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New consensus guidelines from the Clinical and Laboratory Standards Institute for antimicrobial susceptibility testing of infrequently isolated or fastidious bacteria.

The Clinical and Laboratory Standards Institute (CLSI) recently published a new laboratory guideline for antimicrobial susceptibility testing of infrequently encountered or fastidious bacteria not covered in previous CLSI publications. The organisms include Aeromonas species, Bacillus species, and Vibrio species that may cause infections following environmental exposure. Fastidious organisms that may cause endocarditis or medical device infections include Abiotrophia and Granulicatella species; coryneform bacteria; Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella group gram-negative rods; and the instrinsically vancomycin-resistant gram-positive organisms Erysipelothrix, Lactobacillus, Leuconostoc, and Pediococcus species. Organisms not previously covered in depth in CLSI guidelines include Branhamella catarrhalis, Campylobacter jejuni, Campylobacter coli, Listeria species, and Pasteurella species. Clinically important drug resistance has been reported for each of these organisms. The guidelines provide recommendations for when it may be important to test these organisms, how standard methods may be easily adapted for testing, and appropriate interpretive criteria for results. Communication with infectious diseases clinicians prior to performing such testing is emphasized.

Anti-Bacterial Agents↗

Effect of vancomycin on intestinal flora of patients who previously received antimicrobial therapy.

To evaluate the ecological disturbances of peroral vancomycin administration following cephalosporin administration, 20 healthy volunteers received cefuroxime axetil tablets (250 mg) perorally twice a day for 1 week, and 10 of these volunteers subsequently received vancomycin capsules (125 mg) perorally four times daily for 7 days. The concentration of vancomycin in feces after 1 week of vancomycin administration was high (mean +/- SD, 520 +/- 197 mg/kg), which correlated with the ecological disturbances noted in the vancomycin recipients. Vancomycin administration resulted in a rapid decrease in the numbers of intestinal Enterococcus faecium, Enterococcus faecalis, and Enterococcus durans (P < or = .05), while there was a significant emergence of motile enterococci with decreased susceptibility to vancomycin (Enterococcus gallinarum and Enterococcus casseliflavus; minimum inhibitory concentration, 4-16 mg/L) (P < or = .01). Because of vancomycin administration, there was also a significant overgrowth of vancomycin-resistant Pediococcus species and lactobacilli as well as of Klebsiella species, Citrobacter species, and Enterobacter species (P < or = .01). The numbers of bifidobacteria and Bacteroides species were significantly reduced during vancomycin administration. None of the enterococcal strains carried vanA or vanB. Twenty-two of the 27 motile enterococci carried the vanC-1 gene specific for E. gallinarum, whereas five strains carried the vanC-2(C-3) gene, thus implicating that they were E. casseliflavus or Enterococcus flavescens.

Administration, Oral↗

Vancomycin binding to cell walls of non-streptococcal vancomycin-resistant bacteria.

The availability of peptidoglycan for binding to vancomycin was investigated in intrinsically vancomycin-resistant clinical isolates of Leuconostoc mesenteroides, Lactobacillus viridescens and Pediococcus pentosaceus. Intrinsic vancomycin resistance expressed by L. mesenteroides and P. pentosaceus was the result of poor binding of vancomycin to native or SDS-treated cell walls. Pre-exposure of L. viridescens to vancomycin decreased the subsequent binding of vancomycin to the cell walls, suggesting an alternative mechanism to that found in high-level vancomycin-resistant enterococci and intrinsically-resistant L. mesenteroides and P. pentosaceus. Vancomycin binding was sensitive to substitution of larger side chains at the C-terminus. Variations of peptidoglycan peptide types found in L. mesenteroides, L. viridescens and P. pentosaceus could account for their intrinsic vancomycin resistance.

Amino Acid Sequence↗

In-vitro activity of the new ketolide antibiotic HMR 3647 against gram-positive bacteria.

The comparative in-vitro activity of HMR 3647, a new ketolide antibiotic, was investigated against 492 clinical isolates of gram-positive bacteria, including multiply resistant strains, by an agar-dilution technique. All streptococci tested were inhibited by the new ketolide at concentrations < or = 0.5 mg/L. HMR 3647 was more potent than erythromycin against staphylococci. For enterococci the new compound yielded an MIC90 of 8 mg/L. Erysipelothrix spp., Pediococcus spp., Leuconostoc spp., Lactobacillus spp., JK diphtheroids and Listeria monocytogenes were also susceptible to the new ketolide.

Anti-Bacterial Agents↗

The in vitro activity of daptomycin against 514 Gram-positive aerobic clinical isolates.

The in vitro activity of daptomycin was assessed in comparison with that of vancomycin and penicillin against a wide range of Gram-positive aerobic clinical isolates. MICs were determined by an agar dilution method on Mueller-Hinton agar (NCCLS/EUCAST) and on Isotonic agar adjusted to contain 50 mg/L free calcium (BSAC). Both media were enriched with 5% horse blood for fastidious organisms. Daptomycin MICs for all 172 staphylococci, including methicillin-susceptible and methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus, were 0.03-0.5 mg/L. For 99 of the 100 enterococci (Enterococcus faecalis, n = 50; Enterococcus faecium, n = 50), including 37 vancomycin-resistant isolates, they were 0.25-2 mg/L. For all 108 beta-haemolytic streptococci, including Streptococcus pyogenes and Streptococcus agalactiae, daptomycin MICs were 0.016- 0.25 mg/L; for 101 alpha-haemolytic streptococci, including Streptococcus pneumoniae and 'viridans' streptococci, they were 0.016-2 mg/L. For miscellaneous vancomycin-resistant isolates including Lactobacillus spp., Lactococcus spp., Leuconostoc spp., Pediococcus spp. and isolates of Enterococcus casseliflavus and Enterococcus gallinarum, daptomycin MICs were 0.03-2 mg/L; MICs for the seven isolates of Listeria monocytogenes were 0.25-4 mg/L. There was little difference between the results on Mueller-Hinton agar and on supplemented Isotonic agar The discrepant results occasionally obtained tended to be one dilution higher on supplemented Isotonic agar. Daptomycin was active (MICs < or = 2 mg/L) against all the isolates tested with the exception of one isolate each of E. faecium and L. monocytogenes (MICs = 4 mg/L). Our results indicate that daptomycin MICs are independent of methicillin and vancomycin MICs.

Anti-Bacterial Agents↗

Narasin used as a feed additive in conventional rearing of broilers can co-select for vancomycin-resistant Enterococcus faecium through the NarAB ionophore resistance mechanisms.

OBJECTIVES: To investigate the role of the NarAB resistance mechanism in the selection of vancomycin-resistant Enterococcus faecium (VREfm) and assess the impact of ionophore feed additives, particularly narasin, on the emergence of VREfm in broiler chickens. MATERIALS AND METHODS: Three isogenic E. faecium strains with different antimicrobial resistance determinants were created by mutagenesis and conjugation and used in a controlled animal experiment. Ross 308 broiler chickens were inoculated with either a rifampicin-resistant, a rifampicin- and vancomycin-resistant or a rifampicin-, vancomycin- and narasin-resistant strain and fed diets supplemented with selected ionophores. Bacterial populations were analysed on selective Slanetz and Bartley agar to determine the presence and selection of VREfm and other vancomycin-resistant species. Bacterial inoculation strains and isolates were whole genome sequenced for species identification and to identify genetic resistance mechanisms. RESULTS: Narasin was shown to select for VREfm in broilers, with NarAB being essential for co-selection. Intrinsically vancomycin-resistant Pediococcus acidilactici and Enterococcus gallinarum were identified as part of the broilers' vancomycin-resistant resident microbiota. Notably, among the P. acidilactici isolates that were susceptibility tested, strains resistant to both vancomycin and narasin were only found in broilers fed narasin, supporting that narasin promotes the growth of narasin-resistant populations. CONCLUSION: Narasin use in broiler feed can co-select for vancomycin-resistant bacteria, including VREfm, through the NarAB mechanism. These findings emphasize the concerns associated with the use of particular ionophores in poultry and suggest that vancomycin and narasin resistance may be more widespread in the broiler microbiota than previously recognized. Further research is needed to understand the implications for antimicrobial resistance and human health.

Animals↗

Cloning and expression of the glutamate racemase gene of Bacillus pumilus.

A glutamate racemase gene (murI) was found in Bacillus pumilus cells and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B. pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG. The deduced amino acid sequence shows significant similarity with those of glutamate racemases from E. coli (ratio of identical residues, 28%), Pediococcus pentosaceus (44%), and Staphylococcus haemolyticus (49%). B. pumilus MurI was expressed as a fusion protein connected to the N-terminal 12 residues of beta-galactosidase; the fusion protein showed glutamate racemase activity, and resembled the enzyme of P. pentosaceus in physicochemical and enzymological properties.

Amino Acid Isomerases↗

Purification, characterization, and primary structure of a novel cell wall hydrolytic amidase, CwhA, from Achromobacter lyticus.

A novel bacteriolytic enzyme CwhA (cell wall hydrolytic amidase) was purified by ion exchange and gel-filtration chromatographies from a commercial bacteriolytic preparation from Achromobacter lyticus. CwhA exhibited optimal pH at 8.5 and lysed CHCl(3)-treated Escherichia coli more efficiently than Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, and Pediococcus acidilactici. The enzyme was inhibited by 1,10-phenanthroline strongly and by EDTA to a lesser extent, suggesting that it is probably a metalloenzyme. Amino acid composition and mass spectrometric analyses for the CwhA-derived M. luteus muropeptides revealed that CwhA is N-acetylmuramoyl-L-alanine amidase [EC 3.5.1. 28]. The complete amino acid sequence of CwhA was established by a combination of Edman degradation and mass spectrometry for peptides obtained by Achromobacter protease I (API) digestion and cyanogen bromide (CNBr) cleavage. The enzyme consists of a single polypeptide chain of 177 amino acid residues with one disulfide bond, Cys114-Cys121. CwhA was found to be homologous to N-acetylmuramoyl-L-alanine amidase from bacteriophage T7 (BPT7). Its sequence identity with BPT7 is 35%, but the amino acid residues functioning as zinc ligands in BPT7 are absent in CwhA. These results suggest that CwhA is a new type of N-acetylmuramoyl-L-alanine amidase.

Alcaligenes↗

Concomitant septic and gouty arthritis--an analysis of 30 cases.

OBJECTIVES: To analyse the clinical features and outcomes of gouty patients with concomitant septic arthritis in a medical centre. METHODS: From the hospital database, we collected 30 hospitalized cases with concomitant septic arthritis and gouty arthritis from 1987 to 2001. All patients had positive bacterial culture and monosodium urate crystals in the affected joints. Medical records of the patients were analysed in detail. RESULTS: The mean age of patients was 52.8+/-12.5 yr. One-third of patients were afebrile at presentation, 30% had a normal blood leucocyte count and 10% had a synovial fluid leucocyte count less than 6000/mm3. The knee joint was the most common site of involvement, followed by the ankle, shoulder and wrist joints. Most patients had long-standing disease and subcutaneous tophi. Subcutaneous tophi rupture with secondary wound infection is the most common route of infection. Causative micro-organisms were Staphylococcus aureus (16 cases, 7 of whom were oxacillin-resistant), Streptococcus sp. (5 cases), Pediococcus sp. (1 case), and Gram-negative bacilli (9 cases). Fourteen patients received surgical debridement, among them two patients had an arthrodesis owing to severe joint destruction and one received above-knee amputation. Two patients died. One died of septic complications and the other died of acute myocardial infarction. CONCLUSIONS: Septic arthritis coexistent with gout presented a diagnostic difficulty. An early diagnosis requires a high level of suspicion. Prompt aspiration and analysis of the synovial fluid is imperative, regardless of the absence of fever or leucocytosis. Culture of the aspirated synovial fluid is warranted in gouty attack, even when it has a low white cell count or the Gram stain reveals no organisms.

Adult↗

Rapid micromeasurement of lactate in whole blood.

A new lactate sensor makes it possible to measure the lactate content of whole blood directly in less than 1 min, using only a 10-microL blood sample. The procedure works equally well with plasma, serum, spinal fluid, other body fluids, or tissue homogenates. The instrument is calibrated with lactate standards between 0 and 15 mMol/L. The sensor, a polarographic enzyme electrode, gives a current which is a linear function of the lactate concentration. There is no interference from glucose, pyruvate, alcohol, ascorbate, anticoagulants, lidocaine, acetaminophen, or other drugs and metabolites commonly encountered in critically ill patients. The lactate sensor is composed of a peroxide sensor and an enzyme transducer membrane. The lactate is stoichiometrically converted to pyruvate and hydrogen peroxide by lactate oxygen oxidoreductase derived from Pediococcus species. The oxygen required for the enzymatic oxidation is supplied via an air-permeable silicone elastomeric membrane used for stirring. Comparison of our new electroenzymatic method with the Boehringer-Mannheim photoenzymatic method gives correlations of 0.997 for both whole blood and plasma.

Alcohol Oxidoreductases↗

Description of Paralactobacillus selangorensis gen. nov., sp. nov., a new lactic acid bacterium isolated from chili bo, a Malaysian food ingredient.

Paralactobacillus selangorensis gen. nov., sp. nov. is described. This organism, isolated from a Malaysian food ingredient called chili bo, is an obligatory homofermentative, rod-shaped lactic acid bacterium. The G+C content is 46.1-46.2+/-0.3 mol%. Earlier 16S rRNA studies showed that this organism constitutes a new taxon distantly related to the Lactobacillus casei-Pediococcus group. A phenotypic description that distinguishes Paralactobacillus selangorensis from other genera of lactic acid bacteria is presented. The type strain of Paralactobacillus selangorensis is LMG 17710T.

Acetic Acid↗

Lipoquinones of some spore-forming rods, lactic-acid bacteria and actinomycetes.

The respiratory quinones of 73 strains of Gram-positive bacteria including spore-forming rods, lactic-acid bacteria and actinomyctes were examined. Menaquinones with seven isoprenoid units (MK-7) were the main quinone type found in representatives of the genus Bacillus and in Sporolactobacillus inulinus. However, a strain of B. thuringiensis produced MK-8 in addition to MK-7, and strains of B. lentus and B. pantothenticus appeared to produce MK-9 and MK-8, respectively, with no MK-7. In the clostridia and lactic-acid bacteria, no quinones were found, except in Pediococcus cerevisiae NCTC 8066 and Lactobacillus casei subsp. rhamnosus ATCC 7469, which contained menaquinones, and Streptococcus faecalis NCTC 775 and HIM 478-1, which contained demethylmenaquinones, in relatively low concentrations. Menaquinones were also found in the actinomycetes (except Actinomyces odontolyticus and Bifidobacterium bifidum which did not produce any quinones) and in Protaminobacter alboflavus ATCC 8458, the so-called Actinobacillus actinoides ATCC 15900 and Noguchia granulosis NCTC 10559.

Actinomycetales↗

Numerical taxonomy of Streptococcus.

A numerical taxonomic study of strains of Streptococcus, together with representatives of allied genera, showed 28 reasonably distinct phenons. The major areas, with their phenons, were: (a) enterococcal species group (S. faecalis, S. faecium, 'S. avium' and a proposed new species 'S. gallinarum'); (b) paraviridans species group (S. bovis, S. equinus, S. salivarius, 'S. casseliflavus', S. mutans, S. raffinolactis and an unidentified Oral Group I); (c) lactic species group (S. lactis including S. cremoris); (d) thermophilic species group (S. thermophilus); (e) viridans species group (S. mitis, S. sanguis, a proposed new species 'S. oralis' and 'S. milleri'); (f) pyogenic species group (S. agalactiae, S. pyogenes, S. equi, 'S. equisimilis' including 'S. zooepidemicus, and a cluster of Lancefield Group B strains of human origin); (g) parapyogenic species group (S. uberis, 'S. dysgalactiae', and a cluster of strains of Lancefield Groups R, S and T). Species of Aerococcus, Gemella, Leuconostoc and Pediococcus are very closely related to the streptococci.

Enterococcus faecalis↗

Antibodies to a synthetic 1-9-N-terminal amino acid fragment of mature pediocin PA-1: sensitivity and specificity for pediocin PA-1 and cross-reactivity against Class IIa bacteriocins.

Polyclonal antibodies specific for pediocin PA-1 (PedA1) were generated by immunization of rabbits with a chemically synthesized 1-9-N-terminal amino acid fragment of this bacteriocin (PH1) conjugated to the carrier protein keyhole limpet haemocyanin (KLH). The PH1 fragment holds a highly conserved amino acid sequence with closely related Class IIa bacteriocins. The sensitivity and specificity of the PH1-KLH-generated rabbit polyclonal antibodies were evaluated by the development of various ELISAs, such as a non-competitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), a competitive direct ELISA (CD-ELISA) and a sandwich ELISA (S-ELISA), and by protein slot-blotting and Western blotting. NCI- and CI-ELISA were valuable for detecting the existence of PedA1-specific antibodies in the sera of immunized rabbits. The limit of detection of PedA1 in MRS medium was found to be 0.5 microg ml(-1) in NCI-ELISA, while CI-ELISA on plates coated with purified PedA1 increased the affinity of the PH1-KLH-generated antibodies for PedA1; the limit of detection of PedA1 was less than 0.01 microg ml(-1) and 50% binding inhibition was achieved with 0.1 microg PedA1 ml(-1). Similarly, the limits of detection of PedA1 in MRS medium were found to be 5 microg ml(-1) by protein slot-blotting and 0.01 microg ml(-1) by Western blotting. Most importantly, PH1-KLH-generated polyclonal antibodies detected the presence of PedA1 in the supernatants of the producing strains of Pediococcus acidilactici 347, Z102, A172, X13 and P20, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing closely related or different bacteriocins. The approaches taken for the selection of the bacteriocin peptide fragment, the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of adequate specificity for other bacteriocins of interest in the food industry.

Amino Acid Sequence↗

Lactobacillus thermotolerans sp. nov., a novel thermotolerant species isolated from chicken faeces.

Five strains of thermotolerant lactic acid bacteria (G 12, G 22, G 35T, G 43 and G 44) isolated from chicken faeces were characterized taxonomically. The strains were facultatively anaerobic, Gram-positive, catalase-negative, non-motile, non-spore-forming rods. They were heterofermentative lactobacilli that produced DL-lactic acid. Growth of the strains occurred at 45 degrees C but not at 15 degrees C. The optimum temperature for growth was 42 degrees C, as determined from the specific growth rate. The highest permissive temperatures for growth were 50 degrees C for strain G35T and 48 degrees C for the other four strains. DNA G+C content of the strains was between 49 and 51 mol%. Complex fatty acid patterns of the strains showed the presence of C14:0, C16:0, sometimes C18:0, C18:1 and C19:0 cyclo in the cell walls. Phylogenetic analysis of the 16S rRNA gene sequences of the five strains placed them in the Lactobacillus caseil Pediococcus group, with Lactobacillus fermentum as their closest relative (about 95% sequence similarity). DNA-DNA hybridization data indicated that the thermotolerant strains were not L. fermentum. Taken together, the findings of this study show that the five strains isolated from chicken faeces represent a novel species within the genus Lactobacillus, for which the name Lactobacillus thermotolerans is proposed (G 35T = DSM 14792T =JCM 11425T).

Animals↗

Lactobacillus satsumensis sp. nov., isolated from mashes of shochu, a traditional Japanese distilled spirit made from fermented rice and other starchy materials.

Gram-positive, rod-shaped, motile lactic acid bacteria (strains NRIC 0603, NRIC 0604T, NRIC 0605 and NRIC 0606) were isolated from shochu mashes using an enrichment culture approach. These strains clustered in the Lactobacillus casei-Pediococcus group and were closely related to Lactobacillus nagelii and Lactobacillus mali on the basis of 16S rRNA gene sequence similarity. Levels of DNA-DNA relatedness revealed genotypic separation of the four isolates from the above two species. The isolates are therefore considered to represent a novel species, for which the name Lactobacillus satsumensis is proposed. The type strain is NRIC 0604T (=JCM 12392T=DSM 16230T).

Alcoholic Beverages↗

Lactobacillus concavus sp. nov., isolated from the walls of a distilled spirit fermenting cellar in China.

Two novel Lactobacillus strains, C-5-1(T) and HB5, were isolated from the walls of a distilled spirit fermenting cellar in Hebei province, China. The strains were Gram-positive, non-spore-forming, non-motile curved rods and were facultatively anaerobic. They produced 5% (-)-D/95% (+)-L lactic acid exclusively from glucose fermentation, but were catalase-negative. Both strains fermented N-acetylglucosamine, arbutin, salicin, cellobiose, maltose, trehalose and beta-gentiobiose. The mean DNA G+C content of the two strains was 46.9+/-0.8 mol% (46.4 mol% for the type strain, C-5-1(T)). Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain C-5-1(T) was clustered in the Lactobacillus casei/Pediococcus phylogenetic group and was closely related to Pediococcus dextrinicus JCM 5887(T) (97.9% similarity), followed by Lactobacillus algidus JCM 10491(T) (93.9% similarity). The DNA-DNA relatedness between the type strain C-5-1(T) and P. dextrinicus JCM 5887(T) and L. algidus JCM 10491(T) was only 5.4 and 4.3%, respectively. Therefore, based on phenotypic, genotypic and phylogenetic analyses, a novel Lactobacillus species, Lactobacillus concavus sp. nov., is proposed. The type strain is C-5-1(T) (=AS 1.5017(T)=LMG 22739(T)).

Base Composition↗

Microspectrofluorometric Measurement of Chloroplast DNA in Dividing and Expanding Leaf Cells of Spinacia oleracea.

Absolute DNA amounts of individual chloroplasts from mesophyll and epidermal cells of developing spinach leaves were measured by microspectrofluorometry using the DNA-specific stain, 4,6-diamidino-2-phenyl indole, and the bacterium, Pediococcus damnosus, as an internal standard. Values obtained by this method showed that DNA amounts of individual chloroplasts from mesophyll cells fell within a normal distribution curve, although mean DNA amounts changed during leaf development and also differed from the levels in epidermal chloroplasts. There was no evidence in the data of plastids containing either the high or low levels of DNA which would be indicative of discontinuous polyploidy of plastids, or of division occurring in only a small subpopulation of chloroplasts. By contrast, the distribution of nuclear DNA amounts in the same leaf tissues in which cell division was known to be occurring showed a clear bimodal distribution. We consider that the distribution of chloroplast DNA in the plastid population shows that there is no S-phase of chloroplast DNA synthesis, all chloroplasts in the population in young leaf cells synthesize DNA, and all chloroplasts divide.

Journal Article↗