 against anaerobic cocci,Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, otherBacteroides spp. and fusobacteria was determined by an agar dilution method. This activity was compared with that of ciprofloxacin, ofloxacin, piperacillin, cefoxitin, imipenem, clindamycin and metronidazole. BAY y 3118, imipenem, clindamycin and metronidazole were the most active agents tested. The in vitro activity of BAY y 3118 against anaerobic bacteria was superior to that of ciprofloxacin and ofloxacin.     

The role of immunoglobulins in amniotic fluid growth inhibition ofBacteroides fragilis,herpes simplex,coxsackie B 5 and cytomegalovirus

In serum and amniotic fluid (AF) from 30 pregnant women, antibodies against the anaerobeBacteroides fragilis were determined by enzyme-linked immunosorbent assay (ELISA) and the results were compared with those of previously performed tests ofB. fragilis growth inhibition by AF. The neutralizing effects of serum and AF against herpes simplex virus (HSV), cytomegalovirus (CMV) and coxsackie B 5 virus were also investigated, and antibody titres were determined by ELISA. No correlation could be demonstrated between the growth-inhibitory capacity of AF and the presence of antibodies againstB. fragilis in serum or AF. Neutralizing antibodies against HSV and coxsackie B 5 virus were found in 73% and 51%, respectively, of the sera and in 36% and 13%, respectively, of both serum and corresponding AF. In no case could an AF neutralizing effect upon HSV, coxsackie B 5 or CMV be demonstrated without a corresponding positive serum neutralization. It is concluded that the growth-inhibitory effect of AF on viruses in antibody-mediated, while AF inhibition ofB. fragilis, and possibly other bacteria as well, seems to depend upon other antimicrobial mechanisms.     

Wound infection rates after intraincisional plus systemic antibiotic prophylaxis in an animal model

The effect of intraincisional application of antibiotics in addition to systemic antibiotic prophylaxis was evaluated in a randomized blind study using a guinea pig model of surgical wound infection. Wounds were contaminated with 10⁷ Escherichia coli plus 10⁸ Bacteroides fragilis. Wound infection was defined as accumulation of pus draining spontaneously or after opening the wound. Systemic antibiotic prophylaxis with gentamicin plus clindamycin significantly reduced the wound sepsis rate from 83% in the control group to 20% in the treated group (p < 0.001). Intraincisional application of antibiotics in addition to systemic prophylaxis did not further reduce the rate of wound sepsis (p > 0.8).     

Effect of subinhibitory antibiotic concentrations on the phagocytosis ofStaphylococcus aureus

The effect of subinhibitory antibiotic concentrations on the phagocytosis ofStaphylococcus aureus was studied by pretreating³H-thymidine labelled bacteria with one-third the minimal inhibitory concentration of clindamycin, doxycyclin, cefotiam, vancomycin, piperacillin and penicillin G, respectively. Pretreatment with clindamycin and doxycyclin resulted in enhanced uptake of the bacteria by polymorphonuclear leukocytes compared to the untreated control. The augmented phagocytosis was still observed at 1/32 the MIC of clindamycin and 1/64 the MIC of doxycyclin, and when the serum was diluted to a concentration of 1 %. Pretreatment of the bacteria with penicillin, cefotiam, piperacillin and vancomycin had no effect on phagocytosis. Inhibitors of bacterial protein synthesis induce alterations ofStaphylococcus aureus leading to increased phagocytosis, whereas antibiotics acting on cell wall synthesis are without effect.     

Amniotic fluid activity againstBacteroides fragilis and group B streptococci

Summary The antimicrobial capacity of amniotic fluid (AF) againstB. fragilis, group B streptococci andEscherichia coli, the latter as a reference indicator, was investigated in samples from 30 patients. In order to study trimester as well as individual variations in the antibacterial activity, 10 individual samples from each trimester of pregnancy (AF₁, AF₂, AF₃), were separately tested. WithB. fragilis all trimester samples showed initial inhibition lasting for 8 h, the bacteria then regaining growth capacity in all AF₁, and the majority of AF₂ and AF₃ specimens. However, 4 AF₂ and 3 AF₃ samples showed continued bacteriostatic activity for up to 24 h. Group B streptococci exhibited uninhibited growth in all trimester samples. The growth ofE. coli in AF₁ and AF₂ was not inhibited, whereas in AF₃ a temporary arrest was observed at 8 h, this being followed by growth to control levels at 24 h.The findings may explain the frequent isolation ofB. fragilis from cases of septic abortions during the first trimester. Individual variation in the effectiveness of the antibacterial activity againstB. fragilis in AF₂ and AF₃ may explain why this microorganism, which is often implicated in severe maternal and neonatal infections, is occasionally recovered from the mother's endocervix without signs of disease. The inefficacy of AF in defence against group B streptococci correlates well with the frequent isolation of these bacteria from cases of severe congenital infections associated with premature delivery.