Diabetes mellitus (DM) is a metabolic disease whose prevalence has steadily risen in the United States and globally over the last three decades. In addition to its direct effects on hyperglycemia, DM has a variety of associated complications, ranging from an increased risk of cardiovascular disease to an increased risk for multiple infectious diseases. Because of these complications, DM remains a major source of disease burden in the United States and globally. Even as the prevalence of DM has risen, so too have awareness of the disease and the armamentarium of medications available for its treatment. These changes in both awareness and the ability to treat the disease have ostensibly had an impact. Rates of some of the most important complications of DM, including cerebrovascular accident (CVA) and myocardial infarction (MI) have declined in the United States while at the same time, the prevalence of DM has leveled off.

In this dissertation, I focus on three separate, but linked issues about DM. All of the work I present here was done either at the state level in California or at the level of the city of San Francisco. In chapter 1, I examine whether the decreases that have been observed for non-infectious complications of DM are also true for its infectious complications in California overall. In chapter 2, in a cohort of patients in San Francisco, I examine the potential role of medications that are frequently used to treat other conditions in persons with DM in driving the increased risk of infection in persons with DM as well as the findings I describe in chapter 1. Finally, in chapter 3, working with the same cohort studied in chapter 2, I examine the role of distance to the primary care clinic, a potentially modifiable factor, in determining diabetes control.

In chapter 1 of this dissertation, I show that in California, rates of hospitalization for infectious diseases among persons with DM have steadily increased. This result contrasts both with what has been reported for non-infectious disease complications of DM nationally as well as with my own findings for two important non-infectious disease complications of DM, CVA and MI. My results suggest that other factors beyond the level of control of DM could drive the risk of infection among persons with DM. In chapter 2, I show that while one such potential factor, collateral effects of medications used to treat other conditions (e.g. hyperlipidemia,hypertension) in persons with DM, does not appear to drive the increase risk of infection in persons with DM, two medication classes, angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) could actually reduce the risk of developing in infection. Finally, in chapter 3, I show that minimizing the distance between patients and their primary care clinics has the potential to result in a significant improvement in control of DM, as measured by the glycated hemoglobin level. Together, the three chapters of this dissertation provide a comprehensive view of DM in California as a whole and in San Francisco in particular. My findings shed new light on one of DM’s most important complications, infectious diseases, while at the same suggesting two potential methods to limit the development of these and potentially other DM-related complications.

Background

Objectives

Methods

Results

Conclusion

Host-Factor Enhancement of Therapy for Tuberculosis

by

Michael David Schump

Doctor of Philosophy in Infectious Diseases and Immunity

University of California, Berkeley

Professor Lee W. Riley, Chair

Tuberculosis (TB) is a disease of major public health importance and improvements to its treatment could greatly benefit efforts aimed at eliminating the disease. Current treatment options for TB are limited in effectiveness and have numerous fundamental failings due to the necessarily lengthy duration of therapy and toxicity of the antimicrobial drugs deployed, among other issues. The studies described herein where undertaken with the goal of developing adjunctive treatments or modifications of existing treatments which could improve the treatment course, outcome, or both for standard TB antimicrobial chemotherapy.

Three areas of research are discussed beginning with adaptive immune augmentation through therapeutic vaccination, proceeding to investigations of innate immune adjuvant therapy and concluding with host environment mediated improvement of selectivity index of TB antimicrobial compounds.

A post-treatment, therapeutic vaccine was studied with the goal of developing a tool which could prevent relapse or reactivation disease. Though the project was a follow up to a study which demonstrated exceptional protection, the vaccine candidate did not demonstrate any detectable efficacy in three parallel murine infection experiments. Possible reasons and implications of this failure are discussed.

Because correlates of protection for adaptive immunity to TB are poorly understood and have not proven to be tractable for intervention, innate immune enhancement was investigated. Autophagy, a cell-intrinsic process with antimicrobial capabilities, was selected due to its well described tuberculocidal activity and pharmacologic manipulability. However, despite the apparent capacity of some test compounds to increase autophagic flux, none demonstrated robust restriction of mycobacterial growth in murine or human macrophages. That study did, however, lead to the serendipitous discovery that pH based drug partitioning can increase the selectivity index of antimicrobial drugs against M. tuberculosis inside cultured macrophages.

Drug resistant infections with Gram negative bacteria have become increasingly common. Some of these Gram negative bacteria are pan resistant. To better understand the spread of drug resistant infections we used E. coli as a model system. By using this system we investigated both the spread of drug resistance genes and bacteria that are drug resistant.

Drug resistance genes and their mobile genetic elements are frequently identified from environmental saprophytic organisms. It is widely accepted that the use of antibiotics in agriculture selects for drug resistant microorganisms, which are then spread from the farm environment to humans through the consumption of contaminated food products. We wished to identify novel drug resistance genes from microbial communities on retail food products. To do this, we created metagenomic plasmid libraries from microbiota isolated from retail spinach samples. From these libraries we identified five unique plasmids that increased resistance to antimicrobial drugs. These plasmids were identified in E. coli that grew on plates that contained ampicillin (pAMP), aztreonam (pAZT), ciprofloxacin (pCIP), trimethoprim (pTRM), and trimethoprim-sulfamethoxazole (pSXT). We identified open reading frames with similarity to known classes of drug resistance genes in the DNA inserts of all five plasmids. These drug resistance genes conferred resistance to fluoroquinolones, cephalosporins, and trimethoprim, which are classes of antimicrobial drugs frequently used to treat human Gram negative bacterial infections. These results show that novel drug resistance genes are found in microbiota on retail produce items. Food saprophytes may serve as an important reservoir for new drug-resistance determinants in human pathogens.

The clinical management of infections caused by E. coli, including meningitis, is greatly complicated when the organism becomes resistant to broad-spectrum antibiotics. We sought to characterize the antimicrobial susceptibility, multilocus sequence type (MLST), and presence of known drug resistance genes of E. coli that caused meningitis between 1996 and 2011 in Salvador, Brazil. We then compared these findings to E. coli isolates from community acquired urinary tract infections (UTI) that occurred during the same time period and in the same city. We found 19% of E. coli from cases of meningitis and less than 1% of isolates from UTI to be resistant to third-generation cephalosporins. The sequence types of E. coli from cases of meningitis included ST 131, ST 69, ST 405, and ST 62, which were also found among isolates from UTI. These sequence types of E. coli have previously been isolated from produce items and food animals. Additionally, among the E. coli isolates that were resistant to third-generation cephalosporins, we found genes that encode the extended-spectrum beta-lactamases CTX-M-2, CTX-M-14, and CTX-M-15. These observations demonstrate that, compared to E. coli isolated from cases of community acquired UTI, those isolated from cases of meningitis are more resistant to third-generation cephalosporins, even though the same sequence types are shared between the two forms of extraintestinal infections.

The results of our investigation of retail food products indicate that drug resistant genes are frequently found in these products and that these food items may be a reservoir for drug resistance genes found in human pathogens. We also found that many of the sequence types of E. coli that cause both UTI and meningitis have previously been isolated from retail food products. These findings suggest that bacteria that are considered to be normal flora of food products may play an important role of the spread of drug resistant genes and drug resistant bacteria.

Bacterial resistance to antimicrobial agents is not new—for millions of years bacteria have evolved resistance mechanisms to counteract the antimicrobial agents produced by competing bacteria and fungi. The impact of antimicrobial resistance on human health, however, is fairly new and was first realized in the 1940s when bacterial resistance to the first clinically used antibiotic, penicillin, was discovered. Since then the antimicrobial and antimicrobial resistance arms race between scientists and bacteria has ensued, to the point where there is a resistance mechanism to virtually every clinically used antibacterial agent. The prevalence of antimicrobial resistance is projected to increase, such that scientists fear we are nearing the post-antibiotic era, where even simple bacterial infections will be impossible to treat with currently available drugs.

The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) strive to educate the community, scientists, medical professionals, and policy makers on the problems associated with antimicrobial resistance. In 2013, the CDC released a report—Antibiotic Resistance Threats in the United States—detailing the organisms and resistance mechanisms that require action now. More recently, in 2017, the WHO released a similar priority list for antibiotic-resistant bacteria. At the top of the CDC and WHO lists were Gram-negative bacteria (GNB) with carbapenem resistance, and also GNB producing extended-spectrum ß-lactamases (ESBLs). These organisms are GNB with resistance to broad-spectrum ß-lactam antibiotics, including resistance to the last-resort ß-lactam drugs, the carbapenems. In its report, the CDC described four core actions that should be taken to prevent or slow the spread or occurrence of antimicrobial resistance. These included: 1) preventing infections to reduce the occurrence and spread of resistance, 2) improving surveillance of antimicrobial-resistant infections, 3) improving antibiotic prescribing practices and promoting antimicrobial stewardship, and 4) developing new antimicrobial agents and new diagnostic tests for antimicrobial-resistant bacteria.

The dissertation, herein, focuses specifically on issues relating to ß-lactam-resistant GNB. It addresses the need for better surveillance and understanding of the GNB responsible for ß-lactam-resistant infections, as well as the need for rapid diagnostic tests that detect ß-lactam-resistant GNB, specifically in regards to urinary tract infections (UTIs).

In the first chapter, we assess the lineages of uropathogenic E. coli (UPEC) causing ß-lactam-resistant and susceptible community-acquired urinary tract infections (CA-UTIs), to understand the contribution of specific lineages, drug-resistance genes, and plasmids to the prevalence and dissemination of ß-lactam-resistant CA-UTIs in a community. We analyzed 273 consecutively collected UPEC isolated from patients presenting to a university health center with symptoms of UTI. UPEC were subtyped by MLST and fimH typing to establish lineage, and were tested by PCR and sequencing for carriage of ß-lactamase genes and plasmid incompatibility/replicon groups. We found that the distribution of lineages/sublineages among ß-lactam-resistant and susceptible UPEC was different, indicating that selective pressure from ß-lactam drugs is not exerted equally on UPEC. A limited set of circulating UPEC sequence types, sublineages, ß-lactamase genes, and Inc type plasmids likely harboring these genes, contributed to a substantial proportion of ß-lactam-resistant infections in this community. Understanding factors that affect the prevalence and dissemination of antimicrobial resistance may provide insights into strategies to slow the spread of resistance, and inform better empirical treatment decisions. Additionally, population-based studies like these provide essential information for the development of diagnostic tests that target antimicrobial resistance mechanisms.

The development of non-nucleic acid-based strategies for detecting antimicrobial resistance directly from urine specimens are the focus of the second and third chapters. In the second chapter, we describe our first milestone towards development of a lateral flow assay (LFA) for detecting ß-lactam resistance in GNB causing CA-UTIs. The aim of this project is to develop an LFA that detects clinically relevant ß-lactamases found in GNB causing CA-UTIs, to create a diagnostic test that would inform physicians whether their patients could safely receive narrow-spectrum ß-lactam drugs for treatment. LFAs are rapid antibody-based tests; as such, extensive antibody testing is an essential component in their creation. In this chapter, we describe the development of a sensitive and specific anti-CTX-M sandwich ELISA to universally detect the CTX-M ß-lactamases. The CTX-M ß-lactamases are a challenging target for antibody-based and nucleic acid-based approaches, since these ß-lactamases are a relatively sequence-diverse class of enzymes. Sandwich ELISAs represent the first major milestone in our antibody development workflow, and the CTX-M ß-lactamases represent one of several important ß-lactamase targets for our diagnostic test.

In the third chapter, we investigate the utility of a biochemical-based strategy, DETECT, as a diagnostic tool to identify ß-lactamase activity in Gram-negative uropathogens. The aim of this project was to develop a rapid diagnostic test that detects the activity of a variety of clinically relevant ß-lactamases, to provide physicians with resistance information that would guide the selection of appropriate ß-lactam therapy for patients with suspected UTI. In this chapter, we stringently studied the first-generation DETECT system to define its capacity to detect different classes of ß-lactamases. We found that DETECT was able to identify the activity of expanded-spectrum beta-lactamases, namely CTX-M and CMY. Insights gained from this work are directing future iterations of the DETECT diagnostic system, to reach alternative ß-lactamase targets that could impact clinical decision-making.

Tuberculosis (TB) faces difficult challenges for its treatment and control. Both the diagnosis of TB and Mycobacterium tuberculosis (M. tuberculosis) drug susceptibility testing take weeks and clinicians often do not know if the patient is taking an appropriate set of drugs until complications or even death occur. Consequently, early determination of a successful drug therapy response in individuals infected with M. tuberculosis is urgently needed. Since the M. tuberculosis cell wall is comprised of a diverse repertoire of lipids, we examined the possible role of these lipids as antigens for serologic response during M. tuberculosis infection. This dissertation is focused on the examination of lipid-antibody response as a potential biomarker used to monitor treatment response in M. tuberculosis infected hosts. Briefly, Chapter 1 describes the current pitfalls of monitoring tuberculosis treatment with current methods, including acid-fast bacilli (AFB) smear and culture conversion. Chapter 1 also covers the definition of biomarkers and the rationale to use M. tuberculosis cell wall lipids to develop an anti-lipid-antibody based test. Evidence from a similar biomarker-based test for syphilis is presented. The chapter also discusses the biological basis which guided the lipid-antibody biomarker search and discovery.

Chapter 2 describes the use of a serum bank from patients with pulmonary TB provided by the World Health Organization-Tropical Diseases Research consortium (WHO-TDR) to identify M. tuberculosis lipid candidates as targets of antibody response. These samples were used to look for an antibody response to multiple mycobacterial lipids resolved by thin-layer chromatography immunoblot (TLC-I). This approach allowed us to identify M. tuberculosis cardiolipin by mass spectrometry and we determined that the IgM antibody response to cardiolipin can be used as a biomarker of infection.

In Chapter 3, we investigate the biological evidence behind the production of anti-phospholipid IgM antibody during TB infection and anti-TB treatment. For this purpose, we used the Cornell mouse model of infection to monitor the change in IgM antibody response against four phospholipids including cardiolipin (CL), phosphatidyl choline (PTC), phosphatidyl ethanolamine (PE) and phosphatidyl inositol (PI) over the course of M. tuberculosis infection and treatment. We separated BALB/c mice into three groups including acute infection (AI), chronic infection (CI), and healthy control (HC). Both AI and CI groups were infected via the aerosol route with M. tuberculosis strain H37Rv at day 0. The AI group was treated from 4 to 12 weeks post-infection, while the CI group was treated from 20 to 28 weeks post-infection. We also measured the levels of pro-inflammatory cytokines, IL-5 and MCP-1. We observed that in treated AI mice, anti-phospholipid IgM antibody levels decreased compared to those of healthy mice at all time points. Anti-PTC IgM antibodies remained significantly higher in CI mice than in AI mice at all time points post-infection. The anti-PTC IgM antibody levels in CI mice decreased to levels similar to those of AI and HC mice at 32 weeks post-infection. The anti-phospholipid IgM antibody levels correlated with the bacterial load in the lungs, with treated mice showing fewer M. tuberculosis colony-forming units (CFU) after eight weeks of treatment. Furthermore, IL-5 was mainly produced by the site of infection in the lung and decreased with anti-tuberculosis treatment within the CI mice group.

Finally, in Chapter 4, we examine the use of anti-phospholipid IgM antibody changes as a biomarker for treatment response in patients with smear positive pulmonary TB. Serum samples were obtained from pulmonary TB patients at the start and end of the intensive phase of treatment (40 doses of anti-TB combination therapy) enrolled from Kampala, Uganda in a CDC-TB Trials Consortium randomized clinical trial. The samples were screened for IgM antibody levels against five commercially available phospholipids by an in-house ELISA assay. The lipid antigens included CL, PI, PE, PTC, and sphingolipid (SL). IgM antibody levels to CL, PE, PI, PTC and SL significantly decreased following anti-TB drug treatment in patients without lung cavities on their baseline chest radiograph. In contrast, patients with cavitary TB showed an overall increase in the anti-phospholipid IgM antibody response following anti-TB drug treatment, notably with a significant increase in anti-PE antibody levels. Thus, anti-lipid IgM response appears to be a useful biomarker for treatment response, especially in those with non-cavitary disease.

Chapter 5 summarizes the conclusions in support of using the anti-phospholipid IgM antibody response as a useful biomarker for monitoring TB treatment response. This novel biomarker test would greatly facilitate TB management in resource-poor settings. The development of a point of care (POC) test based on anti-phospholipid IgM antibody will be an affordable and highly sensitive alternative to microscopy or culture testing for monitoring treatment response in individuals with TB. Chapter 5 also gives examples of three platforms that might be used for the development of such a POC test. However, we note that it is necessary to explore the specificity of this assay further by testing patients with HIV infection, latent TB infection, and non-TB pulmonary diseases.

Background

Methods

Results

Conclusions

While some microbial encounters can be pathogenic, most interactions between humans and microorganisms do not result in disease. Our gastrointestinal tract harbors approximately 100 trillion organisms, spanning about 1000 species that live in harmony with us. While we provide them with food and shelter, this complex intestinal bacterial community supplies essential nutrients, metabolizes indigestible compounds, protects us from pathogenic organisms and helps develop our immune system, thereby establishing a symbiotic relationship. Bacteroides are one of the most abundant bacterial genera living in the human gut and has long been a model system for studying the molecular mechanisms of commensal-host interactions. In the course of searching for surface structures that have immunomodulatory properties on the host, we serendipitously discovered a previously unknown surface protein BT1927 from Bacteroides that is capable of completely covering the surface of the bacteria by creating a tessellated surface layer. Interestingly, the formation of the entire proteinaceous capsule is controlled by inversion of a single promoter, making it phase-variable. Although only ~ 0.1% of the population produce this capsule in laboratory culture, analyzing the fecal metagenome sequence data from the Human Microbiome Project revealed that ~ 8% of the population produce a homologous capsule in humans, suggesting its significance in vivo. From a series of functional studies, we found that the BT1927-expressing subpopulation is profoundly resistant to complement-mediated killing, due in part to the BT1927-mediated blockade of C3b deposition. Our results show that the Bacteroides capsule is capable of a far greater degree of structural variation than previously known, perhaps to cope with the varying environmental conditions in the human intestine.