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A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.
Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.
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Seventeen Serratia marcescens strains were isolated from patients admitted to a hospital in the Tokyo metropolitan area. In order to study their genetic diversity, pulsed-field gel electrophoresis (PFGE) was performed. Clear bands were not observed by the standard PFGE technique following the instruction manual from the company. To obtain clear results, it was necessary to include some additional steps to the preparation of PFGE samples. We added an inactivation step for DNase using formaldehyde, and the bacterial strains were embedded in thinner plug molds than usual. These modifications were effective with all strains, and the complete PFGE pattern was obtained in the samples treated with formaldehyde and using thin plug molds. We established a rapid method to obtain a high-quality PFGE result for S. marcescens.
Serratia marcescens, a gram-negative rod belonging to the family of the Enterobacteriaceae may be the cause of various ophthalmic infections, especially in patients with decreased resistance. The course of these ocular infections is always severe. This is the first description of a metastatic S. marcescens endophthalmitis. It developed in a patient with chronic lymphatic leukemia after prostatectomy. The purpose of this paper is to draw attention to S. marcescens, an organism of increasing importance as an ocular pathogen.
Serratia marcescens is an important nosocomial pathogen which is often resistant to multiple antimicrobial agents. An imipenem-resistant S. marcescens isolate from a urine specimen was found to carry a bla(VIM-2) gene cassette on a class 1 integron. This finding indicates that bla(VIM-2) is presently spreading even to Serratia spp. in Korea, which could compromise the usefulness of carbapenem in the treatment of multi-resistant Gram-negative bacilli infections. Clinical laboratory should be able to detect the VIM-2-producing isolates with even low carbapenem MIC.
OBJECTIVES: Serratia marcescens, an important cause of nosocomial infections, shows intrinsic resistance to a wide variety of antimicrobial agents (multidrug resistance). Multidrug efflux pumps are often involved in the multidrug resistance in many bacteria. A study was undertaken to characterize the multidrug efflux pumps in S. marcescens. METHODS: The genes responsible for the multidrug resistance phenotype in S. marcescens were cloned into Escherichia coli KAM32, a drug-hypersusceptible strain, for further analysis. RESULTS: We cloned sdeXY genes and determined the nucleotide sequence. Clones that carried the sdeXY genes displayed reduced susceptibility to several antimicrobial agents including erythromycin, tetracycline, norfloxacin, benzalkonium chloride, ethidium bromide, acriflavine and rhodamine 6G. A protein similarity search using GenBank revealed that SdeY is a member of the resistance nodulation cell-division (RND) family of multidrug efflux proteins and SdeX is a member of the membrane fusion proteins. Introduction of sdeXY into E. coli cells possessing tolC, but not in cells lacking tolC, resulted in multidrug resistance. We observed energy-dependent ethidium efflux in cells of E. coli KAM32 possessing sdeXY and tolC. CONCLUSIONS: SdeXY is the first RND-type multidrug efflux pump to be characterized in multidrug-resistant S. marcescens.
Although Serratia marcescens is a well-known nosocomial pathogen, investigation of its hospital ecology has been limited by the lack of available typing techniques. During an investigation of the occurrence of this organism in a neonatal intensive care unit, we evaluated a number of such techniques. Using a selective medium, we conducted prospective surveillance of neonatal rectal colonization and environmental contamination with S. marcescens. In 8 months of surveillance, 5.1% (20 of 394) of the infants admitted to the unit became colonized. Most sink surfaces and drains were also culture positive. Differences between isolates could not be detected in biotypes from a commercial identification system (MicroScan) or by antibiograms, total protein fingerprints, or plasmid profiles. Serogrouping and genomic DNA restriction endonuclease analysis revealed the presence of six strains that colonized infants and a similar number of environmental strains. These two methods were concordant, with the exception that genomic DNA analysis demonstrated lack of relatedness between some strains within the same serogroup. DNA restriction endonuclease analysis was practical and reliable. The differences this method detected between environmental and neonatal strains provided strong evidence that the environment was not an important reservoir for S. marcescens in our neonatal intensive care unit.
Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 g/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.
Serratia marcescens bacteremia has become ubiquitous recently. S. marcescens bacteremia, either hospital- or community-acquired, can no longer be treated as insignificant. We reviewed 23 episodes of S. marcescens bacteremia in 1985. Among them, 17 patients (74%) were hospital-acquired infections, while 6 (26%) were community-acquired. Nine patients died, and the case fatality rate was 39%. Eleven patients (48%) had no clinically apparent source of infection, 5 (22%) had urinary tract infection, 3 (13%) had pneumonia, 2 (9%) had biliary tract infection, 1 (4%) had intra-abdominal infection, and 1 (4%) had skin and soft-tissue infection. Nosocomial isolates are often resistant to many antibiotics. Amikacin and the beta-lactamase-stable (third generation) cephalosporins are superior to gentamicin in the treatment of nosocomial S. marcescens bacteremia. We here emphasize that the awareness and treatment of S. marcescens bacteremia in daily clinical practice is unequivocally critical.
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.
Using Serratia marcescens 2170 harbouring plasmid pTroy11 (encoding for LamB), and lambda (lambda:: Tn5) we constructed a collection of 25 auxotrophic mutations induced by Tn5 insertions. These mutants permitted the use an easy screening method for generalized transducing bacteriophages. Out of twelve bacteriophages isolated from natural sources only one (phage 3M) was able to transduce all Tn5 insertions tested. The preliminary characterization of bacteriophage 3M indicates that it belongs to the Myoviridae family.
Serratia marcescens S6 produces a pI 9.7 carbapenem-hydrolyzing beta-lactamase that is probably encoded by the chromosome (Y. Yang, P. Wu, and D. M. Livermore, Antimicrob. Agents Chemother. 34:755-758, 1990). A total of 11.3 kb of genomic DNA from this strain was cloned into plasmid pACYC184 in Escherichia coli. After further subclonings, the carbapenem-hydrolyzing beta-lactamase gene (blaSme-1) was sequenced (EMBL accession number Z28968). The gene corresponded to an 882-bp open reading frame which encoded a 294-amino-acid polypeptide. This open reading frame was preceded by a -10 and a -35 region consistent with a putative promoter sequence of members of the family Enterobacteriaceae. This promoter was active in E. coli and S. marcescens, as demonstrated by primer extension analysis. N-terminal sequencing showed that the Sme-1 enzyme had a 27-amino-acid leader peptide and enabled calculation of the molecular mass of the mature protein (29.3 kDa). Sequence alignment revealed that Sme-1 is a class A serine beta-lactamase and not a class B metalloenzyme. The earlier view that the enzyme was zinc dependent was discounted. Among class A beta-lactamases, Sme-1 had the greatest amino acid identity (70%) with the pI 6.9 carbapenem-hydrolyzing beta-lactamase, NMC-A, from Enterobacter cloacae NOR-1. Comparison of these two protein sequences suggested a role for specific residues in carbapenem hydrolysis. The relatedness of Sme-1 to other class A beta-lactamases such as the TEM and SHV types was remote. This work details the sequence of the second carbapenem-hydrolyzing class A beta-lactamase from an enterobacterial species and the first in the genus Serratia.
Serratia marcescens RZ has been previously shown to possess pronounced cell-surface hydrophobicity, as evidenced by its affinity for hydrocarbons and polystyrene. The present report suggests the involvement of a 70 kDa protein, serraphobin, in this phenomenon. The 70 kDa protein was recovered from both the cell surface and culture supernatant of hydrophobic wild-type cells, but was either totally absent or present in minor quantities in hydrophobicity-deficient mutants. Similarly, loss of hydrophobicity of RZ cells following growth at 39 degrees C was accompanied by loss of the protein. Serraphobin was capable of binding to hexadecane droplets following a brief mixing procedure, and could be desorbed by solidifying and melting the hexadecane phase.
The Serratia marcescens extracellular lipase (LipA) is an enzyme applicable to enantioselective hydrolysis of racemic substrates. The enzyme is secreted through an ATP-binding cassette (ABC) exporter, the Lip system, encoded by the lipBCD genes. The S. marcescens recombinant carrying pLIPE121, which encodes the lipA gene in pUC19, exhibited a higher LipA production level than the wild-type strain. However, the level was lower than expected, and secretion was suggested to be a bottleneck. lipBCD plasmids were introduced into S. marcescens recombinants harboring lipA plasmids and the effectiveness of the lipBCD plasmids in elevating LipA productivity was investigated. S. marcescens strains harboring both lipA and lipBCD plasmids showed sevenfold greater extracellular LipA activity than the strain harboring the lipA plasmid alone. A high level of extracellular LipA production (1,300 kU/ml) and high plasmid stability (enough to carry out large-scale cultivation) were observed under non-selective conditions. Addition of L-proline and Tween 80 was effective in increasing cell growth of the recombinant, which led to high LipA production. In batch cultivation using a 30-l jar fermentor, LipA production was achieved at a high level of 5,200 kU/ml. This is the first report describing utilization of ABC exporter for the overproduction of an industrially important extracellular protein.
Serratia marcescens is an unusual cause of community-acquired meningitis in adults. We report a case of S. marcescens meningitis occurring 29 years after a head injury and preceded by 3 years of intermittent nasal discharge of cerebrospinal fluid (CSF). One month before admission, the patient had received treatment with cefadroxil. This case illustrates the risk of Gram-negative bacillary meningitis in patients with a CSF leak when they are treated with antibiotics. When patients have a chronic clear nasal discharge, one should look for a past medical history of head injury before prescribing antibiotics. In the presence of a fistula, any antibiotherapy may lead to the selection of resistant organisms which may be difficult to treat. Due to the high risk of meningitis and the fact that spontaneous closure in delayed CSF rhinorrhoea is unlikely, surgical repair of any associated fistulae is mandatory.
Dried Serratia marcescens (ATTC strain 14041) cells were exposed to various partial pressures of oxygen and nitrogen. The colony-forming ability of the organisms was rapidly destroyed during exposure to oxygen but was unimpaired by exposure to purified nitrogen. The degree of inactivation depended upon temperature, time, and the partial pressure of oxygen, regardless of whether pure oxygen or dry air was used. The inactivation by oxygen followed the expression -1nN/N(0) = k[O(2)](1/3)t(1/2), where N(0) and N are the number of viable organisms before and after exposure respectively, [O(2)] is oxygen concentration, t is time, and k is the rate constant. At 25 C, k was 276 +/- 36 moles(-1/3) cc(1/2) hr(-1/3) for oxygen pressures between 5.5 and 258 torr. In the temperature range between -78 and 40 C, the rate constant may be expressed as k = 10(5.95+/-04.2) exp[(-430 +/- 26) cal/RT] moles(-1/3) cc(1/3) hr(-1/2).