John A. Griswold

The role of quorum sensing in the in vivo virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections. The cell-density-dependent signaling mechanisms known as quorum sensing play a role in several of these infections including corneal, lung and burn wound infections. In addition, the quorum-sensing systems contribute to the ability of P. aeruginosa to form biofilms on medically important devices. The quorum-sensing systems accomplish their effect by controlling the production of different virulence factors and by manipulating the host immune response.

Pelvic fracture from major blunt trauma. Outcome is determined by associated injuries

Pelvic hemorrhage has been implicated as the cause of death in 50% of patients who die following pelvic fractures. To establish correlates of morbidity and mortality from pelvic fractures due to blunt trauma, we reviewed 236 patients treated during 4 years. The average age of the 144 men and 92 women was 31.5 years, the average Injury Severity Score was 21.3, the average blood requirement was 5 units, and the average hospital stay was 16.8 days. One hundred fifty-two patients (64.4%) were injured in motor vehicle accidents, 33 (14%) had motor vehicle-pedestrian accidents, 16 (6.8%) had crush injuries, 12 (5.1%) each had either motorcycle accidents or falls, and 11 (4.6%) had miscellaneous accidents. Eighteen patients (7.6%) died, with seven (38.9%) deaths due to hemorrhage. Only one death was caused by pelvic hemorrhage. Other deaths were due to hemorrhage from other sites (6), head injury (5), sepsis or multiple-organ failure (4), pulmonary injury (1), and pulmonary embolus (1). None of the septic deaths was related to a pelvic hematoma. Multivariate multiple regression analysis showed that the severity of injury was correlated with indices of severity of pelvic fractures such as fracture site (p less than 0.0001), fracture displacement (p less than 0.005), pelvic stability (p less than 0.0001), and vector of injury (p less than 0.01). However death could not be predicted on the basis of these indices of severity (p greater than 0.28). Of the nine patients who underwent pelvic arteriography, three required embolization of actively bleeding pelvic vessels, but seven had intra-abdominal hemorrhage that required laparotomy, and eight developed a coagulopathy. Massive bleeding from pelvic fractures was uncommon, and the major threat of hemorrhage was from nonpelvic sites. Furthermore, although injury severity was correlated with the severity of the pelvic fracture, hospital outcome was determined by associated injuries and not by the pelvic fracture.

An In Vivo Polymicrobial Biofilm Wound Infection Model to Study Interspecies Interactions

Chronic wound infections are typically polymicrobial; however, most in vivo studies have focused on monospecies infections. This project was designed to develop an in vivo, polymicrobial, biofilm-related, infected wound model in order to study multispecies biofilm dynamics and in relation to wound chronicity. Multispecies biofilms consisting of both Gram negative and Gram positive strains, as well as aerobes and anaerobes, were grown in vitro and then transplanted onto the wounds of mice. These in vitro-to-in vivo multi-species biofilm transplants generated polymicrobial wound infections, which remained heterogeneous with four bacterial species throughout the experiment. We observed that wounded mice given multispecies biofilm infections displayed a wound healing impairment over mice infected with a single-species of bacteria. In addition, the bacteria in the polymicrobial wound infections displayed increased antimicrobial tolerance in comparison to those in single species infections. These data suggest that synergistic interactions between different bacterial species in wounds may contribute to healing delays and/or antibiotic tolerance.

Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling.

ABSTRACT Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.

Trauma in pregnancy: The role of interpersonal violence

OBJECTIVE Our purpose was to determine what role interpersonal violence as intentional injury plays in the pregnant trauma victim. STUDY DESIGN We performed a retrospective review of medical records. RESULTS During a 9-year period in a single university medical and trauma center, 203 pregnant women were treated for a physically traumatic event. Sixty-four women (31.5%) were victims of intentional injury, in most cases by the husband or boyfriend. Although the mean Injury Severity Score was higher in women with fetal death than in women with successful pregnancy outcomes (7.25 vs 1.74, respectively; p < 0.01), 5 of the 8 women with fetal losses incurred these despite an apparent absence of physical injury (maternal Injury Severity Score = 0). CONCLUSIONS Interpersonal violence during pregnancy is a frequent and increasingly common cause of maternal injury. The inconsistent relationship between Injury Severity Score and serious fetal injury or death is underscored by the loss of 5 fetuses despite an Injury Severity Score of 0.

Pseudomonas aeruginosa Autoinducer Enters and Functions in Mammalian Cells

Quorum sensing (QS) is a cell density-dependent signaling mechanism used by many bacteria to control gene expression. Several recent reports indicate that the signaling molecules (autoinducers) that mediate QS in Pseudomonas aeruginosa may also modulate gene expression in host cells; however, the mechanisms are largely unknown. Here we show that two P. aeruginosa autoinducers, N-3-oxododecanoyl-homoserine lactone and N-butyryl-homoserine lactone, can both enter eukaryotic cells and activate artificial chimeric transcription factors based on their cognate transcriptional activators, LasR and RhlR, respectively. The autoinducers promoted nuclear localization of chimeric proteins containing the full LasR or RhlR coding region, and the LasR-based proteins were capable of activating transcription of a LasR-dependent luciferase gene. Responsiveness to autoinducer required the N-terminal autoinducer-binding domains of LasR and RhlR. Truncated proteins consisting of only the C-terminal helix-turn-helix DNA-binding domains of both proteins attached to a nuclear localization signal efficiently translocated to the nucleus in the absence of autoinducer, and truncated LasR-based proteins functioned as constitutively active transcription factors. Chimeric LasR proteins were only activated by their cognate autoinducer ligand and not by N-butyryl-L-homoserine lactone. These data provide evidence that autoinducer molecules from human pathogens can enter mammalian cells and suggest that autoinducers may influence gene expression in host cells by interacting with and activating as-yet-unidentified endogenous proteins.

Lower Extremity Fracture Fixation in Head-injured Patients

Compared with nonsurgical management or delayed repair, early fracture fixation can reduce the incidence of pulmonary complications in patients with long-bone fractures of the lower extremities. Blunt trauma victims often have multiple nonskeletal injuries that might influence the risk of pulmonary complications, and when head injuries are present it has been a common practice to delay nonemergent operations for several days to protect the injured brain. We conducted a retrospective review of 114 patients with multiple trauma whose injuries included head trauma and a fracture of the neck or shaft of the femur or shaft of the tibia to determine if delayed stabilization of lower extremity fractures increased the risk of pulmonary complications or reduced the risk of cerebral complications. Forty-six patients underwent surgical fixation of their fractures within 24 hours of injury (early fixation), 26 patients had their fractures repaired more than 24 hours after injury (late fixation), and 42 patients did not undergo surgical fracture fixation. The risk of pulmonary complications was not related to the timing of surgical fracture fixation but was strongly influenced by the severity of injuries to the head and to the chest (p less than 0.001). Furthermore, a delay in fracture fixation did not protect the injured brain; the risk of CNS events was determined by the severity of the head injury (p less than 0.0001). Early fracture fixation in patients with head injury may be appropriate because it simplifies patient care and does not seem to worsen the head injury, but it does not prevent pulmonary complications in these high-risk patients.

Gallium Maltolate Treatment Eradicates Pseudomonas aeruginosa Infection in Thermally Injured Mice

ABSTRACT Gallium (Ga) is a semimetallic element that has demonstrated therapeutic and diagnostic-imaging potential in a number of disease settings, including cancer and infectious diseases. Galliums biological actions stem from its ionic radius being almost the same as that of ferric iron (Fe3+), whereby it can replace iron (Fe) in Fe3+-dependent biological systems, such as bacterial and mammalian Fe transporters and Fe3+-containing enzymes. Unlike Fe3+, ionic gallium (Ga3+) cannot be reduced, and when incorporated, it inactivates Fe3+-dependent reduction and oxidation processes that are necessary for bacterial and mammalian cell proliferation. Most pathogenic bacteria require Fe for growth and function, and the availability of Fe in the host or environment can greatly enhance virulence. We examined whether gallium maltolate (GaM), a novel formulation of Ga, had antibacterial activity in a thermally injured acute infection mouse model. Dose-response studies indicated that a GaM dose as low as 25 mg/kg of body weight delivered subcutaneously was sufficient to provide 100% survival in a lethal P. aeruginosa-infected thermally injured mouse model. Mice treated with 100 mg/kg GaM had undetectable levels of Pseudomonas aeruginosa in their wounds, livers, and spleens, while the wounds of untreated mice were colonized with over 108P. aeruginosa CFU/g of tissue and their livers and spleens were colonized with over 105P. aeruginosa CFU/g of tissue. GaM also significantly reduced the colonization of Staphylococcus aureus and Acinetobacter baumannii in the wounds of thermally injured mice. Furthermore, GaM was also therapeutically effective in preventing preestablished P. aeruginosa infections at the site of the injury from spreading systemically. Taken together, our data suggest that GaM is potentially a novel antibacterial agent for the prevention and treatment of wound infections following thermal injury.

Computed tomography in the management of blunt thoracic trauma.

Computed tomographic (CT) scanning has proved to be valuable in evaluating the head and abdomen of victims of blunt trauma; CT scans of the thorax often are obtained on patients with blunt torso trauma, but their value for this purpose is unclear. We conducted a prospective study to evaluate the role of chest CT scanning in thoracic trauma. Hemodynamically stable patients at least 18 years old with an estimated Abbreviated Injury Scale--Thorax score of 2 or greater underwent a contrast-enhanced CT scan of the chest, usually in conjunction with CT scans of the head, abdomen, or both. Thirteen patients were dead on arrival, 14 required emergency surgical procedures, and 13 were too unstable to undergo chest CT scan. Thirty-three patients were not included because they refused to participate or the protocol was not followed. Forty-six men (69%) and 21 women with a mean age of 42.7 years completed the study. Sixty-one were injured in motor vehicle crashes, four were injured in falls, and one each was injured by assault and by crushing forces. Injury Severity Scores ranged from 4 to 45, with a mean of 20.5. Four patients died (6%), three from head injury and one from multiple organ dysfunction. Chest roentgenography (CXR) was superior to CT scanning in identifying rib fractures, but CT scanning was more sensitive than CXR for pneumothorax, fluid collections, and infiltrates (p < 0.001); CT scanning also was more specific for aortic injury. Despite this quantitative superiority, the abnormalities missed by CXR but identified by CT scanning infrequently led to a change in management.(ABSTRACT TRUNCATED AT 250 WORDS)

Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice.

Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. The impaired healing properties of these wounds, which often develop debilitating bacterial infections, significantly increase the rate of lower extremity amputation in diabetic patients. We hypothesize that bacterial biofilms, or sessile communities of bacteria that reside in a complex matrix of exopolymeric material, contribute to the severity of diabetic wounds. To test this hypothesis, we developed an in vivo chronic wound, diabetic mouse model to determine the ability of the opportunistic pathogen, Pseudomonas aeruginosa, to cause biofilm-associated infections. Utilizing this model, we observed that diabetic mice with P. aeruginosa-infected chronic wounds displayed impaired bacterial clearing and wound closure in comparison with their non-diabetic littermates. While treating diabetic mice with insulin improved their overall health, it did not restore their ability to resolve P. aeruginosa wound infections or speed healing. In fact, the prevalence of biofilms and the tolerance of P. aeruginosa to gentamicin treatment increased when diabetic mice were treated with insulin. Insulin treatment was observed to directly affect the ability of P. aeruginosa to form biofilms in vitro. These data demonstrate that the chronically wounded diabetic mouse appears to be a useful model to study wound healing and biofilm infection dynamics, and suggest that the diabetic wound environment may promote the formation of biofilms. Further, this model provides for the elucidation of mechanistic factors, such as the ability of insulin to influence antimicrobial effectiveness, which may be relevant to the formation of biofilms in diabetic wounds.