A trilocus sequence typing scheme for hospital epidemiology and subspecies differentiation of an important nosocomial pathogen, Enterococcus faecalis.

In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes, ace and salA (encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance, lsa (encoding a putative ABC transporter), for subspecies differentiation of Enterococcus faecalis. Each of the alleles was analyzed using 50 E. faecalis isolates representing 42 diverse multilocus sequence types (ST(M); based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis [PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.

Further characterization of the epa gene cluster and Epa polysaccharides of Enterococcus faecalis.

We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model.

Mechanism for sortase localization and the role of sortase localization in efficient pilus assembly in Enterococcus faecalis.

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate.

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

Time-kill and synergism studies of ceftobiprole against Enterococcus faecalis, including beta-lactamase-producing and vancomycin-resistant isolates.

Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of Enterococcus faecalis (including 16 beta-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10(7) CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two beta-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 microg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of

Characterization of the beta-lactamase gene from {\it Enterococcus faecalis\/} strain HH22

The plasmid-encoded, constitutively produced $\beta$-lactamase gene from Enterococcus faecalis strain HH22 was genetically characterized. A restriction endonuclease map of the 5.1 kb EcoRI fragment encoding the enterococcal $\beta$-lactamase was prepared and compared with the restriction map of a cloned staphylococcal $\beta$-lactamase gene (from the naturally-occurring staphylococcal $\beta$-lactamase plasmid pI258). Comparison and hybridization studies showed that there were identical restriction sites in the region of the $\beta$-lactamase structural gene but not in the region surrounding this gene. Also the enterococcal $\beta$-lactamase plasmid did not encode resistance to mercury or cadmium which is encoded by the small, transducible staphylococcal $\beta$-lactamase plasmids. The nucleotide sequence of the enterococcal gene was shown to be identical to the published sequences of three of four staphylococcal type A $\beta$-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred-forty nucleotides upstream of the $\beta$-lactamase start codon were also determined for the inducible staphylococcal $\beta$-lactamase gene on pI258; this sequence was identical to that of the constitutively expressed enterococcal gene indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The gene for the enterococcal $\beta$-lactamase and an inducible staphylococcal $\beta$-lactamase were each cloned into a shuttle vector and then transformed into enterococcal and staphylococcal recipients. The major difference between the two host backgrounds was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene but no qualitative difference was seen between the two genera. Also a difference in the level of resistance to ampicillin was seen between the two backgrounds with the cloned enzymes by MIC and time-kill studies. The location of the enzyme was found to be host dependent since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell bound enzyme in the enterococcus. Based on the identity of the enterococcal $\beta$-lactamase and several staphylococcal $\beta$-lactamases, these data suggest recent spread of $\beta$-lactamase to enterococci and also suggest loss of a functional repressor. ^

Identification of antigen-encoding genes of Enterococcus faecalis during human infections and characterization of a gene cluster for the biosynthesis of an antigenic polysaccharide

Genomic libraries of two Enterococcus faecalis strains, OG1RF and TX52 (an isolate from an endocarditis patient), were constructed in Escherichia coli and were screened with serum from a rabbit immunized with surface proteins of an E. faecalis endocarditis isolate and sera from four patients with enterococcal endocarditis. Thirty-eight immunopositive cosmid clones reacted with at least two of the patient sera and contained distinct inserts based on their DNA restriction patterns. These were chosen for further subcloning in a pBluescript SK ($-$) vector. Each sublibrary was screened with one of the five sera. Analysis of sequences from the immunopositive subclones revealed similarities to a range of proteins, including bacterial virulence factors, transporters, two-component regulators, metabolic enzymes, and membrane or cell surface proteins. Fourteen subclones did not show significant similarity to any sequence in the databases and may contain novel genes. Thirteen of the immunopositive cosmid clones did not yield immunopositive subclones and one such cosmid clone, TX5159, produced an antigenic polysaccharide in Escherichia coli. The insert of TX5159 was found to contain a multicistronic gene cluster containing genes similar to those involved in the biosynthesis and export of polysaccharides from both Gram-positive and Gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of TX5159. RT-PCR of genes within the cluster with total RNA from OG1RF showed that these genes are transcribed. The polysaccharide was detected in two recently reported E. faecalis mucoid strains using specific antibody, but not in the other strains tested. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide. ^

Conjugal Transfer of the Tn916-like Transposon TnFO1 from Enterococcus faecalis Isolated from Cheese to Other Gram-positive Bacteria

A Tn916-like transposon (TnFO1) was found in the multiple antibiotic resistant Enterococcus faecalis strain FO1 isolated from a raw milk cheese. In this strain, the tetracycline determinant was localized by DNA-DNA hybridization with a tetM nucleotide probe on the chromosome and on a 30-kb plasmid. The transposon TnFO1 was identified and characterized by DNA-DNA hybridization experiments with the five internal HincII fragments of Tn916. The tetracycline resistance determinant was identified by its complete nucleotide sequence as TetM. Transposon TnFO1 was also detected in its circular form by DNA-DNA hybridization and PCR amplification. Both ends including the joining region of the closed circular transposon TnFO1 were sequenced. TnFO1 could be transferred by conjugation from Enterococcus faecalis into Enterococcus faecalis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Listeria innocua, Leuconostoc mesenteroides and Staphylococcus aureus, and from Lactococcus lactis subsp. lactis biovar. diacetylactis into Listeria innocua. Pulsed-field electrophoresis of genomic DNA from E. faecalis FO1 transconjugants showed that transposon TnFO1 integrated at different sites.

Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Na 1 Nachlass Max Horkheimer, 11 - Korrespondenzen u.a. mit Edward M. David (p. I 6, 1-183)

1 Brief von Fritz Dannenbaum an Max Horkheimer, 26.11.1935; 2 Briefe zwischen F. Darnbacher und Max Horkheimer, 24.01.1936, 22.01.1936; 3 Briefe zwischen dem Dartmouth College Hanover N.H. und Max Horkheimer, 1939-1941. 06.05.1939; 14 Briefe zwischen Edward M. David und Max Horkheimer, 1941-1942; 1 Brief von der Day Cornell University, Ithaca N.Y. an Max Horkheimer, 24.02.1939; 2 Briefe zwischen Margaret Deaner und Max Horkheimer, 01.04.1935, 04.04.1935; 1 Brief von Max Horkheimer an Hanna Deinhard, 05.01.1949; 1 Brief von Deiters an Max Horkheimer, 26.11.1935; 2 Briefe zwischen Laura Demick und Max Horkheimer, 22.12.1941, 29.12.1941; 2 Briefe zwischen Grace Dertz und Max Horkheimer, 18.10.1934, 11.10.1934; 1 Brief von Max Horkheimer an Dekan, 20.06.1940; 1 Brief von Max Dessoir an Max Horkheimer, 29.08.1937; 1 Brief von Max Horkheimer an Deutschland / deutsches Reich Minister für Wissenschaft, Kunst und Volksbildung, Berli, 21.04.1933; 4 Briefe zwischen John Dewey und Max Horkheimer, 1940-1941, 20.02.1941; 1 Brief von Max Horkheimer an Walter Strauss, 20.02.1941; 3 Briefe zwischen Frederic Dewhurst und Max Horkheimer, 1939, 08.02.1939; 5 Briefe zwischen dem Soziograph Jul Diederich und Max Horkheimer, 1934-1938; 1 Brief von Max Horkheimer an Hugo Sinzheimer, 20.10.1934; 15 Briefe zwischen William Dieterle, Charlotte Dieterle und Max Horkheimer, 1940-1944; 1 Brief von der Dillmann-Oberschule Stuttgart an Max Horkheimer, 13.12.1949; 1 Brief vonMax Horkheimer an W. R. Dittmar, 10.02.1938; 2 Briefe zwischen Sofie Doernberg, Paul Doernberg und Margot von Mendelssohn, 1942, 06.04.1942; 4 Briefe zwischen Willy Dörter und Max Horkheimer, 1936-1937, 20.02.1936; 1 Brief von Georg Glaser anMax Horkheimer; 3 Briefe zwischen Hans Venedey und Max Horkheimer, 22.01.1938, 1938; 28 Briefe sowie Zeitungsausschnitte zwischen Juliette Favez und Max Horkheimer, 1934-1938; 1 Brief von Juliette Favez ann karl Dörter, 02.10.1934; 2 Briefe von Max Horkheimer an Hans Klaus Brill, Januar 1938; 3 Briefe zwischen Karl Dörter und Max Horkheimer, 1934-1937, 12.10.1934; 4 Briefe zwischen Andrés Sternheim und Max Horkheimer, 1937, 16.06.1937; 1 Brief von Else Klee an Max Horkheimer, 13.06.1935;