- Volume 37, Issue 2, 1992

Susceptibilities to cefotaxime (Ctx) and ceftazidime (Caz) were examined for 90 recent clinical isolates of Enterobacter cloacae from Greek hospitals. Most (68%) of the isolates were resistant to both drugs, and all were resistant to cefoxitin. β-Lactamase activities against cephaloridine in crude extracts from Ctx-Caz-resistant isolates were high, irrespective of whether or not the cells were grown with cefoxitin as an inducer of the chromosomal β-lactamase, indicating stable derepression of the gene for the enzyme. On the other hand, double disk antagonism tests showed that all the Ctx-Caz-sensitive isolates possessed inducible expression of this β-lactamase. Iso-electric focusing revealed the presence of five forms of the chromosomal β-lactamase, randomly distributed amongst the Ctx-Cazresistant and -sensitive isolates. Plasmid-mediated β-lactamases of TEM and PSE types also were found in many isolates. These data indicate that the extremely high prevalence of Ctx-Caz-resistant E. cloacae isolates in Greek hospitals is attributed to the dissemination of mutants which constitutively overproduce the class-I chromosomal β-lactamase. Over 90% of these isolates exhibited cross-resistance to aminoglycosides, suggesting the accumulation of unrelated antibiotic resistance mechanisms.

Genetic variability among 41 penicillinase non-producing Neisseria gonorrhoeae strains, isolated in Spain, with different levels of resistance to penicillin was investigated by multilocus enzyme electrophoresis. Based on the results obtained by analysis at seven enzyme loci, the strains were separated into 17 electrophoretic types. The average number of alleles/enzyme locus was 2.85; the mean genetic diversity/locus was 0.49 for individual isolates and 0.516 for electrophoretic types. The results showed that these gonococcal strains were, genetically, a highly variable group of organisms.

The effect of monoclonal antibodies (MAbs) injected alone or in combination on brucella splenic infection in CD-1 mice was tested 7 and 21 days after a challenge with virulent Brucella abortus 544. Passive immunisation of mice with anti-25–27-kDa MAb alone, or mixed with protective anti-16.5 and anti-36–38-kDa MAbs, or with MAbs of the same specificity which were previously demonstrated to have no activity on CD-1 mice, produced a significant reduction of spleen counts of B. abortus (p < 0.01). Other combinations of MAbs did not reduce splenic infection in comparison with the untreated control group. BALB/c mice were used to test the possible interference of the immune response of CD-1 mice against MAbs that were produced in BALB/c mice. No reduction of splenic infection was shown with anti-25–27-or-36–38-kDa MAbs, whereas anti-lipopolysaccharide (LPS) MAb which was produced in CBA mice was effective. Combination of anti-protein MAbs with the anti-LPS MAb produced only the effect of the anti-LPS MAb at 7 and 21 days after challenge.

The phage type, antimicrobial resistance pattern, colicinogenic activity and DNA plasmid content of 172 strains of Salmonella typhimurium isolated in Rome from 1984 to 1986 were determined; 142 isolates were from patients with enteritis, 30 were from asymptomatic subjects. Most of the phage types identified were isolated during 2 or 3 of the study years; others, e.g., PT141, PT 204c and PT 194 were isolated during 1 year only, and only the last of these was isolated in large numbers. Phage typing proved most valuable in identifying epidemiologically related strains; DNA plasmid analysis was most useful in characterising further cultures of the same phage type, especially those isolated during suspected epidemics.

One hundred and forty-two coagulase-negative staphylococci (CNS) isolated from dialysate effluent or skin of patients receiving continuous ambulatory peritoneal dialysis (CAPD) were typed by extended antibiogram (16 antibiotics) and biotype (26 reactions). These isolates were then typed by supplementary methods to determine the most suitable typing method for an epidemiological study of antibiotic resistance. These included phage typing, reverse phage typing, plasmid typing, whole-cell protein typing by SDS-PAGE with analysis by densitometry, and immunoblotting. The percentage of isolates typed successfully by the supplementary methods were: phage typing 20%, reverse phage typing 0%, plasmid typing 66%, SDS-PAGE 100%, immunoblotting 100%. The discrimination of each method was: phage typing 20%, plasmid typing 37%, SDS-PAGE 69%, immunoblotting 57%. Reproducibility was 88% for phage typing and 97% for plasmid typing. The reproducibility of the whole-cell protein typing was 83% if the same extracts were used but only 43% when separate protein extracts were analysed on separate occasions. However, strain relatedness was highly reproducible. The determination of an antibiogram-biotype profile was not a sufficiently accurate typing method for an epidemiological study of antibiotic resistance. Whole-cell protein typing by SDS-PAGE or immunoblotting was technically demanding but was the most effective of the supplementary methods for detecting erroneous discrimination and false matching produced by antibiogram-biotype combinations.

Cytopathic effects of broth-culture filtrates from eight clinical isolates and one reference strain of Helicobacter pylori on three cultured mammalian cell lines were investigated. All the strains, including NCTC 11637, produced cytotoxic factors that caused intracellular vacuolation on these cell lines. AGS and SflEp cells were more sensitive than HEp-2 cells. To examine the role of urease in the cytotoxic effect, a urease-negative mutant was produced. Filtrates from both wild-type and mutant strains produced similar vacuolation on Sfl Ep cells in the absence of urea, suggesting that H. pylori produces a cytotoxic substance other than urease. In contrast, ammonia alone, or jack bean urease with urea, also induced rounding and detachment of Sfl Ep cells, whereas ammonium salts induced the production of small vacuoles. The combination of the broth filtrate of the wild-type strain and urea induced vacuolation followed by rounding and detachment of Sfl Ep cells. Evidence is presented that the latter changes are due to ammonia produced during incubation. Nevertheless, the amounts produced were less than that needed to induce cytopathic effects by itself. These results suggest that the cytotoxic substance induces intracellular vacuolation, and that the vacuolated cells are more susceptible to killing by ammonia. Thus both the cytotoxic substance and urease may contribute to the lethal cytotoxicity of H. pylori in vitro.

Non-motile variants of Helicobacter pylori (strain 26695) occurred with a frequency of 1.6 (SD 0.4) × 10−4 variants/cell/division cycle, and reversion to the motile form occurred with a frequency of < 10−7 variants/cell/division cycle. The two forms remained > 90% pure for up to 50 cell divisions and differed only in the presence or absence of motility and flagella. Bacteria were recovered from nine of 10 gnotobiotic piglets inoculated orally with motile H. pylori, but from only two of eight inoculated with the non-motile variant. The motile form survived for 21 days in infected piglets, but the non-motile variant survived for only 6 days. Bacteria recovered from piglets inoculated with the non-motile variant were non-motile. These data support the hypothesis that motility is a colonisation factor for H. pylori.

Several Pasteurella multocida strains were examined for their ability to produce extracellular enzymes that cleave immunoglobulin A and G (Ig A and Ig G) molecules. Two strains isolated from human pulmonary and genital infections produced proteases that cleaved human IgA and IgG, colostral IgA and human myeloma IgA1 and IgA2. Human IgM was not degraded by these enzymes. Examination of cleavage digests showed two main fragments with different electrophoretic mobilities. The two P. multocida strains produced a protease that cleaved IgA and IgG heavy chains outside the hinge region, and differed in this respect from the hinge-cutting proteases of other bacteria. Protease production may be a virulence mechanism for P. multocida strains.

Haemophilus influenzae grown to exponential phase or stationary phase in medium with a low initial concentration of haemin (0.25 μg/ml) was virtually devoid of cytochromes. Compared with bacteria grown in the presence of excess haemin (10 μg/ml), the haemin-limited organisms failed to respire formate and succinate and, generally, the respiratory rates with other substrates were reduced. However, growth rates were not affected by the haemin supply. Haemin-limited growth was associated with a reduced efficiency of glucose utilisation, in terms of glucose growth yields, and affected the net levels of excreted organic acids. Haemin limitation resulted in reduced acetate and increased succinate accumulation in the culture medium and the novel presence of d-lactate. These results indicate that, in contrast to the phenotype expressed in vitro during conventional cultivation of H. influenzae, the haemin-limited phenotype, which may be expressed in vivo, is characterised by a lack of cytochromes and a shift towards a more anaerobic type of metabolism.

Enterotoxigenic Escherichia coli (ETEC) strain P2200 of porcine origin possessed eight possibly plasmid-determined characters (K88+ Raf+ Hly+Col+SmrTcrSurSTa+) and six plasmid DNA bands of 4.2–93 kb. Analysis of the spontaneous loss of characters and the results of matings with other E. coli strains revealed that the K88, Raf, Hly, Smr, Tcr and Sur characters could be transferred, and that the presence of the K88 and Raf characters was associated with an 83-kb plasmid. The presence and location of the STaI gene was investigated in several ETEC strains of bovine or porcine origin. Hybridisation with a non-radioactive polynucleotide probe associated the STaI gene with a plasmid in each strain; these plasmids were of 32–142 kb. In contrast, plasmids from a P2200 STa− variant and plasmids from two STa− variants of the bovine ETEC strain B41* (strain B41 obtained from a different source) did not hybridise with the probe. One of the B41*STa− variants had lost the STa plasmid, whereas the second variant retained a plasmid of the same size which did not hybridise. In contrast, a third B41*STa− variant retained a plasmid of the same size that still hybridised with the STaI probe. Plasmid DNA restriction fragment analysis, followed by hybridisation with the STaI probe, showed that the STaI gene was associated with 8.3-, 6.8- and 3.5-kb plasmid fragments in strain B41, and with 4.9-, 6.8- and 3.5-kb plasmid fragments in strain B41*, following digestion with EcoRI, BamHI, or EcoRI + BamHI, respectively. The STaI probe hybridised also with 12.2-kb EcoRI and 4.6-kb BamHI fragments of the plasmid from the third B41*STa− variant, that unexpectedly had given an initial positive hybridisation result. Different plasmid restriction fragment profiles were seen for strains B41 and B41*, the B41*STa− variants and the transconjugant strains, thereby providing further evidence that molecular rearrangements of these plasmids can occur spontaneously.