UDP-glucuronic acid decarboxylases of Bacteroides fragilis and their prevalence in bacteria

Xylose is rarely described as a component of bacterial glycans. UDP-xylose is the nucleotide-activated form necessary for incorporation of xylose into glycans and is synthesized by the decarboxylation of UDP-glucuronic acid (UDP-GlcA). Enzymes with UDP-GlcA decarboxylase activity include those that lead to the formation of UDP-xylose as the end product (Uxs type) and those synthesizing UDP-xylose as an intermediate (ArnA and RsU4kpxs types). In this report, we identify and confirm the activities of two Uxs-type UDP-GlcA decarboxylases of Bacteroides fragilis, designated BfUxs1 and BfUxs2. Bfuxs1 is located in a conserved region of the B. fragilis genome, whereas Bfuxs2 is in the heterogeneous capsular polysaccharide F (PSF) biosynthesis locus. Deletion of either gene separately does not result in the loss of a detectable phenotype, but deletion of both genes abrogates PSF synthesis, strongly suggesting that they are functional paralogs and that the B. fragilis NCTC 9343 PSF repeat unit contains xylose. UDP-GlcA decarboxylases are often annotated incorrectly as NAD-dependent epimerases/dehydratases; therefore, their prevalence in bacteria is underappreciated. Using available structural and mutational data, we devised a sequence pattern to detect bacterial genes encoding UDP-GlcA decarboxylase activity. We identified 826 predicted UDP-GlcA decarboxylase enzymes in diverse bacterial species, with the ArnA and RsU4kpxs types confined largely to proteobacterial species. These data suggest that xylose, or a monosaccharide requiring a UDP-xylose intermediate, is more prevalent in bacterial glycans than previously appreciated. Genes encoding BfUxs1-like enzymes are highly conserved in Bacteroides species, indicating that these abundant intestinal microbes may synthesize a conserved xylose-containing glycan.     

Clinical Importance of Infections due to Bacteroides fragilis and Role of Antibiotic Therapy

Out of 200 infections due to Bacteroides fragilis occurring over a period of three years 133 were related to the intestinal tract, 55 to the genitourinary tract, and the remainder were in bedsores and ulcers; 56% occurred in patients undergoing major intestinal surgery.B. fragilis was isolated in pure culture from 56% of the infections. In mixed culture it was most commonly associated with Klebsiella and Enterobacter species. Other anaerobic bacteria were isolated in 9% of the mixed cultures.Altogether 131 (65·5%) of the patients recovered without antibiotic therapy or further surgery, but 59 (29·5%) developed complications and 10 (5%) died. The commonest complication was abscess formation, and the incidence was highest with infections associated with malignancy (44%) and lowest with obstetric infections (5%). The mortality was 5% overall but in the presence of bacteraemia it rose to 33%.Only 43 patients received appropriate chemotherapy. Clindamycin was the most effective antibiotic, having a recovery rate of 78%, but this rate was little better than in untreated patients (65%). The role of prophylactic antibiotic therapy in preventing bacteroides infection remains to be studied.The incidence of the isolation of bacteroides from wound infections after major intestinal surgery rose from 13% in 1970 to 81% in 1973. This increase was due to both the accurate collection and care of specimens while in transit to the laboratory and the use of selective media for the isolation of bacteroides in laboratory culture. The importance of these precautions is emphasized.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

Prevalence of the Bacteroides fragilis Group and Enterotoxigenic Bacteroides fragilis in Immunodeficient Children

Members of the Bacteroides fragilis group are indigenous to the human and animal intestinal microbiota and they are responsible for several endogenous infections. Enterotoxigenic B. fragilis (ETBF) has been associated with acute diarrhea in children and farm animals. Immunodeficient patients are more predisposed to different opportunistic infections, including anaerobic infections. In this study, 130 stool samples were analysed from 56 immunodeficient and 74 healthy children. Enterotoxin production was detected by cytotoxicity assay on HT-29 cells and by PCR. B. fragilis sensu strictu was prevalent in both groups and ETBF species was detected from a single stool sample belonged to an immunodeficient child with AIDS.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis.

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

Therapeutic efficacy of moxifloxacin in a murine model of severe systemic mixed infection with Escherichia coli and Bacteroides fragilis

The therapeutic efficacy of moxifloxacin was studied in an experimental murine model of a systemic aerobic/anaerobic mixed infection and compared to therapies with either imipenem or ciprofloxacin plus metronidazole. Groups of 20 mice each were intravenously (iv) infected with approximately 2.5 x 10(6) colony forming units (CFU) of Escherichia coli and 2 x 10(7)CFU of Bacteroides fragilis. Iv therapy was started 24 h post-infection (pi) with either moxifloxacin, imipenem, or ciprofloxacin plus metronidazole, for 3 days. A control group of 20 mice was left untreated. Survival rate at day seven pi was recorded, mice were then sacrificed and bacterial organ contents of livers and kidneys were determined. All mice treated survived at day seven, while six animals of the untreated group died. B. fragilis was not detected in any of the treated mice. E. coli was found in two of the moxifloxacin-treated mice and in two and five of the ciprofloxacin plus metronidazole and imipenem-treated animals, respectively. The results indicate that a therapy of severe mixed aerobic/anaerobic infections with moxifloxacin might be feasible and possibly be as efficacious as current therapy regimens with ciprofloxacin plus metronidazole or imipenem.     

A study of the candidate virulence factors of Bacteroides fragilis

Bacteroides fragilis strains were classified as virulent or avirulent on the basis of their clearance from the subcutaneous tissues of mice. To determine the factors which may contribute to the virulence of B. fragilis strains, we studied encapsulation, hydrophobicity, growth rate, serum sensitivity, agglutination with erythrocytes of different origin, and neuraminidase production. The strains of the virulent group displayed a higher growth rate in broth and a lower sensitivity to the bactericidal activity of serum than the strains of the avirulent group. They also agglutinated different types of erythrocytes more strongly than did the avirulent strains. No significant differences were found between the two groups of strains as regards encapsulation, hydrophobicity and neuraminidase activity.     

Heterologous expression of the Bacteroides ruminicola xylanase gene in Bacteroides fragilis and Bacteroides uniformis

A cloned xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 was transferred by conjugation into the colonic species Bacteroides fragilis and Bacteroides uniformis by using the Escherichia coli-Bacteroides shuttle vector pVAL-1. The cloned gene was expressed in both species, and xylanase specific activity in crude extracts was found to be at least 1400-fold greater than that found in the B. ruminicola strain. Analysis of crude extract proteins from the recombinant B. fragilis by SDS-PAGE demonstrated a new 60,000 molecular weight protein. The xylanase activity expressed in both E. coli and B. fragilis was capable of degrading xylan to xylooligosaccharides in vitro. This is the first demonstration that colonic Bacteroides species can express a gene from a ruminal Bacteroides species.     

Antibacterial activity of the metabolic by-products of a Veillonella species and Bacteroides fragilis

A Veillonella species and Bacteroides fragilis were isolated from the cecal contents of adult chickens. When growth on an agar medium supplemented with 0.4% glucose and adjusted to pH 6.5, mixed cultures containing Veillonella and B. fragilis inhibited the growth of Salmonella typhimurium; Salmonella enteritidis, Escherichia coli 0157:H7 and Pseudomonas aeruginosa. Decreasing the glucose concentration of the agar decreased the inhibitory activity of the mixed culture. Mixed cultures grown on agar media supplemented with 0.5% glucose and adjusted to pH 6.5, 7.0 or 7.5 also inhibited the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 and P. aeruginosa. However, increasing the pH of the agar decreased the inhibitory activity of the mixed culture. Pure cultures of Veillonella or B. fragilis did not inhibit the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 or P. aeruginosa on any of the agar supplemented with different concentrations of glucose or on any of the agar adjusted to different pH levels. The inhibitory activity of the mixed culture was correlated with the concentration of volatile fatty acids that were formed as B. fragilis metabolized glucose to produce succinate and acetate and as the succinate produced by B. fragilis was decarboxylated by Veillonella to produce propionate.     

Nutritional requirements of Bacteroides fragilis and Bacteroides vulgatus with reference to iron and virulence

Abstract The determinant for resistance to the antiseptics acriflavine, benzalkonium chloride and cetrimide and the intercalating dye, ethidium bromide has been physically mapped on pSK57, a penicillinase plasmid detected in strains of methicillin-resistant Staphylococcus aureus . Restriction endonuclease analysis and DNA-DNA hybridization indicate that this determinant is identical to that which encodes these resistances on the plasmid pSK1, which is the most frequently detected gentamicin resistance plasmid in methicillin-resistant S. aureus isolates from Australian hospitals.     

Susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species

The susceptibility of laboratory mice to intranasal and contact infection with mouse hepatitis virus (MHV)-related coronaviruses was tested in infant CD1 mice. One day old mouse pups were inoculated intranasally with respiratory MHV-S, enteric MHV-Y, rat sialodacryoadenitis virus (SDAV), human coronavirus OC43 (HCV-OC43) or bovine coronavirus (BCV). Twenty-four hours later, they were placed in direct contact with age matched sham inoculated pups. Indices of infection in virus inoculated mice included lesions by histopathology and viral antigen by immunoperoxidase histochemistry in brain, lung, liver and intestine at 3 days after inoculation. Indices of infection in contact mice included mortality or seroconversion by 21 days after exposure. Infant mice were susceptible to infection with all five viruses. Transmission by direct contact exposure occurred with MHV and SDAV, but not HCV or BCV. Furthermore, adult mice were not susceptible to infection with HCV. Tissue distribution of lesions and antigen varied markedly among viruses, indicating that they do not induce the same disease as MHV. This study demonstrates that although these coronaviruses are antigenically closely related, they are biologically different viruses and disease patterns in susceptible infant mice can be used to differentiate viruses.     

Update on resistance of Bacteroides fragilis group and related species with special attention to carbapenems 2006-2009

The susceptibility trends for the species of the Bacteroides fragilis group against various antibiotics were determined using data from 4 years [2006-2009] on 1957 isolates referred by 8 medical centers participating in a National Survey for the Susceptibility of B. fragilis. The antibiotic test panel included doripenem, ertapenem, imipenem, meropenem, ampicillin:sulbactam, piperacillin:tazobactam, cefoxitin, clindamycin, moxifloxacin, tigecycline, chloramphenicol and metronidazole. MICs were determined using agar dilution methods following CLSI recommendations. Genetic analysis of isolates from 2008 with elevated MICs (>2 μg/mL) to one or more of the carbapenems to detect presence of the cfiA gene was performed using PCR methodology. The results showed an increase in the resistance rates to the β-lactam antibiotics. High resistance rates were seen for clindamycin and moxifloxacin (as high as 60% for clindamycin and >80% for moxifloxacin), with relatively stable low resistance (5.4%) for tigecycline. For carbapenems, resistance in B. fragilis was 1.1%-2.5% in 2008-9. One isolate resistant to metronidazole (MIC 32 μg/mL) was observed as well as isolates with elevated MICs to chloramphenicol (16 μg/mL). Genetic analysis indicated that the cfiA gene was present in some but not all of the isolates with high MICs to the carbapenems. These data indicate that there continue to be changes in susceptibility over time, and that resistance can be seen among the carbapenems. High antibiotic resistance rates tend to be associated with specific species.     

Pathway of Succinate and Propionate Formation in Bacteroides fragilis

Cell suspensions of Bacteroides fragilis were allowed to ferment glucose and lactate labeled with (14)C in different positions. The fermentation products, propionate and acetate, were isolated, and the distribution of radioactivity was determined. An analysis of key enzymes of possible pathways was also made. The results of the labeling experiments showed that: (i) B. fragilis ferments glucose via the Embden-Meyerhof pathway; and (ii) there was a randomization of carbons 1, 2, and 6 of glucose during conversion to propionate, which is in accordance with propionate formation via fumarate and succinate. The enzymes 6-phosphofrucktokinase (pyrophosphate-dependent), fructose-1,6-diphosphate aldolase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, and methylmalonyl-coenzyme A mutase could be demonstrated in cell extracts. Their presence supported the labeling results and suggested that propionate is formed from succinate via succinyl-, methylmalonyl-, and propionyl-coenzyme A. From the results it also is clear that CO(2) is necessary for growth because it is needed for the formation of C4 acids. There was also a randomization of carbons 1, 2, and 6 of glucose during conversion to acetate, which indicated that pyruvate kinase played a minor role in pyruvate formation from phosphoenolpyruvate. Phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase, and malic enzyme (nicotinamide adenine dinucleotide phosphate-dependent) were present in cell extracts of B. fragilis, and the results of the labeling experiments agreed with pyruvate synthesis via oxaloacetate and malate if these acids are in equilibrium with fumarate. The conversion of [2-(14)C]- and [3-(14)C]lactate to acetate was not associated with a randomization of radioactivity.     

感染性心内膜炎患者致病菌分布及死亡因素分析

目的 探讨感染性心内膜炎（IE）患者致病菌分布、致病菌耐药性及患者死亡因素。方法 回顾性分析我院2014年11月至2018年3月收治的80例感染性心内膜炎患者的临床资料。根据是否发生死亡将患者分成存活组（75例）与病死组（5例）。采集两组患者血液标本进行细菌培养、鉴定及药敏试验,并对患者死亡因素进行统计。结果 80例IE患者共检测出96株病原菌,其中革兰阳性菌63株,占65.63%;革兰阴性菌27株,占28.13%;真菌6株,占6.25%。药敏试验显示链球菌属及金黄色葡萄球菌对青霉素耐药率最高,分别为90.32%和93.33%;其次为红霉素,分别为70.97%和73.33%。单因素分析结果显示,肝脏肿大、血红蛋白（<90 g/L）及抗生素的使用与IE患者的死亡有关（均P<0.05）。结论 IE患者病原菌以革兰阳性菌为主,其主要菌属对青霉素耐药率最高,要加强对肝脏肿大、血红蛋白及抗生素相关因素的防治,降低IE患者的病死率。     

Enumeration of enterotoxigenic Bacteroides fragilis in municipal sewage

Of 237 isolates of Bacteroides fragilis from sewage influent at the Bozeman, Mont., wastewater treatment plant, 22 (9.3%) were enterotoxigenic, as indicated by the ability to elicit fluid accumulation in the lamb ileal loop test. It appears that enterotoxigenic B. fragilis is endemic in the human population at a moderately high level.     

Analysis of fatty acids from lipopolysaccharides of Bacteroides thetaiotaomicron and Bacteroides fragilis]

The aim of this study was to determine and to compare fatty acids occurring in lipopolysaccharides (LPS) isolated from B. thetaiotaomicron and B. fragilis strains of different origin. Lipopolysaccharides of three B. thetaiotaomicron strains and four B. fragilis strains were isolated by phenol-water extraction according to the procedure of Westphal and Jann (1965). Water-phase LPS fractions were then treated with nucleases and purified by ultracentrifugation as described by Gmeiner (1975). Fatty acid methyl esters, obtained by methanolysis of LPS, were analysed in gas-liquid chromatography combined with mass spectrometry (GLC-MS). Trimethylsilylated hydroxyl groups of fatty acid methyl esters were identified with GLC-MS using a method of selective ion monitoring (SIM). Lipopolysaccharides of B. thetaiotaomicron and B. fragilis strains contained long-chain (15-18 carbon atoms) fatty acids. The broad spectrum of simple long-chain and branched-chain fatty acids as well as 3-hydroxy fatty acids were detected. The main fatty acid of analyzed bacterial species was 3-hydroxy-hexadecanoic acid (3OH C16:0). Several 3-hydroxy fatty acids were detected in LPS of examined strains. Fatty acids occurring in LPS of B. thetaiotaomicron and B. fragilis strains appeared to be qualitatively similar. Quantitative differences in fatty acids composition of lipopolysaccharides isolated from strains of different origin were observed.