Jeffrey C. Burnham

Entrapment and lysis of the cyanobacterium Phormidium luridum by aqueous colonies of Myxococcus xanthus PCO2

A Myxococcus xanthus isolate from a farm drainage ditch, designated strain PCO2, is capable of rapidly inducing lysis of both agar and liquid-grown cultures of the cyanobacterium, Phormidium luridum, var. olivacea. Microscopic studies of the predator-prey interaction demonstrate that lysis of the cyanobacterium occurs within clumps and spherules formed by the cells of M. xanthus PCO2. In the earliest stage, one sees the formation of irregular microclumps of bacteria and cyanobacterial filaments. As these clumps mature, colonies 1 to 6 mm in diameter develops. The center of these densely green colonies contains cyanohacteria in various stages of degradation, while the periphery is almost exclusively a tightly woven mass of myxobacterial cells. Electron microscopy shows that long extrusions from the outer membrane of the M. xanthus PCO2 cells are involved in the formation both of initial clumps and of mature colonial spherules. These extrusions appear to efficiently entangle the cyanobacterial filaments in the culture environment. Predator-to-prey ratios of 1/10, 1/100 and 1/1,000 have resulted in cyanobacterial lysis. Because the entrapment and lysis of P. luridum filaments by M. xanthus PCO2 appears to be independent of any other heterotrophic nutritional requirement, as well as of environmental agitation, this system has potential as a biological control technique for undesirable aquatic cyanobacteria.

Myxococcal predation of the cyanobacterium Phormidium luridum in aqueous environments

Two strains of Myxococcus xanthus, and a strain of Myxococcus fulvus were compared with respect to their ability to entrap and lyse trichomes of the cyanobacterium Phormidium luridum var. olivaceae. All of these isolates form colonial aggregates and spherules in either axenic culture with a tryptone-salts medium or in a mixed culture with viable cyanobacterial cells as the sole source of nutrients. Light microscopy showed evidence of swarming activity on the surface of all three myxococci with the accompanying formation of fruiting structures. Extended incubation of mixed cultures showed the myxococci to be capable of long-term control of the cyanobacterial population with predator-prey population cycling occurring on average every 9 days. Serial transfer of mixed cultures into either fresh autotrophic medium or cyanobacterial cultures of 107 per ml showed the persistence of predatory activity. Myxococcal densities were shown to return repeatedly to initial virulent levels. Predator inoculum levels could be reduced to 50 cells per 100 ml in a cyanobacterial culture of 107 per ml. These in vitro data enhance the potential of the myxococcus predatory colony as a biological control agent for in situ cyanobacteria.

Effect of low concentrations of quinolone antibiotics on bacterial virulence mechanisms.

Recent studies have shown that exposure to quinolone antibiotics at or below the minimal inhibitory concentration (MIC) results in reduction in the level of production or total elimination of certain factors that contribute to the virulence of bacteria. This study was designed to determine whether low concentrations of enoxacin, lomefloxacin, and ciprofloxacin altered the morphology or affected the production of various virulence factors in several different genera of bacteria. The factors studied were nuclease and a toxin production in Staphylococcus aureus, cell size, pili and fimbriae production, and adherence of Salmonella typhimurium, Escherichia coli, and Pseudomonas aeruginosa to urinary epithelial cells and dog bladder cells, and the major virulence factor in Yersinia pseudotuberculosis. In addition, the effect of growth in low levels of enoxacin on phagocytosis of S. aureus by human polymorphonuclear leukocytes (PMNs) was studied. Following exposure to subinhibitory levels of quinolones tested, significant reduction in activity or complete elimination was seen in all of those factors measured. Minor differences were noted in the efficiency of elimination among the three quinolones tested. At as low as 1/8 MIC there is significant enhancement of phagocytic activity by human PMNs. These data suggest that exposure to quinolones at concentrations below the MIC disrupts the regulatory mechanisms that control cell morphology, adherence as well as exocellular enzyme production and plasmid maintenance. This may mean that certain virulent organisms that survive exposure to quinolone antibiotics may be less likely to produce or maintain the disease state in susceptible hosts.

Trichinella spiralis: Behavior, structure, and biochemistry of larvae following exposure to components of the host enteric environment

Four layers are present on the surface of infective larvae of Trichinella spiralis isolated from host muscle in pepsin-HCl. Trypsin treatment of pepsin-HCl isolated worms caused partial degradation and removal of large patches of the two outer surface layers. Following exposure to bile, only traces of the outer layers remained on the worms surface. These changes in the worm surface were accompanied by a shift from Type I behavior, typical of pepsin-HCl isolated larvae, to Type II behavior, (snakelike) following exposure to either trypsin or bile. Worm behavior was also temperature dependent. Type I behavior was typical of worms maintained at room temperature regardless of treatment, while Type II behavior displayed by worms held at 37 C was treatment dependent. The absorption of in vitro glucose or beta-methyl-D-glucoside was lowest in pepsin-HCl isolated first stage infective larvae, significantly higher in trypsin treated worms and greatest in worms following exposure to bile. Sugar uptake by worms isolated from the host small intestine after 1 hr of enteral infection was similar to that seen in worms isolated from host muscle in pepsin-HCl. Sugar uptake in vitro in worms 2 hr following enteral infection was similar to worms following exposure to bile. The highest levels of sugar absorption in vitro occurred in worms which had resided in the small intestine for 3 hr. The lowest rates of incorporation of label into worm tissues was seen in 1 hr enteral and pepsin-HCl isolated worms. Infective larvae treated with trypsin or bile incorporated significantly greater amounts of label than the two former groups. The highest levels of incorporation of label into worm tissues was seen in 3 hr enteral worms. These findings support the view that trypsin, bile, and temperature serve as environmental cues which lead to alteration of the parasites behavioral and nutritional status.

EXTRACELLULAR LYSIS OF THE BLUEGREEN ALGA PHORMIDIUM LURIDUM BY BDELLOVIBRIO BACTERIOVORUS1

When either cells of the bacterium, Bdellovibrio bacteriovorus (Stolp & Starr), strain 15143, or a heat‐resistant lytic factor derived from these cells is added to viable cultures of Phormidium luridum var. olivacea Boresch all the algal cells underwent gradual lysis. This effect was obtained with a mean initial bdellovibrio:algal cell ratio of 7.5:1. When P. luridum was mixed with the bdellovibrio cultures the algal chlorophyll content showed an 8‐fold decrease. Concomitantly, this interspecies interaction caused, a 75% inhibition of algal photosynthesis after‐4 h. Heat, treatment of the B. bacteriovorus culture supernatant fluid increased its ability to inhibit photosynthesis approximately 14%. Light, microscopy showed pale granules and intracellular spaces to form in the P. luridum within 16 h after adding the bdellovibrio lytic factor. Subsequent morphological changes included the development of large intracellular spaces, intercellular spaces, spheroplast formation and finally Complete lysis of the algal cells.

THE EFFECT OF SELENITE ON THE PHYSIOLOGICAL AND MORPHOLOGICAL PROPERTIES OF THE BLUE‐GREEN ALGA PHORMIDIUM LURIDUM VAR. OLIVACEA1

Phormidium luridum cultures were treated with sodium selenite in concentrations ranging from 10−6 M to 10−2 M. In contrast to the increasing culture turbidity of control and 10−6 M selenite cultures, the turbidity of the other selenite cultures declined in proportion to time and selenite concentration. Chlorophyll extraction revealed similar results. Photosynthetic activity was inhibited within 6 hr in all cultures except control and 10−6 M selenite. Phormidium at concentrations greater than 10−6 M selenite showed a gradual loss of the bright green color and turned semitransparent. Cell‐associated granules of reduced selenium were observed at higher selenite concentrations. Other structural changes observed were the presence of intracellular and intercellular spaces, spheroplast formation, and gradual cell lysis. Protein analyses of total cell samples and supernatant fractions confirmed cellular breakdown of selenite‐treated algal cells.

AN IMPROVED METHOD OF CELL ENUMERATION FOR FILAMENTOUS ALGAE AND BACTERIA1

A new simple method for estimating the number of individual cells per milliliter in suspensions of filamentous microorganisms is described. This 2‐part procedure utilizes a standard microscopic counting chamber and is independent of filament length or individual cell size. A statistical analysis of the method is also presented.

Elevated phosphorus in psoriatic skin determined by energy dispersive x-ray micro-analysis

Scanning electron‐microscopy in combination with secondary electron imaging and x‐ray energy spetrometry (electron‐probe micro‐analysis) was performed on the stratum corneum of involved and uninvolved, psoriatic skin. The significant, elemental alterations included an increase in phosphorus and calcium in the involved skin regions. A composite, chemical, elemental profile of normal and uninvolved, psoriatic stratum corneum is presented.

Scanning electron microscopic evidence of bacterial overgrowth in intertrigo.

A scanning electron microscopic (SEM) study of intertrigo is presented. Marked changes of the keratinocyte surface characteristics and in the number and distribution of bacteria in the various stages of intertrigo are reported. Bacterial overgrowth was significant. This study confirms the value of SEM in the study of the skin surface and in cutaneous microbiology.

Development of the male genitalia of Trichinella spiralis during the enteral phase of infection in the mouse: an SEM study.

The cycle of four molts of Trichinella spiralis is typical of all nematodes, culminating in adulthood (Villella, 1958, J. Parasitol. 44:41; Ali Khan, 1966, J. Parasitol. 52: 248-259; Kozek, 1971, J. Parasitol. 57: 1015-1028). Data regarding the timing of each molt have recently been reviewed (Despommier, 1983. In Trichinella and trichinosis, W. C. Campbell (ed.). Plenum Press, New York, pp. 75-152). The precise timing of each of the four molts varied according to the three aforementioned studies. However, those investigations did show that each molt occurs in rapid succession following the first (i.e., 8-10 hr following oral infection with infective Lis isolated by pepsin-HCl). Kozek (1971, loc. cit.) performed a detailed light microscopic examination of the morphology of the male and female reproductive system throughout the worms enteral development. We used SEM to examine further the development of the male genitalia and related structures through the cycle of molts. Adult, male, 3-mo-old, CFW strain mice were each infected with 200 infective L,s of Trichinella spiralis, then a portion of this group was sacrificed at time points corresponding to the midpoints of each stage, as calculated from Villella (1958, loc. cit.), Ali Kahn (1966, loc. cit.), and Kozek (1971, loc. cit.). Worms were collected by thermal migration (Despommier, 1973, J. Parasit. 59: 933-935) at 13 (L2), 17 (L3), 24 (L4), 27, and 72 (adults) hr PI. Worms were fixed in 6% glutaraldehyde in 0.05 M sodium cacodylate, pH 6.8, for 12 hr at 5 C, washed in distilled water and then postfixed in 1% osmium tetroxide in 0.05 M sodium cacodylate, pH 6.8, for 12 hr at 23 C. They were washed again in distilled water and dehydrated in ethanol followed by replacement of ethanol with Freon TF. The worms were then critical-point dried from CO2. All specimens were coated with gold-palladium prior to observation. At least 10 male worms were viewed for each time point. The L,s (Fig. 1) had no visible appendages or papillae; a finding consistent with all previous light microscopic observations. The L2 (Fig. 2), in contrast, possessed the rudiments of both copulatory appendages (ca) and the conical papillae (cp). The conical papillae have not been previously observed at this stage of the worms development (Kozek, 1971, loc. cit.). The L3 (Fig. 3) possessed well-formed copulatory appendages and rudimentary sets of both types of papillae (conical and tuberculate, arrow). Tuberculate papillae have not been reported at this stage prior to the present study. By the fourth stage (Fig. 4), the copulatory appendages and conical papillae were almost fully developed, while the tuberculate papillae remained undifferentiated. Following the final molt (Fig. 6), all accessory male genitalia structures were present and completely developed. The pair of tuberculate papillae were fully elongated and lay external and caudal to the conical papillae. The nomenclature of each set of papillae was established based upon light microscopical observations. As can clearly be seen in the present study, the tuberculate papillae were more conical in appearance than the conical papillae, which themselves appeared as laterally-flattened, triangular projections. The cloacal orifice varied in appearance depending upon which stage was observed. The cloacal opening of the L, appeared as a simple dorsoventral elongate opening. In contrast, from L2 to the adult stage, the cloacal opening was roughly triradiate in appearance (Fig. 3). No additional accessory structure associated with the male genitalia were observed on the adult worm in the present study.