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 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.     

正常口腔细菌与拔牙创干槽症的关系

本实验就11名非腐败坏死型拔牙创干槽症与8名腐败坏死型干槽症患者的拔牙创口的细菌进行了分离鉴定。结果表明两种类型的干槽症均为口腔正常菌群的混合感染,但在与某些特殊细菌的关系上却有较明显的差异,特别是与口腔专性厌氧菌增加的关系较大。本文提示患区各种细菌的比例与正常菌群的比例差异越大,则病变越重。本文的结果也说明了一些破坏正常生理结构的外科手术最终都将导致微生态失调或菌群失调,引起手术部位的病变。     

青年咽部菌群分布状况

目的调查健康青年上呼吸道的微生物群分布,寻找优势菌群,为微生态方法治疗呼吸道感染性疾病提供科学依据。方法随机自愿原则选取沈阳医学院学生61名,采集咽后壁标本分离培养并生化鉴定。结果青年咽后壁共检出19个菌属,42个菌种。需氧菌的95％可信区间为（4．7×10^6,6．8×10^6）CFU／mL,厌氧菌的95％可信区间为（5．9×10^6,8．4×10^6）CFU／mL;需氧菌菌群密度为4．8816±1．0251,厌氧菌菌群密度为5．1347±0．9118。需氧菌中链球菌属检出率为100％,其次为奈瑟球菌属（77．4％）、葡萄球菌属（33．9％）。链球菌中缓症链球菌和口腔链球菌、奈瑟球菌属中的灰色奈瑟球菌的检出率与构成比均较高。厌氧菌中检出率与构成比较高是的韦荣球菌属、拟杆菌属、孪生球菌和放线杆菌属。结论青年上呼吸道菌群种类复杂多样;需氧菌中的口腔链球菌、缓症链球菌、灰色奈瑟球菌,厌氧菌中的韦荣球菌属、拟杆菌属、孪生球菌属为优势菌群,对于维护上呼吸道微生态平衡可能起重要的作用。     

Anaerobic bacteria in clinical infections

The findings of 275 cultures from routine clinical specimens obtained from lesions in different sites of body, during a period of 11 months, are presented. The clinical specimens were obtained from surgical wounds, abdominal infections, orthopaedic operations, biliary tract infections and pleuropulmonary infections. The total number of positive cultures including both aerobes and anaerobes was 203 out of 275 (73.8%). Of the 38 cultures positive for anaerobes, 29 (76.3%) grew both aerobic and anaerobic bacteria, while in nine (23.7%) cultures only anaerobes were found. A total of 42 strains of anaerobic bacteria were isolated. The majority of them were found in clinical specimens obtained from abdominal infections (62%), while a low percentage (3.6%) was found in specimens from orthopaedic operations. Strains belonging to the genus Bacteroides were the most frequently isolated anaerobes, accounting for 35.7% of the total, followed by Clostridia 28.5%, Peptostreptococci 23.8% and Prevotella 12%.     

Presence of anaerobic bacteroides in aerobically grown microbial granules

Microbial granules were grown in a column-type sequential aerobic sludge blanket reactor inoculated with activated sludge flocs taken from a wastewater treatment plant and containing a medium with glucose as the main carbon source. The reactor selected for granules that could settle rapidly by employing a short settling time of 2 min. Matured granules with diameters between 2 and 3 mm were examined for anaerobic bacteria as their presence can signal the onset of diffusion limitation problems that can potentially diminish granule stability due to the bacterial production of fermentation gases and organic acids under anaerobic conditions. To detect the anaerobes in the granules, clones were constructed from 16S rRNA PCR amplicons. Two sequence types associated with a strict anaerobe Bacteroides spp. were identified from these clones. Fluorescence in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM) demonstrated that cells of Bacteroides spp. were concentrated at a depth of approximately 800 mm below the surface of the granule. Cell enumeration using flow cytometry showed that the percentage of labeled cells of Bacteroides spp. compared to total bacterial cells in the granules was 0.56%. This is the first study to use a suite of culture-independent techniques to report the presence of a defined species of anaerobic bacteria in aerobically grown microbial granules.     

Catalase and Superoxide Dismutase: Distribution, Properties, and Physiological Role in Cells of Strict Anaerobes

This review considers the distribution of the main enzymes of antioxidative defense, superoxide dismutase (SOD) and catalase, in various groups of strictly anaerobic microorganisms: bacteria of the genus Clostridium, Bacteroides, sulfate-reducing and acetogenic bacteria, methanogenic archaea, etc. Molecular and biochemical properties of purified Fe-containing SODs, cambialistic SODs, and heme catalases are presented. The physiological role and origin of the enzymes of antioxidative defense in strict anaerobes are discussed. Physiological responses (induction of SOD and catalase) to factors provoking oxidative stress in the cells of strict anaerobes able to maintain viability under aerobic conditions are also considered.     

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.     

Evaluation of culture results of specimens from patients with suspected anaerobic infection.

In order to establish the etiological agents of anaerobic infections, 154 clinical specimens collected from patients with suspected anaerobic infection were cultured under both aerobic and anaerobic conditions. Bacteria were isolated from 111 (72%) of these specimens. Only aerobes were recovered from 48 (43%), only anaerobes from 31 (28%) and both aerobes and anaerobes from 32 (29%) of the 111 specimens. A total 83 anaerobic bacteria including Bacteroides fragilis group (28%), Porphyromonas (19%), Peptostreptococci (10%), Prevotella (17%), Clostridia (12%), Fusobacteria (10%), Veillonella (2%) and Eubacteria (2%) were identified. Anaerobes were most commonly isolated from peritoneal fluid followed by joint fluid, abscess and endometrial materials, blood, soft-tissue biopsy and draining material.     

Selective enumeration of Bacteroides vulgatus and B. distasonis organisms in the predominant human fecal flora by using monoclonal antibodies.

The genus Bacteroides represents about one-third of the isolates from human fecal samples. The proportions of the different species are difficult to estimate because there is no method for rapid identification of mixtures of anaerobes. Monoclonal antibodies against Bacteroides vulgatus and B. distasonis were prepared. They did not react with the other Bacteroides species of the B. fragilis group. These reagents allowed direct enumeration of B. vulgatus and B. diastasonis organisms in human fecal samples. Anaerobic bacteria resistant to 1-h contact with air were enumerated in fecal human samples, a filter was layered on the colonies, and then B. vulgatus colonies were identified by an immunoassay performed with the prepared monoclonal antibodies. Healthy human adult volunteers were tested. Most of them harbored B. vulgatus at high levels, while the B. distasonis levels were always lower. Kinetic studies suggested that time variations for each volunteer were small. The simplified quantification of Bacteroides strains at the species level described here will prove useful in complementing our knowledge of the factors which may influence the predominant human fecal flora.     

Anaerobic microbiology in 198 cases of pleural empyema: a Bulgarian study

The aim of the study was to evaluate the incidence of anaerobic bacteria in 198 patients with pleural empyema and the susceptibility of isolates to eight antibacterial agents. Isolates were identified by the Crystal anaerobes identification system, API System rapid ID 32 A and/or routine methods. Susceptibility was tested by Sceptor MIC system for anaerobic bacteria and limited agar dilution method. Anaerobic bacteria were found in 74.2% of the patients and included 247 strains within 21 genera. The predominant anaerobes were Gram-positive anaerobic cocci (52 isolates), Fusobacterium (51), microaerophilic streptococci (24), Prevotella (19) and Bacteroides species (11). Common species/groups were Fusobacterium nucleatum (in 27.2% of specimens yielding anaerobes), Micromonas micros (8.2%), Finegoldia magna (7.5%), Bacteroides fragilis group (6.8%), Peptostreptococcus anaerobius (6.1%) and F. necrophorum (5.4%). No resistance to chloramphenicol and ampicillin/sulbactam was detected. The susceptibility rates of Gram-negative anaerobic isolates to penicillin, cefoxitin, clindamycin, clarithromycin, metronidazole and tetracycline were 63.8%, 90.2%, 87.8%, 58.6%, 98.8% and 71%, and those of Gram-positive anaerobes were 79.2%, 100%, 84.3%, 68.4%, 41.9% and 75%, respectively. The wide diversity of isolated anaerobic genera and species and the susceptibility patterns of the isolates emphasize the role of the anaerobic microbiology in cases of pleural empyema.     

Empirical antibiotic therapy of wounds complicated by anaerobic non-clostridial infection

Antibacterial activity of 14 drugs against clinical strains of asporogenic anaerobes causing wound infections in the soft tissues i. e. Bacteroides fragilis and Bacteroides melaninogenicus as well as anaerobic gram-positive++ cocci was assayed with the method of serial dilutions in agar. It was shown that among the investigated species B. fragilis had the most marked resistance since out of the 14 drugs only 8 were sufficiently active against it i.e. carbenicillin, levomycetin, lincomycin, dioxidine, metronidazole, thinidazole, nitrazole and erythromycin. The choice of drugs for treating infections caused by B. melaninogenicus and anaerobic grampositive cocci unlike those caused by B. fragilis offered no difficulty since practically++ all the investigated drugs were highly active against the causative agents. There was observed relationship between the frequency of asporogenic anaerobes and the wound genesis. The characteristic features of the species composition connected with localization of the suppurative foci were indicated. The detected specific antimicrobial profiles of the asporogenic anaerobes causing wound infections and the peculiarity of their participation in development of purulent infections of the soft tissues provided a differential approach to empirical antibacterial therapy prior to the pathogen bacteriological investigation and availability of the antibioticograms.     

Salicylates and reversible hearing loss

Skin and soft tissue infections were studied in 21 seriously ill narcotic addicts who had been admitted to hospital. Subcutaneous abscesses were present in 14 patients; cellulitis was noted in 3, pyomyositis in 2 and necrotizing fasciitis in 2. In four patients there was septicemia. Infections in 14 patients (66.6 percent) were associated with anaerobic bacteria, which were the exclusive isolates in 6 patients. In seven patients (33.3 percent) isolates were exclusively aerobic bacteria and in eight both aerobes and anaerobes were present. The anaerobic isolates were clostridia (six), peptostreptococci (five), bacteroides (five), peptococci (three), and one of each of Veillonella, Propionibacterium, Eubacterium, Fusobacterium and Actinomyces. Staphylococcus aureus, generally thought to be the most common cause of subcutaneous infections in addicts, was found only in four (19 percent) patients. The other aerobic isolates were Klebsiella (five) and Enterobacter (four) species. When clinical features or the Gram stain of pus suggest that anaerobic bacteria may be present, antibiotic therapy should be directed against both aerobic and anaerobic bacteria until culture results are available.     

Qualitative and quantitative differences in normal vaginal flora of conventionally reared mice, rats, hamsters, rabbits, and dogs

We examined quantitatively the vaginal flora of conventionally reared mice, rats, hamsters, rabbits and dogs, species that are widely used as laboratory animals. Vaginal specimens were examined according to the method of analyzing intestinal flora (Mitsuoka's procedure). The total number of bacteria (aerobes and anaerobes) and the prevalence of specific bacteria were determined. The total number of bacteria was highest during estrus and lowest during diestrus or anestrus in mice, rats, hamsters, and dogs. The most predominant bacteria during estrus were streptococci in mice; gram-negative rods (GNR), streptococci, and members of the family Bacteroidaceae in rats; GNR, Bacteroidaceae and gram-positive anaerobic cocci in hamsters, and Bacteroidaceae in dogs. The increase in the total number of bacteria during estrus was caused by an increase of predominant bacteria in the vagina. Aerobes were more predominant than anaerobes in mice, and number of aerobes was comparable to that of anaerobes in rats and dogs. On the other hand, in hamsters, anaerobes were more predominant than aerobes and the total number of bacteria was highest among the laboratory animals (mice, rats, hamsters, rabbits, and dogs). However, in rabbits, bacteria were not isolated from about 90% of the vaginal specimens. Rabbits do not have cyclic reproductive stages and are usually in precoital status in the laboratory. In precoital rabbits, vaginal epithelium manifests few signs of secretion. Therefore, we suspect that the vaginal environment in precoital rabbits is comparable to that during diestrus or anestrus in mice, rats, hamsters, and dogs. These results suggest that the vaginal flora of laboratory animals is influenced by the estrous cycle, and probably by mucous secretion. Our data imply that vaginal flora differ among laboratory animals species, and researchers need to take into consideration the estrous cycle of laboratory animals when studying their vaginal flora.     

PCR-based diagnostics for anaerobic infections

Conventional methods to identify anaerobic bacteria have often relied on unique clinical findings, isolation of organisms, and laboratory identification by morphology and biochemical tests (phenotypic tests). Although these methods are still fundamental, there is an increasing move toward molecular diagnostics of anaerobes. In this review, some of the molecular approaches to anaerobic diagnostics based on the polymerase chain reaction (PCR) are discussed. This includes several technological advances in PCR-based methods for the detection, identification, and quantitation of anaerobes including real-time PCR which has been successfully used to provide rapid, quantitative data on anaerobic species on clinical samples. Since its introduction in the mid-1980s, PCR has provided many molecular diagnostic tools, some of which are discussed within this review. With the advances in micro-array technology and real-time PCR methods, the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual anaerobic species but also on whole communities.     

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.     

How obligatory is anaerobiosis?

Historically many bacteria have been classified as obligate anaerobes. They have been construed as wholly intolerant of oxygen, a feature that was originally ascribed to their lack of superoxide dismutases and catalases. Clostridial species were regarded as classic examples. We now know that this view is quite wrong: enzymes that scavenge superoxide, hydrogen peroxide and even oxygen itself abound in anaerobes. In the current issue of Molecular Microbiology, Hillmann et al. demonstrate that full production of these proteins can allow Clostridium acetobutylicum to survive and even grow in oxygenated culture medium. Evidently, oxidative defences in anaerobes can be robust. In all likelihood, they are critical for the movement of bacteria through aerobic environments to new anaerobic habitats.     

Detection of conjugal transfer systems in oral, black-pigmented Bacteroides spp.

Oral, black-pigmented Bacteroides spp. are important pathogens in oral anaerobic infections and dental disease. We detected conjugation systems in isolates of Bacteroides denticola and Bacteroides intermedius that transferred tetracycline resistance (Tetr) and penicillin resistance to Bacteroides buccae and to Bacteroides fragilis, an intestinal Bacteroides species. A cloned Tetr gene from B. fragilis hybridized to the transferable Tetr locus in the oral strains, indicating that genetic exchange occurs between these two groups of anaerobes.     

Changes in sensitivity of clinical strains of bacteria to dioxidine from 1984 to 1988

Dioxidine sensitivity of 7291 strains of aerobic bacteria and 163 strains of anaerobic bacteria was assayed with the disk diffusion method. The sensitivity of the aerobes was studied in the time course from 1984 to 1988. It was shown that during the 5-year period, the sensitivity of gram-positive bacteria to dioxidine gradually decreased. At the same time no increase in resistance of gram-negative organisms to dioxidine was observed. A high dioxidine sensitivity of obligate anaerobes, i.e. Clostridium spp., Bacteroides spp., Fusobacterium spp., anaerobic cocci and others was demonstrated.     

The occurrence of microorganisms in intraoral abscesses

The aim of this study was to assess the occurrence of microorganisms in 39 intraoral abscesses. The samples were place in transport medium. The materials were inoculated on adequate enrichment and selective media and cultivated under anaerobic and aerobic conditions. The microorganisms were found in all samples (100%), Anaerobic bacteria most frequently were recovered. The predominant anaerobes were from genus Prevotella, Bacteroides, Fusobacterium and Peptostreptococcus. Among aerobic bacteria, the most frequent were gram-positive cocci. The microaerophilic bacteria and fungi most rarely were isolated from pus samples.