Hypermutator strains of Pseudomonas aeruginosa reveal novel pathways of resistance to combinations of cephalosporin antibiotics and beta-lactamase inhibitors.

Hypermutation due to DNA mismatch repair (MMR) deficiencies can accelerate the development of antibiotic resistance in Pseudomonas aeruginosa. Whether hypermutators generate resistance through predominantly similar molecular mechanisms to wild-type (WT) strains is not fully understood. Here, we show that MMR-deficient P. aeruginosa can evolve resistance to important broad-spectrum cephalosporin/beta-lactamase inhibitor combination antibiotics through novel mechanisms not commonly observed in WT lineages. Using whole-genome sequencing (WGS) and transcriptional profiling of isolates that underwent in vitro adaptation to ceftazidime/avibactam (CZA), we characterized the detailed sequence of mutational and transcriptional changes underlying the development of resistance. Surprisingly, MMR-deficient lineages rapidly developed high-level resistance (>256 µg/mL) largely without corresponding fixed mutations or transcriptional changes in well-established resistance genes. Further investigation revealed that these isolates had paradoxically generated an early inactivating mutation in the mexB gene of the MexAB-OprM efflux pump, a primary mediator of CZA resistance in P. aeruginosa, potentially driving an evolutionary search for alternative resistance mechanisms. In addition to alterations in a number of genes not known to be associated with resistance, 2 mutations were observed in the operon encoding the RND efflux pump MexVW. These mutations resulted in a 4- to 6-fold increase in resistance to ceftazidime, CZA, cefepime, and ceftolozane-tazobactam when engineered into a WT strain, demonstrating a potentially important and previously unappreciated mechanism of resistance to these antibiotics in P. aeruginosa. Our results suggest that MMR-deficient isolates may rapidly evolve novel resistance mechanisms, sometimes with complex dynamics that reflect gene inactivation that occurs with hypermutation. The apparent ease with which hypermutators may switch to alternative resistance mechanisms for which antibiotics have not been developed may carry important clinical implications.

Disseminated Mycobacterium marinum in Human Immunodeficiency Virus Unmasked by Immune Reconstitution Inflammatory Syndrome.

Distinguishing disseminated Mycobacterium marinum from multifocal cutaneous disease in persons with human immunodeficiency virus/AIDS can present a diagnostic challenge, especially in the context of immune reconstitution inflammatory syndrome (IRIS). In this work, we demonstrate the utility of flow cytometry and whole genome sequencing (WGS) to diagnose disseminated M. marinum unmasked by IRIS following initiation of antiretroviral therapy. Flow cytometry demonstrated robust cytokine production by CD4 T cells in response to stimulation with M. marinum lysate. WGS of isolates from distinct lesions was consistent with clonal dissemination, supporting that preexisting disseminated M. marinum disease was uncovered by inflammatory manifestations, consistent with unmasking mycobacterial IRIS.

From the Pipeline to the Bedside: Advances and Challenges in Clinical Metagenomics.

Next-generation sequencing (NGS) technologies have revolutionized multiple areas in the field of infectious diseases, from pathogen discovery to characterization of genes mediating drug resistance. Consequently, there is much anticipation that NGS technologies may be harnessed in the realm of diagnostic methods to complement or replace current culture-based and molecular microbiologic techniques. In this context, much consideration has been given to hypothesis-free, culture-independent tests that can be performed directly on primary clinical samples. The closest realizations of such universal diagnostic methods achieved to date are based on targeted amplicon and unbiased metagenomic shotgun NGS approaches. Depending on the exact details of implementation and analysis, these approaches have the potential to detect viruses, bacteria, fungi, parasites, and archaea, including organisms that were previously undiscovered and those that are uncultivatable. Shotgun metagenomics approaches additionally can provide information on the presence of virulence and resistance genetic elements. While many limitations to the use of NGS in clinical microbiology laboratories are being overcome with decreasing technology costs, expanding curated pathogen sequence databases, and better data analysis tools, there remain many challenges to the routine use and implementation of these methods. This review summarizes recent advances in applications of targeted amplicon and shotgun-based metagenomics approaches to infectious disease diagnostic methods. Technical and conceptual challenges are considered, along with expectations for future applications of these techniques.

Efflux pump-mediated resistance to new beta lactam antibiotics in multidrug-resistant gram-negative bacteria.

The emergence and spread of bacteria resistant to commonly used antibiotics poses a critical threat to modern medical practice. Multiple classes of bacterial efflux pump systems play various roles in antibiotic resistance, and members of the resistance-nodulation-division (RND) transporter superfamily are among the most important determinants of efflux-mediated resistance in gram-negative bacteria. RND pumps demonstrate broad substrate specificities, facilitating extrusion of multiple chemical classes of antibiotics from the bacterial cell. Several newer beta-lactams and beta-lactam/beta-lactamase inhibitor combinations (BL/BLI) have been developed to treat infections caused by multidrug resistant bacteria. Here we review recent studies that suggest RND efflux pumps in clinically relevant gram-negative bacteria may play critical but underappreciated roles in the development of resistance to beta-lactams and novel BL/BLI combinations. Improved understanding of the genetic and structural basis of RND efflux pump-mediated resistance may identify new antibiotic targets as well as strategies to minimize the emergence of resistance.

Dynamic Emergence of Mismatch Repair Deficiency Facilitates Rapid Evolution of Ceftazidime-Avibactam Resistance in Pseudomonas aeruginosa Acute Infection.

Strains of Pseudomonas aeruginosa with deficiencies in DNA mismatch repair have been studied in the context of chronic infection, where elevated mutational rates ("hypermutation") may facilitate the acquisition of antimicrobial resistance. Whether P. aeruginosa hypermutation can also play an adaptive role in the more dynamic context of acute infection remains unclear. In this work, we demonstrate that evolved mismatch repair deficiencies may be exploited by P. aeruginosa to facilitate rapid acquisition of antimicrobial resistance in acute infection, and we directly document rapid clonal succession by such a hypermutating lineage in a patient. Whole-genome sequencing (WGS) was performed on nine serially cultured blood and respiratory isolates from a patient in whom ceftazidime-avibactam (CZA) resistance emerged in vivo over the course of days. The CZA-resistant clone was differentiated by 14 mutations, including a gain-of-function G183D substitution in the PDC-5 chromosomal AmpC cephalosporinase conferring CZA resistance. This lineage also contained a substitution (R656H) at a conserved position in the ATPase domain of the MutS mismatch repair (MMR) protein, and elevated mutational rates were confirmed by mutational accumulation experiments with WGS of evolved lineages in conjunction with rifampin resistance assays. To test whether MMR-deficient hypermutation could facilitate rapid acquisition of CZA resistance, in vitro adaptive evolution experiments were performed with a mutS-deficient strain. These experiments demonstrated rapid hypermutation-facilitated acquisition of CZA resistance compared with the isogenic wild-type strain. Our results suggest a possibly underappreciated role for evolved MMR deficiency in facilitating rapid adaptive evolution of P. aeruginosa in the context of acute infection.IMPORTANCE Antimicrobial resistance in bacteria represents one of the most consequential problems in modern medicine, and its emergence and spread threaten to compromise central advances in the treatment of infectious diseases. Ceftazidime-avibactam (CZA) belongs to a new class of broad-spectrum beta-lactam/beta-lactamase inhibitor combinations designed to treat infections caused by multidrug-resistant bacteria. Understanding the emergence of resistance to this important new drug class is of critical importance. In this work, we demonstrate that evolved mismatch repair deficiency in P. aeruginosa, an important pathogen responsible for significant morbidity and mortality among hospitalized patients, may facilitate rapid acquisition of resistance to CZA in the context of acute infection. These findings are relevant for both diagnosis and treatment of antimicrobial resistance emerging in acute infection in the hypermutator background and additionally have implications for the emergence of more virulent phenotypes.

Risk Factors for Viridans Group Streptococcal Bacteremia in Neutropenic and Non-neutropenic Patients: A Single Center Case-Case-Control Study.

BACKGROUND: Viridans group streptococcal (VGS) bacteremia is common among neutropenic patients. Although VGS bacteremia occurs in non-neutropenic patients, risk factors are not well established. We conducted a case-case-control study to identify risk factors for VGS among neutropenic and non-neutropenic patients. METHODS: Patients with VGS bacteremia between January 2009 and December 2014 in our 200-bed clinical research hospital were identified using microbiology records. Neutropenic and non-neutropenic patients at the time of positive culture were matched 1:1 to controls on the basis of neutrophil count (ANC), ward, and length of stay. We extracted demographic, laboratory, medication, and other clinical data from chart reviews. Data were analyzed using McNemar's test, Wilcoxon signed-rank test, and conditional logistic regression modeling. RESULTS: Among 101 patients, 63 were neutropenic and 38 non-neutropenic at the time of VGS bacteremia. In multivariable analysis of neutropenic patients, only lower ANC predicted VGS bacteremia (odds ratio [OR], 0.16; 95% confidence interval [CI], 0.05-0.59; P = 0.006). Recent use of vancomycin was protective (OR, 0.23; 95% CI, 0.07-0.73; P = 0.013). No clinical factors were associated with VGS in the non-neutropenic cases. CONCLUSIONS: Only lower ANC nadir increased the risk for VGS bacteremia in the neutropenic group, and vancomycin was protective. Other previously described factors (chemotherapy, radiation, oral conditions) related to neutropenia were not independently associated with VGS bacteremia. No tested clinical factors predicted infection in the non-neutropenic group. Our results suggest that VGS bacteremia should be anticipated when making antimicrobial choices in profoundly neutropenic patients, and merit further exploration in non-neutropenic patients.