Aminoglycoside resistance in Gram-negative blood isolates from various hospitals in Belgium and the Grand Duchy of Luxembourg. Aminoglycoside Resistance Study Group.

A total of 1102 consecutive clinical blood isolates, including 897 Enterobacteriaceae and 205 non-fermenting bacilli, were obtained from 13 university and university-affiliated hospitals, which were divided into a Northern and a Southern group. Resistance to gentamicin, tobramycin, netilmicin, amikacin and isepamicin was determined using a microdilution technique according to NCCLS procedures. The overall mean resistance level was 5.9% for gentamicin, 7.7% for tobramycin, 7.5% for netilmicin, 2.8% for amikacin and 1.2% for isepamicin. Resistance to amikacin and isepamicin was significantly higher in the Northern hospitals than in the Southern hospitals. In total, 157 isolates were found not to be susceptible to aminoglycosides. By PCR, 179 aminoglycoside resistance mechanisms, i.e. 150 genes encoding modifying enzymes and 29 permeability mechanisms, were detected in 148 isolates. A resistance mechanism could not be detected in nine isolates. Moreover, in a further 14 isolates the resistance profile was not fully explained by the detected genes. The aac(6')-I genes were found to be the most predominant resistance mechanism in both the Northern and Southern isolates, followed by aac(3) genes and permeability resistance. A total of 29 non-susceptible isolates harboured a combination of genes, 72.4% of which were a combination with the aac(6')-lb gene. The majority of these combinations were broad-spectrum combinations which represented 9.0% of the resistance mechanisms in non-susceptible Enterobacteriaceae and 19.3% in the non-fermenting bacilli.

Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Im [corrected] associated with a sulI-type integron.

The amikacin resistance gene aac(6')-Im [corrected] from Citrobacter freundii Cf155 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 521 bp located down-stream from the 5' conserved segment of an integron. The sequence of this aac(6')-Im [corrected] gene corresponded to a protein of 173 amino acids which possessed 64.2% identity in a 165-amino-acid overlap with the aac(6')-Ia gene product (F.C. Tenover, D. Filpula, K.L. Phillips, and J. J. Plorde, J. Bacteriol. 170:471-473, 1988). By using PCR, the aac(6')-Im [corrected] gene could be detected in 8 of 86 gram-negative clinical isolates from two Belgian hospitals, including isolates of Citrobacter, Klebsiella spp., and Escherichia coli. PCR mapping of the aac(6')-Im [corrected] gene environment in these isolates indicated that the gene was located within a sulI-type integron; the insert region is 1,700 bases long and includes two genes cassettes, the second being ant (3")-Ib.

Changes in major populations of methicillin-resistant Staphylococcus aureus in Belgium.

A total of 102 epidemic methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 13 Belgian hospitals during two periods (1981-1985 and 1991-1992) were tested for phage-type, for the presence of aminoglycoside-modifying enzymes (AME), and examined by arbitrarily primed polymerase chain reaction (AP-PCR). All isolates, but five, belonged to a few distinct phage-types of group III. Most isolates expressed a combination of AAC(6')-APH(2") with APH(3')III, and ANT(4',4") or both. Both phage-typing and AME suggested a change in the MRSA population between the two periods but the AP-PCR method revealed only slight differences.

The role of lipopolysaccharide anionic binding sites in aminoglycoside uptake in Stenotrophomonas (Xanthomonas) maltophilia.

Aminoglycoside resistance was investigated in six clinical isolates of Stenotrophomonas (Xanthomonas) maltophilia by studying the uptake kinetics and by using a radiochemical method to detect aminoglycoside modifying enzymes. Furthermore, the lipopolysaccharides (LPS) were extracted and characterized by SDS-PAGE and chemical analysis. Dansyl-polymyxin displacement experiments confirmed the availability of anionic binding sites. Growing cells of the isolates bound dansyl-polymyxin but were not lysed.

Detection by polymerase chain reaction of genes encoding aminoglycoside-modifying enzymes in methicillin-resistant Staphylococcus aureus isolates of epidemic phage types. Belgian Study Group of Hospital Infections (GDEPIH/GOSPIZ).

The polymerase chain reaction (PCR) was used to identify the aacA-aphD, aphA3 and aadC genes, encoding the aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(3')III and ANT(4'4"), respectively, and the methicillin resistance determinant mecA, in epidemic aminoglycoside and methicillin-resistant isolates of Staphylococcus aureus. In total, 37 isolates collected in the period 1980-1985 and 81 isolates from the period 1991-1992 were obtained from 10 different Belgian hospitals. Epidemic isolates from the earlier period were characterised by phage type C (6/47/54/75) of phage group III, whereas two other epidemic phage types of group III-types A (77) and B (47/54/75/77/84/85)--were commonest in isolates from the second period. The bifunctional AAC(6')-APH(2") was the enzyme encountered most frequently. The prevalence of APH(3')III decreased significantly in the 1991-1992 period, while ANT(4',4") was found solely in isolates from this period. Resistance mechanisms were more complex in isolates from the 1991-1992 period and the mecA gene was detected in all isolates. The PCR results corresponded well with those obtained in the radiochemical phosphocellulose paper binding assay. Isolates from the 1991-1992 period were shown to express significantly higher levels of acetyltransferase activity than isolates from the 1980s.

Use of the polymerase chain reaction (PCR) for the detection of aacA genes encoding aminoglycoside-6'-N-acetyltransferases in reference strains and gram-negative clinical isolates from two Belgium hospitals.

Genes encoding aminoglycoside 6'-N-acetyltransferases, were identified using the polymerase chain reaction (PCR). Four sets of primers delineating DNA fragments of 209 bp, 250 bp, 260 bp and 347 bp, specific for the four known aacA genes, and probes within these fragments, were constructed based on the nucleotide sequences of the aacA genes. The specificity of the primers was evaluated using reference strains encoding various aminoglycoside-modifying enzymes. The primers reacted with their corresponding aacA genes and did not cross-react with genes coding for other aminoglycoside-modifying enzymes. One hundred and sixty-one aminoglycoside resistant clinical isolates showing AAC(6')I activity were tested using the PCR assays. The gene described by Tran Van Nhieu & Collatz (1987) was the most frequently identified aacA gene. One strain of Citrobacter freundii contained two distinct aacA genes. However, in 46% of the strains, the majority being Serratia spp. and Acinetobacter spp. none of the specific amplified DNA fragments for any of the known aacA genes could be detected.

Serotypes and extended spectrum beta-lactam resistance in aminoglycoside resistant Pseudomonas aeruginosa isolates from two Belgian general hospitals: a seven year study.

A total of 1896 isolates of Pseudomonas aeruginosa resistant to aminoglycosides and isolated during the period 1983-1989 in two Belgian general hospitals were included in this study. The most frequently encountered O serotypes were O4, O11, O12 and non-typable isolates. The majority of the isolates showed resistance to extended spectrum beta-lactam antibiotics (cefotaxime, ceftriaxone and cefepime). However, a low degree of resistance was found for ceftazidime. By contrast, amikacin and isepamicin, remained active on a significant number of aminoglycoside resistant isolates. In both hospitals, impermeability and AAC(3)II enzyme production were the most prevalent aminoglycoside resistance mechanisms. There were marked differences between the two hospitals with regard to the distribution of the O-serotypes and resistance profiles.

Identification of the aadB gene coding for the aminoglycoside-2"-O-nucleotidyltransferase, ANT(2"), by means of the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to identify the gene encoding the aminoglycoside-2"-O-nucleotidyltransferase, ANT(2"). Two primers, delineating a DNA fragment of 188 bp, and a specific probe within this fragment were constructed, based on the nucleotide sequence of the aadB gene encoding this enzyme. Reference strains producing different aminoglycoside-modifying enzymes were used to evaluate the specificity and the sensitivity of the test. Strains producing the ANT(2") enzyme showed the expected 188 bp DNA fragment after amplification. The oligonucleotide primers did not interact with genes encoding other aminoglycoside-modifying enzymes. Evaluation of a one-step single colony technique demonstrated that it was an acceptable alternative to the classic PCR test. The PCR method was used successfully to detect the presence of the aadB gene in 17 gentamicin-resistant clinical isolates.

Comparative in vitro activity of cefepime and four extended-spectrum beta-lactams on 1,251 aminoglycoside-resistant gram-negative hospital strains.

Cefepime was the most active compound on the Enterobacteriaceae with a MIC90 of 0.26 microgram/ml and a resistance rate of 0.1%. Ceftazidime was the most active drug on the non-fermenting bacilli (MIC90 9.65 micrograms/ml; resistance rate 3%). Amikacin- and gentamicin-resistant strains showed a decreased susceptibility to the beta-lactams, though the Enterobacteriaceae and the non-fermenters remained fairly sensitive to cefepime and ceftazidime, respectively. Aminoglycoside-3-N-acetyltransferase was the most prevalent enzyme and was often associated with intermediate resistance or resistance to beta-lactams. Non-fermenters showing aminoglycoside impermeability were very often intermediately resistant or resistant to beta-lactams.

Dual effects of pertussis toxin on in vitro invasive behavior of metastatic lymphoma variants.

A simple monolayer invasion assay (MIA) was recently developed using confluent fibroblastic cells as a target and variants of the BW5147 murine T-cell lymphoma as invading cells. Metastatic variants were consistently invasive in the MIA whereas non-metastatic cells were not. In this paper it is reported that pertussis toxin (PT) treatment of a highly metastatic and invasive variant caused a marked delay of invasion in the MIA at concentrations from 50 pg/ml upwards. Surprisingly, PT treatment of the non-metastatic, non-invasive parental BW5147 cells induced a moderate but significant level of invasion. Morphometric analysis showed that PT provoked an increased pseudopodal activity in cells in which it also caused increased invasive potential, and a decreased motility in cells with decreased invasiveness. This finding strengthens the perception that invasive potential and the capability to perform shape changes are related characteristics in these lymphoma cells.

Rapid purification of Bordetella pertussis toxin by alternating affinity and hydrophobic chromatography.

Pertussis toxin was purified to homogeneity from a 2-day culture supernatant of Bordetella pertussis by stepwise elution from three columns of, consecutively, Blue Sepharose, phenyl Sepharose, and hydroxyapatite. The toxin was eluted from Blue Sepharose and hydroxyapatite by high ionic strength and from phenyl Sepharose with low ionic strength and with 17% glycerol. Toxin fractions from one chromatographic column were immediately charged on the next column, saving laborious and time-consuming concentration or dialysis steps. Based on peptide composition (after sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and on HPLC profile (under nondenaturing conditions), the toxin was already practically pure after two steps, the third hydroxyapatite column serving only to separate the whole native toxin from any free S1 subunit. Recovery was estimated from the capacity of the preactivated toxin (and any preexisting free S1 subunit) to catalyze the ADP-ribosylation of the guanine nucleotide binding protein Ni in rat pancreatic plasma membranes: of the total capacity initially present in the culture medium, 23% could be recovered as pure native toxin with the present procedure. Besides, the nondenaturing HPLC method used to check the purity of the native toxin appeared to be superior to classical acidic polyacrylamide gel electrophoresis.

Specific non-immunoglobulin G antibodies and cell-mediated response to herpes simplex virus antigens in women with cervical carcinoma.

Non-immunoglobulin G-neutralizing antibodies to herpes simplex virus (HSV) type 2 were assayed in sera adsorbed with Staphylococcus aureus Cowan I. They were present in 8% of women with normal cervical smear and in 20, 41, and 74% of women with atypia, dysplasia, and cervical carcinoma, respectively. Lymphocytes of the patients were tested for in vitro transformation by killed HSV type 1 and HSV type 2 (HSV-2), as well as by mitomycin C-treated hamster cells transformed by HSV or other viruses or not transformed. Specific stimulation by the HSV-transformed cells occurred in 2, 22, and 40% of women with normal cervical smear, dysplasia, and carcinoma of the cervix, respectively. This frequency rose to 82% during treatment with irradiation and decreased to 0% after surgery. When HSV-2 virions were used as antigens to stimulate the lymphocytes, similar differences were found between the various groups, but they were less clear-cut, since 16% of the control women had lymphocytes responding to HSV. Non-immunoglobulin G antibodies to HSV-2 were not present in blood at the same time as cell response to HSV-2-transformed cells. There was also a negative correlation between neutralizing activities of the sera and the indices of lymphocyte stimulation, indicating a regulation between humoral and cell-mediated responses.

[Study of a "RFA" locus coding for cell wall lipopolysaccharide core biosynthesis in "Shigella flexneri" F6S (author's transl)]. / Etude d'un locus génétique RFA impliqué dans la biosynthèse du "core" du lipopolyoside de la paroi de Shigella flexneri F6S

The rfa-7 lipopolysaccharide core mutation carried by the R mutant F680, derived from Shigella flexneri F6S serotype 5b, has been mapped by conjugation and transduction experiments. The results show a chromosomal distance of about 1 min between rfa-7 and mtl. Such a position would be similar to those of rfa genes in Escherichia coli K12 and Salmonella typhi-murium LT2. Conjugational transfer of E. coli K12 rfa+ genes to mutant F680 restored S. flexneri O-specificity. Chemical analyses performed on the lipopolysaccharide of such a rfa+ hybrid suggest that this strain can attach the S. flexneri serotype 5b O-specific polysaccharide to the E. coli K12 core.

[Chemotypes of "Shigella flexneri" R mutants and related phage receptors. II. -- Localization of phage receptors (author's transl)]. / Chimiotypes de mutants R de Shigella flexneri et récepteurs de phages. II. -- Localisation des récepteurs de phages

Receptor sites for phages FP3, V, P1kcvir, H+, C21, T4, T3, T7 and 6SR have been investigated, by comparing the lytic activity of these phages on R mutants of strain F6 (F6R) and of various serotypes (FH) of Shigella flexneri with their inhibition by the lipopolysaccharides isolated from these mutants. The results suggest the following localizations for the receptor sites: phage FP3: lipid A-KDO; phage V: heptose or glucose; phage C21: heptose-glucose; phages H+, P1kcvir, T4 and T3: glucose; phage T7: glucose-galactose; phage 6SR: complete core structure.

[Chemotypes of "Shigella flexneri" R mutants and related phage receptors. I. -- Chemical study of the lipopolysaccharides (author's transl)]. / Chimiotypes de mutants R de Shigella flexneri et récepteurs de phages. I. -- Etude chimique des lipopolyosides

The F6R rough mutants isolated from Shigella flexneri F6S, serotype 5b, and the FH rough mutants, derived from other serotypes of S. flexneri, were chemotyped according to the chemical analysis of their lipopolysaccharides. Further, the following stages of lipopolysaccharide core biosynthesis in S. flexneri have been established: --(KDO)3--heptose--heptose--glucose--galactose; the last three stages are: either --glucose--glucosamine--glucose, or --glucosamine--glucose--glucose. The results of the chemical study of the R lipopolysaccharides are compatible with the assumption of the existence of a similar core in all considered S. flexneri serotypes.