Isolation and characterization of a Serratia marcescens with insecticidal activity from Polyphylla olivieri (Col.: Scarabaeidae)

Members of the genus Serratia are known for their abilities to infect insects. In this study, a red‐pigmented S. marcescens was isolated and characterized from the infected larvae of Polyphylla olivieri using bacterial cultivation, phylogenetic analysis as well as bioassays against larvae of the two insect pests, Plodia interpunctella and Ephestia kuehniella. Comparative 16S rRNA and groEL gene sequence BLAST analyses strongly suggested that the isolated strain should be placed in the genus Serratia, sharing high sequence similarities with several strain of S. marcescens associated with insects. Phylogenetic analysis placed the isolated bacterium with other S. marcescens bacteria in a clade with high bootstrapping values. To assess pathogenicity of the S. marcescens isolate, the bacterial cells were either injected into the haemolymph of the fifth‐instar larvae or added to the diets of insects. Survival curves of the control insects and those challenged with six different concentrations of S. marcescens showed that the S. marcescens isolate significantly reduced survival rates of the larvae. The LC₅₀s of the bacterium on P. interpunctella and E. kuehniella were 1992.26 and 1.09 × 10⁴ (CFU/μl) for injection bioassays at 6 h post‐injection, and 4.48 × 10⁴ and 1.96 × 10⁵ (CFU/10 μl) for feeding bioassays at 24 h post‐feeding, respectively. Injection of the bacterial culture supernatant into the larvae led to continuous bleeding from the site of injection, while injection of heat‐treated culture supernatant of the bacterium did not cause continuous bleeding. Together, our results showed the possibility of using this S. marcescens isolate in microbial control of the insect pests after addressing the safety concerns. Moreover, it might be considered as a source of useful bioactive molecules and genes with application in insect control and biotechnology via developing insect‐resistant plants.     

Efflux systems in Serratia marcescens

A widespread bacterium Serratia marcescens (family Enterobacteriaceae) is an opportunistic pathogen and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from the cells by efflux systems is one of the mechanisms responsible for microbial resistance to these compounds. Among enterobacteria, efflux systems of Escherichia coli and Salmonella enterica ser. Typhimurium have been studied most extensively. Few efflux systems that belong to different families have been reported for S. marcescens. In this review, we analyzed available literature about S. marcescens efflux systems and carried out the comparative analysis of the genes encoding the RND type systems in different Serratia species and in other enterobacteria. Bioinformatical analysis of the S. marcescens genome allowed us to identify the previously unknown efflux systems based on their homology with the relevant E. coli genes. Identification of additional efflux systems in S. marcescens genome will promote our understanding of the physiology of these bacteria, will detect new molecular mechanisms of resistance, and will reveal their resistance potential.     

Relationship between <Emphasis Type="Italic">Psoroptes cuniculi</Emphasis> and the Internal Bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The bacterium Serratia marcescens isolated from surface-sterilised Psoroptes cuniculi was found sensitive to the antibiotic Amikacin. Mites placed in this antibiotic for 48–72 h and then washed by centrifugation were found to be alive and S. marcescens-free. Two experimental infestations were undertaken in order to verify the ability of the S. marcescens-free mites to infect and to give ear skin lesions in healthy rabbits and to evaluate the differential ability of the S. marcescens-free and S. marcescens-infected mites to give ear skin lesions. All rabbits were found to be infested, but only rabbits infested with S. marcescens-free mites presented crusts in their ears, whereas mites and/or eggs were only detected in the ear cerumen of all rabbits infested with S. marcescens-infected mites. S. marcescens was isolated only from P. cuniculi mites taken from these latter rabbits. Results indicate that P. cuniculi mites do not need S. marcescens to live and to be able to infest a healthy rabbit. In addition, S. marcescens was not isolated from eggs and newly born larvae of S. marcescens-infected P. cuniculi, demonstrating that in a population of P. cuniculi this bacterium is not transmitted transovarially.     

Serratia marcescens SYBC08 catalase isolated from sludge containing hydrogen peroxide shows increased catalase production by regulation of carbon metabolism

A high‐catalase‐producing strain, which was isolated from sludge containing hydrogen peroxide, was identified as Serratia marcescens SYBC08 by 16S rDNA sequence analysis. Serratia spp. was reported as non‐spore‐forming bacterium (except S. marcescens spp. sakuensis), but in our study electron microscopic observation revealed that the strain did produce spores. The content of the main fatty acid C_16:0 (14.8%) was significantly different from that of S. marcescens spp. sakuensis (33.2%) and S. marcescens spp. marcescens DSM 30121^T (34.8%), and the biochemical characteristics were not identical to those of S. marcescens spp. sakuensis. We speculate that the relatively high catalase activity and the spore structures may enable the strain to survive in a hydrogen peroxide environment. The most suitable carbon and nitrogen sources for the catalase production by S. marcescens SYBC08 were citric acid and corn steep liquor powder. A strategy of carbon metabolism regulation to enhance the catalase production was exploited. In the 7‐L fermenter, catalase production (20 353 U/mL) obtained in the presence of glucose and citric acid was 1.68‐ and 1.31‐fold higher than that obtained in the presence of glucose or citric acid, at equimolar carbon concentration. This production yield was much higher than that of many catalase‐producing strains, but only slightly lower than the production by Micrococcus luteus (34 601 U/mL). The results suggest that the new spore‐forming S. marcescens SYBC08 is a potential candidate for the production of catalase.     

Role of <Emphasis Type="Italic">Serratia marcescens</Emphasis> ACE2 on diesel degradation and its influence on corrosion

A facultative anaerobic species Serratia marcescens ACE2 isolated from the corrosion products of diesel transporting pipeline in North West, India was identified by 16S rDNA sequence analysis. The role of Serratia marcesens ACE2 on biodegradation of diesel and its influence on the corrosion of API 5LX steel has been elucidated. The degrading strain ACE2 is involved in the process of corrosion of steel API 5LX and also utilizes the diesel as an organic source. The quantitative biodegradation efficiency (BE) of diesel was 58%, calculated by gas-chromatography–mass spectrum analysis. On the basis of gas-chromatography–mass spectrum (GC–MS), Fourier Transform infrared spectroscopy (FTIR) and X-ray diffractometer (XRD), the involvement of Serratia marcescens on degradation and corrosion has been investigated. This basic study will be useful for the development of new approaches for detection, monitoring and control of microbial corrosion.     

The Survival of Ingested <Emphasis Type="Italic">Serratia marcescens</Emphasis> in Houseflies (<Emphasis Type="Italic">Musca domestica</Emphasis> L.) After Electrocution with Electric Fly Killers

Electric fly killers (EFKs) are commonly used to control flying insects that enter food establishments. For establishment of the incidence of pathogen-bearing insects in food establishments, insect samples obtained from EFK trays could be used. The principal difficulty with this approach is that the survival time of microorganisms on or within insect corpses after electrocution is unknown. This study determined the survival of Serratia marcescens (as a representative of the enteric bacteria) within houseflies following their electrocution by a commercial EFK. S. marcescens was successfully ingested by houseflies and survived on and within the corpses after electrocution for up to 5 weeks. Maximal levels of bacteria were recovered 24 h postelectrocution. The study also demonstrates the ability of ingested S. marcescens to out-compete resident microbial flora within houseflies. The findings are intended to pave the way for further research to determine the incidence of pathogen-laden flying insects in food establishments. Received: 30 April 2002 / Accepted: 3 July 2002     

Genetic studies of the ribosomal proteins in Escherichia coli. 8. Mapping of ribosomal protein components by intergeneric mating experiments between Serratia marcescens and Escherichia coli

Summary Ribosomal protein compositions of Serratia marcescens and Escherichia coli K12 were analyzed by using carboxymethyl cellulose column chromatography. Nine 50S and nine 30S ribosomal proteins of E. coli K12 could be distinguished from those of S. marcescens on the chromatogram.Episomes of E. coli K12, which cover the streptomycin(str) region of the chromosome, were transferred to S. marcescens. Chromatographic analyses were made on the ribosomal proteins extracted from these hybrid strains. At least nine 30S and six 50S ribosomal proteins of E. coli-type could be detected in the ribosomes of the hybrid strains in addition to the ribosomal proteins of S. marcescens.     

Modeling 1,2-dichloroethane biodegradation by <Emphasis Type="Italic">Klebsiella oxytoca va 8391</Emphasis> immobilized on granulated activated carbon

A mathematical model of the biodegradation of xenobiotics by microbial cells attached to particles of granulated activated carbon was developed. The model allowed the quantitative evaluation of different characteristics of the biofilm behavior: retarded microbial growth, increased concentration of immobilized cells compared to suspended cultures, potential cell detachment from the solid support and consequent independent growth of free cells. The applicability of the model was demonstrated for our own experimental data for 1,2- dichloroethane (DCA) biodegradation by Klebsiella oxytoca VA 8391 cells attached to granulated activated carbon. Two types of reactors, recirculated batch and continuous flow bioreactor, were studied. It was shown that in all investigated cases, the major contribution to DCA biodegradation was provided by the immobilized cells. Furthermore, immobilized cells were found to tolerate much higher substrate concentration and dilution rates in continuous culture than the free cells.     

Cloning of rpsO, the gene for ribosomal protein S15 of Escherichia coli

Summary The gene for Escherichia coli ribosomal protein S15 (rpsO) was cloned on the vector pBR322 from F-prime JCH55 DNA. The recombinant plasmid was transformed to Serratia marcescens cells and it was proved that E. coli S15 was synthesized and incorporated into ribosome particles in S. marcescens cells. A DNA fragment containing rpsO was also inserted into the vector pRF3, which changes its copy number depending on the growth temperature in a temperature-sensitive polA host. By use of this recombinant plasmid it was shown that the relative synthesis rate of S15 increased about twice even when the copy number of the plasmid increased more than twenty-fold.     

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.     

Protoplast-like structures formation from two species of enterobacteriaceae by fosfomycin treatment

A procedure for protoplasts formation from Escherichia coli and Serratia marcescens by treatment with fosfomycin alone is described. This method gives high and low yields of stable protoplasts from E. coli and S. marcescens respectively. In the last case numerous spheroplasts were obtained. Electron micrographs of intact cells, protoplasts and spheroplasts are shown.     

Role of prodigiosin and chitinases in antagonistic activity of the bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis> against the fungus <Emphasis Type="Italic">Didymella applanata</Emphasis>

The molecular features of antagonism of the bacterium Serratia marcescens against the plant pathogenic fungus Didymella applanata have been studied. The chitinases and the red pigment prodigiosin (PG) of S. marcescens were isolated and characterized. Specific antifungal activity of the purified PG and chitinases against D. applanata was tested in vitro. The antagonistic properties of several S. marcescens strains exhibiting different levels of PG and chitinase production were analyzed in vitro with regard to D. applanata. It was found that the ability of S. marcescens to suppress the vital functions of D. applanata depends mainly on the level of PG production, whereas chitinase production does not provide the bacterium with any competitive advantage over the fungus.     

Selective blockage of Serratia marcescens ShlA by nickel inhibits the pore‐forming toxin‐mediated phenotypes in eukaryotic cells

Serratia marcescens is an opportunistic pathogen with increasing incidence in clinical settings. This is mainly attributed to the timely expression of a wide diversity of virulence factors and intrinsic and acquired resistance to antibiotics, including β‐lactams, aminoglycosides, quinolones, and polypeptides. For these reasons, S. marcescens has been recently categorised by the World Health Organization as one priority to strengthen efforts directed to develop new antibacterial agents. Therefore, it becomes critical to understand the underlying mechanisms that allow Serratia to succeed within the host. S. marcescens ShlA pore‐forming toxin mediates phenotypes that alter homeostatic and signal transduction pathways of host cells. It has been previously demonstrated that ShlA provokes cytotoxicity, haemolysis and autophagy and also directs Serratia egress and dissemination from invaded nonphagocytic cells. However, molecular details of ShlA mechanism of action are still not fully elucidated. In this work, we demonstrate that Ni²⁺ selectively and reversibly blocks ShlA action, turning wild‐type S. marcescens into a shlA mutant strain phenocopy. Combined use of Ni²⁺ and calcium chelators allow to discern ShlA‐triggered phenotypes that require intracellular calcium mobilisation and reveal ShlA function as a calcium channel, providing new insights into ShlA mode of action on target cells.     

Prodigiosin is not a determinant factor in lysis of Leishmania (Viannia) braziliensis after interaction with Serratia marcescensd-mannose sensitive fimbriae

In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120 min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37 °C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28 °C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365.     

Ethyl Methanesulfonate Mutagenesis–Enhanced Mineral Phosphate Solubilization by Groundnut-Associated <Emphasis Type="Italic">Serratia marcescens</Emphasis> GPS-5

Twenty-three bacterial isolates were screened for their mineral phosphate–solubilizing (MPS) ability on Pikovskaya and National Botanical Research Institute’s phosphate (NBRIP) agar. The majority of the isolates exhibited a strong ability to solubilize hydroxyapatite in both solid and liquid media. The solubilization in liquid medium corresponded with a decrease in the pH of the medium. Serratia marcescens GPS-5, known for its biocontrol of late leaf spot in groundnut, emerged as the best solubilizer. S. marcescens GPS-5 was subjected to ethyl methanesulfonate (EMS) mutagenesis, and a total of 1700 mutants, resulting after 45 minutes of exposure, were screened on buffered NBRIP medium for alterations in MPS ability compared with that of the wild type. Seven mutants with increased (increased-MPS mutants) and 6 mutants with decreased (decreased-MPS mutants) MPS ability were isolated. All seven increased-MPS mutants were efficient at solubilizing phosphate in both solid and liquid NBRIP medium. Among the increased-MPS mutants, EMS XVIII Sm-35 showed the maximum (40%) increase in the amount of phosphate released in liquid medium compared with wild-type S. marcescens GPS-5, therefore, it would be a useful microbial inoculant in groundnut cultivation. EMS III Sm W, a nonpigmented mutant, showed the lowest solubilization of phosphate among the 6 decreased-MPS mutants.     

Multi-copy repression of Serratia marcescens nuclease expression by dinI

The dinI homolog of S. marcescens was cloned from a plasmid library by virtue of its ability to inhibit nuclease expression from the S. marcescens nucA gene integrated in the genome of E. coli. The S. marcescens DinI protein is 68% identical to DinI of E. coli. It has a similar effect on other SOS regulated genes and likely exerts it effect on nuclease expression, which is most pronounced as the cells entered stationary phase, through inhibition of basal SOS expression. Received: 12 April 2001 / Accepted: 14 May 2001     

Serratia marcescens as a rapid indicator of Microctonus hyperodae oviposition activity in Listronotus maculicollis and potential application of the technique to host-specificity testing

Listronotus maculicollis (Dietz) (Coleoptera: Curculionidae) is a potential novel host of the braconid parasitoid Microctonus hyperodae Loan, but initial studies have shown that levels of parasitism are lower than in the natural host L. bonariensis (Kuschel). A novel bacterial indicator test was used to determine whether the lower level of parasitism was due to behavioural factors, lack of oviposition, or host resistance. The incidence of ovipositor penetration by the parasitoid M. hyperodae into adult L. maculicollis was measured by immersing the ovipositor of the parasitoid in the facultative pathogen, Serratia marcescens Bizio. Adult weevils were then exposed to parasitoids for up to 72 h and rapid mortality used as an indicator of oviposition penetration. Survival was assessed after six days and surviving weevils were dissected and examined for parasitoid larvae. Mortality among L. maculicolis exposed to parasitoids treated with S. marcescens was significantly higher (P<0.001) than the controls but significantly lower (P<0.001) than in the natural host, L. bonariensis. Dissection of weevils exposed to uncontaminated parasitoids revealed that parasitism in L. maculicolis was significantly (P<0.001) less than parasitism in L. bonariensis. Serratia marcescens-induced mortality plus parasitism of surviving weevils in the parasitoid plus bacteria treatments produced a similar overall effect. Application of bacteria to the parasitoid ovipositor provided a rapid, simple test for ovipositor penetration, which shows potential for separation of behavioural and physiological defence mechanisms in parasitoid/host range studies.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Enhanced product formation in continuous fermentations with microbial cell recycle*

The effect of partial recycle of microbial cells on the operation of a chemostat has been investigated for two fermentations. Stable steady states without partial cell recycle were obtained for the conversion of D -sorbitol to L -sorbose by Gluconobacter oxydans subsp. suboxydans 1916B and for the conversion of glucose to 2-ketogluconic acid by Serratia marcescens NRRL B-486. The employment of partial cell recycle dramatically increased product formation rates for both fermentations.