Browsing by Author "Singh, Pradeep K."

Now showing 1 - 6 of 6

Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge: A model for the study of chronic wounds
(2010-08) Zhao, Ge; Hochwalt, Phillip C.; Usui, Marcia L.; Underwood, Robert A.; Singh, Pradeep K.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.
Chronic wounds are a major clinical problem that lead to considerable morbidity and mortality. We hypothesized that an important factor in the failure of chronic wounds to heal was the presence of microbial biofilm resistant to antibiotics and protected from host defenses. A major difficulty in studying chronic wounds is the absence of suitable animal models.The goal of this study was to create a reproducible chronic wound model in diabetic mice by the application of bacterial biofilm. Six-millimeter punch biopsy wounds were created on the dorsal surface of diabetic (db/db) mice, subsequently challenged with Pseudomonas aeruginosa (PAO1) biofilms 2 days postwounding, and covered with semiocclusive dressings for 2 weeks. Most of the control wounds were epithelialized by 28 days postwounding. In contrast, none of biofilm-challenged wounds were closed. Histological analysis showed extensive inflammatory cell infiltration, tissue necrosis, and epidermal hyperplasia adjacent to challenged woundsâ€”all indicators of an inflammatory nonhealing wound. Quantitative cultures and transmission electron microscopy demonstrated that the majority of bacteria were in the scab above the wound bed rather than in the wound tissue. The model was reproducible, allowed localized cutaneous wound infections without high mortality, and demonstrated delayed wound healing following a biofilm challenge. This model may provide an approach to study the role of microbial biofilms in chronic wounds as well as the effect of specific biofilm therapy on wound healing.
Development of a chronic wound in a diabetic (db/db) mouse by infection with biofilm
(2008) Zhao, Ge; Hochwalt, Phillip C.; Usui, Marcia L.; Underwood, Robert A.; Singh, Pradeep K.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.
Chronic wounds such as diabetic ulcers, pressure sores, and venous stasis ulcers are a major source of morbidity and mortality. Since chronic ulcers are not homogeneous, systematic study of ulcer therapies is difficult. Our goal is to create a standard chronic wound model. It has been shown that specialized microbial colonies known as biofilm are present in chronic human wounds. The microorganisms present in biofilm are protected from the host defenses, topical antiseptics, and systemic antibiotics. For this reason, biofilm infections persist and may contribute to poor wound healing. We hypothesize that application of biofilm to an already characterized diabetic mouse wound model may create a reproducible chronic wound. Bacterial biofilm was developed by incubating planktonic Pseudomonas aeruginosa (PAO-1) and transferring to polycarbonate membrane filters placed on LB agar plates. The biofilm (~10^8 CFU) was transferred to 6mm wounds created on the dorsal skin of diabetic mice and the wounds were covered with Tegaderm® dressing. The biofilm was transferred to the wounds at several different time points following wounding. The mice died if the biofilm was transferred soon after wounding; however, mice that were allowed to recover from the surgery before biofilm inoculation developed a purulent wound that persisted for weeks. In addition, the timing of Tegaderm® application proved to be a critical variable for purulence and quality of the wound. Ultimately, the development of a reproducible chronic wound in a diabetic mouse will allow in vivo testing of potential wound healing therapies.
Entropically driven aggregation of bacteria by host polymers promotes antibiotic tolerance in Pseudomonas aeruginosa
(Proceedings of the National Academy of Sciences, 2018-10) Secor, Patrick R.; Michaels, Lia A.; Ratjen, Anina; Jennings, Laura K.; Singh, Pradeep K.
Bacteria causing chronic infections are generally observed living in cell aggregates suspended in polymer-rich host secretions, and bacterial phenotypes induced by aggregated growth may be key factors in chronic infection pathogenesis. Bacterial aggregation is commonly thought of as a consequence of biofilm formation; however the mechanisms producing aggregation in vivo remain unclear. Here we show that polymers that are abundant at chronic infection sites cause bacteria to aggregate by the depletion aggregation mechanism, which does not require biofilm formation functions. Depletion aggregation is mediated by entropic forces between uncharged or like-charged polymers and particles (e.g., bacteria). Our experiments also indicate that depletion aggregation of bacteria induces marked antibiotic tolerance that was dependent on the SOS response, a stress response activated by genotoxic stress. These findings raise the possibility that targeting conditions that promote depletion aggregation or mechanisms of depletion-mediated tolerance could lead to new therapeutic approaches to combat chronic bacterial infections.
Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive InfectionIn Vivo
(American Society for Microbiology, 2017-01) Secor, Patrick R.; Michaels, Lia A.; Smigiel, Kate S.; Rohani, Maryam G.; Jennings, Laura K.; Hisert, Katherine B.; Arrigoni, Allison; Braun, Kathleen R.; Birkland, Timothy P.; Lai, Ying; Hallstrand, Teal S.; Bollyky, Paul L.; Singh, Pradeep K.; Parks, William C.
Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.
The Depletion Mechanism Actuates Bacterial Aggregation by Exopolysaccharides and Determines Species Distribution & Composition in Bacterial Aggregates
(Frontiers Media SA, 2022-06) Secor, Patrick R.; Michaels, Lia A.; Bublitz, DeAnna C.; Jennings, Laura K.; Singh, Pradeep K.
Bacteria in natural environments and infections are often found in cell aggregates suspended in polymer-rich solutions, and aggregation can promote bacterial survival and stress resistance. One aggregation mechanism, called depletion aggregation, is driven by physical forces between bacteria and high concentrations of polymers in the environment rather than bacterial activity per se. As such, bacteria aggregated by the depletion mechanism will disperse when polymer concentrations fall unless other adhesion mechanisms supervene. Here we investigated whether the depletion mechanism can actuate the aggregating effects of Pseudomonas aeruginosa exopolysaccharides for suspended (i.e. not surface attached) bacteria, and how depletion affects bacterial inter-species interactions. We found that cells overexpressing the exopolysaccharides Pel and Psl remained aggregated after short periods of depletion aggregation whereas wild-type and mucoid P. aeruginosa did not. In co-culture, depletion aggregation had contrasting effects on P. aeruginosa’s interactions with coccus- and rod-shaped bacteria. Depletion caused S. aureus (cocci) and P. aeruginosa (rods) to segregate from each other and S. aureus to resist secreted P. aeruginosa antimicrobial factors resulting in species co-existence. In contrast, depletion aggregation caused P. aeruginosa and Burkholderia sp. (both rods) to intermix, enhancing type VI secretion inhibition of Burkholderia by P. aeruginosa, leading to P. aeruginosa dominance. These results show that in addition to being a primary cause of aggregation in polymer-rich suspensions, physical forces inherent to the depletion mechanism can promote aggregation by some self-produced exopolysaccharides and determine species distribution and composition of bacterial communities.
Time course study of delayed wound healing in a biofilm-challenged diabetic mouse model
(2012-05) Zhao, Ge; Usui, Marcia L.; Underwood, Robert A.; Singh, Pradeep K.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.
Bacterial biofilm has been shown to play a role in delaying wound healing of chronic wounds, a major medical problem that results in significant health care burden. A reproducible animal model could be very valuable for studying the mechanism and management of chronic wounds. Our previous work showed that Pseudomonas aeruginosa (PAO1) biofilm challenge on wounds in diabetic (db/db) mice significantly delayed wound healing. In this wound time course study, we further characterize the bacterial burden, delayed wound healing, and certain aspects of the host inflammatory response in the PAO1 biofilm-challenged db/db mouse model. PAO1 biofilms were transferred onto 2-day-old wounds created on the dorsal surface of db/db mice. Control wounds without biofilm challenge healed by 4 weeks, consistent with previous studies; none of the biofilm-challenged wounds healed by 4 weeks. Of the biofilm-challenged wounds, 64% healed by 6 weeks, and all of the biofilmchallenged wounds healed by 8 weeks. During the wound-healing process, P. aeruginosa was gradually cleared from the wounds while the presence of Staphylococcus aureus (part of the normal mouse skin flora) increased. Scabs from all unhealed wounds contained 107 P. aeruginosa, which was 100-fold higher than the counts isolated from wound beds (i.e., 99% of the P. aeruginosa was in the scab). Histology and genetic analysis showed proliferative epidermis, deficient vascularization, and increased inflammatory cytokines. Hypoxia inducible factor expression increased threefold in 4-week wounds. In summary, our study shows that biofilm-challenged wounds typically heal in approximately 6 weeks, at least 2 weeks longer than nonbiofilm-challenged normal wounds. These data suggest that this delayed wound healing model enables the in vivo study of bacterial biofilm responses to host defenses and the effects of biofilms on host wound healing pathways. It may also be used to test antibiofilm strategies for treating chronic wounds.