Clinical relevance and virulence factors of pigmented Serratia marcescens

Pigmented Serratia marcescens isolated in a Brazilian hospital were studied with respect to frequency of isolation, serotyping, antibiotic resistance and virulence factors. The serotype most frequent was O6:K14 (53%) and all isolates were resistant to ampicillin, cephalothin and tetracycline. The majority of the isolates (92%) were resistant to the action of human serum and all produced cytotoxins on Vero, CHO, HEp-2 and HeLa cells. These isolates were virulent for mice (LD(50)=10(7) bacteria ml(-1)) and showed virulence factors, but were isolated with low frequency (3. 4%) and caused infection in only 31% of cases. Analysis of serotyping, phage typing and chromosomal DNA revealed at least 13 unrelated strains among pigmented S. marcescens. In conclusion, this work describes a low frequency of isolation of pigmented S. marcescens from clinical specimens, indicating that non-pigmented strains are clinically more significant.     

Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

Overproduction of Serratia marcescens metalloprotease (SMP) from the recombinant Serratia marcescens strains

Summary To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacI^q, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.     

Extracellular haemolytic activity of Serratia marcescens

Blood agar medium with dialysis membrane mounted between two layers of agar was applied to study the haemolytic activity of 28 strains of Serratia marcescens. Two kinds of lytic substances differing with their ability to pass through dialysis membrane were found. Haemolytic activity was not detected in cell-free filtrates from liquid cultures. The discrepancies between haemolytic activity in blood agar media and activity of liquid cultures were observed. Stable attachment of bacterial cells to the erythrocytes was not necessary to lysis. The possibility of extracellular haemolysin is discussed.     

Respiration and dehydrogenase activity of pigmentless variants of Staphylococcus aureus]

Respiration and dehydrogenase activity were studied in four pigmentless mutants of Staphylococcus aureus 209-P. The rate of oxygen uptake from the incubation medium was higher in the mutants than in the parent strain. The rate of respiration and the activity of dehydrogenase in the pigmentless mutants were found to depend on the permeability of the cell membrane for substrates of respiration; carotenoids are presumed to be involved in this permeability.     

Cytotoxic activity of Serratia marcescens clinical isolates

Twenty Serratia marcescens isolates from clinical specimens were examined for their cytotoxic activity on four cell lines (HEp-2, Vero, CHO, J774). Most of the isolates were found to be cytotoxic to CHO (70%), Vero (75%) and HEp-2 cells (90%). CHO cells were the most sensitive to cell-free supernatants, followed by HEp-2 and Vero cells. Two strains produced cytotonic toxins which caused elongation of CHO cells. Moreover, twelve isolates (60%) revealed cytotoxic potential to macrophage cell line J774. The results indicate that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.     

New R-plasmids in strains of Serratia marcescens with multiple drug resistance

In 4 S. marcescens polyresistant strains isolated from patients conjugative plasmids transferred to Escherichia coli have been detected. Two of these strains carry each one plasmid which codes resistance to 10 different antibiotics, including aminoglycosides which rarely occur in our country, and belongs to group IncC. The third strain is the host of 2 plasmids. One of them is similar to the above-mentioned 2 plasmids with respect to the incompatibility group and a set of markers, but additionally codes resistance to cephalosporins; the second plasmid has been determined as belonging to group IncM, unstable and capable of rendering the cells highly resistant only to aminoglycosides. And, finally, the fourth strain also carries 2 plasmids: one of them is unstable and belongs, supposedly, to group IncI alpha, and the second plasmid is stable and belongs to group IncM. The plasmid of group IncI alpha differs from all other plasmids of our Serratia by its capacity of rendering the cells highly resistant to chloramphenicol.     

Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity

Enrichment for nonhydrophobic mutants of Serratia marcescens yielded two types: (i) a nonpigmented mutant which exhibited partial hydrophobic characteristics compared with the wild type, as determined by adherence to hexadecane and polystyrene; and (ii) a pigmented, nonhydrophobic mutant whose colonies were translucent with respect to those of the wild type. The data suggest that the pronounced cell surface hydrophobicity of the wild type is mediated by a combination of several surface factors.     

pH-dependent modulation of alkaline phosphatase activity in Serratia marcescens

Serratia marcescens is an opportunistic pathogen responsible for causing nosocomial infections, corneal ulcer, necrotizing fasciitis, cellulites, and brain abscess. Alkaline phosphatase (APase) is believed to play an important role in the survival of several intracellular pathogens and their adaptation. We have studied the effect of low phosphate concentration and acid pH on the APase activities of S. marcescens. In a low phosphate medium, some strains of S. marcescens synthesize two different types of APases, a constitutive (CAPase) and an inducible (IAPase). Both the CAPase and IAPase isoenzymes completely lost their enzyme activities at pH 2.3, within 10 min of incubation at 0°C. Acid-treated IAPase isoenzymes I, II, III, and IV solutions when adjusted to pH 7.8 showed recovery of 70%, 52%, 72%, and 60% of the lost activities, respectively. When the pH of the CAPase reaction mixture was raised to pH 7.8, the enzyme activity regained only 5% of its initial activity. Variations in protein concentration also affected the pH-dependent reversible changes of the IAPase activity. The higher the protein concentration, the faster the inactivation of enzyme activity observed at acidic pH at 0°C. Conversely, the lower the protein concentration, the higher the rate of reactivation of enzyme activity observed for IAPase at alkaline pH. Protein interaction studies revealed a lack of similarity between CAPase and IAPase, suggesting separate genetic origin of these potentially virulent genes of S. marcescens. Received: 4 December 2001 / Accepted: 7 January 2002     

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

The cytolytic and haemolytic activity of Serratia marcescens is determined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein. In the absence of ShlB, inactive ShlA* remains in the periplasm of Escherichia coli transformed with an shlA-encoding plasmid, which indicates that ShlB converts ShlA* to active ShlA. ShlA* in a periplasmic extract and partially purified ShlA* were activated in vitro by partially purified ShlB. When both proteins were highly purified, ShlA* was only activated by ShlB when phosphatidylethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA* activation. Lyso-PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysaccharide and various detergents could not substitute for PE. Although radioactively labelled PE was so tightly associated with ShlA that it remained bound to ShlA after heating and SDS–PAGE, it was not covalently linked to ShlA as PE could be removed by thin-layer chromatography with organic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 ± 2.2. Active ShlA was inactivated by treatment with phospholipase A₂, which indicated that PE is also required for ShlA activity. ShlA-255 (containing the 255 N-terminal amino acids of ShlA) reversibly complemented ShlA* to active ShlA and was inactivated by phospholipase A₂, which demonstrated that PE binds to the N-terminal portion of ShlA; this region has previously been found to be involved in ShlA secretion and activation. Electrospray mass spectroscopy of ShlA-255 determined a molar mass that corresponded to that of unmodified ShlA-255. An E. coli mutant that synthesized only minute amounts of PE did not secrete ShlA but contained residual cell-bound haemolytic activity. Since PE binds strongly to ShlA* in the absence of ShlB without converting ShlA* to haemolytic ShlA, ShlB presumably imposes a conformation on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic membrane.     

Cation transport in Serratia marcescens and Serratia marinorubra

The sodium, potassium, and magnesium ion contents of Serratia marcescens and those of its salt-tolerant relative, S. marinoruba, were determined by atomic-absorption spectrometry. The intracellular K(+) and Mg(2+) contents of both microorganisms were found to be dependent on the ionic strength of the growth or suspending medium. The Mg(2+) content of S. marinoruba was generally greater than that of S. marcescens. The Na(+) content of the cells was normally low and did not increase as the cells aged or when the cells were grown in media of high ionic strength. The transport of K(+) by resting cells suspended in hypertonic solution was studied by chemical and light-scattering techniques and was found to be more rapid in S. marcescens than in S. marinorubra. The slower rate of K(+) transport in S. marinorubra is probably related to the lower glycogen reserves found in resting cells of this microorganism. K(+) transport was found to have a pH optimum of 5.5 to 6.1 for S. marcescens, and the K(m) for K(+) was approximately 1.6 mm. Na(+) and Mg(2+) were not taken up by the cells, although the presence of Mg(2+) tended to decrease rates of K(+) uptake. Tris-(hydroxymethyl)aminomethane, routinely used for resuspending the cells, was apparently taken up by the cells at pH >7.