The Hydrolysis of Dextran by Gram Negative Non-sporing Anaerobic Bacilli

The ability of Gram negative anaerobic bacilli to hydrolyse dextran was determined in liquid and solid media containing Blue Dextran 2000. Released blue chromophore in the liquid medium was detected spectrophotometrically. Results obtained with 334 strains of Bacteroidaceae grown on the solid medium indicated that most strains did not hydrolyse the substrate. Hydrolysis of Blue Dextran 2000 occurred with certain strains of Bacteroides thetaiotaomicron , B. melaninogenicus ss. melaninogenicus, B. oralis, B. ovatus and B. ochraceus.     

Antibiotic susceptibility patterns as aids in classification and characterization of gram-negative anaerobic bacilli

Patterns of susceptibility of gram-negative anaerobic bacilli to antibiotics have been found to be distinctive and of significant help in classification and identification. Five major groups of gram-negative anaerobic bacilli have been defined on the basis of morphological and biochemical criteria. Antibiotic sensitivity patterns conform to these groupings and provide additional taxonomic criteria. The Bacteroides fragilis group is resistant to penicillin G, whereas the other groups are generally sensitive. B. fragilis strains are relatively sensitive to erythromycin, whereas the Sphaerophorus necrophorus group is resistant. B. melaninogenicus strains, B. oralis, and Fusobacterium are all more sensitive to kanamycin and neomycin than the other two groups. Kanamycin is more active against Fusobacterium strains than neomycin, but less active against all other groups. Colistin or polymyxin B is useful for distinguishing between the resistant B. fragilis and the sensitive S. necrophorus. Antibiotic susceptibility determinations may be more readily performed in clinical laboratories than certain biochemical tests recommended for differentiation of the gram-negative anaerobic bacilli and may serve as helpful adjuncts to morphological and biochemical observations in classifying and characterizing these organisms. The use of standardized procedures for antibiotic susceptibility tests is essential if comparable results are to be obtained in different laboratories.     

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.     

Comparison of the post-antibiotic effect (PAE) induced by ceftizoxime, ceftriaxone, cefoxitin, ampicillin-sulbactam, and ticarcillin-clavulanate against selected isolates of Bacteroides fragilis and B. thetaiotaomicron

The exposure of bacteria to various groups of antimicrobials at different concentrations can produce damage to the bacteria that persists even after removal of the antimicrobial agent. The post antibiotic effect (PAE) of beta-lactams on aerobic gram-negative bacilli is relatively short (<1 h), however, little information is available regarding anaerobic gram-negative bacilli. We studied the PAE of ceftizoxime, ampicillin-sulbactam, ticarcillin-clavulanate, cefoxitin, and ceftriaxone against strains of Bacteroides fragilis and B. thetaiotaomicron at antimicrobial concentrations 4x, 8x, and 16x the MIC values using colony count determinations of treated and untreated cultures. Against B. fragilis H931, ceftizoxime-induced PAE values were 2 h, 3 h 24 min, and 11 h 36 min at 4xMIC, 8xMIC, and 16xMIC while for the B. thetaiotaomicron isolates PAEs ranged from 2 h 27 min to 6 h 12 min at the same concentrations. Cefoxitin PAE values were 3 h 6 min and 2 h 18 min for the clinical isolates at 16xMIC and 3 h 24 min and 1 h 12 min against the laboratory strains at 16xMIC respectively, and for ceftriaxone 1 h 12 min and 5 h 12 min, respectively, for the B. thetaiotaomicron D933 and B. fragilis H931 strains at 16xMIC. With ampicillin-sulbactam, the longest PAE values were observed at 16xMIC with all the test isolates of B. fragilis and B. thetaiotaomicron. PAE values induced by ticarcillin-clavulanate overall were the shortest for the two clinical isolates. These studies indicate substantial PAE values for beta-lactams against selected anaerobes which may be an important factor in the dosing regimen of these test agents.     

A numerical taxonomic study of anaerobic gram-negative bacilli classified as Bacteroides ureolyticus isolated from patients with non-gonococcal urethritis

A numerical taxonomic study of 64 strains of anaerobic Gram-negative bacilli isolated from men with non-gonococcal urethritis, two unclassified laboratory strains of 'corroding bacilli', and 12 other strains of anaerobic Gram-negative bacilli, including nine received as anaerobic curved rods and three as 'Bacteroides corrodens' (B. ureolyticus), isolated from women with bacterial vaginosis, was undertaken. Seventeen reference anaerobic strains belonging to the genera Bacteroides, Fusobacterium, Mobiluncus, Mitsuokella and Wolinella were included. Morphological, biochemical and physiological characteristics were examined in 103 tests. The resemblance between the 95 strains was calculated using the SSM, SJ and DP coefficients for cluster analyses based on the UPGMA method. All three approaches gave similar groupings, and the estimated average probability of test error was 2.46%. The strains fell into 10 phenons. The unclassified strains from men and three from women with lower genital-tract infections, and the laboratory strains of 'corroding bacilli' clustered in one phenon with the reference strains of B. ureolyticus, indicating that they correspond to B. ureolyticus. The other unclassified strains of anaerobic curved rods clustered as a distinct phenon. They correspond to species of the newly described genus Mobiluncus. The taxonomic data and the compilation of diagnostic tables serve as a useful guide for the laboratory identification of clinical isolates regarded as B. ureolyticus.     

Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.     

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.     

Siderophore and chitinase producing isolates from the rhizosphere of Nicotiana glauca Graham enhance growth and induce systemic resistance in Solanum lycopersicum L.

A screening for Plant Growth Promoting Rhizobacteria (PGPR) was carried out in the rhizosphere of wild populations of Nicotiana glauca Graham in south-eastern Spain. Nine hundred and sixty strains were isolated and grouped in four parataxonomic groups: Gram positive endospore forming bacilli, Gram positive non-endospore forming bacilli, Gram negative bacilli and others. Two groups were selected to continue the study: Gram negative bacilli since it was the most abundant, and Gram positive sporulated bacilli, seeking their sporulating capacity as an advantage for inoculants formulation. The ability of these to release siderophores and chitinases in vitro was evaluated. Ninety six isolates were siderophore producers, and 56 of them were also able to produce chitinases. Fifty percent of these were tested for growth promotion in tomato. The best results were obtained with 5 Gram negative bacilli and one Gram positive sporulated bacilli; 5 strains increased all growth parameters while one of them, N21.4, severely compromised plant growth. The ability of these 6 strains to induce systemic resistance against the leaf pathogen Xanthomonas campestris in tomato was evaluated. Five of them effectively reduced disease symptoms (up to 50%). The six strains were identified by 16s rDNA sequencing resulting in 3 Pseudomonas, 1 Bacillus and 2 Stenotrophomonas; it’s striking that 2 Pseudomonas protected up to 50% while the other increased disease incidence. This indicates that systemic induction is strain specific and not necessarily related to production of siderophores and chitinases.     

Characterization of the Bacteroides fragilis pathogenicity island in human blood culture isolates

Bacteroides fragilis is an important anaerobic pathogen accounting for up to 10% of bacteremias in adult patients. Enterotoxin producing B. fragilis (ETBF) strains have been identified as enteric pathogens of children and adults. In order to further characterize the B. fragilis pathogenicity island (BfPAI) and using PCR assays for bft- and mpII-metalloprotease genes, we determined the frequency of B. fragilis strains with pattern I (containing the BfPAI and its flanking region), pattern II (lacking both the BfPAI and the flanking region), and pattern III (lacking the BfPAI but containing the flanking region) in 63 blood culture isolates. The results were compared to 197 B. fragilis isolates from different clinical sources. We found 19% of blood culture isolates were pattern I (ETBF), 43% were pattern II (NTBF) and 38% were pattern III (NTBF). Comparatively, B. fragilis isolates from other clinical sources were 10% pattern I, 47% pattern II and 43% pattern III. This suggests that the pathogenicity island and the flanking elements may be general virulence factors of B. fragilis.     

Antibiotic resistance among anaerobic Gram-negative bacilli: lessons from a French multicentric survey

Temporal changes of antibiotic susceptibilities among anaerobes in France are followed in our laboratory since 1992. For Bacteroides strains, resistance increased from 1992 to 1998 for amoxicillin-clavulanic acid, cefotetan and clindamycin. The present study evaluates the situation in 2000 for 434 Gram-negative anaerobic clinical isolates (obtained from 9 large university hospitals) by testing amoxicillin and ticarcillin alone or combined with clavulanic acid, cefoxitin, cefotetan, imipenem, clindamycin and metronidazole (using the NCCLS-approved method for MIC determination. The main genera tested included Bacteroides (359 strains of the fragilis group), Prevotella (40 strains), Fusobacterium (23 strains) and miscellaneous species (8 strains). Resistance rates within the B. fragilis group were: amoxicillin-clavulanic acid 5.6%, ticarcillin 33%, ticarcillin-clavulanic acid 2%, cefoxitin 13%, cefotetan 44%, clindamycin 33%, imipenem 1% and metronidazole <1%, respectively. Only one strain of B. fragilis was resistant to metronidazole (MIC=64 mg/L); due to the presence of the nimA gene on the chromosome. Resistance to imipenem or metronidazole was only found among the B. fragilis species. These two former drugs excepted, B. fragilis was less resistant to antibiotics than the other species. beta-lactamase production was detected for 357/359 strains of the fragilis group, 26/40 stains of Prevotella and 3/23 strains of Fusobacterium. Dynamic changes of antibacterial resistance are occurring within the B. fragilis group: decreased resistance to amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, imipenem while resistance for cefoxitin, cefotetan, clindamycin continues to increase. Regular antibiotic surveys are needed as a source of information to guide the empirical therapy of anaerobic infections.     

Some Properties of the Nonsporing Anaerobes from Poultry Caeca

S ummary : A study has been made of the ability of 48 strains of anaerobic bacteria, representing 20 different groups of Gram negative and Gram positive nonsporing rods and peptostreptococci, isolated from poultry caeca, to attack certain substrates that may be of ecological importance. The organisms were predominantly saccharolytic; only the strains of Propionibacterium acnes showed proteolytic activity. None of the strains hydrolysed amorphous cellulose or xylan. A few were weakly pectolytic. Starch was hydrolysed by all the strains of Bacteroides fragilis and by one strain of Eubacterium. Two groups of Gram negative bacteria (Bacteroidaceae) and one of the peptostreptococci attacked uric acid. The resistance of the anaerobes to inhibitory substances (bacitracin, virginiamycin, kanamycin, neomycin, vancomycin and nalidixic acid) was determined. Selective media for the isolation of certain of the groups of Gram negative anaerobes have been developed.     

Extracellular Deoxyribonuclease Production by Anaerobic Bacteria

The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common. All strains of Fusobacterium, including eight species, as well as Bacteroides fragilis and B. melaninogenicus, displayed enzyme activity. Whereas the gram-positive bacteria were generally less productive, all strains of Clostridium perfringens, Peptostreptococcus intermedius, and P. anaerobius specifically produced deoxyribonuclease. The test is taxonomically valuable, particularly in the characterization of gram-positive cocci, since a deoxyribonuclease-producing coccus indicates P. intermedius or P. anaerobius. Additionally, possession of the enzyme may prove to be a useful correlate of the potential pathogenicity of anaerobes.     

Recovery of Bacteroides fragilis group from clinical specimens following antimicrobial therapy.

Over a period of 14 years (1973-1987), 3165 specimens submitted to the microbiology laboratory demonstrated the recovery of anaerobic bacteria. A total of 988 Bacteroides fragilis group isolates were recovered (0.3 isolates per specimen). Bacteroides fragilis accounted for 62% of the total of all B. fragilis group isolates, Bacteroides thetaiotaomicron for 15%, Bacteroides vulgatus for 8%, Bacteroides ovatus for 7%, Bacteroides distasonis for 6%, and Bacteroides uniformis for 2%. Of the 988 B. fragilis group isolates, 310 (31%) were recovered after the administration of antimicrobial therapy, and 129 (13%) were the single isolate recovered from the infected site at that time. The recovery rate of all members of B. fragilis group after the administration of antimicrobial therapy, when isolated alone or when mixed with other bacteria, was similar. The data illustrate the equal ability of all members of the B. fragilis group to persist in and to contribute to the inflammatory process; and provide further support for their pathogenic role.     

烧伤病区感染常见病原菌及抗生素的选择

目的探讨内江市第一人民医院烧伤病区感染的常见病原菌及其对常用抗生素的耐药性．以指导临床抗生素的选择,提出防治措施。方法对1999年1月～2003年12月该院烧伤病区患者因感染而分离出的658株病原菌进行回顾性分析。结果分离出的658株病原菌中．革兰阴性菌425株(64．6％),革兰阳性菌209株(31．8％)．真菌24株(3．6％)。主要病原菌分别为铜绿假单胞菌、金黄色葡萄球菌、大肠埃希菌、凝固酶阴性葡萄球菌和阴沟肠杆菌。药敏提示除真菌外的大多数病原菌出现多重耐药性。革兰阴性菌对亚胺培南、头孢哌酮-舒巴坦、头孢西丁和妥布霉素较为敏感。革兰阳性球菌对替考拉丁和万古霉素高度敏感。结论烧伤感染中以铜绿假单胞菌为主．其次为金黄色葡萄球菌、大肠埃希菌和凝固酶阴性葡萄球菌。了解病原菌的结构及其变化趋势．对于加强抗感染治疗的针对性和主动性,取得最佳的感染防治效果具有重要意义。     

Numerical taxonomy of some non-saccharolytic and saccharolytic Bacteroides species

Various Bacteroides spp. were examined by physiological tests, presence of specific enzymes, antibiotic sensitivity, menaquinone composition and a few miscellaneous tests. The data matrix containing 58 strains and 55 unit characters was examined using Gower's similarity coefficients (SG) and included matching negative character states and multistate characters. The highly saccharolytic strains were separated from the less saccharolytic and non-fermentative strains at the 55% similarity level; while at the slightly higher level of 63% strains of Capnocytophaga (formerly Bact. ochraceus) were recovered as a compact phenon distinct from other saccharolytic species. The phenogram was divided into 6 clusters at 72% similarity level. Most of the 'Bact. fragilis group' of species clustered in one phenon while Bact. melaninogenicus ssp. melaninogenicus, Bact. bivius and a new species, Bact. denticola, formed another group. Another phenon comprised the saccharolytic non-pigmented species closely related to Bact. oralis such as Bact. buccalis and Bact. pentosaceus. The less saccharolytic strains of Bact. melaninogenicus ssp. intemedius and Bact. disiens were recovered in a distinct phenon. The low affinity (less than 55% similarity) between the two subspecies of Bact. melaninogenicus emphasised the need for reclassifying these taxa into separate species. The non-fermentative and very weakly saccharolytic strains formed good taxospecies. The separation of this cluster into three subclusters is in excellent agreement with chemotaxonomic data now available.     

Numerical taxonomy of some non-saccharolytic and saccharolytic Bacteroides species

Various Bacteroides spp. were examined by physiological tests, presence of specific enzymes, antibiotic sensitivity, menaquinone composition and a few miscellaneous tests. The data matrix containing 58 strains and 55 unit characters was examined using Gower's similarity coefficients (S _G ) and included matching negative character states and multistate characters. The highly saccharolytic strains were separated from the less saccharolytic and non-fermentative strains at the 55% similarity level; while at the slightly higher level of 63% strains of Capnocytophaga (formerly Bact. ochraceus ) were recovered as a compact phenon distinct from other saccharolytic species. The phenogram was divided into 6 clusters at 72% similarity level. Most of the ' Bact. fragilis group' of species clustered in one phenon while Bact. melaninogenicus ssp. melaninogenicus, Bact. bivius and a new species, Bact. denticola , formed another group. Another phenon comprised the saccharolytic non-pigmented species closely related to Bact. oralis such as Bact. buccalis and Bact. pentosaceus. The less saccharolytic strains of Bact. melaninogenicus ssp. intermedius and Bact. disiens were recovered in a distinct phenon. The low affinity (less than 55% similarity) between the two subspecies of Bact. melaninogenicus emphasised the need for reclassifying these taxa into separate species. The non-fermentative and very weakly saccharolytic strains formed good taxospecies. The separation of this cluster into three subclusters is in excellent agreement with chemotaxonomic data now available.     

The Classification of Bacteroides melaninogenicus and Related Species

One hundred and seventy-five strains of Bacteroides melaninogenicus , 17 strains of B. oralis and six strains of B. ochraceus were studied in a series of biochemical, chemical tolerance and antibiotic disc resistance tests and by the gas-liquid chromatographic analysis of the acid end products of metabolism. Strains of B. melaninogenicus ss. asaccharolyticus formed a distinct group with clear differences from other B. melaninogenicus strains. B. melaninogenicus ss. intermedius strains formed a homogeneous group that could be readily identified. B. ochraceus was distinguished from other Bacteroides spp. by its ability to grow in air enriched with CO₂. Bacteriodes melaninogenicus ss. melaninogenicus and B. oralis gave very similar patterns of results with the tests used and invariably were indistinguishable; the capacity to produce black-pigmented colonies on blood-containing media may not be a valid criterion for dividing these similar strains into two species.     

儿童乳牙根管感染的细菌学分析

对18例3～8岁儿童乳牙的根管感染以无菌技术进行定量取样,按种于12种选择性培养基和2种非选择性培养基上,进行需氧、微需氧和厌氧培养,并对细菌菌落计数。对牙髓拟杆菌和牙龈拟杆菌作半定量免疫荧光染色计数;并对其中9例病牙进行了菌相分析。检出的所有细菌中,厌氧菌占绝对优势;其中检出率较高的菌为:产黑色素拟杆菌属,厌氧革兰氏阳性球菌,微需氧革兰氏阳性球菌等.本试验证明,儿童乳牙根管感染是以厌氧菌为主的混合感染,其中以产黑色素拟杆菌属等最常见.     

Antimicrobial susceptibilities of Bacteroides fragilis and Bacteroides thetaiotaomicron strains isolated from clinical specimens and human intestinal microbiota

Species of Bacteroides fragilis group bacteria are the most prevalent pathogens and have the highest resistance rates to antimicrobial agents among anaerobic bacteria. Infections due to these micro-organisms often originate from patient's own intestinal microbiota. The objective of the study was to determine and compare the susceptibility profiles of clinical and intestinal B. fragilis and B. thetaiotaomicron strains against certain antimicrobials. Isolates were identified by conventional methods and API-20 A. Susceptibility tests were performed according to recommendations of NCCLS (M 11-A4) agar dilution methods. Beta-lactamase production was determined with nitrocefin discs. Forty-five clinical isolates (33 B. fragilis and 12 B. thetaiotaomicron) were from following sites: blood (n:8), intra-abdominal abscess (n:7), soft tissue (n:26), and miscellaneous foci of infection (n:4). Fifty B. fragilis and 60 B. thetaiotaomicron isolates from intestinal microbiota of individuals with no history of antimicrobial treatment within last 30 days were also examined. Beta-lactamase production was detected in 93% of clinical and 99% of intestinal isolates. The organisms including intestinal isolates were uniformly susceptible to metronidazole. The MIC90s of other antibiotics and resistance rates of all clinical isolates to those antibiotics were as follows: 256 microg/mL (93%) for ampicillin, 128 microg/mL (13%) for piperacillin, 64 microg/mL (11%) for cefoxitin, 1 microg/mL (2%) for amoxicillin-clavulanate, 0.5 microg/mL (2%) for imipenem, >256 microg/mL (36%) for clindamycin, 8 microg/mL (2%) for chloramphenicol. Intestinal isolates demonstrated similar resistance rates and MIC90s. Metronidazole, imipenem, amoxicillin-clavulanate seem to be effective drugs against these bacteria in Turkey.     

Susceptibility to Phosphomycin as a Differential Character for Gram Negative Anaerobic Bacilli

On the basis of their sensitivity to phosphomycin, various species of anaerobic Gram negative bacilli examined fell into one of two groups. All strains of Bacteroides were resistant to 500 umg/ml whereas strains of Fusobacterium were uniformly sensitive to 62.5 μg/ml. Disc sensitivity testing to phosphomycin at concentrations of 200–500 μg/ml provides a reliable and rapid means of distinguishing fusobacteria from bacteroides.