Serratia Grimesii Research Articles

Characterization of bacteria isolated from maize roots: Emphasis on Serratia and infestation with corn rootworms (Chrysomelidae: Diabrotica)

Larval corn rootworms (Coleoptera: Chrysomelidae) are soil-dwelling insect pests that damage maize ( Zea mays L.) by consuming root tissues, thus lowering grain yield. Little is known about interactions between rootworms and soil bacteria, including potential impacts of maize rhizobacteria, such as entomopathogenic Serratia spp., on subterranean rootworm pests. We used selective growth medium (caprylate-thallous agar, CT) to quantify and isolate Serratia spp. from: (1) bulk soil, (2) roots of four field-grown maize genotypes, half of which were infested with rootworm eggs from a reared colony, and (3) non-diseased, larval rootworms from the same colony. Phenotypic testing and 16S rRNA gene sequencing were used to identify bacteria, including non- Serratia spp., that were successfully isolated on the CT solid medium. We also isolated and identified Serratia spp. associated with non-diseased and diseased Diabrotica adults. Serratia spp. associated with maize roots were more abundant than those associated with bulk soil, where they were undetectable with our methods except for Serratia grimesii. There was no impact of plant genotype on densities of bacteria isolated from maize roots. S. grimesii was frequently associated with maize roots, regardless of infestation with rootworm eggs. Serratia marcescens biotype A4, Serratia plymuthica and several other Enterobacteriaceae genera were also associated with maize roots. Infesting the soil with rootworm eggs enhance the occurrence of two strains of the S. marcescens biotype A1b, with comparable densities of these orange and pink strains within infested roots. However, both strains were associated with larval rootworms from the reared colony, which may indicate that rootworms were introducing these bacteria into infested maize roots. In addition, within larvae the orange strain was significantly more abundant than the pink strain, and was also associated with diseased rootworm adults. Our studies identified specific Serratia strains associated with diseased rootworms that may have potential as biological control agents, and additional Serratia biotypes associated with the maize rhizosphere that, based on the literature, may function as plant growth promoting agents via antagonistic action against plant-pathogenic fungi.

Grimelysin, a novel metalloprotease from Serratia grimesii, is similar to ECP32

Limited actin proteolysis is the hallmark of bacterial metalloprotease ECP32. While ECP32 has long been considered an Escherichia coli protein, the N-terminal amino acid sequence of the active enzyme described previously, could not been retrieved in the E. coli genome. We cloned, sequenced and characterized Serratia grimesii protease grimelysin and show that grimelysin is similar to the previously described protease ECP32.

Determination of histamine and histamine-forming bacteria in dried milkfish ( Chanos chanos) implicated in a food-borne poisoning

An incident of food-borne poisoning, causing illness in three victims due to ingestion of dried milkfish, occurred in February, 2006, in Tainan Prefecture, southern Taiwan. The leftovers of the victims’ dried milkfish and three other dried milkfish samples, from the same retail store as the leftover fish, were collected and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, aerobic plate count (APC) and total volatile basic nitrogen (TVBN) in the leftover dried milkfish sample were significantly higher than those of the other three milkfish samples. None of the tested milkfish samples contained total coliform and Escherichia coli. Although the histamine contents in the three other milkfish samples were less than 5 mg/100 g, the suspected milkfish sample contained 61.6 mg/100 g of histamine, which is greater than the hazard action level of 50 mg/100 g. Given the allergy-like symptoms of the victims and the high histamine content in the suspected milkfish sample, this food-borne poisoning was strongly suspected to be due to histamine intoxication. Four histamine-producing bacterial strains, capable of producing 11.9–1243 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH), were identified as Staphylococcus sciuri subsp. sciuri, Serratia grimesii, Bacillus cereus and Raoultella ornithinolytica, by 16S rDNA sequencing with PCR amplification. R. ornithinolytica was a potent histamine-former capable of producing more than 800 ppm of histamine in TSBH in the presence of 1.5% or 3.5% NaCl. Therefore, R. ornithinolytica could be the causative bacterium responsible for the production of high content of histamine that eventually led to this dried milkfish poisoning incident.

The first study on the bacterial flora of the european spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

AbstractThe European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera, Scolytidae), is one of the most serious pests of oriental spruce (Picea orientalis L.) in Turkey. In this study, we investigated bacterial flora of D. micans collected from different populations of the forests of Eastern Black Sea Region of Turkey from 2002 to 2004. Seven different bacteria were isolated from healthy, diseased and dead specimens based on the color of colony and morphology. According to morphological, physiological and biochemical properties, metobolic enyzme profile by BIOLOG microtiter plate system, and total cellular fatty acid profile by Microbial Identification System (MIS), isolates were identified as Micrococcus luteus, Bacillus thuringiensis subsp. morrisoni, Serratia grimesii, Enterobacter cloaceae, Enterobacter intermedius, Streptococcus sp. and Pseudomonas putida. This is the first study on the bacterial flora of D. micans.

IDENTIFICATION AND EFFICIENCY OF BACTERIAL STRAINS ISOLATED FROM INFECTED LARVAE OF COTTON PINK BOLLWORM Pectinophora gossypiella AND SPINY BOLLWORM Earias insulana (LEPIDOPTERA: NOCTUIDAE)

From infected pink and spiny bollworm larvae, collected from Qualyobia Gover-norate, Egypt, 13 bacterial isolates belonging to 7 species were detected. The effi-ciency of these bacterial isolates was evaluated on newly hatched pink and spiny bollworm larvae. Three bacterial species, namely, Pseudomonas viridiflava, Serratia grimesii and Cellulomonas flavigena had no efficiency. Meanwhile, four other en-tomopathogenic isolates bacterial species, Pseudomonas pyrrocinia (A1), Serratia marcesens (M3), Serratia rubidaea (E3) and Bacillus thuringiensis (S2) had notice-able efficiency. The efficiency of these isolates was compared to two commercial products, Dipel 2X and Protecto. Biochemical studies showed differences in total proteins bands patterns in uninfected and infected larvae.

Interactive inhibition of meat spoilage and pathogenic bacteria by lysozyme, nisin and EDTA in the presence of nitrite and sodium chloride at 24 °C

To develop a nisin- and lysozyme-based antimicrobial treatment for use with processed ham and bologna, in vitro experiments were conducted to determine whether inhibition enhancing interactions occur between the antimicrobials lysozyme, chrisin (a commercial nisin preparation), EDTA, NaCl and NaNO 2. Inhibitory interactions were observed between a number of agents when used against specific pathogenic and spoilage bacteria. The observed interactions included lysozyme with EDTA ( Enterococcus faecalis and Weissella viridescens), chrisin with EDTA (all Gram-positive organisms), EDTA with NaCl ( Escherichia coli, Salmonella enterica serovar Typhimurium, Serratia grimesii), EDTA with nitrite ( E. coli, Lactobacillus curvatus, Leuconostoc mesenteroides, Listeria monocytogenes, S. Typhimurium), chrisin with nitrite ( Lc. mesenteroides, L. monocytogenes), and NaCl with nitrite ( S. Typhimurium, Shewanella putrefaciens). Previous reports have described interactions between nisin with EDTA that resulted in enhanced antimicrobial effect against Gram-negative bacteria, or lysozyme with nisin against Gram-positive bacteria. These interactions were not observed in these experiments. We observed that unlike previous studies, these experiments were conducted on growing cells in nutrient broth, rather than under conditions of nutrient limitation. We propose that screening of antimicrobials for use in food systems in nutrient-deficient systems is inappropriate and that new protocols should be developed.

Biogenic amines produced by Enterobacteriaceae isolated from meat products

Biogenic amines in ground meat and processed meat products are one of the indicators to determine the poor quality raw material. Major histamine forming bacterium was Escherichia coli (strain EC04 with 65.88 mg/100 ml in Brain Heart Infusion medium), followed by the microorganisms Morganella morganii (strain MM4 with 8.40 mg/100 ml and strain MM7 with 8.28 mg/100 ml) and Proteus mirabilis (strain PM02 with 8.76 mg/100 ml), respectively. The highest putrescine production level was found in Citrobacter freundii and Enterobacter spp. strains, followed by Serratia grimesii, Proteus alcalifaciens, E. coli, Escherichia fergusonii, Morganella morganii, Proteus mirabilis, Proteus penneri and Hafnia alvei, respectively. The most important cadaverine producer was E. coli EC03 with a production level of 45.48 mg/100 ml, and of the strains Escherichia vulnaris EV01, Escherichia fergusonii EF06 produced 37.92 mg/100ml and 35.40 mg/100 ml, respectively. On the basis of our results, it can be concluded that the major biogenic amines produced by Enterobacteriaceae are putrescine, cadaverine, tyramine and histamine, both in culture medium and meat products.

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community.

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log 10 5 colony forming units g −1 fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.

Inhibition of bacterial growth on ham and bologna by lysozyme, nisin and EDTA

Ham and bologna sausages were prepared with or without addition of 500 mg kg −1 lysozyme:nisin, 1:3, and 500 mg. kg −1 EDTA. Sausages were inoculated with one of; Brochothrix thermosphacta, Escherichia coli O157:H7, Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, Listeria monocytogenes, Salmonella typhimurium, Serratia grimesii or Shewanella putrefaciens, vacuum packed and stored for 4 weeks at 8°C. Plate counts were made on selective and nonselective media. Inhibitor treatment reduced initial populations of B. thermosphacta and Lc. mesenteroides on both meats . Treatment of ham and bologna prevented growth of B. thermosphacta, to week 4. Treatment reduced growth of Lb. curvatus on ham and bologna, to week 3. Treatment of bologna reduced growth of Lc. mesenteroides and L. monocytogenes for 2 weeks. Treatment of ham reduced growth of E. coli O157:H7 for 4 weeks. On treated ham the growth of S. typhimurium was increased from week 3. No difference was observed between control and treatment samples with other organisms.

Inhibition of spoilage and pathogenic bacteria on agar and pre-cooked roast beef by volatile horseradish distillates

Bacteria inoculated on agar and roast beef slices were incubated at 12°C in a sealed model system with increasing concentrations of a volatile distillate extracted from fresh horseradish root. The distillate contained approximately 90% allyl isothiocyanate and 9% 2-phenethyl isothiocyanate. Growth of Staphylococcus aureus, Escherichia coli O157:H7, Salmonella typhimurium, Listeria monocytogenes and Serratia grimesii on agar was completely inhibited for seven days in aerobic storage at 12°C when 4000 nl distillate/l air was added to the model system at the outset. Complete inhibition of Lactobacillus sake required 20 000 nl l −1. Bactericidal activity varied between species and increased with distillate concentration. Bacteria inoculated on roast beef were more resistant to the bacteriostatic effect of the distillate and 20 000 nl l −1 were required to completely inhibit growth. Lactobacillus sake was weakly inhibited at this concentration. Bactericidal activity was observed only for Staphylococcus aureus, Escherichia coli and Serratia grimesii. Residual allyl isothiocyanate (AIT) concentrations in the head space above agar decreased to 11.4, 15.7 and 31.1% of original concentrations after 7 days in storage for initial distillate inputs of 2000, 4000 and 20 000 nl l −1, respectively, and to 1.1, 2.4 and 21.2% in the roast beef model system. Differences in bacteriostatic and bactericidal activities were attributed to enhanced depletion of AIT in the meat model system. The safety and shelf-life of packaged, cooked, uncured roast may be enhanced by storage under atmospheres containing AIT-rich horseradish distillates.

Association of a Large Plasmid with Amber Disease of the New Zealand Grass Grub, Costelytra zealandica, Caused by Serratia entomophila and Serratia proteamaculans

Amber disease, a chronic disease of Costelytra zealandica (Coleoptera: Scarabaeidae) larvae, is caused by some isolates of the bacteria, Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae). A large plasmid was visualized in some isolates of these two Serratia species. Further investigation showed that 18 of 19 pathogenic isolates of Serratia spp. were found to have a megaplasmid, estimated to be 105 kb. The one virulent isolate lacking a 105-kb plasmid had two smaller plasmids. The 105-kd plasmid was present in only 1 of 22 naturally occurring, nonpathogenic isolates examined. Plasmid DNA isolated from pathogenic isolates of each species showed identical restriction patterns. Heat-treated derivatives of pathogenic S. entomophila and S. proteamaculans isolates, which lost disease-causing ability, also lost the 105-kb plasmid. Among isolates of 7 other species of Serratia, single isolates of Serratia grimesii and Serratia ficaria were found to have a similarly sized plasmid, but caused no disease symptoms. The implications of the association between the plasmid and amber disease are discussed.

Taxonomic Diversity of the D-Glucose Oxidation Pathway in the Enterobacteriaceae

Tests allowing the screening of large numbers of enterobacterial strains for the presence of the glucose oxidation pathway (glucose, gluconate, and 2-ketogluconate dehydrogenases) were devised or adapted. A total of 506 strains representing 111 taxa (named species or subspecies and unnamed genomic groups) were studied. The members of the genera Budvicia, Edwardsiella, Leminorella, Providencia, and Xenorhabdus and the species Citrobacter freundii, Erwinia carnegeana, Erwinia carotovora, Erwinia chrysanthemi, Erwinia nigrifluens, Erwinia salicis, Moellerella wisconsensis, Proteus penneri, Proteus vulgaris, Yersinia intermedia, Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia ruckeri were negative in all tests. Five species, Erwinia cypripedii, Ewingella americana, Rahnella aquatilis, Serratia marcescens (at 20°), and Tatumella ptyseos produced 2,5-diketogluconate from glucose without a requirement for pyrroloquinoline quinone (PQQ). When PQQ was provided (required for glucose oxidation), Serratia grimesii and Serratia liquefaciens produced 2,5-diketogluconate from glucose at 20°. Escherichia blattae had gluconate- and 2-ketogluconate dehydrogenases without glucose dehydrogenase. The members of the genera Hafnia, Obesumbacterium, and Pragia had only gluconate dehydrogenase. Other species had glucose dehydrogenase (with or without a requirement for PQQ) with or without gluconate dehydrogenase. Classification and identification may take advantage of tests exploring the glucose oxidation pathway.