Mark R. Rodgers

Characterization of Aeromonas Virulence Using an Immunocompromised Mouse Model

An immunocompromised mouse model was used to characterize Aeromonas strains for their ability to cause opportunistic, extraintestinal infections. A total of 34 isolates of Aeromonas (A. hydrophila [n = 12]), A. veronii biotype sobria [n = 7], A. caviae [n = 4], A. enchelia [n = 4], A. allosaccharophila [n = 2], A. salmonicida (n = 4), and A. bestiarum [n = 1]) were introduced by intraperitoneal injection into immunocompetent or chemically compromised (using cyclophosphamide) mice. The ability of each isolate to persist in the liver and spleen tissue was monitored at 24 hours after exposure. A majority of A. hydrophila and A veronii v. sobria strains, but none of the isolates of other Aeromonas species, were capable of persistent colonization (<300 cells/mg spleen and liver tissue at 24 hours). The presence or absence of several putative virulence factors (cytotoxicity to HEp-2, lipase activity, elastase activity, and hemolysis) were determined for each isolate using in vitro tests. There were no correlations between the presence or absence of biochemical test results for putative virulence factors and persistence of the isolate in spleen and liver tissue at 24 hours.

Identification of a flavobacterium strain virulent against Giardia lamblia cysts

We have isolated from a Kentucky stream a bacterial strain capable of killing the cyst form of Giardia lamblia. This bacterium, designated Sun4, is a Gram-negative, aerobic rod which produces a yellow pigment, but not of the flexirubin-type. Although true gliding motility has not been observed in Sun4, this strain does exhibit a spreading colony morphology when grown on R2A agar. Strain Sun4 has been identified by 16S rRNA sequencing and phylogenetic analysis as belonging to the genus Flavobacterium, and is most closely related to Cytophaga sp. strain Type 0092 and associated Flavobacterium columnare strains. Lipid analysis also identified fatty acids characteristic of the Cytophaga–Flavobacterium group of bacteria. In culture, Sun4 is able to degrade casein and cellulose, but not chitin, gelatin, starch, or agar. Degradation of Giardia cysts by Sun4 appears to require direct cellular contact as neither cell-free extracts nor cells separated from the cysts by dialysis membranes showed any activity against cysts. Activity against Giardia cysts is rapid, with Sun4 killing over 90% of cysts within 48 h. Strain Sun4 requires elevated levels of Ca2+ for optimal growth and degradative activity against Giardia cysts. We propose that bacterial strains such as Sun4 could be used as biological control agents against Giardia cysts in drinking water treatment systems.

Evaluating virulence of waterborne and clinical Aeromonas isolates using gene expression and mortality in neonatal mice followed by assessing cell culture’s ability to predict virulence based on transcriptional response

Aims:  To assess the virulence of Aeromonas spp. using two models, a neonatal mouse assay and a mouse intestinal cell culture.

Applicability of UV resistant Bacillus pumilus endospores as a human adenovirus surrogate for evaluating the effectiveness of virus inactivation in low-pressure UV treatment systems

Recent studies have demonstrated the potential to use Bacillus pumilus endospores as a surrogate for human adenovirus (HAdV) in UV disinfection studies. The use of endospores has been limited by observations of batch-to-batch variation in UV sensitivity. This study reports on a propagation method that utilizes a commercially available medium to produce UV tolerant B. pumilus endospores with a consistent UV sensitivity. It is further demonstrated that the endospores of B. pumilus strain (ATCC 27142), produced using this protocol (half strength Columbia broth, 5 days incubation, with 0.1mM MnSO4), display a UV dose-response that is similar to that of HAdV. Endospore stocks could be stored in ethanol for up to two months at 4 °C without a significant change in UV sensitivity. Synergistic endospore damage was observed by pre-heat treatment of water samples followed by UV irradiation. UV tolerant B. pumilus endospores are a potential surrogate of HAdV for UV treatment performance tests in water utilities which do not have in-house research virology laboratories.

Biofilm: Shelter or Barrier in Drinking Water Distribution Systems?

Six biofilm annular reactors were used in three duplicate pairs to simulate a 15.24cm diameter pipe with a flow velocity of 0.3m/s (Re=46,000). The mean residence time was set to be at least 3 hours. These conditions were chosen to reproduce the characteristics of an experiment by Gibbs et al. (2003). The main objective of this study was to check if a given pathogenic microorganism, Klebsiella pneumoniae, could colonize a mature water-distribution system biofilm. To achieve conservative results it was decided to choose parameters representative of the friendliest waterdistribution system environment possible for the bacteria to penetrate the biofilm. This included dechlorinating the tap water used as feed and keeping the reactors at room temperature. The choice of Klebsiella pneumoniae as the microorganism of study was warranted by its ability to produce high amounts of polysaccharides, making the cells likely to stick to the biofilm. The study was split in two parts. In a first step, the spatial variability of the biofilm density and the hydraulic residence time distribution were determined for each reactor. The second study consisted of injecting three 100mL solutions of different concentrations of Klebsiella pneumoniae into respective duplicate reactors. The first experiment showed that at a given sample time the biofilm is not homogeneous within a reactor and this pattern is repeated throughout each reactor. These results were taken into account when designing the second experiment. Composite samples of two coupons were collected to decrease the impact of sampling variability. The second study showed that at injected concentrations of 10 7 and 10 5 microorganisms per milliliter, colonization of the biofilm seemed unlikely. The cell counts dropped very quickly and after 5 days most of the samples showed non-detectable cell counts. In the case of a 10 9 cell/mL injected amount, the cell counts were steady during the first 24 hours before decreasing. This shows the potential for a threshold value in retention of the microorganism by the biofilm. After 5 days, the cell counts had decreased by 4 log units. However, it is not possible to reject the potential for regrowth, since the amounts of K. pneumoniae cells in the samples were still very high in the last sample event. The sampling period was limited by the number of available coupons. To check for regrowth, future experiments will require a sample design that includes a longer sampling period.