Gram-Negative Anaerobes in the Intestinal Flora of Pigs

A differential count of the gram-negative, anaerobic, non sporeforming bacteria in different segments of the pig intestinal tract was performed in 2 groups of pigs. Seventy-seven strains were identified, belonging to 6 groups: Sphaerophorus necrophorus, anhemolytic Sphaerophorus spp., Bacteroides (Eggerthella) spp., Fusobactenum spp., Veillonella/Acidaminococcus spp., and Peptostreptococcus elsdenii. The characters of these groups are described, and quantitative data on their occurrence in a group of normal porkers and a group of pigs with experimental swine dysentery are given.     

Epidemiological characteristics of infections caused by Bacteroides, Prevotella and Fusobacterium species: a prospective observational study

In order to investigate differences among infections due to Gram-negative anaerobic bacteria (Bacteroides, Prevotella and Fusobacterium spp.), clinical, epidemiological, and microbiological data were collected and evaluated from 206 anaerobic infections. The most frequently isolated species was Bacteroides fragilis. The majority of the cases were intra-abdominal infections (49%) followed by skin and soft tissue infections (24.7%). Logistic regression analysis showed that Bacteroides spp. strains were more often isolated from intra-abdominal infections (p?=?0.002), whereas Prevotella spp. were isolated more frequently from cases with shorter duration of hospitalization (p?=?0.026), and less frequently from bloodstream infections (p?=?0.049). In addition, Bacteroides spp. were associated with coinfection due to Enterobacteriaceae species (p?=?0.007), whereas Prevotella spp. were associated with coinfection due to Staphylococcus spp. (p?=?0.002). Patients with an infection due to B.?fragilis, were more frequently admitted in a general surgical ward (p?=?0.017), or have been treated with a 2nd generation cephalosporin before anaerobic infection onset (p?=?0.05). Total mortality was 10.9% and was associated with bacteremia (p?=?0.026), and hematological (p?=?0.028), or solid organ malignancy (p?=?0.007). Metronidazole resistance was detected only among Prevotella spp. (16.2%) and B. fragilis group (0.8%) isolates. In conclusion, this study indicated differences between infections due to the most frequently isolated Gram-negative anaerobic species, differences that may affect the design and implementation of empirical antimicrobial chemotherapy guidelines.     

Beta-lactamases with a wide substrate spectrum in gram negative strictly anaerobic rods

This study was performed to determine the susceptibility of the clinical strains of Gram-negative strictly anaerobic rods to newer beta-lactam antibiotics. Also, the trial was undertaken to detect strains producing extended-spectrum beta-lactamases (ESBLs) and inducible beta-lactamases (IBLs) among Bacteroides spp. and Prevotella spp. rods isolated from hospitalized patients. One hundred strains of Gram-negative, obligatory anaerobic rods were applied in the study. The strains were identified in automatic ATB system using API 20 A strips. beta-lactamase-positive strains were determined with disc nitrocefin test. ESBL-producing strains were detected with double disc test according to Jarlier et al. (1988). Clavulanate was applied as the inhibitor of these beta-lactamases (AMO/CLAV disc). ESBL-positive strains were confirmed with the use of E test (TZ/TZL strip). Inducible beta-lactamases were determined by double disc method according to Sanders and Sanders (1979). Cefoxitin was the inducer of these beta-lactamases (FOX disc). Among 93 Bacteroides spp. strains and 7 Prevotella spp. strains, 91 strains (91%) produced beta-lactamases. Two ESBL-producing strains (2%) were detected. Strains producing inducible beta-lactamases (IBL) were not found. A high activity of the examined beta-lactam antibiotics against strains of Gram-negative anaerobes was found. The majority of strains were susceptible to piperacillin (95%), piperacillin combined with tazobactam (99%), ticarcillin combined with clavulanic acid (99%), meropenem (97%) and imipenem (99%). The obtained results indicate the necessity of ESBL determination among strains of the genus Bacteroides, isolated from clinical specimens. Newer beta-lactam antibiotics, especially penicillins in combination with beta-lactamase inhibitors and carbapenems, are useful in empiric therapy of infections caused by Bacteroides spp. and Prevotella spp. anaerobic rods.     

Draft genome sequence of Bacteroides faecis MAJ27T, a strain isolated from human feces

Despite the ecological importance of the dominant gut bacteria Bacteroides, few genomes have been defined. The Gram-negative, strictly anaerobic intestinal bacterium Bacteroides faecis MAJ27(T) was isolated from the feces of a healthy adult. Here, the draft genome sequence of the type strain B. faecis MAJ27 (6.11 Mbp) is reported.     

Dehydrogenase Patterns in the Study of Bacteroidaceae

Enzyme patterns were obtained by starch-gel electrophoresis of cell-free extracts from organisms in the family Bacteroidaceae. Esterases, phosphatases, and lactic and succinic dehydrogenases were detected but offered no possibility for classification purposes. Alpha-glycerophosphate dehydrogenase and 6-phosphogluconate dehydrogenase were not detected. Zymograms of malic, glutamic, and glucose-6-phosphate dehydrogenases separated Bacteroides species from Sphaerophorus and Fusobacterium species. Malic dehydrogenase and glucose-6-phosphate dehydrogenase were found in Bacteroides but not Sphaerophorus and Fusobacterium. These dehydrogenase zymograms placed three gram-negative, nonsporeforming anaerobic rod isolates with the Bacteroides species. A close correlation was found between the classification of Bacteroidaceae by zymogram analysis and a numerical taxonomy scheme previously published from this laboratory.     

Some Morphological and Physiological Characteristics of Gram Negative Anaerobic Bacteria isolated from the Alimentary Tract of the Pig

S ummary . A collection of 100 strains of Gram negative anaerobic bacteria isolated from the alimentary tract of the pig has been divided into four groups on morphological and physiological grounds. The four groups resemble the genera Bacteroides, Sphaerophorus, Veillonella and Peptostreptococcus as described in Bergey's Manual (Breed, Murray & Smith, 1957).     

Characteristic of clinical strains of gram-negative obligate anaerobes

The aim of the study was to assess prevalence and antibiotic susceptibility profiles ofGram-negative strictly anaerobic bacteria isolated from clinical specimens taken from hospitalized patients in 2005-2006. Biochemical identification and antibiotic susceptibility were done in an automated system ATB Expression (bioMerieux sa). From 12262 specimens examined 867 strains of obligate anaerobes were isolated. Gram-negative strictly anaerobic bacteria were cultured in number of 138 strains (15,9%). All cultures were performed on Columbia agar and Schaedler agar media (bioMerieux sa) supplemented with 5% sheep blood and incubated at 37 degrees C for 48-120 h in 85% N2, 10% H2, 5% CO2. Most frequently isolated was Bacteroides spp. (41,3%). For this group beta-lactamase activity was evaluated by using nitrocefin disc test (Cefinase BBL, Becton Dickinson and Co., Cockeysville, MD, USA). Production of ESBLs was detected with the use of two disc diffusion methods: the double-disc synergy test (DDST) according to Jarlier et al. and the diagnostic disc (DD) test according to Appleton. ESBLs were produced by 5,3% strains of Bacteroides spp. For all Bacteroides spp. strains MIC values were determined by gradient diffusion method Etest (AB BIODISK, Sweden). ESBLs and MIC were performed on Wilkins-Chalgren solid medium supplemented with 5% sheep blood (Difco Lab., USA) and all plates were incubated at 35 degrees C for 48 hours in 85% N2, 10% H2, 5% CO2. Most Gram-negative obligate anaerobes isolated from clinical specimens are still susceptible to imipenem (100%), metronidazole (99,3%) and beta-lactam antibiotics with beta-lactamase inhibitors: piperacillin/tazobactam (99,3%), ticarcillin/clavulanate (99.3%), amoxicillin/clavulanate (97.8%).     

Molecular microbiology of gut bacteria: genetic diversity and community structure analysis

Recently developed molecular biology approaches make possible the detailed genetic, taxonomic and ecological examination of microorganisms from various habitats. Animal gut represents one of the most complex microbial ecosystems with a large degree of microbial biodiversity present. Bacteria inhabiting the gut usually play important roles in metabolic transformations of substrates and sometimes, e.g. in ruminants, they make the basis for an obligate symbiosis with the host. Here we discuss molecular microbiology as a strategy for examination of gut bacteria, concentrating on a typical and in such environment dominant group of strictly anaerobic Gram-negative bacteria from the phylogenetic group Cytophaga/Flexibacter/Bacteroides. The bacteria from the genus Prevotella are the most abundant Gram-negative bacteria in the rumen and form a distinctive phylogenetic cluster, clearly separated from prevotellas isolated from other ecological niches. They may represent a good choice for a model organism in genetic manipulation experiments and for studies of gene transfer mechanisms taking place in the gut. The molecular tools for detection and monitoring of ruminal prevotellas are discussed.     

Anaerobic bacteria in bronchoalveolar lavage fluid (BAL) after thoracic surgery

Purpose of this study was to find out what kind of anaerobic bacteria were in lower respiratory tract and how often they were present there considering patients after thoracic surgery. Also, what is susceptibility of bacteria to antibiotics. Research covered 30 patients after operation. Material for research was bronchoalveolar lavage (BAL) taken during bronchoscopy. Collected sample was cultivated in anaerobic and aerobic conditions. Anaerobic bacteria were found in 28 samples (93%). Totally there were 100 anaerobic bacteria strains. The most common Gram-negative rods were from genus Prevotella (24 strains, 24%) and Bacteroides (15 strains, 15%). Gram-negative bacteria except Bacteroides characterised biggest susceptibility to imipenem, piperacillin/tazobactam, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin, clindamycin and metronidazol. Bacteroides were susceptible to imipenem, piperacillin/tazobactam and metronidazol. Among Gram-positive anaerobic bacteria mostly were isolated from cocci Peptostreptococcus (18 strains, 18%) and were susceptible to all antibiotics. Gram-positive rods were in most cases represented by Actinomyces (12 strains 12%) and were highly susceptible to all antibacterial means except metronidazol (100% is resistant).     

The range of antagonistic effects of Lactobacillus bacterial strains on etiologic agents of bacterial vaginosis

Bacterial vaginosis is caused by uncontrolled sequential overgrowth of some anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia, Bacteroides spp., Peptostreptococcus spp., Mobiluncus sp. usually occurring in stable numbers in the bacterial flora of healthy women. On the other hand, different species of bacteria belonging to the genus Lactobacillus, most frequently L. plantarum, L. rhamnosus and L. acidophilus, form a group of aerobic bacteria dominating in the same environment. The diversity and density of their populations depend on the age and health conditions. Thanks to their antagonistic and adherence properties bacteria of the genus Lactobacillus can maintain a positive balance role in this ecosystem. The aim of this study was to assess the antagonistic properties of Lactobacillus strains isolated from the vagina of healthy women against most common agents of bacterial vaginosis. It was found that nearly all of the tested Lactobacillus strains exerted distinct antagonistic activity against anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia and Peptostreptococcus anaerobius and quite a number also against Gram-negative rods, while only some of them were able to inhibit Gram-positive aerobic cocci as Enterococcus faecalis or Staphylococcus aureus.     

Anaerobic bacteria in the gut of terrestrial isopod crustaceanPorcellio scaber

Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and Enterococcus were retrieved, as well as sequences from 'A1 subcluster' of the wall-less mollicutes. Bacteria from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable, i.e. predominantly oxic, conditions.     

Anaerobic bacteria detected in inflammatory conditions of the respiratory tract

In this study a participation of anaerobic bacteria in respiratory tract diseases is presented. Bronchial washings collected by ++fibrobronchoscope constituted material for the study. Immediately after collection the material was plated onto two media for aerobic bacteria (hemomedium) and anaerobic bacteria (anaeromedium). Then, the samples were centrifuged and a sediment was plated on solid media suitable for aerobic and anaerobic bacteria. Bacterial anaerobic isolates were identified by using API 20E and their sensitivity to antibiotics was tested. From the material described above the most frequently isolated anaerobic bacteria were such as: Streptococcus intermedius, Bacteroides melaninogenicus, Veilonella sp. Among aerobic bacteria the most frequently isolated were Gram-negative rods, Streptococcus faecalis, Branhamella catarrhalis. It is worth to underline that in about 25% of cases anaerobic bacteria were the only isolates.     

Unusual Sphaerophorus Species from the Large Intestine of Man

An obligately anaerobic, gram-negative microorganism identified as a Sphaerophorus species was recovered from the fecal material of two cancer (chronic myelogenous leukemia and idiopathic thrombocythemia) patients receiving cobalt radiation therapy. The organism, isolated on sheep blood-agar, exhibited extreme pleomorphism (rods, filaments, and spheroids) and was a major component of the anaerobic fecal microflora. In one patient the numbers of Sphaerophorus species (designated as isolate 6-13-68), Bacteroides species, and Clostridium perfringens declined after irradiation; however, they were stable in this same patient after a second therapeutic dose of radiation. The numbers of anaerobes in the other patient remained fairly consistent after radiation. The biochemical and morphological characteristics and carbohydrate fermentation reactions of isolate 6-13-68 most closely resembled those of Sphaerophorus ridiculosis.     

Participation of gram-negative anaerobes in infections in hospitalized patients

The aim of the study was to assess the prevalence and antibiotic susceptibility profiles of strictly anaerobic Gram-negative bacteria isolated from clinical samples taken from hospitalized patients from 01.01.2007 to 31.12.2008. The specimens were cultured using media, incubated at 37 degrees C under anaerobic conditions. Biochemical identification and antibiotic susceptibility were done in an automated system ATB Expression (bioMerieux S.A, France). For selected strains of Bacteroides sp. sensitivity was determined using E-test (AB BIODISK, Sweden). Overall 1274 strains of obligate anaerobes were isolated. Gram-negative bacteria were cultured in number of 333 strains. Most frequently isolated was Bacteroides sp. (46,9%) and Prevotella sp. (29,7%). Isolated bacteria are still susceptible to imipenem (100%), metronidazole (100%) and beta-lactam antibiotics with beta-lactamase inhibitors: amoxicillin/clavulanate (97,8%) piperacillin/tazobactam (99,1%), ticarcillin/clavulanate (99,1%).     

Isolation of Peptostreptococcus heliotrinreducens from human polymicrobial abscesses

Five strains of Peptostreptococcus heliotrinreducens were identified out of 256 strains of Gram-positive anaerobic cocci from human sources. They could be distinguished from similar organisms, notably P. micros , by their preformed enzyme profile, colonial morphology and sensitivity to novobiocin and neomycin. They were isolated from polymicrobial abscesses in which other anaerobes, particularly Bacteroides spp., were present.     

Identification of a new ribosomal protection type of tetracycline resistance gene,tet(36), from swine manure pits

Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.     

Microbial Ecology of the Gut in Laboratory Stocks of the Migratory Grasshopper, Melanoplus sanguinipes (Fab.) (Orthoptera: Acrididae)

Mean pH values in pooled samples of foregut, midgut, and hindgut from adult Melanoplus sanguinipes, which had been raised in the laboratory on barley shoots and wheat bran, were 5.15, 6.39, and 5.98, respectively. Homogenates of midgut/hindgut sections and frass (feces) yielded colony counts of bacteria by the spread plate method of 5.7 to 5.9 and 5.3 to 5.5 log₁₀ colonies per mg, respectively; there were no significant differences (P > 0.05) between counts obtained on several media or on media incubated aerobically or anaerobically. There was no evidence of significant populations of protozoa, fungi, or obligately anaerobic bacteria associated with the gut. A total of 168 pure strains of bacteria isolated from the gut sections were characterized and assigned to 11 taxonomic groups, including Enterococcus spp., Serratia liquefaciens, Pseudomonas spp., and Enterobacter spp. Numbers of Enterococcus spp. in the gut were 2 to 3 orders of magnitude higher than those of the other genera. Strains representing only four of the groups were recovered from bran fed to the grasshoppers; the barley shoots, which were raised in sterile soil, appeared virtually sterile. Examination of the gut wall by scanning electron microscopy revealed the presence of epimural bacteria in the foregut and hindgut but not in the midgut. The distribution of epimural cocci and bacilli differed with the gut section examined. Numerous spherical to ovoid structures up to 10 μm in diameter, which were not identified, were associated with the microvillous surface of the midgut epithelium. Acetate was present in gut, hemolymph, and frass, and it was shown that representative isolates of Enterococcus spp. and Enterobacter agglomerans produced acetate when incubated in an aqueous suspension of bran. The egestion time of solid digesta, as measured with methylene blue-stained barley shoots, was 3.0 to 5.7 h. The results show that M. sanguinipes supported extensive indigenous populations of luminal and epimural bacteria in the gut which were composed predominantly of facultatively anaerobic species; the relatively short egestion time, indicating rapid passage of digesta through the gut, was consistent with the microscopic appearance of digesta residues in frass and could account, at least in part, for the absence of a significant population of obligately anaerobic bacteria from the gut.     

Succinate accumulation in pig large intestine during antibiotic-associated diarrhea and the constitution of succinate-producing flora

Succinate was the major organic acid detected in the hindgut content of pigs suffering from antibiotic-associated diarrhea. Antibiotic-associated diarrhea was induced by an oral dose of polymyxin B sulfate (3,000,000 units/day) or an intramuscular injection of enrofloxacin (0.6 g enrofloxacin/day). In the large intestine of enrofloxacin-treated pigs, Gram-negative facultative anaerobic rods phylogenetically related to Escherichia coli and Gram-positive facultative anaerobic non-spore-forming rods phylogenetically related to Lactobacilli were isolated as succinate producers. Succinate-producing Lactobacilli were only isolated as the succinate producer in polymyxin B sulfate-treated pigs. In contrast to antibiotic-associated diarrhea pigs, bacteria belonging to Bacteroidaceae, Fusobacteria, and Enterobacteriaceae were detected as succinate producers in a non-treated pig. In antibiotic-associated diarrhea conditions, antibiotic-resistant Enterobacteria, E. coli in particular, and Lactobacilli may contribute to an abnormal succinate accumulation and may affect water absorption in the hindgut that relates to an expression of antibiotic-associated diarrhea.     

Effect of Bile and Desoxycholate on Gram-Negative Anaerobic Bacteria

The bile tests for characterizing gram-negative anaerobic bacilli were reevaluated in prereduced anaerobically sterilized peptone-yeast-glucose broth, in thioglycollate broth, and on blood agar plates. Blood agar plates were unsatisfactory. The combination of 20% bile with 0.1% desoxycholate inhibited Fusobacterium, Bacteroides melaninogenicus, and B. oralis and sometimes Sphaerophorus necrophorus, but not B. fragilis or other Sphaerophorus species studied. Ten per cent bile with 0.05% desoxycholate was less satisfactory. There was no significant difference between fresh and commercial powdered bile. Desoxycholate (0.1% in thioglycollate broth) inhibited B. fragilis, Fusobacterium, B. melaninogenicus, B. oralis, and S. necrophorus, but not S. varius or S. mortiferus/S. ridiculosus. The bile and desoxycholate tests are simple to perform and helpful for characterization and classification of gram-negative anaerobic bacilli.     

Uric Acid-Degrading Bacteria in Guts of Termites [Reticulitermes flavipes (Kollar)

Uricolytic bacteria were present in guts of Reticulitermes flavipes in populations up to 6 x 10 cells per gut. Of 82 strains isolated under strict anaerobic conditions, most were group N Streptococcus sp., Bacteroides termitidis, and Citrobacter sp. All isolates used uric acid (UA) as an energy source anaerobically, but not aerobically, and NH(3) was the major nitrogenous product of uricolysis. However, none of the isolates had an absolute requirement for UA. Utilization of heterocyclic compounds other than UA was limited. Fresh termite gut contents also degraded UA anaerobically, as measured by CO(2) evolution from [2-C]UA. The magnitude of anaerobic uricolysis [0.67 pmol of UA catabolized/(gut x h)] was entirely consistent with the population density of uricolytic bacteria in situ. Uricolytic gut bacteria may convert UA in situ to products usable by termites for carbon, nitrogen, energy, or all three. This possibility is consistent with the fact that R. flavipes termites from UA, but they do not void the purine in excreta despite the lack of uricase in their tissues.