Search

We report the complete genome sequence of Moraxella osloensis strain YV1, which was isolated from a wastewater treatment plant in Australia. The YV1 genome comprises a 2,615,801-bp chromosome and four plasmids. Moraxella osloensis strain YV1 displays the distinctive morphology of Eikelboom morphotype 1863.

Here, we present the complete whole-genome sequences of three Moraxella osloensis strains with octylphenol polyethoxylate-degrading abilities. These strains were isolated from human skin.

We report the complete genome sequence of Moraxella osloensis strain KMC41, isolated from laundry with malodor. The KMC41 genome comprises a 2,445,556-bp chromosome and three plasmids. A fatty acid desaturase and at least four β-oxidation-related genes putatively associated with 4-methyl-3-hexenoic acid generation were detected in the KMC41 chromosome.

Moraxella osloensis is a gram-negative bacterium associated with Phasmarhabditis hermaphrodita , a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug, Deroceras reticulatum . This bacterium-feeding nematode acts as a vector that transports M. osloensis into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex. We discovered that M. osloensis produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity. Washed or broken cells treated with penicillin and streptomycin from 3-day M. osloensis cultures were more pathogenic than similar cells from 2-day M. osloensis cultures. However, heat and protease treatments and 2 days of storage at 22°C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics. This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells. Moreover, 2 days of storage of the young washed bacterial cells at 22°C enhanced their endotoxin activity if they were not treated with the antibiotics. Furthermore, purified lipopolysaccharide (LPS) from the 3-day M. osloensis cultures was toxic to slugs, with an estimated 50% lethal dose of 48 μg per slug, thus demonstrating that the LPS of M. osloensis is an endotoxin that is active against D. reticulatum . This appears to be the first report of a biological toxin that is active against mollusks.

Using a polyphasic approach (including cellular protein and fatty acid analysis, biochemical characterization, 16S ribosomal DNA sequencing, and DNA-DNA hybridizations), we characterized 51 bacterial isolates recovered from respiratory secretions of cystic fibrosis (CF) patients. Our analyses showed that 24 isolates belong to taxa that have so far not (or only rarely) been reported from CF patients. These taxa include Acinetobacter sp., Bordetella hinzii , Burkholderia fungorum , Comamonas testosteroni , Chryseobacterium sp., Herbaspirillum sp., Moraxella osloensis , Pandoraea genomospecies 4, Ralstonia gilardii , Ralstonia mannitolilytica , Rhizobium radiobacter , and Xanthomonas sp. In addition, one isolate most likely represents a novel Ralstonia species, whereas nine isolates belong to novel taxa within the α- Proteobacteria . Eight of these latter isolates are classified into the novel genus Inquilinus gen. nov. as Inquilinus limosus gen. nov., sp. nov., or as Inquilinus sp. The remaining 17 isolates are characterized as members of the family Enterobacteriaceae . The recovery of these species suggests that the CF lung is an ecological niche capable of supporting the growth of a wide variety of bacteria rarely seen in clinical samples. Elucidation of the factors that account for the association between these unusual species and the respiratory tract of CF patients may provide important insights into the pathophysiology of CF infection. Because accurate identification of these organisms in the clinical microbiology laboratory may be problematic, the present study highlights the utility of reference laboratories capable of identifying unusual species recovered from CF sputum.

The classic silver staining method for detecting bacterial lipopolysaccharides (LPSs) in polyacrylamide gels (C. Tsai and C. E. Frasch, Anal. Biochem. 119:115-119, 1982) was at least 20 times more sensitive than the modified silver staining method (A. Fomsgaard, M. A. Freudenberg, and C. Galanos, J. Clin. Microbiol. 28:2627-2631, 1990) for detecting LPS from the bacterium Moraxella osloensis. However, the classic method is only about three to four times more sensitive than the modified method for detecting LPSs from Escherichia coli J5, EH100, and O111:B4 or Salmonella enterica serovar Typhimurium. The reduction of sensitivity is due to omission of the initial fixing step in the modified method. The retention of LPS fractions in the gels during fixing and/or oxidation may depend on the structures of their lipid A moieties. Lipopolysaccharides (LPSs) play a major role in the pathogenesis of gram-negative infections (1). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining has been used extensively to characterize LPS (3, 11, 12). Tsai and Frasch (11) first reported a highly sensitive classic silver staining method for detecting LPS in polyacrylamide gels. This classic method can detect even less than 5 ng of the rough type of LPS. However, Fomsgaard et al. (2) revealed that the classic method did not stain certain LPS preparations containing a low number of fatty acids, which were washed out of the gels during the initial fixing step (40% ethanol-5% acetic acid, overnight). Thus, they developed a modified silver staining method by omitting the fixing step and increasing the LPS periodic acid oxidation (the second step) time from 5 min to 20 min to restore the ability to detect all LPSs. We purified LPS from Moraxella osloensis, a bacterium associated with a slug-parasitic nematode, Phasmarhabditis hermaphrodita (8, 9). M. osloensis LPS is an active endotoxin against the slug Deroceras reticulatum (10). Analysis of different quantities of M. osloensis LPS or four commercially available LPSs from other bacteria by SDS-PAGE followed by each of the two methods revealed that the modified method was less sensitive than the classic method. M. osloensis LPS was purified by classical phenol-water extraction (13), with modification as described by Gu et al. (3), from 3-day pure cultures of M. osloensis supplied by MicroBio, Ltd., Cambridge, United Kingdom. LPS preparations from Escherichia coli J5, EH100, and O111:B4 strains and Salmonella enterica serovar Typhimurium were purchased from Sigma Chemical Company, St. Louis, Mo. LPS preparations were treated for 5 min at 100°C in 0.05 M Tris-HCl buffer (pH 6.8) containing 2% (wt/vol) SDS, 10% (wt/vol) sucrose, and 0.01% bromophenol blue. Ten microliters of each sample was then loaded on precast Ready Gel Tris-HCl polyacrylamide gels (86 by 68 by 1.0 mm) containing 4 and 15% acrylamide in the stacking and separating gels, respectively (Bio-Rad Laboratories, Inc., Hercules, Calif.). Electrophoresis was performed at 12 mA in the stacking gels and 25 mA in the separating gels until the bromophenol blue had run about 6.7 cm. LPSs in the gels were visualized by either the classic method (11) or the modified method (2). The sensitivities of the two methods were compared by using from 50 ng to 5 μg of M. osloensis LPS (Fig. ​(Fig.1).1). The LPS was revealed to be a rough-type LPS, because only one main band was detected in the gels by both methods. The band patterns obtained at 50 ng by the classic method were equivalent to or better than those obtained at 1 μg by the modified method. Therefore, the classic method is at least 20 times more sensitive than the modified method for detecting M. osloensis LPS. FIG. 1. Comparison of sensitivities of two silver staining methods for detecting M. osloensis LPS. (A) Modified method. (B) Classic method. The lanes contain the following amounts of M. osloensis LPS: 1, 50 ng; 2, 100 ng; 3, 1 μg; 4, 5 μg. The ... The sensitivities of the two methods were also compared by using the same quantities of rough-type LPS from E. coli J5 or EH100 (Fig. ​(Fig.2).2). The band patterns obtained at 50 ng by the classic method were better than those obtained at 100 ng by the modified method. However, the band patterns obtained at 1 μg by the classic method were less clear than those obtained at 5 μg by the modified method. Thus, the classic method is about three to four times more sensitive than the modified method for detecting the two rough-type LPSs. A similar level (three to four times) of difference in LPS detection sensitivities between the two methods was also observed for the smooth-type LPS from E. coli O111:B4 or S. enterica serovar Typhimurium (Fig. ​(Fig.33). FIG. 2. Comparison of sensitivities of two silver staining methods for detecting E. coli J5 or EH100 LPS. (A) Modified method. (B) Classic method. Lanes 1 to 4 contain the following amounts of E. coli J5 LPS: 1, 50 ng; 2, 100 ng; 3, 1 μg; 4, 5 μg. ... FIG. 3. Comparison of sensitivities of two silver staining methods for detecting E. coli O111:B4 or S. enterica serovar Typhimurium LPS. (A) Modified method. (Note that the background was overstained.) (B) Classic method. (Note that a small piece of the gel was ... M. osloensis LPS in the gels was also visualized by the modified method, except that the periodic acid oxidation time was increased from 20 min to 100 min (Fig. ​(Fig.4).4). With the increase in oxidation time, the band patterns obtained at 1 or 5 μg of LPS became better and clearer. It is estimated that the sensitivity of the modified method for detecting M. osloensis LPS increases about one to two times with the increase in the oxidation time from 20 min to 100 min. Thus, increasing the oxidation time of LPS from 5 min to 20 min in the modified method at least should not reduce LPS detection sensitivity. Therefore, the reduction in sensitivity is due to the omission of the initial fixing step in the modified method. FIG. 4. Effects of 20 (A) and 100 (B) min of periodic acid oxidation on sensitivity of the modified silver staining method for detecting M. osloensis LPS. Lanes 1 and 2 contain 1 and 5 μg, respectively, of M. osloensis LPS. The figure was created with ... It is not fully clear why the detection sensitivities of the two methods differ for M. osloensis LPS and the other LPSs tested. The four purchased LPSs have different structures of polysaccharide moiety, but had similar levels (three to four times) of difference in LPS detection sensitivity by the two methods. This could be due to the same structure of lipid A moiety being present in the four LPSs (5, 7). The structure of M. osloensis LPS is unknown. Since the structure of lipid A for a given bacterial genus usually exhibits constant characteristics (6), the structure of lipid A from M. osloensis LPS can be forecast from the known lipid A structure for Moraxella catarrhalis LPS (4). Thus, it is forecast that lipid A from M. osloensis LPS contains seven fatty acids, including four 3-hydroxydodecanoic acids, two decanoic acids, and one dodecanoic acid. In contrast, lipid A from the purchased LPS only contains six fatty acids, including five 3-hydroxytetradecanoic acids and one dodecanoic acid (5, 7). Fomsgaard et al. (2) suggested that the retention of LPS fractions in the gels during fixing and/or oxidization may be a property of the number of fatty acids present in their lipid A moiety. However, since the lipid A from the purchased LPS contains the same number of fatty acids as M. osloensis lipid A, the retention of LPS fractions in the gels during fixing and/or oxidation may depend on the structure of their lipid A moiety (e.g., fatty acid pattern or conformation of lipid A), but not the number of the fatty acids. It is possible that those LPSs containing a low number of fatty acids are fixed weakly or rarely in the gels, whereas M. osloensis LPSs are fixed much more slowly than the other LPSs tested, thus requiring more time (e.g., overnight) to be fixed mostly or completely in the gels. We conclude that each of the two methods has its advantages and disadvantages. The classic method is more sensitive, but time-consuming. Furthermore, it does not detect those LPSs containing a low number of fatty acids. In contrast, the modified method is simpler and faster and detects LPS that would not be stained by the classic method. However, the present results reveal that the modified method has lower LPS detection sensitivity than the classic method for all LPSs tested, especially that from M. osloensis. Therefore, it is suggested that unknown bacterial LPS preparations in the gels be visualized by both methods.

Gentisate:oxygen 1,2-oxidoreductase (decyclizing) (EC 1.13.11.4; gentisate 1,2-dioxygenase) from Moraxella osloensis was purified to homogeneity as shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 154,000 and gives rise to subunits of molecular weight 40,000 in the presence of sodium dodecyl sulfate. Gentisate 1,2-dioxygenase showed broad substrate specificity and attacked a range of halogen- and alkyl-substituted gentisic acids. Maleylpyruvate, the product formed from gentisate, was degraded by cell extracts supplemented with reduced glutathione, but substituted maleylpyruvates were not attacked under these conditions.

A genetic transformation assay for unequivocal identification of strains of Moraxella osloensis is described. In this assay a stable tryptophan auxotroph is transformed to prototrophy by deoxyribonucleic acid (DNA) samples from other strains of M. osloensis but not by DNA samples from unrelated bacteria. The test is simple to perform and definitive results can be obtained in less than 24 h. The procedure, which is suitable for routine diagnosis in a clinical laboratory, involves a rapid method for preparation of crude transforming DNA from small quantities of bacterial cells and permits simultaneous examination of large numbers of isolated cultures. The assay was shown to correctly identify 27 strains previously classified as M. osloensis . Forty-five other gram-negative, oxidase-positive, nonmotile coccobacilli, which might be confused with M. osloensis unless subject to more extensive testing, were shown to be unrelated genetically to M. osloensis . The transformation assay clearly distinguishes M. osloensis from Acinetobacter . Although most strains of M. osloensis are nonfastidious, being able to grow in a mineral medium supplemented with a single organic carbon source, one of the strains tested was only able to grow on fairly complex media and could not be transformed to grow on simple media. Inability to alkalize Simmons citrate agar was shown not to be characteristic of all strains of M. osloensis .

Moraxella osloensis osteomyelitis of the femur developed in a paraplegic man. He responded to treatment with oral ampicillin. Disease in humans caused by this unusual clinical isolate is reviewed.