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PubMed1998

Killing of Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia by protegrins.

Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study, we (i) examine the sensitivity of Gram-negative, anaerobic periodontal pathogens to synthetic protegrins; (ii) determine the relative potencies of protegrin congeners against these bacteria; and (iii) compare the potency of protegrins with other antibiotic peptides, including magainin MSI-78, tachyplesin I, cecropin P1, human defensins HNP-1-3, and clavanin A. Synthetic L- and D-enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3 and 5 (PG-2, PG-3 and PG-5) were tested against Fusobacterium nucleatum, and black-pigmented organisms including Porphyromonas gingivalis and Prevotella intermedia. Strains of both F. nucleatum and the black-pigmented organisms were sensitive to PG-1, and exhibited mean ED99 of 2.2-2.3 micrograms/ml and 3.4-9.9 micrograms/ml, respectively. The D-form was statistically more potent than the L-form against these oral anaerobes, and although this difference in potency is unlikely to be of decisive therapeutic significance, the D-form may be of value given ability to resist microbial and host-derived proteases. PG-1 was more potent than magainin, tachyplesin, cecropin, defensins and clavanin under test conditions. Hypertonic salt concentrations and heat-inactivated serum were found to be inhibitory to the bactericidal activity of PG-1. PG-1 was found to induce morphologic alterations in the ultrastructural appearance of F. nucleatum consistent with damage to the bacterial membranes. We conclude that protegrins may be useful antimicrobial agents in therapy against Gram-negative anaerobic bacteria believed to be involved in chronic, adult forms of periodontal infections.

Amino Acid Sequence↗

PubMed1997

Rescue of a Drosophila NF1 mutant phenotype by protein kinase A.

The neurofibromatosis type 1 (NF1) tumor suppressor protein is thought to restrict cell proliferation by functioning as a Ras-specific guanosine triphosphatase-activating protein. However, Drosophila homozygous for null mutations of an NF1 homolog showed no obvious signs of perturbed Ras1-mediated signaling. Loss of NF1 resulted in a reduction in size of larvae, pupae, and adults. This size defect was not modified by manipulating Ras1 signaling but was restored by expression of activated adenosine 3', 5'-monophosphate-dependent protein kinase (PKA). Thus, NF1 and PKA appear to interact in a pathway that controls the overall growth of Drosophila.

Amino Acid Sequence↗

PubMed2006

Direct molecular approach to monitoring bacterial colonization on vacuum-packaged beef.

Denaturing gradient gel electrophoresis allowed us to monitor total bacterial communities and to establish a pattern of succession between species in vacuum-packaged beef stored at 2 and 8 degrees C for 9 weeks and 14 days. Species-specific PCR was used to confirm the presence of Lactobacillus sakei and Lactobacillus curvatus. Multiplex PCRs using 16S rRNA-specific primers allowed differentiation between Leuconostoc species. These methods provided the desired information about microbial diversity by detecting the main microorganisms capable of colonizing this ecological niche.

Animals↗

PubMed2003

Increasing the oxidative stress response allows Escherichia coli to overcome inhibitory effects of condensed tannins.

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H(2)O(2). The oxidative stress response helps E. coli strains to overcome their inhibitory effect.

Acacia↗

PubMed2004

Two bacteria phylotypes are predominant in the Suiyo seamount hydrothermal plume.

Microbial diversity and populations in a hydrothermal plume that was present inside the caldera of the Suiyo Seamount, a submarine volcano on the Izu-Bonin Arc, were investigated by performing a phylogenetic analysis of the 16S rRNA gene and by using fluorescence in situ hybridization (FISH). Corresponding to transmissivity, an indicator of turbidity, the vertical total cell count as determined by 4',6'-diamidino-2-phenylindole (DAPI) staining varied from 5.6 x 10(4) to 1.1 x 10(5) cells ml(-1), and the apparent plume layer was assessed to be at a depth of 1,050 to 1,200 m inside the caldera and to contain 1.0 x 10(5) to 1.1 x 10(5) cells ml(-1). From microbial samples collected in the plume by an in situ filtration system, the following two major phylogenetic groups, which were closely related to sulfur-oxidizing microbes, were obtained: the SUP05 group belonging to the gamma subclass of the Proteobacteria (13 of 20 clones) and the SUP01 group belonging to the epsilon subclass of the Proteobacteria (5 of 20 clones). Specific oligonucleotide probes for these groups (SUP05-187 and SUP01-63) were designed and were used with various water samples obtained from the Suiyo Seamount. In the apparent plume layer, up to 66% of the total counts of microbial cells were estimated to be Bacteria cells that hybridized to EUB338, and few cells were identified by the archaeal probe ARCH915. Almost all Bacteria cells were hard to identify with the known group-specific probes, such as ALF19, GAM42a, and CF319, while 88 to 90% of the Bacteria cells hybridized with SUP05-187 and >98% of them were considered members of the SUP05 and SUP01 populations. In a low-temperature vent fluid emitted from a bivalve-colonized mound, the SUP05 cells accounted for >99% of the Bacteria cells, suggesting that a portion of the plume cells originated on the surface of the seafloor at a depth of about 1,380 m. From further analysis of cell morphology (i.e., cell size and cell elongation index) we inferred that the SUP05 cells were active in the plume layer at a depth of 1,050 to 1,200 m compared to the activity in a near-bottom layer, while many elongated cells were found between these layers. These findings suggest that the morphology and distribution of SUP05 cells have complex relationships with hydrothermal activities and water circulation. Although growth and production rates remain to be defined, we concluded that this Suiyo Seamount caldera has functioned as a natural continuous incubator for these two phylotypes of Bacteria in an aphotic deep-sea environment.

Colony Count, Microbial↗

PubMed2005

Diversity of bacteria growing in natural mineral water after bottling.

Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes. Sequence analyses identified 11 operational taxonomic units (OTUs), all but one affiliated with the betaproteobacterial order Burkholderiales (6 OTUs) or the class Alphaproteobacteria (4 OTUs). Fluorescence in situ hybridization (FISH) was applied in combination with DAPI (4',6'-diamidino-2-phenylindole) staining, viability staining, and microscopic counting to quantitatively monitor changes in bacterial community composition. A growth curve similar to that of a bacterium grown in a batch culture was recorded. In contrast to the current perception that Gammaproteobacteria are the most important bacterial components of natural mineral water in bottles, Betaproteobacteria dominated the growing bacterial community and accounted for 80 to 98% of all bacteria detected by FISH in the late-exponential and stationary-growth phases. Using previously published and newly designed genus-specific probes, members of the betaproteobacterial genera Hydrogenophaga, Aquabacterium, and Polaromonas were found to constitute a significant proportion of the bacterial flora (21 to 86% of all bacteria detected by FISH). For the first time, key genera responsible for bacterial growth in a natural mineral water were identified by applying molecular cultivation-independent techniques.

Bacteria↗

PubMed2006

Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases beta-lactam susceptibility and virulence.

The twin arginine translocation (Tat) system is used by many bacteria to export fully folded proteins containing cofactors. Here, we show genetically that this system is essential for Mycobacterium tuberculosis, as the tatAC operon and tatB genes could be inactivated only in partially diploid strains. Using comparative genomics, the rv2525c gene of M. tuberculosis was identified as encoding a histidine-rich protein, with a twin arginine signal peptide, and orthologous genes were shown to be present in several but not all actinobacterial species. Conservation of this gene by Mycobacterium leprae, which has undergone reductive evolution, suggested an important role for rv2525c. An rv2525c knockout mutant was constructed, and biochemical analysis indicated that the mature Rv2525c protein is secreted. Upon exposure to antituberculous drugs, rv2525c expression is significantly up-regulated together with those of other genes involved in cell wall biogenesis. Phenotypic comparison of the mutant with the parental strain revealed an increase in susceptibility to some beta-lactam antibiotics and, despite slower growth in vitro, enhanced virulence in both cellular and murine models of tuberculosis. The Tat system thus contributes in multiple ways to survival of the tubercle bacillus.

Amino Acid Sequence↗

PubMed1997

Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys.

In vivo passage of a chimeric simian-human immunodeficiency virus (SHIV-89.6) expressing the human immunodeficiency virus type 1 (HIV-1) tat, rev, vpu, and env genes generated pathogenic viruses (SHIV-89.6P) inducing rapid CD4+ lymphocyte depletion and AIDS-like illness in rhesus monkeys (K. Reimann, J. T. Li, R. Veazey, M. Halloran, I.-W. Park, G. B. Karlsson, J. Sodroski, and N. L. Letvin, J. Virol. 70:6922-6928, 1996). To characterize the molecular changes responsible for this increase in virulence, infectious proviral clones of SHIV-89.6P isolates were derived. Viruses generated from some of these clones caused a rapid and profound decline of CD4+ lymphocytes in a high percentage of inoculated monkeys. Nucleotide changes potentially responsible for the increased virulence of SHIV-89.6P were limited to the env, tat, or long terminal repeat sequences, with most of the observed changes in env. Nucleotide changes in env altered 12 amino acids in the gp120 and gp41 exterior domains, and a 140-bp deletion in env resulted in the substitution of the carboxyl terminus of the SIVmac gp41 glycoprotein for that of the HIV-1 gp41 glycoprotein. The availability of pathogenic proviral clones should facilitate dissection of the molecular determinants of SHIV-89.6P virulence.

Animals↗

PubMed1992

Maturation of follicle-stimulating hormone gonadotropes during the rat estrous cycle.

FSH mRNA is transcribed after the onset of high FSH secretion during proestrus and estrus. Pituitary cell fractions separated by size and density were studied to determine if expression of FSH mRNA activity was predominantly in one subset during the estrous cycle and to determine the source and significance of "silent FSH" cells that secrete FSH, but store too little for detection. Pituitary cells were separated by centrifugal elutriation, plated, and then exposed to 0.1-1 nM [D-Lys6]GnRH for 3 h. Media were assayed for FSH by RIA, and the cells were fixed for immunocytochemistry or in situ hybridization. The percentages of immunoreactive FSH cells in unseparated populations increased from 8% at metestrus to 12% during proestrus. Percentages of cells with FSH beta mRNA showed the same rise; however, peak levels were higher (17%) during proestrus and estrus. Small cells with FSH beta mRNA were more frequent than those with antigens early in the cycle. The largest cell fractions contained 38-44% immunoreactive cells. Only 8-21% of these cells had FSH beta mRNA, except during the morning of proestrus (33%). The distribution analyses showed that the increment in immunoreactive FSH cells during diestrus initially stemmed from smaller subsets; however, over half of immunoreactive FSH cells were large by the evening of proestrus. During the time of active transcription of FSH mRNA, more than half of the cells with FSH beta mRNA were small or medium-sized. Thus, early in the cycle, FSH beta mRNA is transcribed in the smaller cells, which may be the source of the silent FSH cells reported in previous studies. During proestrus, smaller FSH cells also secreted as well if not better than those in the unseparated population or large fractions. When they secreted more than expected from their percentages of FSH cells, this response was interpreted to be due to either the presence of cells that are immunoreactively silent or the possible removal of autocrine or paracrine regulatory factors.

Animals↗

PubMed2000

The distribution of cells containing estrogen receptor-alpha (ERalpha) and ERbeta messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females.

We have used in situ hybridization to compare the distributions of estrogen receptor alpha (ERalpha) and ERbeta messenger RNA (mRNA)-containing cells in the preoptic area and hypothalamus of ewes and rams. Perfusion-fixed brain tissue was collected from luteal phase ewes and intact rams (n = 4) during the breeding season. Matched pairs of sections were hybridized with sheep-specific, 35S-labeled riboprobes, and semiquantitative image analysis was performed on emulsion-dipped slides. A number of sex differences were observed, with females having a greater density of labeled cells than males (P < 0.001) and a greater number of silver grains per cell (P < 0.01) in the ventromedial nucleus for both ER subtypes. In addition, in the retrochiasmatic area, males had a greater (P < 0.05) cell density for ERalpha mRNA-containing cells than females, whereas in the paraventricular nucleus, females had a greater density (P < 0.05) of ERalpha mRNA-containing cells than males. There was a trend (P = 0.068) in the arcuate nucleus for males to have a greater number of silver grains per cell labeled for ERalpha mRNA. In both sexes, there was considerable overlap in the distributions of ERalpha and ERbeta mRNA-containing cells, but the density of labeled cells within each nucleus differed in a number of instances. Nuclei that contained a higher (P < 0.001) density of ERalpha than ERbeta mRNA-containing cells included the preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus, whereas the subfornical organ (P < 0.001), paraventricular nucleus (males only, P < 0.05), and retrochiasmatic nucleus (females only, P < 0.05) had a greater density of ERalpha than ERbeta mRNA-containing cells. The anterior hypothalamic area and supraoptic nucleus had similar densities of cells containing both ER subtypes. The lateral septum and arcuate nucleus contained only ERalpha, whereas only ERbeta mRNA-containing cells were seen in the zona incerta. The sex differences in the populations of ER mRNA-containing cells in the ventromedial and arcuate nuclei may explain in part the sex differences in the neuroendocrine and behavioral responses to localized estrogen treatment in these nuclei. Within sexes, the differences between the distributions of ERalpha and ERbeta mRNA-containing cells may reflect differential regulation of the actions of estrogen in the sheep hypothalamus. Low levels of ERbeta mRNA in the preoptic area and ventromedial and arcuate nuclei, regions known to be important for the regulation of reproduction, suggest that ERbeta may not be involved in these functions.

Animals↗

PubMed2006

Functional deficiency in IL-7 caused by an N-ethyl-N-nitrosourea-induced point mutation.

N-ethyl-N-nitrosourea (ENU)-induced mutagenesis provides a powerful approach for identifying genes involved in immune regulation and diseases. Here we describe a new mutant strain, HLB368, with hereditary leukopenia. At necropsy, the mutant mice had very small thymuses and spleens. All but the inguinal nodes were absent and there were no Peyer's patches. By flow cytometry, the ratios of T-cell subsets were normal, but B-cell development was blocked at the pre-pro-B-cell stage. The development of B1 and marginal zone B cells was relatively normal. The mutation was mapped to chromosome 3 between D3Mit221 and D3Mit224, a region that contains the Il7 gene. cDNA and genomic DNA sequences of Il7 revealed a T-to-C missense transition resulting in a change of Leu to Pro within the leader peptide that would be predicted to inhibit secretion. In keeping with this concept, we found that in vitro treatment of B-cell progenitors from mutant mice with IL-7 induced them to differentiate into pre-BII cells. Phenotypic comparisons of HLB368 with genetically targeted Il7 null mice showed many similarities along with a few differences, indicating that this ENU-induced mutant carries a novel allele. This new strain thus provides a new model for studying the functions of IL-7 on a pure C57BL/6 background.

Animals↗

PubMed1999

Enumeration of antigen-presenting cells in mice infected with Sendai virus.

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.

Amino Acid Sequence↗

PubMed1999

Two mechanisms for the non-MHC-linked resistance to spontaneous autoimmunity.

Genetic susceptibility and resistance to most autoimmune disorders are associated with highly polymorphic genes of the MHC and with non-MHC-linked polygenic modifiers. It is known that non-MHC-linked polymorphisms can override or enhance the susceptibility to an autoimmune disease provided by pathogenic MHC genes, but the mechanisms remain elusive. In this study, we have followed the fate of two highly diabetogenic beta cell-specific T cell receptors (Kd and I-Ag7 restricted, respectively) in NOR/Lt mice, which are resistant to autoimmune diabetes despite expressing two copies of the diabetogenic MHC haplotype H-2g7. We show that at least two mechanisms of non-MHC-linked control of pathogenic T cells operate in these mice. One segregates as a recessive trait and is associated with a reduction in the peripheral frequency of diabetogenic CD8+ (but not CD4+) T cells. The other segregates as a dominant trait and is mediated by IL-4- and TGF-beta1-independent immune suppressive functions provided by lymphocytes that target diabetogenic CD4+ and CD8+ T cells, without causing their deletion, anergy, immune deviation, or ignorance. These results provide explanations as to how non-MHC-linked polymorphisms can override the susceptibility to an autoimmune disease provided by pathogenic MHC haplotypes, and demonstrate that protective non-MHC-linked genes may selectively target specific lymphoid cell types in cellularly complex autoimmune responses.

Amino Acid Sequence↗

PubMed1993

Possible involvement of c-kit receptor and its ligand in increase of mast cells in neurofibroma tissues.

Many mast cells are present in the tumor tissues of neurofibromatosis 1. We investigated the mechanism of the mast cell increase. Since the stem cell factor (SCF) induces development of mast cells and since the receptor of SCF is encoded by the c-kit gene, we examined the expression of SCF mRNA and c-kit mRNA in neurofibroma tissues. In situ hybridization demonstrated strong expression of c-kit messenger RNA in mast cells in the neurofibroma, but the expression of SCF mRNA was not demonstrable by in situ hybridization in either neurofibroma tissues or control normal skin tissues. When RNA extracted from neurofibroma tissues or normal skin tissues was reverse transcribed and then amplified by the polymerase chain reaction, the amount of SCF cDNA was greater in neurofibroma tissues than in normal skin tissues. The results suggest that SCF and the c-kit receptor are associated with the increase of mast cells in neurofibroma tissues.

Adolescent↗

PubMed1995

High level expression and export of beta-glucuronidase from murine mucopolysaccharidosis VII cells corrected by a double-copy retrovirus vector.

Retrovirus vectors were constructed to transfer and express the cDNA of the human lysosomal acid hydrolase beta-glucuronidase (GUSB) under control of the human GUSB promoter. Expression of the transcription unit (minigene) was evaluated in a GUSB-negative cell line established from a mouse with the lysosomal storage disease mucopolysaccharidosis (MPS) type VII. A vector designed to transfer single copies of the minigene (N2H beta H) expressed normal levels of GUSB activity in the deficient cells. GUSB expression was increased to several times greater than normal by inserting the minigene into a double-copy vector (DCH beta H), which places one copy of the transcription unit upstream of the retrovirus promoter in both the 3' and 5' long terminal repeats (LTRs) of the integrated provirus. The specific activity of GUSB and a control normal lysosomal enzyme, alpha-galactosidase (GLA), were higher in normal and in vector-corrected cells from confluent cultures than in subconfluent dividing cells. The ratios of GUSB to GLA were similar at all phases of cell growth, but the level of GUSB expression from the double copy vector was several-fold higher than from the single copy vector. To determine if this effect was controlled by the GUSB promoter, a vector was constructed using the thymidine kinase (TK) promoter to drive the human GUSB cDNA (NTK beta H). The levels of GUSB in cells corrected with this vector exhibited the same cell density dependent pattern as when the GUSB promoter was used, indicating that the variation in enzymatic activity was not a function of the GUSB promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Cytotoxic T lymphocytes in asymptomatic long-term nonprogressing HIV-1 infection. Breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load.

Although vigorous activated and memory CTL have been associated with HIV-1 infection, data are lacking regarding the breadth of epitopes recognized in a given individual and the relationship to the viral quasispecies present in vivo. In this study we performed a detailed analysis of the HIV-1-specific CTL response in a seropositive person with documented HIV-1 infection of 15 yr duration, stable CD4 counts above 500 cells/ml, and viral load persistently below 500 molecules of RNA/ml of plasma. Epitope mapping studies revealed the presence of HLA class I-restricted CTL responses to six different epitopes in p17, p24, RT, Env, and Nef, which conferred broadly cross-reactive recognition of reported HIV-1 variants. Sequence analysis of autologous viruses revealed the absence of immune escape variants within five of the six epitopes. Despite consistently low viral RNA levels in plasma and viral DNA levels in PBMC, in vivo-activated circulating CTL were detected against three of the epitopes. Five of the six epitopes, including the three dominant epitopes, have been detected in persons with progressive disease, suggesting that nonprogressors may not target unique epitopes. This study demonstrates that HIV-1-specific CTL can be highly activated and broadly directed in the setting of an extremely low viral load, and that neither high viral load nor antigenic diversity is required for the generation of a multispecific CTL response. Although the detection of strong CTL responses, low viral load, and lack of immune escape are consistent with the hypothesis that CTL may contribute to lack of disease progression in this individual, the contribution of these responses to maintenance of the asymptomatic state remains to be determined.

Amino Acid Sequence↗

PubMed2000

A systematic approach to the validation of process control parameters for monoclonal antibody production in fed-batch culture of a murine myeloma.

A systematic approach to the validation of control ranges of control parameters for a cell culture process producing a monoclonal antibody is described. Specifically, the structure and functional activity of a monoclonal IgG1 antibody produced at the outer limits of numerical ranges of fed-batch culture control parameters such as pH and temperature were examined, with the aim of providing assurance that antibody produced under varying culture conditions was of consistent quality based on a carefully defined set of specifications. An experimental design was created using a half-fractional factorial design for fed-batch culture incorporating half of the thirty two possible combinations of five selected control parameters at high and low levels. Statistical analysis of all data gathered from the study allowed an assessment of the effects of the process control parameters at either high or low outer limits on fed-batch culture response variables such as growth rate and specific antibody productivity. Measured values for the responses of growth rate and specific antibody productivity throughout this study ranged from 0.22-0.44 d(-1) and 6.4-32 microg monoclonal antibody/10(6) cells/d respectively. Analytical characterisation of monoclonal antibody purified from each fed-batch culture considered the purity, structure and biological activity of the glycoprotein. All antibody preparations were identical to each other and to the current antibody reference standard or control. Glycosylation analysis of certain samples from the study demonstrated that the distribution of glycoforms of the antibody was not affected by the varying process control conditions of the fed-batch cultures.

Amidohydrolases↗

PubMed2003

Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 2. Pheno- and genotypic criteria.

This paper reviews the methodology applied for the identification and characterisation of enterococci and covers phenotypic, genotypic and phylogenetic techniques. Although conventional phenotypic typing schemes are useful for rapid and simple identification of enterococcal species for routine applications, other methods like standardised sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), multilocus enzyme electrophoresis (MLEE), antimicrobial susceptibility testing, serotyping, pyrolysis mass spectrometry (pyMS) and vibrational spectroscopic methods allow a more in-depth characterisation of enterococci. Many of the recently described enterococcal species exhibit deviations from hitherto so-called classical enterococci with regard to their phenotypical properties. Therefore, genotypic methods have to be used to clarify their possible assignment to the genus Enterococcus. In this review, special emphasis is given on recently developed polymerase chain reaction (PCR)-based typing methods such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), specific and random amplification (SARA) and modifications of PCR-ribotyping as well as pulsed-field gel electrophoresis (PFGE) and partial sequence analysis. The use of PCR and probes for genus and species identification of enterococci is also considered like the application of sequence data of conserved DNA regions (e.g., ribosomal ribonucleic acid (rRNA) genes) in the case of species identification.

Bacterial Typing Techniques↗