Browsing by Author "Ehrlich, Garth D."

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The application of biofilm science to the study and control of bacterial infections
(2003-11) Costerton, J. William; Veeh, Richard Harold; Shirtliff, Mark E.; Pasmore, M.; Post, C.; Ehrlich, Garth D.
Unequivocal direct observations have established that the bacteria that cause device-related and other chronic infections grow in matrix-enclosed biofilms. The diagnostic and therapeutic strategies that have served us so well in the partial eradication of acute epidemic bacterial diseases have not yielded accurate data or favorable outcomes when applied to these biofilm diseases. We discuss the potential benefits of the application of the new methods and concepts developed by biofilm science and engineering to the clinical management of infectious diseases.
Arginine or nitrate enhances antibiotic susceptibility of Pseudomonas aeruginosa in biofilms
(2006-01) Borriello, Giorgia B.; Richards, Lee A.; Ehrlich, Garth D.; Stewart, Philip S.
Arginine enhanced the killing of Pseudomonas aeruginosa by ciprofloxacin and tobramycin under anaerobic, but not aerobic, growth conditions. Arginine or nitrate also enhanced the killing by these antibiotics in mature biofilms, reducing viable cell counts by a factor of 10 to 100 beyond that achieved by antibiotics alone.
Biofilm-related infections of cerebrospinal fluid shunts
(2006-04) Fux, C. A.; Quigley, Mark; Worel, A. M.; Post, C.; Zimmerli, S.; Ehrlich, Garth D.; Veeh, Richard Harold
Cerebrospinal fluid (CSF) shunts carry a high risk of complications. Infections represent a major cause of shunt failure. Diagnosis and therapy of such infections are complicated by the formation of bacterial biofilms attached to shunt surfaces. This study correlated the pathophysiology and clinical course of biofilm infections with microscopic findings on the respective shunts. Surface irregularities, an important risk factor for shunt colonisation with bacteria, were found to increase over time because of silicone degradation. Scanning electron-microscopy (SEM) documented residual biological material (dead biofilm), which can further promote extant bacterial adhesion, on newly manufactured shunts. Clinical course, and SEM both documented bacterial dissemination against CSF flow and the monodirectional valve. In all cases, biofilms grew on both the inner and outer surfaces of the shunts. Microscopy and conventional culture detected all bacterial shunt infections. Analyses of 16S rDNA sequences using conserved primers identified bacteria in only one of three cases, probably because of previous formalin fixation of the samples.
Intelligent implants to battle biofilms
(2004) Ehrlich, Garth D.; Hu, Fen Z.; Lin, Qiao; Costerton, J. William; Post, J. C.
Mucosal biofilm formation on middle-ear mucosa in a nonhuman primate model of chronic suppurative otitis media
(2005-08) Dohar, Joseph E.; Hebda, Patricia A.; Veeh, Richard Harold; Awad, Marie; Costerton, J. William; Hayes, J. T.; Ehrlich, Garth D.
Background: An increased awareness of bacterial biofilms and their formation has led to a better understanding of bacterial infections that occur in the middle ear. Perhaps the best studied pathogen for its propensity toward biofilm formation is Pseudomonas aeruginosa, also the primary pathogen in chronic suppurative otitis media (CSOM). Objective: The aim of this study was to determine whether P. aeruginosa forms a biofilm in the middle ear in the setting of CSOM in a nonhuman primate model. Methods: Cynomolgus monkeys underwent perforation of the tympanic membrane and inoculation of the middle ear with a known biofilm-forming strain of P. aeruginosa. The contralateral ear was used as an internal control and was neither perforated nor infected. At the end of the study period, both ears were irrigated to remove planktonic bacteria, and the middle ear mucosa was removed and examined ultrastructurally using scanning electron microscopy (SEM) for determination of the presence or absence of biofilm formation. Main Outcome Measure: The identification of middle ear biofilm containing rod-shaped bacteria. Results: SEM revealed that P. aeruginosa formed bacterial biofilm in vivo on the middle ear mucosal surface, seen only in the infected ear. Interestingly, biofilm formation caused by cocci was also seen in both the experimental as well as the control ear. Conclusion: P. aeruginosa forms biofilms in the middle ear in CSOM in primates. To our knowledge, this is the first report of disease-associated bacterial biofilm in a nonhuman primate model of CSOM. Such a model lays a foundation for much needed study into the role of biofilms in the pathophysiology of CSOM. Should CSOM be caused by biofilms, which is uncertain at this time, development of novel strategies for treatment and prevention may be possible. The finding of both rods and cocci forming biofilms also warrants further investigation.
Mucosal biofilm formation on middle-ear mucosa in the chinchilla model of otitis media
(2002-04) Ehrlich, Garth D.; Veeh, Richard Harold; Wang, Xue; Costerton, J. William; Hayes, Jay D.; Hu, Fen Z.; Daigle, Bernie J.; Ehrlich, Miles D.; Post, J. C.
CONTEXT Chronic otitis media with effusion (OME) has long been considered to be a sterile inflammatory process. The previous application of molecular diagnostic technologies to OME suggests that viable bacteria are present in complex communities known as mucosal biofilms; however, direct imaging evidence of mucosal biofilms associated with OM is lacking. OBJECTIVE. To determine whether biofilm formation occurs in middle-ear mucosa in an experimental model of otitis media. DESIGN and MATERIALS. A total of 48 research-grade, young adult chinchillas weighing 500g were used for 2 series of animal experiments: one to obtain specimens for scanning electron microscopy and the other to obtain specimens for confocal laser scanning microscopy using vital dyes. In each series, 21 animals were bilaterally injected with viable Haemophilus influenzae bacteria and 1 was inoculated to account for expected mortality. Three served as negative controls. Effusions and mucosal specimens were collected from 2 infected animals that were euthanized at 3, 6, 12, and 24 hours and at days 2, 4, 5, 10, 16, and 22 after inoculation. MAIN OUTCOME MEASURES. Images were analyzed for biofilm morphology, including presence of microcolony formation and for presence of bacteria on tissue surfaces. RESULTS. Scanning electron microscopy demonstrated that biofilm formation was evident in all specimens from animals beginning 1 day after infection and was present through 21 days. Confocal laser scanning microscopy indicated that bacteria within the biofilms was viable. CONCLUSION. These preliminary findings provide evidence that mucosal biofilms form in an experimental model of otitis media and suggest that biofilm formation may be an important factor in the pathogenesis of chronic otitis media with effusion.
Nanoscale Structural and Mechanical Properties of Nontypeable Haemophilus influenzae Biofilms
(2009-02) Arce, Fernando Teran; Carlson, Ross P.; Monds, James; Veeh, Richard Harold; Hu, Fen Z.; Stewart, Philip S.; Lal, Ratnesh; Ehrlich, Garth D.; Avci, Recep
Nontypeable Haemophilus influenzae (NTHI) bacteria are commensals in the human nasopharynx, as well as pathogens associated with a spectrum of acute and chronic infections. Two important factors that influence NTHI pathogenicity are their ability to adhere to human tissue and their ability to form biofilms. Extracellular polymeric substances (EPS) and bacterial appendages such as pili critically influence cell adhesion and intercellular cohesion during biofilm formation. Structural components in the outer cell membrane, such as lipopolysaccharides, also play a fundamental role in infection of the host organism. In spite of their importance, these pathogenic factors are not yet well characterized at the nanoscale. Here, atomic force microscopy (AFM) was used in aqueous environments to visualize structural details, including probable Hif-type pili, of live NTHI bacteria at the early stages of biofilm formation. Using single-molecule AFM-based spectroscopy, the molecular elasticities of lipooligosaccharides present on NTHI cell surfaces were analyzed and compared between two strains (PittEE and PittGG) with very different pathogenicity profiles. Furthermore, the stiffness of single cells of both strains was measured and subsequently their turgor pressure was estimated.
New methods for the detection of orthopedic and other biofilm infections
(2011-03) Costerton, J. William; Post, J. C.; Ehrlich, Garth D.; Hu, Fen Z.; Kreft, R.; Nistico, L.; Kathju, S.; Stoodley, Paul; Hall-Stoodley, Luanne; Maale, G.; James, Garth A.; Sotereanos, N.; DeMeo, P.
The detection and identification of bacteria present in natural and industrial ecosystems is now entirely based on molecular systems that detect microbial RNA or DNA. Culture methods were abandoned in the 1980s because direct observations showed that <1% of the bacteria in these systems grew on laboratory media. Culture methods comprise the backbone of the Food and Drug Administration-approved diagnostic systems used in hospital laboratories, with some molecular methods being approved for the detection of specific pathogens that are difficult to grow in vitro. In several medical specialties, the reaction to negative cultures in cases in which overt signs of infection clearly exist has produced a spreading skepticism concerning the sensitivity and accuracy of traditional culture methods.We summarize evidence from the field of orthopedic surgery, and from other medical specialties, that support the contention that culture techniques are especially insensitive and inaccurate in the detection of chronic biofilm infections. We examine the plethora of molecular techniques that could replace cultures in the diagnosis of bacterial diseases, and we identify the new Ibis technique that is based on base ratios (not base sequences), as the molecular system most likely to fulfill the requirements of routine diagnosis in orthopedic surgery.
Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms
(2004-06) Borriello, Giorgia B.; Werner, Erin M.; Roe, Frank L.; Kim, Aana M.; Ehrlich, Garth D.; Stewart, Philip S.
The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 µm into the biofilms, which averaged 210 µm thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band, approximately 30 µm wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h-1. These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.
Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis
(2010) Folsom, James P.; Richards, Lee A.; Roe, Frank L.; Ehrlich, Garth D.; Parker, Albert E.; Mazurie, Aurélien J.; Stewart, Philip S.
BACKGROUND: Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and published data sets.RESULTS: Biofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. Transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. Similar rankings were obtained from data sets published in the GEO database (www.ncbi.nlm.nih.gov/geo). By comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. These biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. Genes associated with elaboration of type IV pili were strongly expressed in the biofilm. The biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps. Using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be 0.08 h-1.CONCLUSIONS: Collectively these data underscore the oxygen-limited, slow-growing nature of the biofilm population and are consistent with antimicrobial tolerance due to low metabolic activity.
Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm
(2002-02) Sauer, K.; Camper, Anne K.; Ehrlich, Garth D.; Costerton, J. William; Davies, David Gwilym
Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.
Role of biofilms in neurosurgical device-related infections
(2005-10) Baxton Jr, Ernest E.; Ehrlich, Garth D.; Hall-Stoodley, Luanne; Stoodley, Paul; Veeh, Rick; Fux, C. A.; Hu, Fen Z.; Quigley, Matthew; Post, J. C.
Bacterial biofilms have recently been shown to be important in neurosurgical device-related infections. Because the concept of biofilms is novel to most practitioners, it is important to understand that both traditional pharmaceutical therapies and host defense mechanisms that are aimed at treating or overcoming free-swimming bacteria are largely ineffective against the sessile bacteria in a biofilm. Bacterial biofilms are complex surface-attached structures that are composed of an extruded extracellular matrix in which the individual bacteria are embedded. Superimposed on this physical architecture is a complex system of intercellular signaling, termed quorum sensing. These complex organizational features endow biofilms with numerous microenvironments and a concomitant number of distinct bacterial phenotypes. Each of the bacterial phenotypes within the biofilm displays a unique gene expression pattern tied to nutrient availability and waste transport. Such diversity provides the biofilm as a whole with an enormous survival advantage when compared to the individual component bacterial cells. Thus, it is appropriate to view the biofilm as a multicellular organism, akin to metazoan eukaryotic life. Bacterial biofilms are much hardier than free floating or planktonic bacteria and are primarily responsible for device-related infections. Now that basic research has demonstrated that the vast majority of bacteria exist in biofilms, the paradigm of biofilm-associated chronic infections is spreading to the clinical world. Understanding how these biofilm infections affect patients with neurosurgical devices is a prerequisite to developing strategies for their treatment and prevention.