beta-N-acetylhexosaminidases from Serratia marcescens.

Two forms of beta-N-acetylhexosaminidase from Serratia marcescens with an optimum pH of 5.0 and 6.5, respectively, to 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside were separated by DEAE-cellulose chromatography and Sephacryl S-200 chromatography. On the basis of their molecular weights, thermal stability, substrate specificity and isoelectric points, the form with an acidic pH optimum resembled hexosaminidase B, whereas the form with a neutral pH optimum resembled hexosaminidase C. Lectin binding studies showed that the acidic form does not bind to concanavalin-A-Sepharose, Tetragonolobus purpurea-agarose, wheat germ-agglutinin-Sepharose or Ricinus communis-agglutinin-agarose, whereas the neutral form binds to the last two lectin columns.     

Immobilization of Chloroperoxidase from Serratia marcescens

A bacterial non-heme chloroperoxidase from Serratia marcescensW 250 was immobilized in calcium alginate gel. Methods for stabilization of the immobilized enzyme were developed, and some kinetic parameters of the immobilized preparations were determined. The enzyme encapsulated into the gel granules in the presence of potassium ferricyanide followed by treatment with glutaraldehyde demonstrated the highest stability under the reaction conditions.     

Novel tetracycline resistance determinant isolated from an environmental strain of Serratia marcescens

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.     

Immobilization of chloroperoxidase from Serratia marcescens

A bacterial non-heme chloroperoxidase from Serratia marcescens W 250 was immobilized in calcium-alginate gel. Methods for stabilization of the immobilized enzyme were developed, and some kinetic parameters of the immobilized preparations were determined. The enzyme encapsulated into the gel granules in the presence of potassium ferricyanide followed by treatment with glutaraldehyde demonstrated the highest stability under the reaction conditions.     

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.     

Purification of Cytosine Deaminase from Serratia marcescens

Cytosine deaminase was purified about 900-fold from the cell-free extract of Serratia marcescens. The purification procedure included heat treatment, ammonium sulfate fractionation, ethyl alcohol fractionation, DEAE-cellulose and hydroxylapatite column chromatography, and Sephadex G–200 gel filtration.

The enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 580,000 and the molecule was composed of equimolecular weight of 8 subunits.

The enzyme catalyzed the stoichiometric conversion of cytosine into uracil and ammonia.     

Morphological and genetic analysis of three bacteriophages of Serratia marcescens isolated from environmental water

Increases in multidrug-resistant strains of Serratia marcescens are of great concern in pediatrics, especially in neonatal intensive care units. In the search for bacteriophages to control infectious diseases caused by multidrug-resistant S. marcescens , three phages (KSP20, KSP90, and KSP100) were isolated from environmental water and were characterized morphologically and genetically. KSP20 and KSP90 belonged to morphotype A1 of the family Myoviridae , and KSP100 belonged to morphotype C3 of the family Podoviridae . Analysis of the DNA region coding virion proteins, together with their morphological features, indicated that KSP20, KSP90, and KSP100 were related to the P2-like phage (temperate), T4-type phage (virulent), and phiEco32 phage (virulent), respectively. Based on amino acid sequences of the major capsid protein, KSP90 formed a new branch with a Stenotrophomonas maltophilia phage, Smp14, in the T4-type phage phylogeny. Both Smp14 and phiEco32 have been reported as potential therapeutic phages. These results suggest that KSP90 and KSP100 may be candidate therapeutic phages to control S. marcescens infection.     

Stimulation and inhibition of polymorphonuclear leukocytes phagocytosis by lipoamino acids isolated from Serratia marcescens

Abstract The role of the lipoamino acids (serratamolide and ornithine lipid), membrane lipid components of Serratia marcescens , was examined in phagocytosis and phagosome-lysosome fusion of human peripheral polymorphonuclear leukocytes. A mutant strain of Serratia marcescens (NS 38-09) lacking serratamolide was actively phagocytosed by human PMN, while the wild-type strain (NS 38) producing serratamolide was more resistant to phagocytosis by human PMN. Phagocytosis of killed Staphylococcus aureus coated with lipoamino acid (serratamolide), showed that they were more resistant to phagocytosis by PMN, while the cells coated with ornithine lipid or serratamic acid were phagocytosed more actively. Staphylococci coated with phosphatidylethanolamine or phosphatidylglycerol had no significant effect on phagocytosis by PMN. Phagosome-lysosome fusion by PMN labelled with acridine orange was examined by fluorescence microscopy. The fusion indices of lipoamino acid-coated staphylococci were the same as that of controls. Further, ornithine lipid-coated staphylococci stimulated the release of superoxide anion from PMN slightly, but serratamolide did not. These results suggested that serratamolide may contribute to the virulence of S. marcescens in vitro.     

Size and UV germicidal irradiation susceptibility of Serratia marcescens when aerosolized from different suspending media

Experimental systems have been built in laboratories worldwide to investigate the influence of various environmental parameters on the efficacy of UV germicidal irradiation (UVGI) for deactivating airborne microorganisms. It is generally recognized that data from different laboratories might vary significantly due to differences in systems and experimental conditions. In this study we looked at the effect of the composition of the suspending medium on the size and UVGI susceptibility of Serratia marcescens in an experimental system built in our laboratory. S. marcescens was suspended in (i) distilled water, (ii) phosphate buffer, (iii) 10% fetal calf serum, (iv) phosphate-buffered saline (saline, 0.8% sodium chloride), and (v) synthetic saliva (phosphate-buffered saline with 10% fetal calf serum). At low humidity (36%), S. marcescens suspended in water-only medium was the most susceptible to UVGI, followed by those in serum-only medium. The count median diameters (CMDs) for culturable particles from water-only and serum-only media were 0.88 and 0.95 micro m, respectively, with the measurements based on their aerodynamic behavior. The bacteria suspended in phosphate buffer, synthetic saliva, and phosphate-buffered saline had similar UVGI susceptibility and CMD at 1.0, 1.4, and 1.5 micro m, respectively. At high humidity (68%) the CMD of the particles increased by 6 to 16%, and at the same time UVGI susceptibility decreased, with the magnitude of decrease related to the type of suspending medium. In conclusion, the choice of suspending medium influenced both size and UVGI susceptibility of S. marcescens. These data are valuable for making comparisons and deciding on the use of an appropriate medium for various applications.     

Properties of Serratia marcescens strains isolated in septic diseases of newborn infants

As the result of the study of blood and liquor samples from 120 newborns, Serratia marcescens was isolated in 21 cases (17.5 %). 8 strains were isolated from the environment of these patients. Almost all strains isolated from both the patients and the environment (with the exception of one environmental strain) belonged to serotype 04. The isolated S. marcescens strains were resistant to penicillin, ampicillin, streptomycin, kanamycin, oxacillin, methicillin, ceporin and moderately sensitive to polymixin. 2 strains from the environment and 9 strains from the patients were mildly sensitive to gentamicin. In one hospital all isolated strains were found to have 2 transmissive R plasmids with the molecular weight 40 and 60 megadaltons. The presence of R plasmids with the same molecular weight in all S. marcescens strains isolated in this hospital, as well as their serological identity, suggest that in all patients infection originated from a common source.     

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.     

Identification and susceptibility to antimicrobial agents of strictly anaerobic bacteria isolated from hospitalized patients

The aim of this study was to identify anaerobic strains isolated in 2001 from clinical specimens obtained from patients of Warsaw hospital and to evaluate a susceptibility of these strains to antimicrobial agents. In 2001 two hundred and twenty five clinical strains of obligate anaerobes were cultured, which were identified in the automatic ATB system (bioMérieux, France) using biochemical tests API 20 A. Drug-susceptibility of strains was determined also in ATB system with the use of ATB ANA strips. C. difficile strains were isolated on selective CCCA medium. Toxins A/B of C. difficile directly in stool specimens were detected by means of ELISA test (TechLab, USA). Fifty four strains of Gram-negative anaerobes (B. fragilis strains dominated) and 171 strains of Gram-positive anaerobes (the greatest number of strains belonged to genus Peptostreptococcus) were cultured from clinical specimens. In the cases of antibiotic-associated diarrhea 28 C. difficile strains were isolated and C. difficile toxins A/B were detected in 39 stool samples. The most active in vitro antimicrobials against Gram-negative anaerobes were metronidazole, imipenem, ticarcillin combined with clavulanic acid and piperacillin with tazobactam. Gram-positive, clinical strains of anaerobes were the most susceptible in vitro to beta-lactam antibiotics combined with beta-lactamase inhibitors (amoxicillin/clavulanate, piperacillin/tazobactam, ticarcillin/clavulanate) and imipenem.