A method for typing Listeria monocytogenes strains by classification of listeriocins and phage receptors.

A method for typing Listeria monocytogenes (L.m.) strains was developed which is based on two different approaches. First, strains were classified according to the phage receptors specific to their mitomycin-induced prophages. The frequency of lysogenic strains ranged between 63 and 80%, depending on the serotype. For non-lysogenic strains, a reversed phage-receptor analysis was applied based on the susceptibility of the strains to be typed against bacteriophages from selected, inducible indicator strains. As a second method, typing of mitomycin-induced listeriocins was shown to be practicable as well. By this approach, it was possible to distinguish 11 different listeriocins when using a set of 7 selected indicator strains. Only two of these listeriocins were probably produced by L.m. strains of serotype 4b. A combination of the two approaches described raises the number of types. When applied to 20 human isolates of L.m. belonging to serotype 4b, 17 different types were demonstrated. 46 L.m. strains of serotype 1/2b could be differentiated into 38 types of which 18 clinical isolates represented 17 different types. 26 L.m. strains of serotype 1/2a isolated from various food products showed a large heterogeneity, with 22 different types. The power of discrimination of the method outlined recommends its application in epidemiological investigations.

[Significance of plasmid virulence factors for transmission of nosocomial infection]. / Die Bedeutung plasmidischer Virulenzfaktoren für die Ausbreitung nosokomialer infektionen.

The genetical basis of germ change especially for the gain of the resistance, and virulence plasmids have been treated not only with regard to clinical consequences but also have been classified in their evolutionary importance. Mechanisms of virulence have been illustrated on example of origin of siderophors, the increase of virulence through cytotoxical damage, on the obtained ability of adhesion and colonisation as well as by enhancement of equipment with enzyms. The patient with reduced resistance has been put out as selection condition.

Haemolytic activity and characteristics of plasmid and chromosomally borne hly genes isolated from E. coli of different origin.

20 hly plasmids of different origin and source and 8 recombinant plasmids containing either chromosomally or plasmid encoded hly determinants were characterized with emphasis on their haemolytic activity. Remarkable differences among the hly genes tested were found in their haemolytic activity and in the regulation of the haemolytic activity by the extracellular iron concentration. Under iron-limiting growth conditions the haemolysin secretion encoded by 6 hly plasmids, but not by the remaining 14 hly plasmids and the chromosomal hly genes, was significantly induced. The 6 plasmids all derived from human isolates, belonged to the incompatibility groups FIV and FVI and showed similar restriction patterns. Two hly determinants, encoding an inducible haemolysin secretion, cloned from plasmids isolated in Berne and Paris, respectively, showed identical restriction maps, indicating a common origin. Compared to the hly determinant of a plasmid encoding a noninducible haemolysin secretion, distinct differences, mainly in the flanking regions, were observed.

The chromosomal fur gene regulates the extracellular haemolytic activity encoded by certain hly plasmids.

The haemolytic activity encoded by thirteen hly-plasmids of different origin and sources was examined as a function of the Fe3+-concentration in E. coli fur+ and E. coli fur- strains, respectively. In E. coli fur+ the relatively low haemolytic activity of five hly-plasmids isolated in Berne and one isolated in Paris was increased significantly under iron-limiting growth conditions. Contrastingly, in E. coli fur- strains containing the same plasmids, a considerably higher amount of secreted haemolysin was detected. This activity could not be further increased by limiting the extracellular iron concentration. Seven other hly-plasmids expressed similar and non-inducible amounts of secreted haemolysin in both E. coli fur+ and E. coli fur- strains. These results indicate that the extracellular haemolytic activity encoded by certain hly-plasmids was controlled by the chromosomally encoded fur gene.

An approach of the in vivo antibacterial activity of benzoxonium chloride and comparison with other buccopharyngeal disinfectants.

Different pharmaceutical forms of N-benzyl,N-dodecyl-N,N-di(2-hydroxyethyl) ammonium chloride (benzoxonium chloride, Orofar) have been tested on the overall buccopharyngeal bacterial count in healthy volunteers, and compared to competitors. Gargle solutions are most effective in reducing initial bacterial count. An open repeated dose study has been performed on 38 patients with sore throat to assess the safety and the efficacy of benzoxonium chloride tablets and gelsolets at doses of 6 mg and 10 mg daily for 4 to 6 days. Pathogens disappeared or were clearly reduced and the syndrome resolved. Tolerance was good. Differences in methodologies are discussed.

Occurrence and properties of hly-plasmids in E. coli from patients of the Berne area.

1818 E. coli wild-type strains from patients of the region of Berne were screened for their haemolytic property and for carriage of hly-plasmids. Among 885 strains of fecal and 993 strains of urinary tract isolates 136 (15%) and 259 (28%) were haemolytic respectively. In five fecal, but none of the urinary strains the haemolytic activity was encoded by transmissible plasmids. In spite of independent origin, the five isolated hly-plasmids proved to be very similar. They all showed a high transfer-frequency according to their derepressed state and coded for F-sexpili. Four belonged to the rare F VI- and one to the F IV-incompatibility group. Restriction patterns after Eco R1- and Hind III-digestion were identical in three and similar in the other two plasmids. Comparing the plasmids from Berne with hly-plasmids isolated in Essex, similarities as well as specific differences were detected.

The development of chromosomally coded beta-lactamase production by Escherichia coli after in vitro selection with ampicillin, cefoxitin and ceftriaxone.

Subclones of Escherichia coli K12 921 which produced a chromosomal beta-lactamase were naturally selected in vitro with ampicillin, cefoxitin and ceftriaxone. The selected subclones were identical with respect to beta-lactamase production, resistance spectrum and resistance level, regardless of the selection agent. A ceftriaxone selection following an ampicillin selection led to higher ceftriaxone resistance. The beta-lactamase which was formed hydrolysed ceftriaxone to a measurable extent. After incubation of ceftriaxone with the beta-lactamase, the hydrolysed degradation product was demonstrated by thin-layer chromatography.

Relation between the hemolytic property and iron metabolism in Escherichia coli.

The hemolytic activity of wild-type strains of Escherichia coli was measured by a standardized method in liquid broth. The system also allowed us to investigate the influence of various Fe3+ concentrations in the cultures on the amount of secreted hemolysin. We found that the hemolysin secretion of all strains was clearly reduced after addition of FeCl3. However, the influence of additional iron chelators showed remarkable differences. The hemolytic activity of Hly plasmid-containing strains isolated in Berne significantly increased. Most of the strains with a chromosomal hemolysin determinant showed a similar effect, but to a lesser degree. Contrary to this, Hly plasmid-containing strains isolated in Essex and some strains with chromosomal hemolysin determinant were either not affected or even showed reduced hemolysin secretion after limitation of free iron ions in the broth. Our results suggest that hemolysin secretion in E. coli is related to the bacterial iron metabolism, and hemolysin secretion is differentially regulated among E. coli strains.

[Selection of R plasmid-carrying E. coli pathogens in a mixed population by subinhibitory antibiotic concentrations]. / Selektion R-Plasmid-tragender E. coli-Keime in einer Mischpopulation durch subinhibitorische Antibiotika-Konzentrationen.

The effect of subinhibitory concentrations of tetracyclin, chloramphenicol, gentamicin and ampicillin in a mixed culture of E. coli was investigated in a chemostat in order to determine minimal selection concentration of these drugs. The mixed culture consisted of three clones of E. coli K12 921. One of these clones was plasmid free, one harboured the F'lac plasmid and the third one an R-factor. The antibiotics tetracyclin, chloramphenicol and gentamicin selected the antibiotic resistant R-factor bearing strain at a concentration of 1/10 of the minimal inhibition concentration. Subinhibitory concentrations of ampicillin failed to select the R-factor bearing strain because this strain produced a secreted betalactamase.

Thoughts of the medical microbiologist on the use of antimicrobial drugs in animals.

The restriction in feed-antibiotics as carried out in our country so far did not lead to the expected decrease of R-factors in the faecal flora of the animals staying in stable. Possible explanations on this subject are given in this report. The question arises whether the best solution to the problem would not be a complex separation of antibiotics used in human medicine from those used in veterinary medicine, as already discussed in the Swann report. A further spreading of plasmids and transposons should be stopped because their influence on the microcosmos is not presumable for the future, especially because R-factors get more and more genes for rising the virulence of their host bacteria.

Enzymatic modification of aminoglycoside antibiotics in gentamicin-resistant gram-negative bacteria.

An in vitro evaluation of 32 gentamicin-resistant, Gram-negative bacteria from hygiene-centres in Berne, Switzerland and Bratislava, Czechoslovakia, revealed that 24 strains produced gentamicin-modifying enzymes. Occurrence of acetyltransferases (AAC) was confirmed in 16 strains, adenylyltransferase (AAD) /2''/ was observed in 13 strains while 5 of the gentamicin-resistant strains produced both enzymes. All Czechoslovak strains were amikacin-susceptible, in amikacin-resistant Pseudomonas aeruginosa strains from Switzerland the presence of AAC/6'/ was found. In the majority of gentamicin-resistant, netilmicin-susceptible strains the occurrence of AAD/2''/ was observed. Gentamicin-resistance in the modifying enzymes producing strains was due either to production of an acetyltransferase or the adenylyltransferase except Swiss strains of Serratia marcescens where a simultaneous production of both types of the enzymes was noted. Twenty eight strains produced an enzyme modifying aminoglycosides of any kind.

R-selection of subbacteriostatic tetracyclin-concentrations.

We investigated the smallest R-factor selecting tetracyclin concentration in a chemostat. All experiments were performed with a miscellaneous culture of three clones of the E. coli K12 921 strain. The first clone was plasmid-free, the second one harboured the R270 (Tc, Su) Inc F II rep and the third one the F'lac-plasmid Inc F I derep. The influence of different tetracyclin concentrations on the development of the viable count of these three clones was investigated in batch and in steady state culture phase. The steady state was kept for about three hours. It served to enforce the selection effect of the antibiotic. A selection effect was already stated with 0.25 microgram Tc/ml. That is to say with 1/10 of the MIC (2.5 micrograms Tc/ml) of the clones used. The importance of these results will be discussed.

[Comparative analysis of tetracycline, doxycycline and minocycline on chromosomal and plasmid tetracycline-resistant and -sensitive strains of Staphylococcus aureus using MIC, microcalorimetric effect and ultrastructural alterations]. / Vergleichende Untersuchung von Tetracyclin, Doxycyclin und Minocyclin bei chromosomal bzw. plasmidisch tetracyclinresistenten und -empfindlichen Staphylococcus-aureus-Keimen auf MHK, mikrokalorimetrischen Effekt und ultrastrukturelle Veränderungen.

A staphylococcus aureus strain of a patient with plasmidical tetracyclinresistance has first been cured from the plasmid and after that selected in nutritive broth by increasing the concentrations of tetracyclin. By this way we found three variants of the same strain: a tetracyclin-sensitive and two tetracyclin-resistant: an R-plasmid-mediated and the other as a result of mutation. These three variants have been tested against tetracyclin, minocyclin and doxycyclin on an agarmedium. Under influence of these three substances a microcalorimetrical investigation of the strains has taken place. We then tested the strains with the electronic microscope for alterations in the ultra-structure by the three substances. Following results have been obtained: 1. The sensitive strains were inhibited in the same extent by all of the three substances. Plasmidical resistant strains were only resistant against tetracyclin but not against minocyclin and even less against doxycyclin. 2. The microcalorimetrical tests confirmed the conclusions from the cultural test. 3. Alterations in ultra-structure were only found under tetracyclin-influence by strains in division. Comparing the three tetracyclines we did not find any significant difference in quality. 4. With all of the three investigation-methods we proved that R-plasmid-mediated tetracyclin-resistance of staphylococci is not effective for minocyclin and even less for doxycyclin.

[Classification of R-factors. Possible errors and their elimination]. / Klassifizierung von R-Faktoren. Fehlermöglichkeiten und deren Beseitigung.

The necessity for classifying plasmids is emphasized. From our results the uniformity of a nosocomial infectious outbreak is only given by the R-factor and not by the infectious agent. Determination of the appropriate pilus groups by sexphages using the HFRT-system and identification of incompatibility as a further means of subdividing the pilus groups into incompatibility groups is described. A distinction is made between incoming- and resident incompatibility. To eliminate possible errors in plasmid classification the following points are considered: 1. Testing in recombination deficient rec A- -strains of E. coli K12 to avoid any recombination of the plasmids. 2. Trypsin addition to remove the possibility of plasmid coded colicin production. 3. A proper choice of the indicator plasmids. 4. Consideration of the spontaneous loss of the indicator plasmid and also an occurrence of transposons coding for an additional pilus type.

Quality of breast milk: its control and preservation.

The numerous problems of bacterial contamination and proper preservation of breast milk have been examined. Both the many advantages of human milk as the optimal nourishment for the newborn baby and the extensive experiences of veterinary medicine in the handling of milk have been carefully considered. Bacterial multiplication has been studied under various storage conditions. Out of several possible procedures a simple, inexpensive, rapid and reliable dip-slide screening method for determining total bacterial colony counts is described. A clear-cut procedure for the optimal collection of breast milk is presented.

Constant infusions vs. intermittent doses of gentamicin against Pseudomonas aeruginosa in vitro.

Comparative studies were performed in vitro to test the advocated superiority of infusion over intermittent injection of aminoglycosides. Pseudomonas aeruginosa was exposed to constant and to continuously decreasing (simulating in vivo kinetics) concentrations of gentamicin. In comparing the effect with similar area-under-the-concentration-vs.-time curves, a substantial difference in killing and regrowth could not be demonstrated. Regrowth occurred only when the gentamicin concentration had continuously decreased below one fourth of the minimal inhibitory concentration for greater than 2 hr. Exposure of P. aeruginosa to gentamicin for 30 min was followed by persistent suppression of bacterial regrowth for 1.4-1.9 hr. Thus, intermittent exposure of P. aeruginosa to gentamicin is as effective as constant exposure in vitro. The demonstrated persistent postantibiotic effect might cover in part the periods between intermittent doses of gentamicin in vivo as well as in vitro.