Stephanie Cagle

Clinical Use of Colistin Induces Cross-Resistance to Host Antimicrobials in Acinetobacter baumannii

ABSTRACT The alarming rise in antibiotic resistance has led to an increase in patient mortality and health care costs. This problem is compounded by the absence of new antibiotics close to regulatory approval. Acinetobacter baumannii is a human pathogen that causes infections primarily in patients in intensive care units (ICUs) and is highly antibiotic resistant. Colistin is one of the last-line antibiotics for treating A. baumannii infections; however, colistin-resistant strains are becoming increasingly common. This cationic antibiotic attacks negatively charged bacterial membranes in a manner similar to that seen with cationic antimicrobials of the innate immune system. We therefore set out to determine if the increasing use of colistin, and emergence of colistin-resistant strains, is concomitant with the generation of cross-resistance to host cationic antimicrobials. We found that there is indeed a positive correlation between resistance to colistin and resistance to the host antimicrobials LL-37 and lysozyme among clinical isolates. Importantly, isolates obtained before and after treatment of individual patients demonstrated that colistin use correlated with increased resistance to cationic host antimicrobials. These data reveal the overlooked risk of inducing cross-resistance to host antimicrobials when treating patients with colistin as a last-line antibiotic. IMPORTANCE Increased use of the cationic antibiotic colistin to treat multidrug-resistant Acinetobacter baumannii has led to the development of colistin-resistant strains. Here we report that treatment of patients with colistin can induce not only increased resistance to colistin but also resistance to host cationic antimicrobials. This worrisome finding likely represents an example of a broader trend observed in other bacteria against which colistin is used therapeutically such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Furthermore, these data suggest that the possible future use of an array of cationic antimicrobial peptides in development as therapeutics may have unintended negative consequences, eventually leading to the generation of hypervirulent strains that are resistant to innate host defenses. The potential for the induction of cross-resistance to innate immune antimicrobials should be considered during the development of new therapeutics. Increased use of the cationic antibiotic colistin to treat multidrug-resistant Acinetobacter baumannii has led to the development of colistin-resistant strains. Here we report that treatment of patients with colistin can induce not only increased resistance to colistin but also resistance to host cationic antimicrobials. This worrisome finding likely represents an example of a broader trend observed in other bacteria against which colistin is used therapeutically such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Furthermore, these data suggest that the possible future use of an array of cationic antimicrobial peptides in development as therapeutics may have unintended negative consequences, eventually leading to the generation of hypervirulent strains that are resistant to innate host defenses. The potential for the induction of cross-resistance to innate immune antimicrobials should be considered during the development of new therapeutics.

Biomarkers on patient T cells diagnose active tuberculosis and monitor treatment response

BACKGROUND The identification and treatment of individuals with tuberculosis (TB) is a global public health priority. Accurate diagnosis of pulmonary active TB (ATB) disease remains challenging and relies on extensive medical evaluation and detection of Mycobacterium tuberculosis (Mtb) in the patients sputum. Further, the response to treatment is monitored by sputum culture conversion, which takes several weeks for results. Here, we sought to identify blood-based host biomarkers associated with ATB and hypothesized that immune activation markers on Mtb-specific CD4+ T cells would be associated with Mtb load in vivo and could thus provide a gauge of Mtb infection. METHODS Using polychromatic flow cytometry, we evaluated the expression of immune activation markers on Mtb-specific CD4+ T cells from individuals with asymptomatic latent Mtb infection (LTBI) and ATB as well as from ATB patients undergoing anti-TB treatment. RESULTS Frequencies of Mtb-specific IFN-γ+CD4+ T cells that expressed immune activation markers CD38 and HLA-DR as well as intracellular proliferation marker Ki-67 were substantially higher in subjects with ATB compared with those with LTBI. These markers accurately classified ATB and LTBI status, with cutoff values of 18%, 60%, and 5% for CD38+IFN-γ+, HLA-DR+IFN-γ+, and Ki-67+IFN-γ+, respectively, with 100% specificity and greater than 96% sensitivity. These markers also distinguished individuals with untreated ATB from those who had successfully completed anti-TB treatment and correlated with decreasing mycobacterial loads during treatment. CONCLUSION We have identified host blood-based biomarkers on Mtb-specific CD4+ T cells that discriminate between ATB and LTBI and provide a set of tools for monitoring treatment response and cure. TRIAL REGISTRATION Registration is not required for observational studies. FUNDING This study was funded by Emory University, the NIH, and the Yerkes National Primate Center.

Neurocognition across the spectrum of mucopolysaccharidosis type I: Age, severity, and treatment.

OBJECTIVES Precise characterization of cognitive outcomes and factors that contribute to cognitive variability will enable better understanding of disease progression and treatment effects in mucopolysaccharidosis type I (MPS I). We examined the effects on cognition of phenotype, genotype, age at evaluation and first treatment, and somatic disease burden. METHODS Sixty patients with severe MPS IH (Hurler syndrome treated with hematopoietic cell transplant and 29 with attenuated MPS I treated with enzyme replacement therapy), were studied with IQ measures, medical history, genotypes. Sixty-seven patients had volumetric MRI. Subjects were grouped by age and phenotype and MRI and compared to 96 normal controls. RESULTS Prior to hematopoietic cell transplant, MPS IH patients were all cognitively average, but post-transplant, 59% were below average, but stable. Genotype and age at HCT were associated with cognitive ability. In attenuated MPS I, 40% were below average with genotype and somatic disease burden predicting their cognitive ability. White matter volumes were associated with IQ for controls, but not for MPS I. Gray matter volumes were positively associated with IQ in controls and attenuated MPS I patients, but negatively associated in MPS IH. CONCLUSIONS Cognitive impairment, a major difficulty for many MPS I patients, is associated with genotype, age at treatment and somatic disease burden. IQ association with white matter differed from controls. Many attenuated MPS patients have significant physical and/or cognitive problems and receive insufficient support services. Results provide direction for future clinical trials and better disease management.

Neurocognitive and neuropsychiatric phenotypes associated with the mutation L238Q of the α-L-iduronidase gene in Hurler–Scheie syndrome

UNLABELLED The lysosomal enzyme α-L-iduronidase hydrolyzes terminal iduronic acid from heparan sulfate and dermatan sulfate, and is an essential step in GAG degradation. Mutations of its gene, IDUA, yield a spectrum of mucopolysaccharidosis (MPS) type I clinical disorders. The IDUA mutation, c.712T>A (p.L238Q) was previously noted as a mild mutation. In a longitudinal study of MPS brain structure and function (Lysosomal Disease Network), we found this mutation in 6 of 14 Hurler-Scheie syndrome patients in the age range of 15 to 25 years. We hypothesized that L238Q, when paired with a nonsense mutation, is significantly more severe than other missense-nonsense combinations. METHODS Of 6 patients with a L238Q mutation, the L238Q allele was paired with a nonsense mutation in 4 patients, paired with a deletion in 1, and with a splice site mutation in another. This group was compared to 6 Hurler-Scheie patients closely matched in age and mutation type. IQ and other neuropsychological tests were administered as part of the protocol. Medical history was compiled into a Physical Symptom Score (PSS). Assessment of IQ, attention, memory, spatial ability, adaptive function and psychological status were measured. RESULTS No group differences were found in mean age at evaluation (17.8 and 19.0 years), duration of ERT, or PSS. By history, all were reported to be average in IQ (4/6 with documentation) in early childhood. All (100%) of the L238Q group had a psychiatric history and sleep problems compared to none (0%) of the comparison group. Significant differences were found in depression and withdrawal on parent report measures. IQ was lower in the L238Q group (mean IQ 74) than the comparison group (mean IQ 95; p<0.016). Attention, memory, and visual-spatial ability scores were also significantly lower. Three occurrences of shunted hydrocephalus, and 4 of cervical cord compression were found in the L238Q group; the comparison group had one occurrence of unshunted hydrocephalus and two of cord compression. DISCUSSION The missense mutation L238Q, when paired with a nonsense mutation, is associated with significant, late-onset brain disease: psychiatric disorder, cognitive deficit, and general decline starting at a later age than in Hurler syndrome with a mutation-related rate of GAG accumulation and its pathologic sequelae. This particular genotype-phenotype may provide insight into the genesis of psychiatric illnesses more broadly. Consideration of methods for early, brain-targeted treatment in these patients might be considered.

Psychological Health in Adults with Morquio Syndrome

Mucopolysaccharidosis type IV (MPSIV), also known as Morquio syndrome, is a progressive genetic condition which predominantly affects skeletal development. Research thus far has focused on physical manifestations, with little attention to psychological characteristics. As a first step in determining the natural occurrence of psychological symptoms in this population, we administered Achenbach measures of psychological functioning (ASEBA ASR and OASR), quality of life (SF-36), and pain severity (BPI) questionnaires to 20 adults with Morquio syndrome. 11/20 subjects (55%) scored within the symptomatic range on at least one or more ASEBA problem scales. These subjects also had higher pain severity scores (p = 0.051) and pain interference scores (p = 0.03) on the BPI. However, subjects with psychological symptoms did not differ significantly on QOL measures from those without psychological symptoms. Overall, subjects scored below the US mean only in physical health QOL (p < 0.001) on the SF-36, not mental health QOL. Implications of this study include the need for greater attention to psychological health in persons with Morquio syndrome, including regular assessment for psychological symptoms in addition to the quality of life measures typically used, as the latter may miss important information. Greater attention to psychological symptoms may help maximize overall health in adults with Morquio syndrome. Comparison with psychological studies on other lysosomal storage diseases suggests these results may be disease specific, rather than the result of living with chronic pain or having an LSD in general.

Induction of human plasmablasts during infection with antibiotic-resistant nosocomial bacteria

OBJECTIVES Nosocomial pathogens such as Acinetobacter baumannii are a growing public health threat, due in part to their increasing resistance to antibiotics. Since some strains are resistant to all available antibiotics, novel therapies are urgently needed. Plasmablasts are short-lived B cells found in the blood that can be collected and harnessed to produce therapeutic antibodies. We set out to determine whether plasmablasts are induced during infection with A. baumannii and other nosocomial pathogens. METHODS We obtained blood samples from patients infected with antibiotic-resistant nosocomial pathogens, and analysed their plasmablast response by flow cytometry. RESULTS We observed a strong induction of plasmablasts in patients with antibiotic-resistant A. baumannii infection. Furthermore, plasmablasts were also induced in response to other drug-resistant nosocomial pathogens. CONCLUSIONS These data suggest that plasmablasts may be broadly harnessed to develop therapeutic antibodies to combat otherwise untreatable antibiotic-resistant infections.

Neutrophil extracellular trap (NET) levels in human plasma are associated with active TB

Neutrophils are increasingly associated with tuberculosis (TB) disease. Neutrophil extracellular traps (NETs), which are released by neutrophils as a host antimicrobial defense mechanism, are also associated with tissue damage. However, a link between NET levels and TB disease has not been studied. Here we investigate plasma NETs levels in patients with active pulmonary tuberculosis using an ELISA assay that is suitable for high-throughput processing. We show that plasma NETs levels at baseline correlated with disease severity and decreased with antibiotic therapy. Our study demonstrates the biologic plausibility of measuring NETs in plasma samples from patients with TB.

High Frequencies of Caspase-3 Expressing Mycobacterium tuberculosis-Specific CD4+ T Cells Are Associated With Active Tuberculosis

Antigen-specific CD4+ T cell responses to Mycobacterium tuberculosis (Mtb) infection are important for host defense against tuberculosis (TB). However, Mtb-specific IFN-γ-producing T cells do not distinguish active tuberculosis (ATB) patients from individuals with asymptomatic latent Mtb infection (LTBI). We reasoned that the immune phenotype of Mtb-specific IFN-γ+CD4+ T cells could provide an indirect gauge of Mtb antigen load within individuals. We sought to identify immune markers in Mtb-specific IFN-γ+CD4+ T cells and hypothesized that expression of caspase-3 Mtb-specific CD4+ T cells would be associated with ATB. Using polychromatic flow cytometry, we evaluated the expression of caspase-3 in Mtb-specific CD4+ T cells from LTBI and ATB as well as from ATB patients undergoing anti-TB treatment. We found significantly higher frequencies of Mtb-specific caspase-3+IFN-γ+CD4+ T cells in ATB compared to LTBI. Caspase-3+IFN-γ+CD4+ T cells were also more activated compared to their caspase-3-negative counterparts. Furthermore, the frequencies of caspase-3+IFN-γ+CD4+ T cells decreased in response to anti-TB treatment. Our studies suggest that the frequencies of caspase-3-expressing antigen-specific CD4+ T cells may reflect mycobacterial burden in vivo and may be useful for distinguishing Mtb infection status along with other host biomarkers.