The role of encapsulated anaerobic bacteria in synergistic infections

Abstract: The effect of encapsulation on the virulence, survival, and protection of anaerobic bacteria from phagocytosis is reviewed. Support for the importance of encapsulated Gram-negative anaerobic rods ( Bacteroides sp., Prevotella sp. and Porphyromonas sp.), anaerobic and facultative Gram-positive cocci (AFGPC) was provided by their higher recovery rate in oropharyngeal infections, abscesses and blood, compared to their number in the normal flora. The pathogenicity of Bacteroides, Fusobacterium, Clostridium , and AFGPC was studied by inoculating them into mice and observing their ability to induce subcutaneous abscesses. Encapsulated Bacteroides, Fusobacteria , and AFGPC generally induced abscesses, whereas non-encapsulated organisms did not. However, many of the strains that had only a minimal number of encapsulated organisms (< 1%) survived in the abscesses, and they became heavily encapsulated when inoculated with other viable or non-viable encapsulated bacteria. Thereafter, these strains were able to induce abscesses when injected alone. Encapsulated Gram-negative anaerobic rods and AFGPC-induced bacteraemia and translocation, and increased the mortality of the infected animals more often than did the non-encapsulated form of the same strains. As determined by using selective antimicrobial therapy and quantitative cultures of abscesses induced in mice, possession of a capsule generally made Gram-negative anaerobic rods more important than their aerobic counterparts. Synergistic potentials were seen between encapsulated Gram-negative anaerobic rods and all tested aerobic bacteria and most AFGPC, and also between most AFGPC and Pseudomonas aeruginosa or Staphylococcus aureus . These studies demonstrated the importance of encapsulated anaerobes in mixed infections.     

Comparison of Methods for Isolation of Anaerobic Bacteria from Clinical Specimens

Five different anaerobic culture methods and several different media were compared for their ability to recover anaerobes from clinical specimens. Specimens were obtained from patients with documented infections, avoiding contamination with normal flora, and immediately placed in an anaerobic transporter. Each specimen was cultured by all methods and on all the various media. The comparative data indicate that anaerobic jars (GasPak and evacuation-replacement types) are just as effective in the recovery of clinically significant anaerobes as the more complex roll-tube and chamber methods employing prereduced media. Liquid media were disappointing as a "back-up" system but chopped-meat glucose was superior to two thioglycolate formulations. Growth of all anaerobes was poorer on selective media, but these media were very helpful in the workup of specimens containing mixed growth of anaerobic and facultative organisms. A variety of different anaerobes was isolated, but no very fastidious or extremely oxygen-sensitive organisms were recovered. This suggests that such organisms may not play a significant role in causing clinical infections.     

Antibiotics and anaerobes of gut origin

Hundreds of bacterial species make up human gut flora. Of these, 99% are anaerobic bacteria. Although anaerobes are part of the normal commensal flora, they can become opportunistic pathogens, causing serious, sometimes fatal infections if they escape from the colonic milieu. Most often, this escape occurs as a result of perforation, surgery, diverticulitis or cancer. Infections involving anaerobic bacteria are often difficult to treat because antibiotic resistance is increasing among the genera, mediated primarily through horizontal transfer of a plethora of mobile DNA transfer factors. Some of these transfer factors can also be transmitted to aerobic bacteria. It is becoming increasingly clear that antibiotic resistance trends have to be carefully monitored, and the transfer factors and mechanisms of transfer understood at a molecular level to avoid negative clinical outcomes when infections involve anaerobic bacteria.     

The presence of black-pigmented Gram-negative anaerobes in the oral cavity of edentolous subjects

Abstract The aim of this study was to investigate whether black-pigmented Gram-negative anaerobes are part of the indigenous oral flora of edentulous subjects with or without dentures. Group I consisted of 11 subjects with dentures (mean age 63.6 years, range 52–75) and Group II consisted of 39 subjects with complete dentures in both jaws (mean age 59.3 years, range 37–80). Two microbial samples for microbiological examination were taken from each subject. One of them was from the dorsum of the tongue and the other was from saliva. Black-pigmented Gram-negative anaerobes > 10⁷ cfu/ml were found in both samples. In 50 edentulous subjects, they were found more commonly from tongue (40%) than from saliva (26%). None of the subjects had Porphyromonas gingivalis . There was no statistically significant difference ( P > 0.05) between the presence of black-pigmented Gram-negative anaerobes on tongue and in saliva in the two groups. Our results suggested that high levels of black-pigmented Gram-negative anaerobes may belong to the indigenous oral flora in edentulous mouths with or without dentures.     

Black-pigmented Gram-negative anaerobes in genito-urinary tract and pelvic infections

Abstract Black-pigmented Gram-negative anaerobes are part of the normal vaginal flora and contribute to a range of superficial and deep genital infections. Prevotella melaninogenica is found in moderate numbers (10⁴⁻⁶ cfu/g) in healthy women; the numbers and detection rates increase in anaerobic vaginosis (where it may be a significant contributor to changed microbial metabolic activity that gives the signs and symptoms of this condition) and in other vaginal infective conditions. Pr. melaninogenica is also part of the mixed flora in deep pelvic infections: endometritis, post-partum and post-abortal uterine infections; salpingitis and tubo-ovarian abscesses; PID and pelvic abscesses. Porphyromonas asaccharolytica is probably not a vaginal commensal, but may be isolated from patients with vaginal or pelvic disease. It is more specifically associated with superficial abscesses (e.g. Bartholin's abscess) and ulcers of the genitalia and perineum. P. asaccharolytica was the commonest species isolated from men with genital ulcers of various primary causes and ranging in severity from superficial balanitis/balanoposthitis to synergic gangrene.     

Postpartum uterine infection in cattle

Postpartum uterine infections results from uterine contamination with bacteria during parturition. The prevalence of uterine infections varies considerably among studies. Uterine infection implies adherence of pathogenic organisms to the mucosa, colonization or penetration of the epithelium, and/or release of bacterial toxins that lead to establishment of uterine disease. The development of uterine disease depends on the immune response of the cow, as well as the species and number (load or challenge) of bacteria. The postpartum uterus has a disrupted surface epithelium in contact with fluid and tissue debris that can support bacterial growth. A variety of species of bacteria, both Gram-positive and Gram-negative aerobes and anaerobes, can be isolated from the early postpartum uterus. Most of these are environmental contaminants that are gradually eliminated during the first 6 weeks postpartum. A normal postpartum cow resolves uterine infection by rapid involution of the uterus and cervix, discharge of uterine content, and mobilization of natural host defenses, including mucus, antibodies and phagocytic cells. Clinical signs of uterine infection vary with the virulence of the causative organisms and the presence of factors that predispose to the disease. The treatment of endometritis and metritis in bovine should be directed towards improving fertility. The antibiotic should be active against the main uterine pathogens and should maintain its activity in the environment of the uterus. Also, should not inhibit the normal defense mechanisms and should be well tolerated and not induce irritation in the endometrium. Effective use of hormones in uterine infection requires knowledge of both normal reproductive endocrinology and the therapeutic characteristics of available hormonal preparations.     

Anaerobic balanoposthitis.

To assess the causative role of non-sporing anaerobes in cass of erosive balanoposthitis, anaerobic culture was performed on purulent discharges from 104 patients with penile ulceration, a foul-smelling discharge, and a mixed and motile bacterial flora. Most of 29 culturally confirmed infections were due to mixed anaerobes and eight to single anaerobes. A rapid response to treatment with metronidazole also confirmed the anaerobic cause of the infection. Thus, acute anaerobic balanoposthitis can be readily diagnosed clinically and is easily treated.     

The place of molecular genetic methods in the diagnostics of human pathogenic anaerobic bacteria. A minireview

Anaerobic infections are common and can cause diseases associated with severe morbidity, but are easily overlooked in clinical settings. Both the relatively small number of infections due to exogenous anaerobes and the much larger number of infections involving anaerobic species that are originally members of the normal flora, may lead to a life-threatening situation unless appropriate treatment is instituted. Special laboratory procedures are needed for the isolation, identification and susceptibility testing of this diverse group of bacteria. Since many anaerobes grow more slowly than the facultative or aerobic bacteria, and particularly since clinical specimens yielding anaerobic bacteria commonly contain several organisms and often very complex mixtures of aerobic and anaerobic bacteria, considerable time may elapse before the laboratory is able to provide a final report. Species definition based on phenotypic features is often time-consuming and is not always easy to carry out. Molecular genetic methods may help in the everyday clinical microbiological practice in laboratories dealing with the diagnostics of anaerobic infections. Methods have been introduced for species diagnostics, such as 16S rRNA PCR-RFLP profile determination, which can help to distinguish species of Bacteroides, Prevotella, Actinomyces, etc. that are otherwise difficult to differentiate. The use of DNA-DNA hybridization and the sequencing of special regions of the 16S rRNA have revealed fundamental taxonomic changes among anaerobic bacteria. Some anaerobic bacteria are extremely slow growing or not cultivatable at all. To detect them in special infections involving flora changes due to oral malignancy or periodontitis, for instance, a PCR-based hybridization technique is used. Molecular methods have demonstrated the spread of specific resistance genes among the most important anaerobic bacteria, the members of the Bacteroides genus. Their detection and investigation of the IS elements involved in their expression may facilitate following of the spread of antibiotic resistance among anaerobic bacteria involved in infections and in the normal flora members. Molecular methods (a search for toxin genes and ribotyping) may promote a better understanding of the pathogenic features of some anaerobic infections, such as the nosocomial diarrhoea caused by C. difficile and its spread in the hospital environment and the community. The investigation of toxin production at a molecular level helps in the detection of new toxin types. This mini-review surveys some of the results obtained by our group and others using molecular genetic methods in anaerobic diagnostics.     

Occurrence of plasmids in black-pigmented Gram-negative anaerobes

Abstract Black-pigmented Gram-negative anaerobes are causative agents of pyogenic infections and are closely linked to various forms of periodontal diseases. Whereas many studies have shown a high incidence of plasmids in intestinal Bacteroides spp., there have been only a few reports of plasmid analyses in pigmented Gram-negative anaerobes. According to previous reports and confirmed in this study, plasmids can be present in Porphyromonas asaccharolytica, Prevotella intermedia, Pr. melaninogenica , and B. levii but have not been detected in P. gingivalis or other black-pigmented species. There were no correlations between plasmids and phenotypes such as resistance to antibiotics or bacteriocinogenicity. The highest carriage rate was found in isolates from cases of chronic otitis media, but the relationship between this site of infection and a high incidence of plasmids could be incidental. The size of plasmids ranged from 1.5 to 29 MDa. Plasmids with molecular weight > 10 MDa were described for the first time in these organisms. Repeated plasmid analyses showed that the plasmid patterns were generally stable.     

R Factor Transmission In Vivo

Experimental infections were induced in weanling pigs orally both with nalidixic acid (NA)-sensitive and -resistant strains of Salmonella choleraesuis var. kunzendorf, designated RC221 and RC221NA, respectively. Prior to the time of infection, cultures of normal bacterial flora were isolated from swine fecal matter and screened for the presence of R factors. A majority of these bacterial isolates harbored transferable resistances. Both strains RC221 and RC221NA have been shown to be competent recipients in vitro of the R factors present in the normal intestinal flora. The property of NA resistance greatly facilitated recovery of the infecting organism. After infection, salmonellae from liver, lung, spleen, lymph node, intestine, and feces were screened for the presence of R factors. Transfer of drug resistance in vivo was a rare occurrence; however, if infected specimens, particularly intestinal, were incubated in nutrient broth prior to plating, R factor transfer occurred, presumably in the test tube. Changes in recipient cultures were frequently observed after introduction of R factors from organisms of pig origin into the S. choleraesuis var. kunzendorf test organisms. Alterations include changes in typing reaction, granular growth in broth, differences in colony form, and reduction of virulence.     

Intestinal Bacteria — The Role They Play in Normal Physiology,Pathologic Physiology,and Infection

Anaerobic bacteria predominate in the normal human fecal flora, out-numbering aerobes at least 100 to one. The two most prevalent organisms are Bacteroides fragilis and Bifidobacterium. Ileostomy flora is, on the other hand, chiefly aerobic and the total count is lower (10⁸ per ml of fluid, compared to 10¹⁰ per gram for feces). In normal people, small bowel bacterial counts are generally 10⁵ per ml or less. The upper small bowel consists primarily of Gram-positive aerobes in small numbers. In the terminal ileum, counts are higher and aerobes and anaerobes are present in equal numbers. In the presence of acute obstruction and certain bowel stasis or other syndromes, the small bowel flora may become relatively profuse and fecal in type. The stomach normally has less than 10³ organisms per ml but counts are higher in gastric samples with pH above 4.0.Intestinal bacteria are important in such processes as conversion of bilirubin to urobilinogen, supply of vitamin K to the host, defense against infection, bile acid deconjugation and conversion, infections related to the bowel, the malabsorption of blind loop and other bacterial overgrowth syndromes, and hepatic coma.     

Pathogenic synergy: mixed infections in the oral cavity

In almost all infections in the oral cavity, mixed populations of bacteria are present. However, recent evidence points to a certain specificity in these infections:Streptococcus mutans is related to caries and black-pigmentedBacteroides species are suspected pathogens in periodontal disease. Periodontal diseases, endodontic infections and submucous abscesses in the oral cavity are probably mixed infections in which anaerobic bacteria together with facultatives or other anaerobes are present. In experimental mixed anaerobic infections black-pigmentedBacteroides strains have been shown to play a key role. Little is known about the pathogenic synergy between the bacteria involved in mixed infections. Important mechanisms could be nutritional interrelationships and interactions with the host defense. Within the group of black-pigmentedBacteroides B. gingivalis seems to be the most virulent species. These bacteria possess a great number of virulence factors, which might be important in the pathogenesis of oral infections.     

Staphylococcus aureus and Staphylococcus epidermidis Virulence Strains as Causative Agents of Persistent Infections in Breast Implants

Staphylococcus epidermidis and Staphylococcus aureus are currently considered two of the most important pathogens in nosocomial infections associated with catheters and other medical implants and are also the main contaminants of medical instruments. However because these species of Staphylococcus are part of the normal bacterial flora of human skin and mucosal surfaces, it is difficult to discern when a microbial isolate is the cause of infection or is detected on samples as a consequence of contamination. Rapid identification of invasive strains of Staphylococcus infections is crucial for correctly diagnosing and treating infections. The aim of the present study was to identify specific genes to distinguish between invasive and contaminating S. epidermidis and S. aureus strains isolated on medical devices; the majority of our samples were collected from breast prostheses. As a first step, we compared the adhesion ability of these samples with their efficacy in forming biofilms; second, we explored whether it is possible to determine if isolated pathogens were more virulent compared with international controls. In addition, this work may provide additional information on these pathogens, which are traditionally considered harmful bacteria in humans, and may increase our knowledge of virulence factors for these types of infections.     

Use of antibiotics in the management of postirradiation wound infection and sepsis

Ionizing gamma irradiation depresses the host defenses and enhances the susceptibility of the immunocompromised host to local and systemic infection due to endogenous or exogenous microorganisms. Trauma and wounding act synergistically and decrease the survival after exposure to irradiation. The current antimicrobial agents suitable for controlling serious infections and their use in post irradiation local and systemic infection with and without trauma are discussed. The experience gained in managing immunocompromised patients following chemotherapy is reviewed. Empiric single agent or combination agent therapy should be directed at the eradication of potential gram-negative as well as gram-positive pathogens. The most important organisms known to cause these infections are Pseudomonas sp. and Enterobacteriaceae. Management of intra-abdominal infections following trauma should include early surgical correlation and antimicrobials directed against the Bacteroides fragilis group and Enterobacteriaceae. Staphylococcus aureus and Streptococcus pyogenes cause most skin and soft tissue infections following trauma. Chemoprophylaxis of enteric sources of systemic infection can be achieved by antimicrobials that selectively inhibit the Enterobacteriaceae sp. and preserve the anaerobic flora. The management of infection in the injured and irradiated host includes supportive and restorative therapy. Supportive therapy includes débridement and cleansing of wounds, fluids, immunoglobulin, and antimicrobials. Restorative therapy includes definite surgery repair and replenishment of the immune system by use of immunomodulators, growth factors, and bone marrow transplantation. Further studies are needed to examine the usefulness of presently available drugs and experimental agents in the irradiated and traumatized host.     

Black-pigmented Gram-negative anaerobes in endodontic infections

Abstract Necrotic dental root canal infections are polymicrobial infections dominated by anaerobic bacteria. The number of different species in one canal is usually low, approx. 4–7 species. The species isolated most frequently belong to the genera Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Eubacterium and Streptococcus . The frequency of isolation of black-pigmented Gram-negative anaerobes in endodontic infections varies from 25% to >50%. Pr. intermedia is the most commonly found pigmented species, followed by Pr. denticola and two Porphyromonas species, P. gingivalis and P. endodontalis . Several studies have shown that P. gingivalis and P. endodontalis are closely related to the presence of acute symptoms in endodontic infections, whereas other black-pigmented Gram-negative anaerobes are not. However, several other species may also be involved in acute infections. Moreover, Porphyromonas species have occasionally been isolated from cases with no symptoms. Although Porphyromonas spp. are clearly related to symptoms at the beginning of therapy, they are not important for the prognosis of the treatment.     

Use of Zebrafish to Probe the Divergent Virulence Potentials and Toxin Requirements of Extraintestinal Pathogenic Escherichia coli

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, α-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.     

The role of microbial interactions in infectious disease

The occurrence of infectious disease is affected by interaction between microorganisms in three ways. The indigenous flora (commensal microorganisms) of some mucous surfaces provide one of the main protective mechanisms against infection by pathogens (disease-producing microbes). The commensal populations interfere with the establishment of pathogens on mucous membranes by evoking anaerobic conditions, by competing for space and nutrients and by producing inhibitors. How, at the beginning of successful infection, pathogens in relatively small numbers overcome this protective activity of the commensal population is unknown. Although not a general phenomenon, some pathogens exacerbate the effects of others. The best examples are the potentiation of bacterial infections by existing viral infections: mucosal adherence and penetration by bacteria are enhanced and phagocytic defences against them weakened. Some microorganisms that are unable to produce significant disease on their own may combine with others to cause serious sickness. The harmful effects of these combinations of microorganisms can be explained by the multifactorial nature of pathogenicity (virulence), i.e. the capacity to produce disease. Although each member of the mixed population cannot alone produce the full complement of factors needed for disease production, the complement can be attained by combining contributions from different members.