Lucy Tompkins

All Publications

• Butyrate Differentiates Permissiveness to Clostridioides difficile Infection and Influences Growth of Diverse C. difficile Isolates. Infection and immunity Pensinger, D. A., Fisher, A. T., Dobrila, H. A., Van Treuren, W., Gardner, J. O., Higginbottom, S. K., Carter, M. M., Schumann, B., Bertozzi, C. R., Anikst, V., Martin, C., Robilotti, E. V., Chow, J. M., Buck, R. H., Tompkins, L. S., Sonnenburg, J. L., Hryckowian, A. J. 2023: e0057022

  Abstract

  A disrupted "dysbiotic" gut microbiome engenders susceptibility to the diarrheal pathogen Clostridioides difficile by impacting the metabolic milieu of the gut. Diet, in particular the microbiota-accessible carbohydrates (MACs) found in dietary fiber, is one of the most powerful ways to affect the composition and metabolic output of the gut microbiome. As such, diet is a powerful tool for understanding the biology of C. difficile and for developing alternative approaches for coping with this pathogen. One prominent class of metabolites produced by the gut microbiome is short-chain fatty acids (SCFAs), the major metabolic end products of MAC metabolism. SCFAs are known to decrease the fitness of C. difficile in vitro, and high intestinal SCFA concentrations are associated with reduced fitness of C. difficile in animal models of C. difficile infection (CDI). Here, we use controlled dietary conditions (8 diets that differ only by MAC composition) to show that C. difficile fitness is most consistently impacted by butyrate, rather than the other two prominent SCFAs (acetate and propionate), during murine model CDI. We similarly show that butyrate concentrations are lower in fecal samples from humans with CDI than in those from healthy controls. Finally, we demonstrate that butyrate impacts growth in diverse C. difficile isolates. These findings provide a foundation for future work which will dissect how butyrate directly impacts C. difficile fitness and will lead to the development of diverse approaches distinct from antibiotics or fecal transplant, such as dietary interventions, for mitigating CDI in at-risk human populations. IMPORTANCE Clostridioides difficile is a leading cause of infectious diarrhea in humans, and it imposes a tremendous burden on the health care system. Current treatments for C. difficile infection (CDI) include antibiotics and fecal microbiota transplant, which contribute to recurrent CDIs and face major regulatory hurdles, respectively. Therefore, there is an ongoing need to develop new ways to cope with CDI. Notably, a disrupted "dysbiotic" gut microbiota is the primary risk factor for CDI, but we incompletely understand how a healthy microbiota resists CDI. Here, we show that a specific molecule produced by the gut microbiota, butyrate, is negatively associated with C. difficile burdens in humans and in a mouse model of CDI and that butyrate impedes the growth of diverse C. difficile strains in pure culture. These findings help to build a foundation for designing alternative, possibly diet-based, strategies for mitigating CDI in humans.

  View details for DOI 10.1128/iai.00570-22

  View details for PubMedID 36692308

• Getting to zero: Impact of a device to reduce blood culture contamination and false-positive central-line-associated bloodstream infections. Infection control and hospital epidemiology Tompkins, L. S., Tien, V., Madison, A. N. 2022: 1-5

  Abstract

  To assess the impact of initial specimen diversion device (ISDD) on inpatient and emergency department blood culture contamination (BCC), central-line-associated bloodstream infection (CLABSI) standardized infection ratios (SIRs), and antibiotic administration.Single-center quasi-experimental prospective cohort study wherein phlebotomists used traditional venipuncture with or without the ISDD while registered nurses (RNs) used traditional venipuncture.BCC events among phlebotomists and RNs were observed and compared from March 17, 2019, through January 21, 2020, defined by contaminant detection in 1 of 4 bottles for matched sets or 1 of 2 bottles in both subsets for coagulase negative staphylococci. CLABSIs throughout this period were recorded and SIRs were calculated. Enhanced oversight took place through July 21, 2019, with chart review assessing antibiotic use for patients with possible BCC.Overall, 24% of blood cultures obtained were from patients in intensive care. Phlebotomists using traditional venipuncture (n = 4,759) had a 2.3% BCC rate; phlebotomists using the ISDD (n = 11,202) had a 0% BCC rate. RNs drew 7,411 BCs with a 0.8% BCC rate. The CLABSI SIR was decreased from 1.103 in 2017 and 0.658 in 2018 to 0.439 in 2019. The CLABSI incidence was 33%-64% of predicted value for each 2019 quarter. This range fell to 18%-37% after the exclusion of likely false-positive results. Among 42 patients with possible BCC under enhanced oversight, 2 patients were treated with prolonged antibiotic courses.ISDD use by phlebotomists was associated with BCC reduction and reduced false-positive CLABSI results. This patient-care quality improvement could constitute sustainable antibiotic stewardship expansion.

  View details for DOI 10.1017/ice.2022.284

  View details for PubMedID 36539993

• Clinical Outcomes of Treated and Untreated C. difficile PCR-Positive/Toxin-Negative Adult Hospitalized Patients: a Quasi-Experimental Noninferiority Study. Journal of clinical microbiology Hogan, C. A., Hitchcock, M. M., Frost, S., Kapphahn, K., Holubar, M., Tompkins, L. S., Banaei, N. 2022: e0218721

  Abstract

  Clostridioides difficile infection (CDI) is routinely diagnosed by PCR, with or without toxin enzyme immunoassay testing. The role of therapy for positive PCR and negative toxin remains unclear. The objective of this study was to determine whether clinical outcomes of PCR+/cycle threshold-based toxin (CT-toxin)- individuals vary by result reporting and treatment strategy. We performed a quasiexperimental noninferiority study comparing clinical outcomes of PCR+/CT-toxin- individuals by reporting PCR result only (most patients treated) with reporting CT-toxin result only (most patients untreated) in a single-center, tertiary academic hospital. The primary outcome was symptomatic PCR+/CT-toxin+ conversion at 8weeks. Secondary outcomes included 7-day diarrhea resolution, hospital length of stay, and 30-day all-cause mortality. A total of 663 PCR+/CT-toxin- test results were analyzed from 632 individuals with a median age of 61years (interquartile range [IQR], 44 to 72) and 50.4% immunocompromised. Individuals in the preintervention group were more likely to have received CDI therapy than those in the intervention group (91.5 versus 15.1%; P < 0.001). Symptomatic toxin conversion at 8weeks and hospital length of stay failed to establish the predefined thresholds for noninferiority. Lack of diarrhea resolution at 7days and 30-day all-cause mortality was similar and established noninferiority (20.0 versus 13.7%; adjusted odds ratio [aOR], 0.57; 90% confidence interval [CI], 0.32 to 1.01; P = 0.1; and 8.6 versus 6.5%; aOR, 0.46; 90% CI, 0.20 to 1.04; P = 0.12). These data support the safety of withholding antibiotics for selected hospitalized individuals with suspected CDI but negative toxin.

  View details for DOI 10.1128/jcm.02187-21

  View details for PubMedID 35611653

• Case-control study evaluating risk factors for SARS-CoV-2 outbreak amongst healthcare personnel at a tertiary care center. American journal of infection control Rosser, J. I., Tayyar, R., Giardina, R., Kolonoski, P., Kenski, D., Shen, P., Steinmetz, L. M., Hung, L., Xiao, W., Bains, K., Morrison, T., Madison, A., Chang, S., Tompkins, L., Pinsky, B. A., Holubar, M. 2021

  Abstract

  BACKGROUND: Despite several outbreaks of SARS-CoV-2 amongst healthcare personnel (HCP) exposed to COVID-19 patients globally, risk factors for transmission remain poorly understood.METHODS: We conducted an outbreak investigation and case-control study to evaluate SARS-CoV-2 transmission risk in an outbreak among HCP at an academic medical center in California that was confirmed by whole genome sequencing.RESULTS: A total of 7/9 cases and 93/182 controls completed a voluntary survey about risk factors. Compared to controls, cases reported significantly more patient contact time. Cases were also significantly more likely to have performed airway procedures on the index patient, particularly placing the patient on high flow nasal cannula, continuous positive airway pressure (CPAP), or bilevel positive airway pressure (BiPAP) (OR=11.6; 95% CI=1.7-132.1).DISCUSSION: This study highlights the risk of nosocomial infection of SARS-CoV-2 from patients who become infectious midway into their hospitalization. Our findings also reinforce the importance of patient contact and aerosol-generating procedures as key risk factors for HCP infection with SARS-CoV-2.CONCLUSIONS: Re-testing patients for SARS-CoV-2 after admission in suspicious cases and using N95 masks for all aerosol-generating procedures regardless of initial patient SARS-CoV-2 test results can help reduce the risk of SARS-COV-2 transmission to HCP.

  View details for DOI 10.1016/j.ajic.2021.09.004

  View details for PubMedID 34536502

• SARS-CoV-2 Neutralization Resistance Mutations in Patient with HIV/AIDS, California, USA. Emerging infectious diseases Hoffman, S. A., Costales, C., Sahoo, M. K., Palanisamy, S., Yamamoto, F., Huang, C., Verghese, M., Solis, D. C., Sibai, M., Subramanian, A., Tompkins, L. S., Grant, P., Shafer, R. W., Pinsky, B. A. 2021; 27 (10)

  Abstract

  We report persistent severe acute respiratory syndrome coronavirus 2 infection in a patient with HIV/AIDS; the virus developed spike N terminal domain and receptor binding domain neutralization resistance mutations. Our findings suggest that immunocompromised patients can harbor emerging variants of severe acute respiratory syndrome coronavirus 2.

  View details for DOI 10.3201/eid2710.211461

  View details for PubMedID 34296992

• Asymptomatic SARS-CoV-2 Transmission from Community Contacts in Healthcare Workers. Annals of surgery Graham, L. A., Maldonado, Y. A., Tompkins, L. S., Wald, S. H., Chawla, A., Hawn, M. T. 2020

  View details for DOI 10.1097/SLA.0000000000003968

  View details for PubMedID 32487801

• Could the prevention of health care-associated infections increase hospital cost? The financial impact of health care-associated infections from a hospital management perspective. American journal of infection control Shepard, J., Frederick, J., Wong, F., Madison, S., Tompkins, L., Hadhazy, E. 2020; 48 (3): 255–60

  Abstract

  BACKGROUND: The financial burden health care-associated infections (HAIs) have on patients, payers, and hospitals is not clear. Although patient safety is the highest priority, administrators require data to justify the expense of HAI reduction programs.METHODS: Chart review was performed to identify HAIs for patients discharged from Stanford Hospital. Using a t test, we tested whether patients with an HAI will have a different daily total hospital cost and length of stay than patients without an HAI. We calculated the change in hospital profit related to HAIs by comparing patients with and without an HAI in the same admit All-Patient Refined Diagnosis Related Group and complexity score.RESULTS: Between October 1, 2015 and September 30, 2018, there were 78,551 inpatient discharges and 1,541 HAIs identified. Daily total hospital cost and length of stay for patients with an HAI versus patients without an HAI was $6,433 ($6,251, $6,615) versus $6,604 ($6,557, $6,651) (P= .073), and 26.30 days (24.89, 27.71) versus 5.69 (5.64, 5.74) (P<.001).DISCUSSION: For each HAI eliminated, data suggests that hospital's cost and revenue would increase $25,008 and $1,518,682, respectively, by backfilling beds with new patients at a 4.62:1 ratio. The reduction of HAIs is profitable for hospitals.CONCLUSIONS: Data from this study suggest that the more HAIs you eliminate and the more capacity you build for the hospital, the higher the total hospital costs will go. This is an essential shift to the current paradigm that will allow for the accurate and continued funding of HAI reduction programs. Although hospital cost appears to increase as HAIs are reduced, hospital profits rise even more.

  View details for DOI 10.1016/j.ajic.2019.08.035

  View details for PubMedID 32089192

• N95 Respirator Alternatives And Conservation Strategies. Anesthesia and analgesia Wong, B. J., Lu, A. C., Tarlow, B. D., Tompkins, L. S., Chawla, A. n., Pearl, R. G., Wald, S. H. 2020

  View details for DOI 10.1213/ANE.0000000000005134

  View details for PubMedID 32701549

• Dual reporting of Clostridioides difficile PCR and predicted toxin result based on PCR cycle threshold reduces treatment of toxin-negative patients without increases in adverse outcomes. Journal of clinical microbiology Hitchcock, M. M., Holubar, M. n., Hogan, C. A., Tompkins, L. S., Banaei, N. n. 2019

  Abstract

  Nucleic acid amplification tests are commonly used to diagnose C. difficile infection (CDI). Two-step testing with toxin enzyme immunoassay is recommended to discriminate between infection and colonization but requires additional resources. Prior studies showed that PCR cycle threshold (CT) can predict toxin positivity with high negative predictive value. Starting Oct. 2016, predicted toxin result (CT-Toxin) based on a validated cut-off was routinely reported at our facility. To evaluate the clinical efficacy of this reporting, all adult patients with positive GeneXpert PCR from Oct. 2016 through Oct. 2017 underwent chart review to measure recurrence of or conversion to a CT-Toxin+ result and 30-day all-cause mortality. There were 482 positive PCR tests in 430 unique patients, 282 CT-Toxin+ and 200 CT-Toxin-. Patient characteristics were similar at testing, though CT-Toxin+ patients had a higher WBC count (12.5 v. 9.3 k/μL; p=0.001). All cases (n=21) of fulminant CDI had a CT-Toxin+ result. Index CT-Toxin+ patients were significantly more likely to have a CT-Toxin+ result within 90 days than CT-Toxin- patients (17.4% [n=49] v. 8.0% [n=16]; p=0.003). Thirty-day all-cause mortality was higher in CT-Toxin- patients (11.1% v. 6.8%; p=0.1), though no deaths in CT-Toxin- patients were directly attributable to CDI. Of 200 CT-Toxin- patients, 51.5% (n=103) were treated for CDI. The rate of conversion to a CT-Toxin+ result (8.8% v. 7.2%; p=0.8) and all-cause mortality (8.8% v. 13.4%; p=0.3) were similar between treated and untreated CT-Toxin- patients. CT-based toxin prediction may identify patients at higher risk for CDI-related complications and reduce treatment among CT-Toxin- patients.

  View details for DOI 10.1128/JCM.01288-19

  View details for PubMedID 31511334

• Infection Rates. Journal of clinical microbiology Truong, C. Y., Gombar, S., Wilson, R., Sundararajan, G., Tekic, N., Holubar, M., Shepard, J., Madison, A., Tompkins, L., Shah, N., Deresinski, S., Schroeder, L. F., Banaei, N. 2017

  Abstract

  Health care-onset health care facility-associated Clostridium difficile infection (HO-CDI) is overdiagnosed for several reasons, including the high prevalence of C. difficile colonization and the inability of hospitals to limit testing to patients with clinically significant diarrhea. We conducted a quasiexperimental study from 22 June 2015 to 30 June 2016 on consecutive inpatients with C. difficile test orders at an academic hospital. Real-time electronic patient data tracking was used by the laboratory to enforce testing criteria (defined as the presence of diarrhea [≥3 unformed stools in 24 h] and absence of laxative intake in the prior 48 h). Outcome measures included C. difficile test utilization, HO-CDI incidence, oral vancomycin utilization, and clinical complications. During the intervention, 7.1% (164) and 9.1% (211) of 2,321 C. difficile test orders were canceled due to absence of diarrhea and receipt of laxative therapy, respectively. C. difficile test utilization decreased upon implementation from an average of 208.8 tests to 143.0 tests per 10,000 patient-days (P < 0.001). HO-CDI incidence rate decreased from an average of 13.0 cases to 9.7 cases per 10,000 patient-days (P = 0.008). Oral vancomycin days of therapy decreased from an average of 13.8 days to 9.4 days per 1,000 patient-days (P = 0.009). Clinical complication rates were not significantly different in patients with 375 canceled orders compared with 869 episodes with diarrhea but negative C. difficile results. Real-time electronic clinical data tracking is an effective tool for verification of C. difficile clinical testing criteria and safe reduction of inflated HO-CDI rates.

  View details for DOI 10.1128/JCM.02319-16

  View details for PubMedID 28250001

• Helicobacter pylori Usurps Cell Polarity to Turn the Cell Surface into a Replicative Niche PLOS PATHOGENS Tan, S., Tompkins, L. S., Amieva, M. R. 2009; 5 (5)

  Abstract

  Helicobacter pylori (Hp) intimately interacts with the gastric epithelial surface and translocates the virulence factor CagA into host cells in a contact-dependent manner. To study how Hp benefits from interacting with the cell surface, we developed live-cell microscopy methods to follow the fate of individual bacteria on the cell surface and find that Hp is able to replicate and form microcolonies directly over the intercellular junctions. On polarized epithelia, Hp is able to grow directly on the apical cell surface in conditions that do not support the growth of free-swimming bacteria. In contrast, mutants in CagA delivery are defective in colonization of the apical cell surface. Hp perturbs the polarized epithelium in a highly localized manner, since wild-type Hp does not rescue the growth defect of the CagA-deficient mutants upon co-infection. CagA's ability to disrupt host cell polarity is a key factor in enabling colonization of the apical cell surface by Hp, as disruption of the atypical protein kinase C/Par1b polarity pathway leads to rescue of the mutant growth defect during apical infection, and CagA-deficient mutants are able to colonize the polarized epithelium when given access to the basolateral cell surface. Our study establishes the cell surface as a replicative niche and the importance of CagA and its effects on host cell polarity for this purpose.

  View details for DOI 10.1371/journal.ppat.1000407

  View details for Web of Science ID 000267085800051

  View details for PubMedID 19412339

  View details for PubMedCentralID PMC2669173

• Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult CLINICAL INFECTIOUS DISEASES Chiu, C. Y., Rouskin, S., Koshy, A., Urisman, A., Fischer, K., Yagi, S., Schnurr, D., Eckburg, P. B., Tompkins, L. S., Blackburn, B. G., Merker, J. D., Patterson, B. K., Ganem, D., DeRisi, J. L. 2006; 43 (8): E71-E76

  Abstract

  A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening.

  View details for Web of Science ID 000240666200029

  View details for PubMedID 16983602

• Investigation of mediastinitis due to coagulase-negative staphylococci after cardiothoracic surgery INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY Van Kerkhove, M. D., Parsonnet, J., Weingart, M., Tompkins, L. S. 2006; 27 (3): 305-307

  Abstract

  Six cases of coagulase-negative staphylococcal mediastinitis were identified in the latter half of 1999. A new preoperative cleansing solution was suspected by hospital staff to be a factor in the outbreak. We evaluated this possible risk factor along with other known and suspected surgical site infection risk factors in this case-control study.

  View details for Web of Science ID 000249035800015

  View details for PubMedID 16532421

• Prolonged incubation and extensive subculturing do not increase recovery of clinically significant microorganisms from standard automated blood cultures CLINICAL INFECTIOUS DISEASES Baron, E. J., Scott, J. D., Tompkins, L. S. 2005; 41 (11): 1677-1680

  Abstract

  An extensive blood culture protocol, including prolonged incubation of cultures, for 215 patients believed to have had endocarditis yielded only 3 clinically relevant results. Twenty-four Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella (i.e., HACEK) organisms were recovered from standard 5-day blood cultures during the same time period. Specialized methods and not extended incubation times are recommended for recovery of fastidious agents of septicemia.

  View details for Web of Science ID 000233018600020

  View details for PubMedID 16267743

• Helicobacter pylori CagA induces a transition from polarized to invasive phenotypes in MDCK cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bagnoli, F., Buti, L., Tompkins, L., Covacci, A., Amieva, M. R. 2005; 102 (45): 16339-16344

  Abstract

  CagA is a bacterial effector protein of Helicobacter pylori that is translocated via a type IV secretion system into gastric epithelial cells. We previously described that H. pylori require CagA to disrupt the organization and assembly of apical junctions in polarized epithelial cells. In this study, we provide evidence that CagA expression is not only sufficient to disrupt the apical junctions but also perturbs epithelial differentiation. CagA-expressing cells lose apicobasal polarity and cell-cell adhesion, extend migratory pseudopodia, and degrade basement membranes, acquiring an invasive phenotype. Expression of the CagA C-terminal domain, which contains the tyrosine phosphorylated EPIYA motifs, induces pseudopodial activity but is not sufficient to induce cell migration. Conversely, the N terminus targets CagA to the cell-cell junctions. Neither domain is sufficient to disrupt cell adhesion or cell polarity, but coexpressed in trans, the N terminus determines the localization of both polypeptides. We show that CagA induces a morphogenetic program in polarized Madin-Darby canine kidney cells resembling an epithelial-to-mesenchymal transition. We propose that altered cell-cell and cell matrix interactions may serve as an early event in H. pylori-induced carcinogenesis.

  View details for Web of Science ID 000233283700039

  View details for PubMedID 16258069

  View details for PubMedCentralID PMC1274241

• Growth phase-dependent response of Helicobacter pylori to iron starvation INFECTION AND IMMUNITY Merrell, D. S., Thompson, L. J., Kim, C. C., Mitchell, H., Tompkins, L. S., Lee, A., FALKOW, S. 2003; 71 (11): 6510-6525

  Abstract

  Iron is an essential nutrient that is often found in extremely limited available quantities within eukaryotic hosts. Because of this, many pathogenic bacteria have developed regulated networks of genes important for iron uptake and storage. In addition, it has been shown that many bacteria use available iron concentrations as a signal to regulate virulence gene expression. We have utilized DNA microarray technology to identify genes of the human pathogen Helicobacter pylori that are differentially regulated on a growth-inhibiting shift to iron starvation conditions. In addition, the growth phase-dependent expression of these genes was investigated by examining both exponential and stationary growth phase cultures. We identified known iron-regulated genes, as well as a number of genes whose regulation by iron concentration was not previously appreciated. Included in the list of regulated factors were the known virulence genes cagA, vacA, and napA. We examined the effect of iron starvation on the motility of H. pylori and found that exponential- and stationary-phase cultures responded differently to the stress. We further found that while growing cells are rapidly killed by iron starvation, stationary-phase cells show a remarkable ability to survive iron depletion. Finally, bioinformatic analysis of the predicted promoter regions of the differentially regulated genes led to identification of several putative Fur boxes, suggesting a direct role for Fur in iron-dependent regulation of these genes.

  View details for DOI 10.1128/IAI.71.11.6510-6525.2003

  View details for Web of Science ID 000186194300049

  View details for PubMedID 14573673

  View details for PubMedCentralID PMC219544

• pH-regulated gene expression of the gastric pathogen Helicobacter pylori INFECTION AND IMMUNITY Merrell, D. S., Goodrich, M. L., Otto, G., Tompkins, L. S., FALKOW, S. 2003; 71 (6): 3529-3539

  Abstract

  Colonization by the gastric pathogen Helicobacter pylori has been shown to be intricately linked to the development of gastritis, ulcers, and gastric malignancy. Little is known about mechanisms employed by the bacterium that help it adapt to the hostile environment of the human stomach. In an effort to extend our knowledge of these mechanisms, we utilized spotted-DNA microarrays to characterize the response of H. pylori to low pH. Expression of approximately 7% of the bacterial genome was reproducibly altered by shift to low pH. Analysis of the differentially expressed genes led to the discovery that acid exposure leads to profound changes in motility of H. pylori, as a larger percentage of acid-exposed bacterial cells displayed motility and moved at significantly higher speeds. In contrast to previous publications, we found that expression of the bacterial virulence gene cagA was strongly repressed by acid exposure. Furthermore, this transcriptional repression was reflected at the level of protein accumulation in the H. pylori cell.

  View details for DOI 10.1128/IAI.71.6.3529-3539.2003

  View details for Web of Science ID 000183116300066

  View details for PubMedID 12761138

  View details for PubMedCentralID PMC155744

• Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA SCIENCE Amieva, M. R., Vogelmann, R., Covacci, A., Tompkins, L. S., NELSON, W. J., FALKOW, S. 2003; 300 (5624): 1430-1434

  Abstract

  Helicobacter pylori translocates the protein CagA into gastric epithelial cells and has been linked to peptic ulcer disease and gastric carcinoma. We show that injected CagA associates with the epithelial tight-junction scaffolding protein ZO-1 and the transmembrane protein junctional adhesion molecule, causing an ectopic assembly of tight-junction components at sites of bacterial attachment, and altering the composition and function of the apical-junctional complex. Long-term CagA delivery to polarized epithelia caused a disruption of the epithelial barrier function and dysplastic alterations in epithelial cell morphology. CagA appears to target H. pylori to host cell intercellular junctions and to disrupt junction-mediated functions.

  View details for Web of Science ID 000183181800045

  View details for PubMedID 12775840

• Use of an open-reading frame-specific Campylobacter jejuni DNA microarray as a new genotyping tool for studying epidemiologically related isolates JOURNAL OF INFECTIOUS DISEASES Leonard, E. E., Takata, T., Blaser, M. J., FALKOW, S., Tompkins, L. S., Gaynor, E. C. 2003; 187 (4): 691-694

  Abstract

  Findings from use of an open-reading frame-specific Campylobacter jejuni DNA microarray to investigate genetic diversity among clinical isolates associated with 5 independent clusters of infection were compared with data from random amplified polymeric DNA (RAPD) and Penner serotyping analyses. The DNA microarray provides a highly specific epidemiological typing tool for analysis of C. jejuni isolates and reveals both divergent and highly conserved gene classes among isolates.

  View details for Web of Science ID 000180884500022

  View details for PubMedID 12599089

• Cag pathogenicity island-specific responses of gastric epithelial cells to Helicobacter pylori infection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Guillemin, K., Salama, N. R., Tompkins, L. S., FALKOW, S. 2002; 99 (23): 15136-15141

  Abstract

  Helicobacter pylori infects over half the world's population and causes a wide range of diseases, including gastritis, peptic ulcer, and two forms of gastric cancer. H. pylori infection elicits a variety of phenotypic responses in cultured gastric epithelial cells, including the expression of proinflammatory genes and changes in the actin cytoskeleton. Both of these responses are mediated by the type IV secretion system (TFSS) encoded by the cag pathogenicity island (cag PAI). We used human cDNA microarrays to examine the temporal transcriptional profiles of gastric AGS cells infected with H. pylori strain G27 and a panel of isogenic mutants to dissect the contributions of various genes in the cag PAI. Infection with G27 induced expression of genes involved in the innate immune response, cell shape regulation, and signal transduction. A mutant lacking the cagA gene, which encodes an effector molecule secreted by the TFSS and required for the host cell cytoskeletal response, induced the expression of fewer cytoskeletal genes. A mutant lacking cagE, which encodes a structural component of the TFSS, failed to up-regulate a superset of host genes, including the cagA-dependent genes, and many of the immune response genes. A mutant lacking the entire cag PAI failed to induce both the cagE-dependent genes and several transiently expressed cagE independent genes. Host cell transcriptional profiling of infection with isogenic strains offered a detailed molecular picture of H. pylori infection and provided insight into potential targets of individual virulence determinants such as tyrosine kinase and Rho GTPase signaling molecules.

  View details for DOI 10.1073/pnas.182558799

  View details for Web of Science ID 000179224800094

  View details for PubMedID 12411577

  View details for PubMedCentralID PMC137556

• Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells CELLULAR MICROBIOLOGY Amieva, M. R., Salama, N. R., Tompkins, L. S., FALKOW, S. 2002; 4 (10): 677-690

  Abstract

  Although intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell-associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco-2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time-lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half-life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori-containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA- mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.

  View details for Web of Science ID 000178480600005

  View details for PubMedID 12366404

• Selection of signature-tagged Legionella pneumophila mutants in Acanthamoeba castellanii 5th International Conference on Legionella Tompkins, L. S., Polesky, A., Ross, J., FALKOW, S. AMER SOC MICROBIOLOGY. 2002: 152–160

  View details for Web of Science ID 000173698000027

• Role of clinical microbiology laboratories in the management and control of infectious diseases and the delivery of health care CLINICAL INFECTIOUS DISEASES Peterson, L. R., Hamilton, J. D., Baron, E. J., Tompkins, L. S., MILLER, J. M., WILFERT, C. M., Tenover, F. C., Thomson, R. B. 2001; 32 (4): 605-610

  Abstract

  Modern medicine has led to dramatic changes in infectious diseases practice. Vaccination and antibiotic therapy have benefited millions of persons. However, constrained resources now threaten our ability to adequately manage threats of infectious diseases by placing clinical microbiology services and expertise distant from the patient and their infectious diseases physician. Continuing in such a direction threatens quality of laboratory results, timeliness of diagnosis, appropriateness of treatment, effective communication, reduction of health care-associated infections, advances in infectious diseases practice, and training of future practitioners. Microbiology laboratories are the first lines of defense for detection of new antibiotic resistance, outbreaks of foodborne infection, and a possible bioterrorism event. Maintaining high-quality clinical microbiology laboratories on the site of the institution that they serve is the current best approach for managing today's problems of emerging infectious diseases and antimicrobial agent resistance by providing good patient care outcomes that actually save money.

  View details for Web of Science ID 000166857900014

  View details for PubMedID 11181125

• Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis INFECTION AND IMMUNITY Polesky, A. H., Ross, J. T., FALKOW, S., Tompkins, L. S. 2001; 69 (2): 977-987

  Abstract

  Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes.

  View details for Web of Science ID 000166528700045

  View details for PubMedID 11159993

• Vacuolating cytotoxin of Helicobacter pylori plays a role during colonization in a mouse model of infection INFECTION AND IMMUNITY Salama, N. R., Otto, G., Tompkins, L., FALKOW, S. 2001; 69 (2): 730-736

  Abstract

  Helicobacter pylori, the causative agent of gastritis and ulcer disease in humans, secretes a toxin called VacA (vacuolating cytotoxin) into culture supernatants. VacA was initially characterized and purified on the basis of its ability to induce the formation of intracellular vacuoles in tissue culture cells. H. pylori strains possessing different alleles of vacA differ in their ability to express active toxin. Those strains expressing higher toxin levels are correlated with more severe gastric disease. However, the specific role(s) played by VacA during the course of infection and disease is not clear. We have used a mouse model of H. pylori infection to begin to address this role. A null mutation of vacA compromises H. pylori in its ability to initially establish infection. If an infection by a vacA mutant is established, the bacterial load and degree of inflammation are similar to those associated with an isogenic wild-type strain. Thus, in this infection model, vacA plays a role in the initial colonization of the host, suggesting that strains of H. pylori expressing active alleles of vacA may be better adapted for host-to-host transmission.

  View details for Web of Science ID 000166528700013

  View details for PubMedID 11159961

  View details for PubMedCentralID PMC97945

• Fecal leukocyte stain has diagnostic value for outpatients but not inpatients JOURNAL OF CLINICAL MICROBIOLOGY Savola, K. L., Baron, E. J., Tompkins, L. S., Passaro, D. J. 2001; 39 (1): 266-269

  Abstract

  The methylene blue stain for fecal leukocytes (FL) is widely used as an adjunct to slower but more accurate tests of diarrheal etiology, such as stool culture (SCx) or toxin assays for Clostridium difficile. Prior studies investigating the utility of FL for predicting SCx and C. difficile toxin assay (CDTA) results did not evaluate the importance of inpatient versus outpatient status. We conducted a study of patients who submitted a stool specimen to the Stanford Hospital Microbiology Laboratory between May 1998 and April 1999. The results for stool specimens that were tested by FL and by a confirmatory test (either SCx or CDTA) were used to determine whether the FL method helped to predict the results of these tests. Of 797 stools that were tested by FL method and at least one confirmatory test, 502 stools were tested by CDTA, and 473 stools were cultured. The FL test was 14% sensitive and 90% specific for C. difficile with a diagnostic threshold of one white blood cell/high-power field (WBC/HPF). The overall likelihood ratio (LR) for a positive CDTA was 1.4 with a 95% confidence interval (CI) of 0. 5 to 3.7 (P = 0.5) and was similar among inpatients and outpatients. In contrast, the presence of >/=1 WBC/HPF was 52% sensitive and 88% specific for the 27 positive SCx results and helped to predict a positive SCx result (LR, 4.2; 95% CI, 2.7 to 6.5; P < 0.001). The sensitivity of >/=1 WBC/HPF was 57%, and its predictive value for SCx was higher among outpatients (outpatient LR, 5.0; 95% CI, 2.9 to 8.6; P < 0.001; inpatient LR, 1.9; 95% CI, 0.3 to 10.8; P = 0.5). Among inpatients, only 4 (1.5%) of the 273 SCx results were positive, and the presence of >/=1 WBC/HPF was insensitive (25%) and did not predict a positive SCx (LR, 1.9; 95% CI, 0.3 to 10.8; P = 0.5). When the data were reanalyzed using a diagnostic threshold of five WBC/HPF for FL, the predictive power of the FL method was similar. Thus, FL was of no value in predicting CDTA positivity, nor was it helpful in predicting SCx results for inpatients. Neither SCx nor the FL method should routinely be performed on samples from inpatients. Among outpatients, presence of FLs should suggest a bacterial diarrhea in clinically compatible cases.

  View details for Web of Science ID 000166468900043

  View details for PubMedID 11136781

• A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Salama, N., Guillemin, K., McDaniel, T. K., Sherlock, G., Tompkins, L., FALKOW, S. 2000; 97 (26): 14668-14673

  Abstract

  Helicobacter pylori colonizes the stomach of half of the world's population, causing a wide spectrum of disease ranging from asymptomatic gastritis to ulcers to gastric cancer. Although the basis for these diverse clinical outcomes is not understood, more severe disease is associated with strains harboring a pathogenicity island. To characterize the genetic diversity of more and less virulent strains, we examined the genomic content of 15 H. pylori clinical isolates by using a whole genome H. pylori DNA microarray. We found that a full 22% of H. pylori genes are dispensable in one or more strains, thus defining a minimal functional core of 1281 H. pylori genes. While the core genes encode most metabolic and cellular processes, the strain-specific genes include genes unique to H. pylori, restriction modification genes, transposases, and genes encoding cell surface proteins, which may aid the bacteria under specific circumstances during their long-term infection of genetically diverse hosts. We observed distinct patterns of the strain-specific gene distribution along the chromosome, which may result from different mechanisms of gene acquisition and loss. Among the strain-specific genes, we have found a class of candidate virulence genes identified by their coinheritance with the pathogenicity island.

  View details for Web of Science ID 000165993700121

  View details for PubMedID 11121067

• Collaborative multidisciplinary workshop report: detection, culture, serology, and antimicrobial susceptibility testing of Chlamydia pneumoniae (vol 181, pg S460, 2000) JOURNAL OF INFECTIOUS DISEASES Tompkins, L. S., Schachter, J., Boman, J., Chernesky, M. A. 2000; 182 (5): 1581

  View details for Web of Science ID 000090106000051

• Collaborative multidisciplinary workshop report: Detection, culture, serology, and antimicrobial susceptibility testing of Chlamydia pneumoniae JOURNAL OF INFECTIOUS DISEASES Tompkins, L. S., SCHACHTER, J., Boman, J., Dowell, S., Gaydos, C. A., Levison, M. E., MAASS, M., Madico, G., Orfila, J., Ouchi, K., Peeling, R. W., Taylor-Robinson, D., STAMM, W. E., Wang, S. P., Blasi, F., Relman, D. 2000; 181: S460-S461

  View details for Web of Science ID 000088023900019

  View details for PubMedID 10839740

• Altered states: Involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segal, E. D., Cha, J., Lo, J., FALKOW, S., Tompkins, L. S. 1999; 96 (25): 14559-14564

  Abstract

  Helicobacter pylori, present in half of the world's population, is a very successful pathogen. It can survive for decades in the human stomach with few obvious consequences to the host. However, it is also the cause of gastric diseases ranging from gastritis to ulcers to gastric cancer and has been classified a type 1 carcinogen by the World Health Organization. We have previously shown that phosphorylation of a 145-kDa protein and activation of signal transduction pathways are associated with the attachment of H. pylori to gastric cells. Here we identify the 145-kDa protein as the H. pylori CagA protein. We also show that CagA is necessary to induce a growth-factor-like phenotype (hummingbird) in host gastric cells similar to that induced by hepatocyte growth factor (HGF). Additionally, we identify a second cellular phenotype induced after attachment by H. pylori, which we call SFA (stress fiber associated). SFA is CagA independent and is produced by type I and type II H. pylori.

  View details for Web of Science ID 000084149700075

  View details for PubMedID 10588744

  View details for PubMedCentralID PMC24475

• The application of molecular biology to medical bacteriology - Yesterday, today, and tomorrow ASM NEWS FALKOW, S., Tompkins, L. S. 1999; 65 (5): 317-321

  View details for Web of Science ID 000080304000013

• The host cell response to Helicobacter pylori attachment Symposium on Helicobacter Pylori - Basic Mechanisms to Clinical Cure Tompkins, L. S., Segal, E., FALKOW, S. SPRINGER. 1998: 77–83

  View details for Web of Science ID 000075410300010

• Induction of host signal transduction pathways by Helicobacter pylori PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segal, E. D., Lange, C., Covacci, A., Tompkins, L. S., FALKOW, S. 1997; 94 (14): 7595-7599

  Abstract

  Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8). H. pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection. Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements. Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H. pylori pathways is independent of the tyrosine phosphorylation step.

  View details for Web of Science ID A1997XJ87600085

  View details for PubMedID 9207137

  View details for PubMedCentralID PMC23867

• Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segal, E. D., FALKOW, S., Tompkins, L. S. 1996; 93 (3): 1259-1264

  Abstract

  The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.

  View details for Web of Science ID A1996TU64000056

  View details for PubMedID 8577751

  View details for PubMedCentralID PMC40067

• Bartonella species infections, including cat-scratch disease, trench fever, and bacillary angiomatosis - What molecular techniques have revealed WESTERN JOURNAL OF MEDICINE Tompkins, L. S. 1996; 164 (1): 39-41

  View details for Web of Science ID A1996TR29000023

  View details for PubMedID 8779201

  View details for PubMedCentralID PMC1303292

• BARTONELLA-HENSELAE AND BARTONELLA-QUINTANA ADHERENCE TO AND ENTRY INTO CULTURED HUMAN EPITHELIAL-CELLS INFECTION AND IMMUNITY Batterman, H. J., Peek, J. A., Loutit, J. S., FALKOW, S., Tompkins, L. S. 1995; 63 (11): 4553-4556

  Abstract

  Bartonella henselae expresses pili phenotypically similar to type 4 pili. B. henselae pilus expression undergoes phase variation with multiple passages. Low-passage-number, piliated B. henselae adhered to and invaded HEp-2 cells to a greater extent than did multiply passaged B. henselae with reduced pilus expression. Pili may be a pathogenic determinant for Bartonella species.

  View details for Web of Science ID A1995TB40000056

  View details for PubMedID 7591104

• CONSTRUCTION AND CHARACTERIZATION OF AN ISOGENIC UREASE-NEGATIVE MUTANT OF HELICOBACTER-MUSTELAE INFECTION AND IMMUNITY SOLNICK, J. V., JOSENHANS, C., SUERBAUM, S., TOMPKINS, L. S., LABIGNE, A. 1995; 63 (9): 3718–21

  Abstract

  Helicobacter mustelae infects the ferret stomach and provides an opportunity to study pathogenic determinants of a Helicobacter species in its natural host. We constructed an isogenic urease-negative mutant of H. mustelae which produced no detectable urease and showed a reduced acid tolerance. This mutant provides an opportunity to further evaluate the role of urease in the pathogenesis of Helicobacter infection.

  View details for Web of Science ID A1995RQ79200072

  View details for PubMedID 7642313

  View details for PubMedCentralID PMC173517

• THE NEW PATH TO PREVENTING ULCERS SCIENCE Tompkins, L. S., FALKOW, S. 1995; 267 (5204): 1621-1622

  View details for Web of Science ID A1995QM39700028

  View details for PubMedID 7886448

• NEW TECHNOLOGY IN THE CLINICAL MICROBIOLOGY LABORATORY - WHAT YOU ALWAYS WANTED TO KNOW BUT WERE AFRAID TO ASK JOURNAL OF INFECTIOUS DISEASES Tompkins, L. S., Tenover, F., Arvin, A. 1994; 170 (5): 1068-1074

  View details for Web of Science ID A1994PN66800002

  View details for PubMedID 7963694

• GROWTH OF LEGIONELLA-PNEUMOPHILA IN ACANTHAMOEBA-CASTELLANII ENHANCES INVASION INFECTION AND IMMUNITY Cirillo, J. D., FALKOW, S., Tompkins, L. S. 1994; 62 (8): 3254-3261

  Abstract

  Legionella pneumophila is considered to be a facultative intracellular parasite. Therefore, the ability of these bacteria to enter, i.e., invade, eukaryotic cells is expected to be a key pathogenic determinant. We compared the invasive ability of bacteria grown under standard laboratory conditions with that of bacteria grown in Acanthamoeba castellanii, one of the protozoan species that serves as a natural host for L. pneumophila in the environment. Amoeba-grown L. pneumophila cells were found to be at least 100-fold more invasive for epithelial cells and 10-fold more invasive for macrophages and A. castellanii than were L. pneumophila cells grown on agar. Comparison of agar- and amoeba-grown L. pneumophila cells by light and electron microscopy demonstrated dramatic differences in the morphology and structure of the bacteria. Analyses of protein expression in the two strains of bacteria suggest that these phenotypic differences may be due to the expression of new proteins in amoeba-grown L. pneumophila cells. In addition, the amoeba-grown bacteria were found to enter macrophages via coiling phagocytosis at a higher frequency than agar-grown bacteria did. Replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease.

  View details for Web of Science ID A1994NY87200029

  View details for PubMedID 8039895

  View details for PubMedCentralID PMC302953

• EFFECTS OF AN ISOGENIC ZN-METALLOPROTEASE-DEFICIENT MUTANT OF LEGIONELLA-PNEUMOPHILA IN A GUINEA-PIG PNEUMONIA MODEL MOLECULAR MICROBIOLOGY Moffat, J. F., Edelstein, P. H., Regula, D. P., Cirillo, J. D., Tompkins, L. S. 1994; 12 (5): 693-705

  Abstract

  To determine the effects, if any, of the Zn-metalloprotease on virulence of Legionella pneumophila infection, an isogenic mutant deficient in protease (encoded by the proA gene) was tested in an Acanthamoeba cell model, in guinea-pig macrophages, and in a guinea-pig pneumonia model. The cloned proA gene was completely inactivated by insertion of a kanamycin-resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA- mutant AA200. AA200 showed no difference in its ability to enter, survive, or grow in Acanthamoeba and explanted guinea-pig macrophages; neither light nor electron microscopy revealed morphological differences in the eukaryotic cells infected with the protease mutant or the wild-type strains. The proA gene was found to be expressed in L. pneumophila during intracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the protease mutant was attenuated when tested in a guinea-pig model of infection employing the intratracheal inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likely to be found in association with macrophage vacuoles than the parent strain. Although deletion of the protease was not sufficient to completely abolish virulence in a guinea-pig model, the mutation caused a delay in the lethal effects of L. pneumophila and attenuated the infection.

  View details for Web of Science ID A1994NP48400002

  View details for PubMedID 8052122

• MOLECULAR ANALYSIS OF UREASE GENES FROM A NEWLY IDENTIFIED UNCULTURED SPECIES OF HELICOBACTER INFECTION AND IMMUNITY Solnick, J. V., Orourke, J., Lee, A., Tompkins, L. S. 1994; 62 (5): 1631-1638

  Abstract

  "Gastrospirillum hominis" is an uncultured gastric spiral bacterium that has recently been shown by 16S rDNA sequence analysis to be a newly recognized species of Helicobacter that infects humans, and it has been provisionally designated "Helicobacter heilmannii." We used PCR to directly amplify the urease structural genes of "H. heilmannii" from infected gastric tissue. DNA sequence analysis identified two open reading frames, ureA and ureB, which code for polypeptides with predicted molecular weights of 25,729 and 61,831, respectively. The urease subunit genes from "H. heilmannii" were cloned and expressed in Escherichia coli. Western blot (immunoblot) analysis showed that antiserum directed against the ureA and ureB gene products from H. pylori was cross-reactive with the corresponding polypeptides from "H. heilmannii." Analysis of the derived amino acid sequences of "H. heilmannii" UreA and UreB demonstrated that "H. heilmannii" urease is more highly related to the urease from H. felis (found in the stomachs of cats and dogs) than to the urease from H. pylori. These data are consistent with 16S rDNA sequence analysis and suggest that "H. heilmannii" is phylogenetically most closely related to H. felis.

  View details for Web of Science ID A1994NH31300017

  View details for PubMedID 8168924

• FURTHER MOLECULAR CHARACTERIZATION OF THE CLONED LEGIONELLA-PNEUMOPHILA ZINC METALLOPROTEASE INFECTION AND IMMUNITY Moffat, J. F., BLACK, W. J., Tompkins, L. S. 1994; 62 (2): 751-753

  Abstract

  On the basis of DNA sequence similarities to other Zn metalloproteases, further studies of the synthesis, processing, and enzymatic structure of the cloned Legionella protease gene, proA, were initiated. TnphoA fusions indicated that the entire proA open reading frame was transcribed and translated, including the 5' leader sequence. The results also suggested that the entire polypeptide was exported to the periplasm before cleavage to produce the mature protease. A site-directed mutation in the putative active site, changing glutamate 378 to asparagine, abolished proteolytic activity and cytotoxicity.

  View details for Web of Science ID A1994MR84100062

  View details for PubMedID 8300238

  View details for PubMedCentralID PMC186173

• IDENTIFICATION AND CHARACTERIZATION OF A HELICOBACTER-PYLORI HEMOLYSIN 1st Bristol-Myers Squibb Symposium on Infectious Disease Research: The Cell and Molecular Biology of Bacterial-Host Cell Interactions Segal, E. D., Tompkins, L. S. LIPPINCOTT-RAVEN PUBL. 1993: 178–82

  View details for Web of Science ID A1993MZ25500004

  View details for PubMedID 8173790

• AN UNCULTURED GASTRIC SPIRAL ORGANISM IS A NEWLY IDENTIFIED HELICOBACTER IN HUMANS JOURNAL OF INFECTIOUS DISEASES Solnick, J. V., Orourke, J., Lee, A., Paster, B. J., Dewhirst, F. E., Tompkins, L. S. 1993; 168 (2): 379-385

  Abstract

  "Gastrospirillum hominis" is an uncultivated spiral bacterium in human gastric mucosa that is larger and more tightly coiled than Helicobacter pylori. In an attempt to determine if this organism is a new species of Helicobacter, its 16S rRNA gene was cloned and sequenced. Gastric mucosa from 2 patients infected with "Gastrospirillum hominis" was fed to specific pathogen-free mice. Electron microscopy of gastric tissue confirmed that the mice became colonized with "Gastrospirillum hominis." The 16S rRNA gene from bacterial target sequences was amplified directly from mouse stomach tissue by the polymerase chain reaction (PCR), cloned into Escherichia coli, and sequenced. Both fragments were 16S rRNA genes from the Helicobacter genus that were most closely related to Helicobacter felis. "Gastrospirillum hominis" is probably a newly recognized Helicobacter infection in humans. Because this is the only Helicobacter organism that infects both humans and small animals, it may be particularly suited for studies of pathogenesis.

  View details for Web of Science ID A1993LN81300018

  View details for PubMedID 8335974

• MOLECULAR STRAIN TYPING OF MYCOBACTERIUM-TUBERCULOSIS TO CONFIRM CROSS-CONTAMINATION IN THE MYCOBACTERIOLOGY LABORATORY AND MODIFICATION OF PROCEDURES TO MINIMIZE OCCURRENCE OF FALSE-POSITIVE CULTURES JOURNAL OF CLINICAL MICROBIOLOGY Small, P. M., McClenny, N. B., Singh, S. P., SCHOOLNIK, G. K., Tompkins, L. S., Mickelsen, P. A. 1993; 31 (7): 1677-1682

  Abstract

  Molecular strain typing by restriction fragment length polymorphism analysis was used to demonstrate that two clusters of Mycobacterium tuberculosis cultures involving six patients resulted from cross-contamination in the mycobacteriology laboratory. Contaminated cultures were processed by the decontamination procedure and were read on the BACTEC instrument following acid-fast bacillus smear-positive specimens from patients with active tuberculosis. Investigation of these episodes suggested opportunities for modification of laboratory procedures to minimize cross-contamination and confirmed the adverse medical and public health consequences of false-positive cultures. Strain-typing results were used in decisions regarding patient care, including the curtailment of unnecessary treatment in one patient. Molecular strain typing appears to be a valuable means of identifying false-positive cultures of M. tuberculosis in selected settings.

  View details for Web of Science ID A1993LJ20100001

  View details for PubMedID 8102372

  View details for PubMedCentralID PMC265613

• ROLE OF FLAGELLA IN ADHERENCE, INTERNALIZATION, AND TRANSLOCATION OF CAMPYLOBACTER-JEJUNI IN NONPOLARIZED AND POLARIZED EPITHELIAL-CELL CULTURES INFECTION AND IMMUNITY Grant, C. C., Konkel, M. E., Cieplak, W., Tompkins, L. S. 1993; 61 (5): 1764-1771

  Abstract

  Previous studies of Campylobacter jejuni have suggested that flagellin is an adhesin for epithelial cells and that motility is a virulence factor of this bacterium. The role of flagella in the interactions of C. jejuni with nonpolarized and polarized epithelial cells was examined with flagellar mutants. Flagellated, nonmotile (flaA flaB+ Mot-) and nonflagellated, nonmotile (flaA flaB Mot-) mutants of C. jejuni were constructed by in vivo homologous recombination and gene replacement techniques. Both classes of mutants were found to adhere to cells of human epithelial origin (INT 407) equally well; however, on the basis of the percentage of the inoculum internalized, internalization of the flaA flaB Mot- mutants was decreased by factors ranging from approximately 30 to 40 compared with the parent. The flaA flaB+ Mot- mutant was internalized by the INT 407 cells at levels six- to sevenfold higher than the flaA flaB Mot- mutants. Both classes of mutants, unlike the parent, were unable to translocate across polarized Caco-2 monolayers. These results indicate that flagella are not involved in C. jejuni adherence to epithelial cells but that they do play a role in internalization. Furthermore, the results suggest that either the motility of C. jejuni or the product of flaA is essential for the bacterium to cross polarized epithelial cell monolayers.

  View details for Web of Science ID A1993KZ17700023

  View details for PubMedID 8478066

  View details for PubMedCentralID PMC280763

• NOSOCOMIAL LEGIONELLOSIS - A REVIEW OF PULMONARY AND EXTRAPULMONARY SYNDROMES AMERICAN JOURNAL OF INFECTION CONTROL Lowry, P. W., Tompkins, L. S. 1993; 21 (1): 21-27

  Abstract

  Surgical patients appear to be at highest risk for acquisition of nosocomial Legionella pneumonia; most appear to become infected during respiratory tract manipulation and mechanical ventilation. Although the lungs are the most common site of nosocomial Legionella infection, an important subset of patients have infection at extrapulmonary sites. We describe 22 cases of extrapulmonary legionellosis reported in the literature. Most of these patients were surgical patients; more than half did not have serious underlying illnesses, and only five (23%) were receiving immunosuppressive agents. A total of 13 extrapulmonary sites of infection were reported, many in the absence of clinical pneumonia; these infections included sinusitis, hip wound infection, and prosthetic valve endocarditis. Five patients (23%) had fatal infections; in four of these cases diagnosis of Legionella infection was made after death, underscoring the need for a high index of clinical suspicion. A large percentage of extrapulmonary Legionella infections may result from direct topical exposure of susceptible tissue to contaminated tap water. Use of tap water must be carefully monitored, particularly in dressing changes and bathing of surgical patients.

  View details for Web of Science ID A1993KN18800005

  View details for PubMedID 8442518

• TRANSFORMATION OF HELICOBACTER-PYLORI BY ELECTROPORATION BIOTECHNIQUES Segal, E. D., Tompkins, L. S. 1993; 14 (2): 225-226

  Abstract

  A highly efficient method of introducing DNA into an important human pathogen is reported here. Electroporation-mediated transformation of a laboratory-passaged isolate of Helicobacter pylori was successfully used to establish genetic mutants at a transformation frequency of > 10(5)/micrograms DNA. This method should be widely applicable to all isolates of H. pylori and may eliminate the variability reported when natural transformation was used on fresh clinical isolates.

  View details for Web of Science ID A1993KL40900023

  View details for PubMedID 8431287

• EVALUATION OF A DNA AMPLIFICATION PROCEDURE FOR DETECTION OF LEGIONELLA-PNEUMOPHILA AND LEGIONELLA-DUMOFFII IN WATER 4TH INTERNATIONAL SYMP ON LEGIONELLA Loutit, J. S., Tompkins, L. S. AMER SOC MICROBIOLOGY. 1993: 176–178

  View details for Web of Science ID A1993BY23Q00059

• DETECTION OF LEGIONELLA BY MOLECULAR METHODS 4TH INTERNATIONAL SYMP ON LEGIONELLA Tompkins, L. S., Loutit, J. S. AMER SOC MICROBIOLOGY. 1993: 163–168

  View details for Web of Science ID A1993BY23Q00055

• HELICOBACTER-PYLORI AND GASTRODUODENAL DISEASE - PATHOGENESIS AND HOST-PARASITE INTERACTION INFECTIOUS AGENTS AND DISEASE-REVIEWS ISSUES AND COMMENTARY Solnick, J. V., Tompkins, L. S. 1992; 1 (6): 294-309

  Abstract

  Helicobacter pylori has been shown to be the cause of chronic active gastritis and the evidence that it is involved in the development of peptic ulcer disease and gastric cancer is compelling. Narrow host range, tissue specificity, and chronic inflammation are hallmarks of infection. The study of virulence determinants has just begun but it seems likely that urease, adhesins, cytotoxins, and mediators of inflammation will prove to be important.

  View details for Web of Science ID A1992KK66600002

  View details for PubMedID 1344668

• The use of molecular methods in infectious diseases. New England journal of medicine Tompkins, L. S. 1992; 327 (18): 1290-1297

  View details for PubMedID 1406820

• CURRENT CONCEPTS - THE USE OF MOLECULAR METHODS IN INFECTIOUS-DISEASES NEW ENGLAND JOURNAL OF MEDICINE Tompkins, L. S. 1992; 327 (18): 1290-1297

  View details for Web of Science ID A1992JV25900008

• CHARACTERIZATION OF HELICOBACTER-PYLORI UREASE MUTANTS INFECTION AND IMMUNITY Segal, E. D., Shon, J., Tompkins, L. S. 1992; 60 (5): 1883-1889

  Abstract

  The association between Helicobacter pylori, gastritis, and peptic ulcer is well established, and the association of infection with gastric cancer has been noted in several developing countries. However, the pathogenic mechanism(s) leading to disease states has not been elucidated. The H. pylori urease is thought to be a determinant of pathogenicity, since the enzyme is produced by all H. pylori clinical isolates. Evidence indicates that some H. pylori strains are more cytotoxic than others, with a correlation between the activity of the urease and the presence of a vacuolating cytotoxin having been made. However, the number of cytotoxins remains unknown at this time. The relationship between the urease and cytotoxicity has previously been examined with chemical inhibitors. To examine the role of the urease and its relationship to cytotoxicity, urease-deficient mutants were produced following ethyl methanesulfonate mutagenesis of H. pylori 87A300. Two mutants (the ure1 and ure5 mutants) which were entirely deficient in urease activity (Ure-) were selected. Characterization of the isolates at the protein level showed that the urease subunits lacked the ability to complex and form the active urease enzyme. The ure1 mutant was shown to be sensitive to the effects of low pH in vitro and exhibited no cytotoxicity to eucaryotic cells, whereas the parental strain (Ure+) produced a cytotoxic effect in the presence of urea. Interaction between the H. pylori Ure+ and Ure- strains and Caco-2 cells appeared to be similar in that both bacterial types elicited pedestal formation and actin condensation. These results indicate that the H. pylori urease may have many functions, among them (i) protecting H. pylori against the acidic environment of the stomach, (ii) acting as a cytotoxin, with human gastric cells especially susceptible to its activity, and (iii) disrupting cell tight junctions in such a manner that the cells remain viable but an ionic flow between the cells occurs.

  View details for Web of Science ID A1992HR06500025

  View details for PubMedID 1563778

  View details for PubMedCentralID PMC257089

• A QUANTITATIVE MODEL OF INTRACELLULAR GROWTH OF LEGIONELLA-PNEUMOPHILA IN ACANTHAMOEBA-CASTELLANII INFECTION AND IMMUNITY Moffat, J. F., Tompkins, L. S. 1992; 60 (1): 296-301

  Abstract

  A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37 degrees C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one-step growth curve and should be useful to study the molecular basis of the host-parasite interaction.

  View details for Web of Science ID A1992GW97400042

  View details for PubMedID 1729191

  View details for PubMedCentralID PMC257535

• RESTRICTION ENZYME AND SOUTHERN HYBRIDIZATION ANALYSES OF PSEUDOMONAS-AERUGINOSA STRAINS FROM PATIENTS WITH CYSTIC-FIBROSIS JOURNAL OF CLINICAL MICROBIOLOGY Loutit, J. S., Tompkins, L. S. 1991; 29 (12): 2897-2900

  Abstract

  Conventional typing schemes for Pseudomonas aeruginosa may not discriminate strains. P. aeruginosa isolates from patients with cystic fibrosis were examined by restriction enzyme analysis and Southern hybridization. There was marked diversity of restriction enzyme analysis patterns among P. aeruginosa DNAs from cystic fibrosis isolates; however, sequential isolates obtained from individual patients showed very little variation over an 8-year period. DNA fragments from the alginate biosynthesis gene complex, the exotoxin A gene, and the elastase gene of P. aeruginosa were used in Southern hybridization analysis. The patterns of hybridization to the elastase and algD gene probes were highly conserved in all isolates, therefore, these DNA fragments are not useful in discriminating strains, in contrast to the exotoxin A gene probe.

  View details for Web of Science ID A1991GP88400046

  View details for PubMedID 1684586

• A CLUSTER OF LEGIONELLA-STERNAL WOUND INFECTIONS DUE TO POSTOPERATIVE TOPICAL EXPOSURE TO CONTAMINATED TAP WATER NEW ENGLAND JOURNAL OF MEDICINE Lowry, P. W., BLANKENSHIP, R. J., GRIDLEY, W., TROUP, N. J., Tompkins, L. S. 1991; 324 (2): 109-113

  View details for Web of Science ID A1991EQ97700007

  View details for PubMedID 1984176

• THE AGENT OF BACILLARY ANGIOMATOSIS - AN APPROACH TO THE IDENTIFICATION OF UNCULTURED PATHOGENS NEW ENGLAND JOURNAL OF MEDICINE Relman, D. A., Loutit, J. S., Schmidt, T. M., FALKOW, S., Tompkins, L. S. 1990; 323 (23): 1573-1580

  Abstract

  Bacillary angiomatosis is an infectious disease causing proliferation of small blood vessels in the skin and visceral organs of patients with human immunodeficiency virus infection and other immunocompromised hosts. The agent is often visualized in tissue sections of lesions with Warthin-Starry staining, but the bacillus has not been successfully cultured or identified. This bacillus may also cause cat scratch disease.In attempting to identify this organism, we used the polymerase chain reaction. We used oligonucleotide primers complementary to the 16S ribosomal RNA genes of eubacteria to amplify 16S ribosomal gene fragments directly from tissue samples of bacillary angiomatosis. The DNA sequence of these fragments was determined and analyzed for phylogenetic relatedness to other known organisms. Normal tissues were studied in parallel.Tissue from three unrelated patients with bacillary angiomatosis yielded a unique 16S gene sequence. A sequence obtained from a fourth patient with bacillary angiomatosis differed from the sequence found in the other three patients at only 4 of 241 base positions. No related 16S gene fragment was detected in the normal tissues. These 16S sequences associated with bacillary angiomatosis belong to a previously uncharacterized microorganism, most closely related to Rochalimaea quintana.The cause of bacillary angiomatosis is a previously uncharacterized rickettsia-like organism, closely related to R. quintana. This method for the identification of an uncultured pathogen may be applicable to other infectious diseases of unknown cause.

  View details for Web of Science ID A1990EK69600001

  View details for PubMedID 2233945

• VISCERAL BACILLARY EPITHELIOID ANGIOMATOSIS - POSSIBLE MANIFESTATIONS OF DISSEMINATED CAT SCRATCH DISEASE IN THE IMMUNOCOMPROMISED HOST - A REPORT OF 2 CASES AMERICAN JOURNAL OF MEDICINE Kemper, C. A., Lombard, C. M., Deresinski, S. C., Tompkins, L. S. 1990; 89 (2): 216-222

  Abstract

  Opportunistic infection with the causative agent of cat scratch disease may be responsible for an unusual vascular proliferative lesion, referred to as bacillary epithelioid angiomatosis, previously described only in human immunodeficiency virus (HIV)-infected patients. We present a case of an HIV-infected patient with bacillary epithelioid angiomatosis involving the liver and bone marrow causing progressive hepatic failure. We also report a case of a cardiac transplant recipient with hepatic and splenic bacillary epithelioid angiomatosis manifesting as a fever of unknown origin, a previously unreported event in a non-HIV-infected patient. These cases represent the first documentation of bacillary epithelioid angiomatosis with visualization of cat scratch-like organisms involving internal organs.

  View details for Web of Science ID A1990DT83100015

  View details for PubMedID 2382668

• LEGIONELLA-PNEUMOPHILA ZINC METALLOPROTEASE IS STRUCTURALLY AND FUNCTIONALLY HOMOLOGOUS TO PSEUDOMONAS-AERUGINOSA ELASTASE JOURNAL OF BACTERIOLOGY BLACK, W. J., Quinn, F. D., Tompkins, L. S. 1990; 172 (5): 2608-2613

  Abstract

  The sequence of the structural gene encoding the Legionella pneumophila extracellular zinc metalloprotease has been determined and was found to possess a single large open reading frame (ORF) of 1,629 nucleotides (nt). This ORF was preceded by consensus promoter (TTAACT . . . 17 nt . . . TATAAC) and ribosome-binding (TAAGGAG) sequences. The deduced polypeptide contained a putative signal sequence and a total of 543 amino acid residues with a computed molecular size of 60,775 daltons, substantially larger than the observed 38,000 daltons of the native and recombinant proteins. A homology search revealed extensive amino acid identity with Pseudomonas aeruginosa elastase, a protein that is also encoded by an ORF substantially larger than that predicted for the mature size of the protein. The structural identity between the L. pneumophila protease and P. aeruginosa elastase was most pronounced in the regions forming the enzymatic active site of elastase. Amino acid residues constituting the active-site cleft of elastase were greater than 75% conserved. Elastase residues that interact with and mediate proteolysis of substrate peptides were 100% conserved. Competitive inhibitors of elastase and the structurally and functionally related thermolysin (phosphoramidon and a phosphoramidate analog, Z-GlyP(O)Leu-Ala), were shown to be equally potent at inhibiting the proteolytic activity of the L. pneumophila protease. These inhibitor studies along with the amino acid sequence similarities provide strong evidence that the L. pneumophila protease and P. aeruginosa elastase share a similar molecular mechanism of proteolysis.

  View details for Web of Science ID A1990DC16700057

  View details for PubMedID 2110146

  View details for PubMedCentralID PMC208904

• TRANSFORMATION OF ACTINOBACILLUS-PLEUROPNEUMONIAE AND ANALYSIS OF R FACTORS BY ELECTROPORATION AMERICAN JOURNAL OF VETERINARY RESEARCH Lalonde, G., Miller, J. F., Tompkins, L. S., OHANLEY, P. 1989; 50 (11): 1957-1960

  Abstract

  An efficient method for DNA transfer is essential for the genetic manipulation of any organism. Such a capacity will be required for the genetic analysis of Actinobacillus pleuropneumoniae as a swine pathogen, as well as for its manipulation for vaccination purposes. For this reason, the use of electroporation as a means of plasmid DNA introduction into this species was examined. The multiple antibiotic-resistant strain 80-8141 of Actinobacillus pleuropneumoniae harbors 3 plasmids: pYG10, pYG15, and pYG12 of 5.0, 2.7, and 2.5 kb, respectively. Electroporation of A pleuropneumoniae strain 4074 with a plasmid extract of strain 80-8141 showed that pYG10 encodes chloramphenicol resistance and that pYG12 encodes ampicillin resistance. Electrical pulse conditions for efficient electroporation of strain 4074 were examined by use of pYG10 DNA isolated from a 4074 transformant. Efficiency, expressed as transformants per microgram of plasmid DNA, increased directly with pulse amplitude. However, high efficiencies were only observed in a narrow window of pulse duration (tau = 12 to 22 ms at 6.25 kV/cm). Longer pulse durations resulted in cell death. Electroporation efficiencies increased with cell density. Yield of transformants increased directly with DNA concentration. Results indicate that electroporation can be used to efficiently transform A pleuropneumoniae and that pYG10 and pYG12 are suitable plasmid vectors for use in the genetic manipulation of this organism.

  View details for Web of Science ID A1989AX74300029

  View details for PubMedID 2619125

• GENETIC, IMMUNOLOGICAL, AND CYTO-TOXIC COMPARISONS OF LEGIONELLA PROTEOLYTIC ACTIVITIES INFECTION AND IMMUNITY Quinn, F. D., KEEN, M. G., Tompkins, L. S. 1989; 57 (9): 2719-2725

  Abstract

  Several strains of Legionella pneumophila and other species of Legionella with proteolytic activities were compared by assays, including Southern hybridizations and Western immunoblots, to determine their proteolytic, hemolytic, and cytotoxic activities. Only proteases from strains of L. pneumophila were both hemolytic and cytotoxic, and proteolytic activities extracted from other species of Legionella possessed only hemolytic activity. A 4.0-kilobase DNA sequence encoding the 38-kilodalton metalloprotease from L. pneumophila Philadelphia 1 that we showed previously was responsible for the observed hemolytic and cytotoxic phenotypes (F. D. Quinn and L. S. Tompkins, Mol. Microbiol., 3:797-805, 1989) was used in Southern hybridizations to probe chromosomal DNA from several strains of L. pneumophila and other Legionella species. The probe hybridized to the chromosomal DNA of all serogroups of L. pneumophila but not to any strains of L. dumoffii, L. micdadei, L. feeleii, or L. jordanis that we examined. Additionally, Western immunoblots done with rabbit antisera made to the cloned L. pneumophila protease demonstrated cross-reactions among 38-kilodalton proteins from strains of L. pneumophila, but no reactions were observed with proteins from other species of Legionella. Similarly, the cloned protease from L. pneumophila reacted with convalescent-phase sera from patients infected with L. pneumophila, but not with antisera isolated from patients infected with other Legionella species. Thus, despite some similarities among the proteolytic activities of members of the genus Legionella, including proteolytic and hemolytic phenotypes, metal requirements for zinc or iron, sensitivity to EDTA, and temperature and pH optima, we documented distinct genetic, immunological, and cytotoxicity differences among the proteolytic activities produced by Legionella species.

  View details for Web of Science ID A1989AL70400019

  View details for PubMedID 2668184

  View details for PubMedCentralID PMC313517

• ANALYSIS OF A CLONED SEQUENCE OF LEGIONELLA-PNEUMOPHILA ENCODING A 38 KD METALLOPROTEASE POSSESSING HEMOLYTIC AND CYTO-TOXIC ACTIVITIES MOLECULAR MICROBIOLOGY Quinn, F. D., Tompkins, L. S. 1989; 3 (6): 797-805

  Abstract

  The DNA encoding the zinc metalloprotease of Legionella pneumophila Philadelphia 1 has been isolated and expressed in Escherichia coli. This protein, which is 38,000 Daltons in size, possesses immunological and biochemical properties identical to those previously described for the purified L. pneumophila protease. Periplasmic extracts of E. coli clones expressing the recombinant protease are also capable of causing the haemolysis of canine erythrocytes and the cytotoxic destruction of CHO cells. Using transposon mutagenesis, it was determined that a maximum of 1.2 kb of DNA encoded all three biological activities. Inactivation of proteolytic activity by transposon insertion occurred concomitantly with losses of the haemolytic and cytotoxic phenotypes. A putative regulatory sequence approximately 200-500 bp upstream of the gene's coding region was identified. A 4.0 kb fragment encoding these activities hybridized to the chromosomal DNA of the parent strain of L. pneumophila Philadelphia 1 as well as clinical isolates of L. pneumophila.

  View details for Web of Science ID A1989AA80200011

  View details for PubMedID 2546010

• SPECIES-SPECIFIC DETECTION OF LEGIONELLA-PNEUMOPHILA IN WATER BY DNA AMPLIFICATION AND HYBRIDIZATION JOURNAL OF CLINICAL MICROBIOLOGY Starnbach, M. N., FALKOW, S., Tompkins, L. S. 1989; 27 (6): 1257-1261

  Abstract

  A sensitive detection system specific for Legionella pneumophila in water was developed. This system is based on amplification of a chromosomal DNA sequence from L. pneumophila by the polymerase chain reaction, followed by detection of the amplified product by hybridization of a radiolabeled oligodeoxynucleotide. After 35 cycles of amplification, a water sample which had been seeded with 35 CFU of L. pneumophila contained sufficient amplified DNA to be detected on dot blots. Bacteria of other genera tested did not generate positive signals under these conditions. Application of this technique to environmental water samples may help identify the natural reservoirs of nosocomial and community-acquired L. pneumophila infections.

  View details for Web of Science ID A1989U715700022

  View details for PubMedID 2754000

  View details for PubMedCentralID PMC267537

• LEGIONELLA PROSTHETIC-VALVE ENDOCARDITIS NEW ENGLAND JOURNAL OF MEDICINE Tompkins, L. S., Roessler, B. J., Redd, S. C., Markowitz, L. E., Cohen, M. L. 1988; 318 (9): 530-535

  Abstract

  Since 1982 seven patients at Stanford University Medical Center have been shown to have prosthetic-valve endocarditis caused by Legionella pneumophila or L. dumoffii. We studied the clinical features of legionella endocarditis at the time of diagnosis and performed a case-control study to analyze risk factors for the infection. All patients with endocarditis had a chronic course (3 to 19 months after surgery) of fever, night sweats, weight loss, and anemia, but no embolic events or immune-complex deposition disease. Five patients required surgical replacement of their infected prosthetic valves. The case-control study revealed that during the early postoperative period, patients who later contracted legionella endocarditis were more likely to have had symptoms and signs attributable to postcardiomyotomy syndrome than were patients who did not contract endocarditis (P less than 0.013). Examination of the legionella isolates by means of molecular techniques demonstrated that the Stanford L. pneumophila isolates were genotypically identical to isolates from the hospital drinking water. L. dumoffii isolates from patients with endocarditis were derived from a single strain apparently unique to this medical center. We conclude that legionella infection was nosocomially acquired in the perioperative period. These cases demonstrate an expanding spectrum of illness caused by legionella species and emphasize the need to consider legionella as a cause of "culture-negative" endocarditis.

  View details for Web of Science ID A1988M298800002

  View details for PubMedID 3340136

• HIGH-VOLTAGE ELECTROPORATION OF BACTERIA - GENETIC-TRANSFORMATION OF CAMPYLOBACTER-JEJUNI WITH PLASMID DNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Miller, J. F., Dower, W. J., Tompkins, L. S. 1988; 85 (3): 856-860

  Abstract

  Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 x 10(6) transformants per microgram of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

  View details for Web of Science ID A1988L992200045

  View details for PubMedID 3277182

  View details for PubMedCentralID PMC279654

• GENE-TRANSFER FROM ESCHERICHIA-COLI TO CAMPYLOBACTER SPECIES - DEVELOPMENT OF SHUTTLE VECTORS FOR GENETIC-ANALYSIS OF CAMPYLOBACTER-JEJUNI JOURNAL OF BACTERIOLOGY LABIGNEROUSSEL, A., Harel, J., Tompkins, L. 1987; 169 (11): 5320-5323

  Abstract

  A shuttle cloning vector (pIL550) has been constructed which can be mobilized from Escherichia coli to Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus by complementation with the transfer functions of an IncP plasmid in trans, with a frequency of 10(-4) transconjugants per donor. We also present evidence for a DNA modification system in C. jejuni.

  View details for Web of Science ID A1987K704200068

  View details for PubMedID 2822671

• MOLECULAR EPIDEMIOLOGY OF LEGIONELLA SPECIES BY RESTRICTION ENDONUCLEASE AND ALLOENZYME ANALYSIS JOURNAL OF CLINICAL MICROBIOLOGY Tompkins, L. S., TROUP, N. J., Woods, T., Bibb, W., MCKINNEY, R. M. 1987; 25 (10): 1875-1880

  Abstract

  As part of an ongoing investigation into nosocomial Legionella infections at Stanford University Medical Center (SUMC), we applied the technique of restriction endonuclease analysis (REA) to determine strain differences among three species, including Legionella pneumophila, Legionella dumoffii, and Legionella micdadei. A total of 26 human and environmental water isolates from SUMC were selected for REA and compared with control strains that were not epidemiologically linked to SUMC. REA results were compared with results of alloenzyme typing, typing by monoclonal antibodies, and plasmid fingerprinting in all but L. micdadei strains. REA and alloenzyme typing showed that SUMC patient isolates were derived from distinct strains of three species. L. pneumophila strains from SUMC patients were genotypically identical to those isolated from potable water. REA was especially useful in proving that SUMC L. dumoffii patient isolates were derived from a single strain and that patients may have been exposed to a common source(s). REA typing correlated well with alloenzyme typing. These methods complement serologic typing of L. pneumophila and provide discriminating capability between strains of other Legionella species such as L. dumoffii, for which serologic types have not been identified. In addition, REA typing is somewhat easier to perform than alloenzyme typing and can be done in clinical laboratories.

  View details for Web of Science ID A1987K084200012

  View details for PubMedID 2822760

  View details for PubMedCentralID PMC269359

• CLONED, RANDOM CHROMOSOMAL SEQUENCES AS PROBES TO IDENTIFY SALMONELLA SPECIES JOURNAL OF INFECTIOUS DISEASES Tompkins, L. S., Troup, N., LABIGNEROUSSEL, A., Cohen, M. L. 1986; 154 (1): 156-162

  Abstract

  We have developed and evaluated a new technique--chromosomal probe fingerprinting--to differentiate between strains of Salmonella species by using sequences of cloned chromosomal DNA as probes to highlight restriction site heterogeneity. Chromosomal probe fingerprint patterns were compared with other strain-typing methods and epidemiological data. Seventeen isolates of Salmonella typhimurium recovered from 11 outbreaks had six unique chromosomal probe fingerprint patterns. Most strains of Salmonella dublin, including some that had identical plasmid profiles and restriction endonuclease analysis patterns, could be distinguished by this method. On the other hand, eight of nine isolates of Salmonella enteritidis had a common chromosomal probe fingerprint pattern, although there were differences in plasmid profiles and the isolates had been collected over a lengthy time interval from widely disparate geographic locations. These results suggest clonal dissemination of some Salmonella serovars.

  View details for Web of Science ID A1986C823200020

  View details for PubMedID 3011919

• DNA METHODOLOGIES IN DIAGNOSTIC MICROBIOLOGY SURVEY AND SYNTHESIS OF PATHOLOGY RESEARCH Tompkins, L. S., FALKOW, S. 1983; 2 (1-2): 134-140

  View details for Web of Science ID A1983RK16200014

• GROWING PROBLEM OF BACTERIAL-RESISTANCE DRUG THERAPY Tompkins, L. S., FALKOW, S. 1979; 9 (5): 39-46

  View details for Web of Science ID A1979GU85000002