Corynebacterium Research Articles

Corynebacterium ureicelerivorans sp. nov., a lipophilic bacterium isolated from blood culture

A lipophilic coryneform bacterium isolated from a blood culture from a patient with signs of septicaemia was characterized by means of phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short-chain mycolic acids, which are consistent with the genus Corynebacterium. The isolate was characterized biochemically by the very rapid (approx. 60 s) positive result that was obtained in a urease test in the API Coryne system. Comparative 16S rRNA gene sequencing demonstrated that the isolate belonged phylogenetically to the genus Corynebacterium. The values for sequence divergence (> or =1.4 %) with respect to known Corynebacterium species, together with phenotypic differences, show that the unidentified bacterium represents a novel member of this genus. On the basis of both the phenotypic and phylogenetic data, this isolate should be classified within a novel species of the genus Corynebacterium, for which the name Corynebacterium ureicelerivorans sp. nov. is proposed. The type strain is IMMIB RIV-2301(T) (=DSM 45051(T)=CCUG 53377(T)).

In vitro activity of linezolid and 12 other antimicrobials against coryneform bacteria

Objectives To compare the in vitro activity of linezolid with 12 other antimicrobials against 190 strains of the coryneform bacteria, including 60 strains of C. amycolatum, 30 of C. striatum, 30 of C. jeikeium, 10 of C. urealyticum, 20 of B. casei, 20 of D. hominis and 20 of T. otitidis. Methods Minimum inhibitory concentrations (MICs) and time–death curves were carried out according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). Results Linezolid was very active against the 130 strains of the Corynebacterium species studied. Only the glycopeptides showed similar efficacy. In contrast, penicillin G, ampicillin, macrolides, lincosamides, fluoroquinolones and aminoglycosides showed generally high MICs. Among the β-lactams, only imipenem was active against the majority of strains of C. striatum and C. amycolatum, and, approximately half of the C. jeikeium and C. urealyticum isolates. Both Dermabacter hominis and Brevibacterium casei showed marked resistance against most of the antimicrobials tested, while Turicella otitidis only showed high MICs against macrolides and clindamycin. For all of them, linezolid, vancomycin and teicoplanin proved effective. The time–death curves showed linezolid to behave as a bacteriostatic agent (approximately 90% death rate). Such activity was more accentuated for C. amycolatum and C. striatum (reduction of 1.3 and 1.7 log 10 CFU/mL, respectively) than for C. jeikeium and C. urealyticum (reduction of 1.0 and 0.8 log 10, respectively). Conclusions Our results indicate that linezolid is active against coryneform bacteria. The efficacy of linezolid is equal to or even superior to that of the glycopeptides.

Taxonomic composition of bacteria associated with cultivated mollusks Crassostrea lugubris and Perna viridis and with the water of the Gulf of Nha Trang lagoon, Vietnam

One hundred and four strains of heterotrophic bacteria have been isolated and characterized from two species of bivalve mollusks cultivated in the Gulf of Nha Trang (Vietnam) and from the water of a mariculture farm. The isolates have been identified on the basis of morphological, physiological, biochemical, and chemotaxonomic properties, as well as by the content of G+C bases in DNA. In the microflora of mollusks, Vibrio alginolyticus was predominant; the pathogenic species V. harveyi and V. splendidus were found as well. Staphylococci and bacilli occupied the second place in abundance after vibrios. In addition, coryneforms and enterobacteria, as well as Pseudomonas spp. and Pseudoalteromonas spp., were revealed. The composition of the water microflora was more diverse as compared with the microflora of mollusks. In the water, Bacillus spp., Vibrio spp., and Pseudomonas spp. were predominant. Brevibacterium spp. and other coryneform bacteria, as well as enterobacteria, occurred in significant amounts. In addition, Pseudoalteromonas spp., Marinococcus sp., Halobacillus sp., Shewanella sp., Sulfitobacter sp., and bacteria of the CFB cluster were noticed. The presence of pathogenic and conditionally pathogenic bacterial species in the water and mollusks is probably the reason for the high death rate of cultivated animals at the mariculture farm.

Molecular identification of Exiguobacterium acetylicum as the aetiological agent of bacteraemia

A case of catheter-related bacteraemia caused by Exiguobacterium acetylicum is reported in an elderly patient. The availability of sequence-based methods facilitated rapid identification and expanded the spectrum of diseases attributed to coryneform bacteria and specifically to the genus Exiguobacterium.

Sources of microbial contamination in tissue culture laboratories in southwestern Nigeria

Microbial contamination is a constant problem, which often compromise development of all in vitro techniques. This study aimed at investigating the source of microbial contamination in tissue culture laboratories in southwestern Nigeria. Nineteen microbial contaminants (consisting of eleven bacteria and eight fungi) were found associated with the tissue culture plants and the laboratory environments. The bacterial contaminants includes, Pseudomonas flourescens,Escherichia coli, Proteus sp, Micrococcus spp, Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Corynebacterium sp and Erwinia sp. While Fungi isolates were Alterneria tenius, Aspergillus niger, Aspergillus fumigatus, Cladosporium sp,Saccharomyces sp, Fusarium oxysporum, Rizopus nigricans and Fusarium culmorum. The rate of occurrence of S. aureus, B. cereus, B. subtilis and E. coli were found to be higher (ranging from 36-46%) in human skin than in all other sampled materials. The laboratory walls and tables also harbored most of the contaminating microbes. The laboratory indoor air was found associated with all the contaminating microbes. Key words: Micro-propagation, plant tissue culture, bacterial and fungal isolates, contamination.

Biodiversity and the survival of autochthonous fermented products.

Many microorganisms isolated and characterized during the past century have received considerable attention from the pharmaceutical and/or food fermentation industry because of their potential biotechnological interest. Lactic acid bacteria (LAB), particularly those belonging to beneficial and nonpathogenic genera (Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, and Streptococcus), have traditionally been used in the food industry. They also play an essential role in the dairy industry due to the tremendous level of human consumption of several important fermented products, mainly cheese and acidified/fermented milks. In the USA, more than 4.6 million tons of cheese and more than 1.3 million tons of yogurt were produced in 2005 [1], whereas in the European Community (EU-25) more than 8 million tons of cheese and 6.9 million tons of acidified/fermented milk were produced in 2003 [3]. These figures correspond to a global sales value that surpasses US$ 28.44 billion, with the global sales value of cheese representing about 30% of total dairy-product sales [2]. Nowadays, more than 500 kinds of cheese are manufactured worldwide, and the many varieties are derived from modifications in cheese-making techniques. However, the fermentation process whereby milk is transformed into cheese has not changed to any great extent since humans discovered the process as a means of preserving food (the fermentation of milk dates back to ~3000 BC). Cheese is a dynamic biochemical product and, unlike many processed food products for which stability is essential, it undergoes significant changes during ripening. The unripe curds of many varieties of cheese have bland and largely similar flavors. However, the sequential actions of cheese microbiota and the interactions among the different microorganisms during the ripening period result in the flavor compounds that characterize each variety. Acidifying lactococci species usually occur as dominant bacteria early in the ripening process. Afterwards their numbers decrease as they give way to lactobacilli, which, due to their higher versatility for fermentation, can survive in cheeses at stages when concentrations of major carbohydrates and free water decrease, whereas the sodium chloride load increases. Apart from the above-mentioned major genera of LAB, other bacteria, including non-starter LAB and non-lactic-acid bacteria, as well as some fungi (yeasts, moulds), staphylococci, micrococci, coryneform bacteria, and propionic acid bacteria, which develop in the product particularly during the maturation phase as secondary microbiota, are often responsible for the intensity of flavor in many dairy products. The production of a wide range of antagonistic primary and secondary metabolites, including organic acids, diacetyl, CO2 and even bacteriocins, affects the complex dynamics of microorganisms in cheese. Thus, a well-founded understanding of the microbial balance in this special living ecosystem is essential to improve older varieties of cheese and to develop new ones. Due to the dairy market’s continuous growth, commercial cultures have conventionally been selected to obtain greater quantities of product, homogeneous quality of the end product, and short production times, with the final goal of satisfying the preferences of consumers. Most varieties of commercially important cheeses are manufactured worldwide by means of commercial starter cultures of generic composition (including acidifying and aromatic diacetyl-producing lactococci strains and sometimes also leuconostocs and lactobacilli strains). In Europe, Asia, Africa, and Latin America, however, there are regions where fermented products are still manufactured traditionally. In global markets, these fermented products are now highly appreciated and are considered to be of premium value because of their flavor characteristics, which are not found in varieties of cheese produced industrially. In Europe, such products are elaborated according to well-established rules governing the production of registered designations of origin. Protected designations of origin (PDOs) apply to foodstuffs that are produced, processed, and prepared in a given geographic area using recognized

P828 Activity of daptomycin against coryneform bacteria isolated from clinical samples

P828 Activity of daptomycin against coryneform bacteria isolated from clinical samples

L-Lysine Fermentation

Amino acids are the basic bioelements of proteins, which are the most important macromolecules for the functions of humans and animals. Out of the 20 L-amino acids, ecumenically found in most of living organisms, L-lysine is one of the 9 amino acids which are essential for human and animal nutrition. L-lysine is useful as medicament, chemical agent, food material (food industry) and feed additive (animal food). Its demand has been steadily increasing in recent years and several hundred thousands tones of L-lysine (about 800,000 tones/year) are annually produced worldwide almost by microbial fermentation. The stereospecificity of amino acids (the L isomer) makes the fermentation advantageous compared with synthetic processes. Mutant auxotrophic or resistant to certain chemicals strains of so-called gram positive coryneform bacteria are generally used, including the genera Brevibacterium and Corynebacterium, united to the genus. The significance of Research and Development increased rapidly since the discovery of fermentative amino acid production in the fifties (S. Kinoshita et al., Proceedings of the International Symposium on Enzyme Chemistry 2:464-468 (1957)), leading to innovative fermentation processes which replaced the classical manufacturing methods of L-lysine like acid hydrolysis. L-Lysine is separated and purified by suitable downstream processes involving classical separation or extraction methods (ultrafiltration or centrifugation, separation or ion exchange extraction, crystallization, drying) and is sold as a powder. Alternatively, spray dried pellets or liquid fermentation broth can be used as animal feed supplement. On behalf of today's strong competition in amino acid industry, Biotechnology companies are continuously aiming in innovative research developments and use complex management concepts and business strategies, towards gaining market leadership in the field of amino acid production.

Agrococcus casei sp. nov., isolated from the surfaces of smear-ripened cheeses

Seven Gram-positive, coryneform bacteria with virtually identical whole-organism protein patterns were isolated from the surface of smear-ripened cheeses. Representatives of these strains were the subject of a polyphasic study designed to establish their taxonomic status. The organisms formed a distinct branch in the Microbacteriaceae 16S rRNA gene tree and were most closely related to members of the genus Agrococcus, sharing sequence similarities of 95.4-98.7 %. The chemotaxonomic profiles of the strains were consistent with their classification in the genus Agrococcus. The combined genotypic and phenotypic data show that the isolates should be classified in the genus Agrococcus as representatives of a novel species. The name Agrococcus casei sp. nov. is proposed for this taxon. Isolate R-17892t2(T) (=DSM 18061(T)=LMG 22410(T)) is the type strain of Agrococcus casei sp. nov.

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.

Coryneform bacteria in semen of chronic prostatitis patients

Our aim was to compare the presence and species composition of coryneform bacteria in chronic prostatitis patients and controls. Semen of 50 men with inflammatory prostatitis and 59 controls (without pelvic pain/discomfort complaints and leukocytospermia) was investigated. First-catch urine was additionally investigated in 36 men (30 with and 6 without prostatitis). Coryneform bacteria were found in semen of 76% men with inflammatory prostatitis and 83% controls. More than half of the isolates were identified as Corynebacterium seminale. Prostatitis patients with severe leukocytospermia (>1 million white blood cells per ml) harboured significantly more Corynebacterium group G (33% vs. 2%, p = 0.0003) and Arthrobacter sp. (17% vs. 2%, p = 0.03) in comparison with controls. Nine species of coryneforms with high concentration (>or=10,000 CFU per ml) were found in prostatitis patients as against only four species in controls. Half of the men harboured corynebacteria in semen as well as in urine, 22% of men in semen only, and 3% in urine only. The total concentration of coryneforms was greater in semen than in urine (median 5000 vs. 100 CFU per ml, p = 0.053). We suggest that although coryneforms are generally considered as saprophytes, they are not uniform and some species (Corynebacterium group G and Arthrobacter sp.) may be associated with inflammatory prostatitis.

Precise metabolic flux analysis of coryneform bacteria by gas chromatography–mass spectrometry and verification by nuclear magnetic resonance

Precise metabolic flux analysis (MFA) by gas chromatography-mass spectrometry (GC-MS) and computer calculation was performed, and the consistency of the estimated results was verified by independently performed nuclear magnetic resonance (NMR) analysis. The precise estimation of flux by the integration method of the mass isotopomer signal, defined as the coefficient of variance (CV) of multiple determination, was investigated, and the results estimated using different data sets with the same magnitude of error were confirmed. The CV of multiple determinations was sufficiently small to discuss and compare the fluxes of a metabolic pathway. The estimated fluxes using the GC-MS data were cross-validated with the NMR data that were independently measured and not used for MFA. The developed method was successfully applied to the MFA of the growth phase of two different glutamate-producing coryneform bacteria, Corynebacterium glutamicum and C. efficiens. The difference in the growth rate between these two bacterial species was discussed while considering the results of MFA, including forward and backward (exchange) fluxes.

Genome characterization, analysis of virulence and transformation ofMicrobacterium nematophilum, a coryneform pathogen of the nematodeCaenorhabditis elegans

A coryneform bacterium designated Microbacterium nematophilum has previously been reported to act as a pathogen for Caenorhabditis elegans. This bacterium is able to colonize the rectum of infected worms and cause localized swelling, constipation and slowed growth. Additional isolates and analysis of this bacterium are described here. Tests of pathogenicity on other Caenorhabditis nematodes show that M. nematophilum infection is lethal to most species in the genus, in contrast to its relatively mild effects on C. elegans. The size and geometry of the pathogen genome have been determined as a closed circular molecule of 2.85 Mb with high G+C content. Bacteria also harbor a 55 kb plasmid, pMN1, which is largely composed of a lysogenic bacteriophage genome. Mutagenesis experiments have yielded stable avirulent mutants of M. nematophilum. As a first step towards molecular genetic analysis, methods for low-efficiency transformation of M. nematophilum have been developed.

Corynebacterium glutamicum as a model bacterium for the bioremediation of arsenic.

Arsenic is an extremely toxic metalloid that, when present in high concentrations, severely threatens the biota and human health. Arsenic contamination of soil, water, and air is a global growing environmental problem due to leaching from geological formations, the burning of fossil fuels, wastes generated by the gold mining industry present in uncontrolled landfills, and improper agriculture or medical uses. Unlike organic contaminants, which are degraded into harmless chemical species, metals and metalloids cannot be destroyed, but they can be immobilized or transformed into less toxic forms. The ubiquity of arsenic in the environment has led to the evolution in microbes of arsenic defense mechanisms. The most common of these mechanisms is based on the presence of the arsenic resistance operon (ars), which codes for: (i) a regulatory protein, ArsR; (ii) an arsenite permease, ArsB; and (iii) an enzyme involved in arsenate reduction, ArsC. Corynebacterium glutamicum, which is used for the industrial production of amino acids and nucleotides, is one of the most arsenic-resistant microorganisms described to date (up to 12 mM arsenite and >400 mM arseniate). Analysis of the C. glutamicum genome revealed the presence of two complete ars operons (ars1 and ars2) comprising the typical three-gene structure arsRBC, with an extra arsC1 located downstream from arsC1 (ars1 operon), and two orphan genes (arsB3 and arsC4). The involvement of both ars operons in arsenic resistance in C. glutamicum was confirmed by disruption and amplification of those genes. The strains obtained were resistant to up to 60 mM arsenite, one of the highest levels of bacterial resistance to arsenite so far described. Using tools for the genetic manipulation of C. glutamicum that were developed in our laboratory, we are attempting to obtain C. glutamicum mutant strains able to remove arsenic from contaminated water.

Meio de cultura semi-seletivo para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens em solo e sementes de feijoeiro

A murcha-de-curtobacterium, causada pela bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), é um importante problema para o cultivo de feijão (Phaseolus vulgaris L.) na região Centro-Sul do Brasil. A principal forma de disseminação da bactéria é por sementes contaminadas. Embora a ocorrência desta doença seja relativamente recente no Brasil, Cff tem sido disseminado rapidamente nas regiões produtoras de feijão do país. Este trabalho teve como objetivo ajustar o meio de cultura CNS (Corynebacterium Nebraskense Selective Medium) para recuperação e detecção de Cff em solo e sementes de feijoeiro. Suspensões provenientes da lavagem de amostras de solo e sementes contaminadas pela bactéria foram plaqueadas no meio de cultura CNS - modificado. Os ajustes realizados no meio de cultura possibilitaram a recuperação eficiente de Cff presente em solo e sementes contaminadas. As modificações estabelecidas para o meio CNS foram a redução da concentração de sulfato de polimixina para 16 mg/l, exclusão do componente ciclohexamida e substituição do fungicida Daconil 2787-F (530 mg/ml de chlorothalonil) pelo Dacostar 500 (500 mg/ml de chlorothalonil).

Actualités de l′épidémiologie et du rôle pathogène des corynébactéries

The corynebacteria are aerobically growing, asporogenous, irregularly shaped gram-positive rods. During the last few years, there has been an increase in the number of publications claiming an association of coryneform bacteria with disease. The well-known species Corynebacterium diphtheriae, agent of diphteria, is still endemic in many countries of all continents but it can be also isolated in France from serious infections such as endocarditis. The related species C. ulcerans can produce a diphtheria-like toxin and was isolated from wounds in humans after cat or dog bites. Many lipophilic species such as C. jeikeium and C. urealyticum are recognized as pathogens responsible for septicaemia and urinary tract infections, respectively, but several reports have recently associated granulomatous mastitis in women to the presence of other lipophilic bacteria notably the recently described species C. kroppenstedtii. Other Corynebacterium species are associated with infections such as C. aurimucosum ( C. nigricans) isolated from female genital infections and C. glucuronolyticum isolated from male genital infections. Some species of the closely related anaerobic genera Actinomyces such as A. neuii can grow in aerobic atmosphere and cause subcutaneous abscesses not histologically related to actinomycosis. Recently, the Actinomyces related genus Actinobaculum has been documented to cause urinary tract infection. Susceptibility testing should always be performed with clinically relevant isolates by using a disk diffusion method but also by determining the MIC and the MBC in the case of penicillin tolerant C. diphtheriae isolates. Recently, the CLSI/NCCLS has published guidelines for the susceptibility testing of Corynebacterium isolates as well as for the interpretive criteria. The species C. jeikeium and C. urealyticum are mainly resistant to antibiotics except to glycopeptides, but other species can show resistance to one class of antibiotics. Mechanism of resistance to β-lactams is probably based on modified cell wall components such as porins. For other antibiotics such as macrolides, modification of targets can explain resistance. Microbiologists should be now aware of the potential pathogenic role of these corynebacteria in order to accurately isolate and identify them from some clinical specimens.

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.

Clavibacter michiganensis subsp. sepedonicus

Clavibacter michiganensis<i> subsp. </i>sepedonicus

A Case of Catheter-Related Bacteremia by Arthrobacter woluwensis

Arthrobacter woluwensis, a catalase-positive coryneform bacterium recognized as an opportunistic pathogen, was repeatedly isolated from the blood of a 56-year-old male patient with metastatic colon cancer. The isolate was identified by various phenotypic tests and by sequencing analysis of 16S rRNA. Antimicrobial susceptibility testing was performed by E-test; the MICs to vancomcyin, cefotamine, and penicillin were 1.5 microg/mL, >64 microg/mL, and 4 microg/mL, respectively. The patient was treated with vancomycin, and the subclavian catheter, which was presumed to be the source of the infection, was removed. Thereafter, repeated blood cultures did not grow the organism. The infections of human caused by A. woluwensis have not been reported previously in Korea, probably because of the difficulty of identifying Arthrobacter strains by conventional biochemical tests.

Diversity of nocardio- and coryneform bacteria in soil adjacent to pig farm treatment installation

Effect of excessive active sludge on soil microbial communities has been studied at «Vostochny» pig farm treatment installation in the Leningrad oblast. Comparative evaluation of genetic and physiological diversity of nocardio - and coryneform bacteria in soil adjacent to the reservoir for active sludge waste accumulation has been carried out.