Characterization of nosocomial Serratia marcescens isolates: comparison of Fourier-transform infrared spectroscopy with pulsed-field gel electrophoresis of genomic DNA fragments and multilocus enzyme electrophoresis.

A total of 66 Serratia marcescens isolates from 46 patients was investigated by macrorestriction using XbaI followed by pulsed-field gel electrophoresis. 7 restriction fragment patterns attributable to more than one patient and 9 individual patterns were identified. The isolates were additionally characterized by multilocus enzyme electrophoresis and Fourier-transform infrared spectroscopy. The macrorestriction patterns and the multilocus enzyme electrophoresis patterns corresponded fairly well while the classifications derived from these methods were not completely congruent. The grouping achieved by Fourier-transform infrared spectroscopy on the basis of high (> 1000) and moderately high heterogeneity values (300) was consistent with the macrorestriction results. Grouping on a lower heterogeneity level did not contribute to further discrimination. In general, Fourier-transform infrared spectroscopy was less discriminatory than the two other methods, but easier to perform. Therefore, laboratories equipped with the necessary devices may use it to rapidly select bacterial isolates for macrorestriction or other well established characterization procedures.

Characterization of urinary Escherichia coli O75 strains.

Forty-four Escherichia coli O75 strains from patients with urinary tract infections were characterized by a variety of methods to obtain evidence of their clonal distribution and uropathogenic properties. By K and H antigen typing, the strains were divided into the following serotypes: O75:K5:H- (18 strains), O75:K95:H- (10 strains), O75:K95:H5 (7 strains), O75:K100:H5 (4 strains), and O75:K-:H55 (5 strains). Generally, biotyping proved to be of no discriminative value. With two exceptions the strains were found to be sensitive to the bactericidal effect of normal human serum. As shown by multilocus enzyme electrophoresis, the whole-cell protein profile (WCPP), and the patterns of the outer membrane proteins and lipopolysaccharides, all but the five O75:H55 strains were genetically closely related to each other and could be classified into one clonal group. The O75:K-:H55 strains proved to be quite different and lacked type 1 fimbriae. All 17 K95 (H-, H5) strains produced hemolysin and P fimbriae. Five of the O75:K5:H- strains were different from the other K5 strains by showing hemagglutinating properties, on the basis of the presence of the OX adhesin. The last two groups are suggested to be uropathogenic and are proposed to represent separate clonal groups or subgroups.

NMR investigation of the 6-deoxy-L-talose-containing O45, O45-related (O45rel), and O66 polysaccharides of Escherichia coli.

The structures of the 6-deoxytalose-containing O-specific polysaccharides from the O45 antigen, an O45-related antigen (O45rel), and the O66 antigen (lipopolysaccharides, LPSs) of Escherichia coli were elucidated by chemical characterization and by one- and two-dimensional 1H and 13C NMR spectroscopy. The O45 and O45-related polysaccharides have the following general structure: [formula: see text] For the O45 antigen, X is alpha-D-FucpNAc and for the O45-related antigen, X is beta-D-GlcpNAc. The structure of the O66 polysaccharide is [formula: see text]

Genomic and biochemical relatedness between Vibrio cholerae serovar O139 and serovar O1 eltor strains.

Vibrio cholerae O139 (Bengal) the new pandemic cholera strain emerging on the Indian subcontinent has revealed considerable homology to Vibrio cholerae O1 EL Tor (strain of the seventh pandemic cholera) in terms of genetic and biochemical properties. Apart from capsule and O139 LPS formation, all strains of V. cholerae O139 were found to be identical to V. cholerae O1 EL Tor strains with respect to genomic restriction fragment length polymorphism, genomic distribution of the pathogenic island, pattern of OMP and multilocus enzymes. However, the analysis of a nonpathogenic V. cholerae O139 isolate from Sri Lanka with a totally different pattern of genetic properties underline that horizontal gene transfer of a piece of DNA encoding biosynthesis of the Vibrio cholerae O139-specific LPS and capsule formation to an O1 El Tor precursor strains must have occurred giving rise to a kind of hybrid V. cholerae O1 El Tor encoding the new serovar-specific O139 antigens.

Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community.

The occurrence and the further spread of high-level glycopeptide-resistant, vanA-positive Enterococcus faecium strains outside of hospitals have been investigated. We could isolate such bacteria directly from thawing liquids of commercially produced frozen poultry (chickens, turkeys; no further data on previous feeding with avoparcin were available). In 5 of 13 samples of raw minced meat of pigs originating from 13 different butcher's shops, glycopeptide-resistant E. faecium (VanA type) could be detected after overnight broth cultivation of these samples. No glycopeptide-resistant enterococci could be isolated from meat samples of chickens that were fed without avoparcin. VanA type E. faecium strains were also identified in 12 fecal samples recovered from 100 nonhospitalized humans in the rural area of Saxony-Anhalt federal county. These results suggest a possible role of the food chain in the spread of glycopeptide-resistant E. faecium. Molecular typing (macrorestriction and multilocus enzyme analysis) reveal a wide dissemination of the vanA gene among strains of different ecological origins.

Comparative classification of Acinetobacter baumannii strains using seven different typing methods.

A group of 49 Acinetobacter baumannii strains obtained from several hospital outbreaks and some sporadic cases were typed by biotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), plasmid typing, multilocus enzyme electrophoresis, whole-cell protein profile, and Fourier-transform infrared (FT-IR) spectroscopy. All these methods have shown a high degree of reproducibility and are capable of recognising strains from the same epidemiological event. However, their power to discriminate between epidemiologically unrelated strains varies, with PFGE being superior to the other methods investigated. FT-IR spectroscopy, which has not yet been used for typing of Acinetobacter strains, proved to be a very rapid and highly reproducible method, but was somewhat limited in its discriminating power.

Application of physico-chemical typing methods for the epidemiological analysis of Salmonella enteritidis strains of phage type 25/17.

Eighty-nine Salmonella enteritidis phage type 25/17 strains isolated from a localized outbreak in the German state Nordrhein-Westfalen (outbreak NWI) could not be further differentiated by biochemotyping and plasmid pattern analysis. They were submitted to a complex typing system consisting of modern physico-chemical analytical procedures. In lipopolysaccharide pattern analysis the strains proved to be homogeneous. In multilocus enzyme electrophoresis, outer membrane and whole cell protein pattern (WCPP) analysis, and Fourier-transform infrared (FT-IR) spectroscopy (increasing extent of differentiation in the given order) strains deviating from each basal pattern were found. The extent of correspondence in these deviations was satisfactory. Forty-six strains of the same sero- and phage type, however, obtained from different outbreaks, were additionally typed. The results obtained with them indicate that the data of the first group were not restricted to strains from outbreak NWI, but of general validity. It was found that both WCPP and FT-IR represent valuable methods for the sub-grouping of bacteria.

Nourseothricin (streptothricin) inactivated by a plasmid pIE636 encoded acetyl transferase of Escherichia coli: location of the acetyl group.

Escherichia coli strains harbouring the plasmid pIE636 are able to synthesize acetylcoenzyme A: streptothricin acetyltransferase (ACSAT). The (enzymatic) N-acetylation of streptothricin F is known to contribute significantly towards the loss of antibacterial activity. 13C-NMR analysis of [14C]N-acetyl-labelled streptothricin F, produced by ACSAT-catalysed acetylation of streptothricin F and subsequent purification by various chromatographical steps, unequivocally revealed streptothricin F to be acetylated at the beta-amino group (C16) (and not at the epsilon-amino group (C19)).

Characterization of a lipopolysaccharide encoded by a recombinant Shigella sonnei plasmid in Escherichia coli K-12.

The genetic information to synthesize the S-specific region of Shigella sonnei phase I lipopolysaccharide (LPS) is localized on a 180 kb plasmid which is lost quite readily. A recombinant plasmid derivative remaining stable in the bacteria was shown to determine the S-specific region of the LPS which is completely identical with that of a S. sonnei phase I strain following transfer in Escherichia coli K-12. However, the length control in polysaccharide biosynthesis is lost at least partially.

Resistance of Escherichia coli to nourseothricin (streptothricin): sensitization of resistant strains by abolition of its outer membrane resistance.

The polycationic antibiotic, nourseothricin, represents a mixture of several streptothricins, mainly D and F. The molecular weight of the latter compound amounts to 486. Obviously, although very slowly, it can pass the outer membrane via the porin pores. It has been shown earlier that nourseothricin is able to generate some kind of channels into the outer membrane through which it can pass the cell wall. On the other hand, there were indications that resistant strains containing a streptothricin-inactivating acetyl transferase possess an additional protecting system, namely a reduced penetrability of the outer membrane. In this study, it could be shown that such strains indeed could be rendered sensitive by damaging the barrier function of the outer membrane.

[Determining the pattern of outer membrane proteins of gram-negative bacteria as a contribution to complex typing]. / Die Bestimmung des Musters der Proteine der äusseren Membran gramnegativer Bakterien als Beitrag zur komplexen Typisierung.

The aim of these investigations was to study relations between the serotype of E. coli strains and the pattern of their outer membrane proteins ("OMP") in sodium dodecylsulfate polyacrylamide gel electrophoresis. Three groups of strains being well characterized at least serologically (01, 02, 018ac containing different K, H, and in part F antigens) were submitted to this analysis. In all cases a nearly complete paralellity between OMP pattern and O:K:H(F:) type was observed, provided that the strains were epidemiologically related. The possibility is discussed that the OMP type could be used as a guide marker for the complex typing of E. coli strains.

Biochemical aspects of the resistance to nourseothricin (streptothricin) of Escherichia coli strains.

In most cases Escherichia coli strains phenotypically resistant against nourseothricin (streptothricin) harbour a plasmid which codes for an acetyltransferase. This enzyme transfers an acetyl group from acetyl-coenzyme A to an amino group of the beta-lysine (peptide) chain of the antibiotic, thus inactivating it. Additionally, the penetrability for nourseothricin of the cell wall is drastically reduced in a high percentage of the resistant strains. Both resistance mechanisms seem to be independent of each other.

Resistance of Escherichia coli to nourseothricin (streptothricin): reduced penetrability of the cell wall as an additional, possibly unspecific mechanism.

The resistance of E. coli strains to the antibiotic nourseothricin is known to be caused by an acetyltransferase acetylating the beta-lysine chain of the antibiotic. In addition, most of the resistant strains exhibit reduced penetrability of the outer membrane, presumably caused by a reduced amount of available negative charges. This was shown using crystal violet, Congo red, or the hydrophobic antibiotic novobiocin as indicators.

Studies on clonal assignment of urinary Escherichia coli O1:K1 strains.

Fifty-nine Escherichia coli strains belonging to two clonal groupings were investigated for major outer membrane proteins, colicin production, and partly for plasmid DNA content. The membrane protein patterns of the 01:K1:H7(H-):F11 and O1:K1:H-:F9 strains obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinctly different from each other and, therefore, are useful for clonal assignment. All of the F11 isolates had one plasmid of about 85 Md in common which is suggested to be characteristic for the clone. Their content of smaller plasmid DNA was heterogeneous and showed geographical differences. The F9 strains showed a plasmid pattern different from the F11 strains. A plasmid of 2.6 Md was found in all of these isolates. Colicin production was found to be useful for clonal assignment only in two thirds of the F9 and not in the F11 strains. Some strains with identical properties seem to be of epidemic importance.

Nourseothricin (streptothricin) inactivated by a plasmid pIE636 encoded acetyl transferase: nature of the inactivated nourseothricin.

Nourseothricin, a mixture of several streptothricins, is inactivated by an acetyl transferase produced by Escherichia coli containing the plasmid pIE636. Nourseothricin inactivated in the presence of 14C-acetate was purified and submitted to partial hydrolysis. In the hydrolysate besides others a radioactive and ninhydrin-reactive substance moving only slightly towards the cathode was found. It proved to be [14C]-acetyl beta-lysine.

Effect of nourseothricin (streptothricin) on the outer membrane of sensitive and resistant Escherichia coli strains.

Nourseothricin (streptothricin) causes disturbances (perforations) in the outer membrane of sensitive E. coli strains allowing lysozyme and deoxycholate, but not the periplasmic alkaline phosphatase to penetrate. EDTA slightly increases, but Mg++ ions slightly decrease this effect. The cell walls of three from four nourseothricin-resistant strains do not become permeable under these conditions, but remain sensitive against TRIS/EDTA. Nourseothricin is supposed to pass the outer membrane of sensitive bacteria via some kind of "self-promoting" pathway. This way can (but need not) be blocked in resistant strains.

Surface hydrophobicity of plasmid-carrying virulent Shigella flexneri and their avirulent variants.

Five pairs of strains of S. flexneri each differing in the colour of their colonies after growth on Congo red agar have been tested for their ability to cause keratoconjunctivitis in the guinea pig eye, for the presence of the 140 Md virulence plasmid, for the presence of the virulence marker antigen, and for their ability to adsorb to hydrophobic surfaces (cellulose nitrate filters and Phenyl Sepharose). The results suggest that the presence of the 140 Md virulence plasmid provides the bacterial surface with a rather high degree of hydrophobicity; exceptions have been found.

Monosaccharide composition of lipopolysaccharides from Campylobacter jejuni and Campylobacter coli.

The monosaccharide composition of the LPS from 5 Campylobacter jejuni strains and 7 Campylobacter coli strains has been studied. All LPS's contained KDO, heptose, glucosamine, glucose, and (with one exception) galactose. All C. jejuni and 3 C. coli LPS's contained greater than 1% galactosamine. 3-Amino-3.6-dideoxyglucose was present in all but one C. coli LPS and in only one C. jejuni LPS.