Extensively Drug-Resistant Pseudomonas aeruginosa Outbreak associated with Artificial Tears.

BACKGROUND: Carbapenemase-producing, carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) are extensively drug resistant bacteria. We investigated the source of a multistate CP-CRPA outbreak. METHODS: Cases were defined as a U.S. patient's first isolation of P. aeruginosa sequence type 1203 with the carbapenemase gene blaVIM-80 and cephalosporinase gene blaGES-9 from any specimen source collected and reported to CDC between January 1, 2022-May 15, 2023. We conducted a 1:1 matched case-control study at the post-acute care facility with the most cases, assessed exposures associated with case status for all case-patients, and tested products for bacterial contamination. RESULTS: We identified 81 case-patients from 18 states, 27 of whom were identified through surveillance cultures. Four (7%) of 54 case-patients with clinical cultures died within 30 days of culture collection, and four (22%) of 18 with eye infections underwent enucleation. In the case-control study, case-patients had increased odds of receiving artificial tears compared to controls (crude matched OR: 5.0, 95% CI: 1.1, 22.8). Overall, artificial tears use was reported by 61 (87%) of 70 case-patients with information; 43 (77%) of 56 case-patients with brand information reported use of Brand A, an imported, preservative-free, over-the-counter (OTC) product. Bacteria isolated from opened and unopened bottles of Brand A were genetically related to patient isolates. FDA inspection of the manufacturing plant identified likely sources of contamination. CONCLUSIONS: A manufactured medical product serving as the vehicle for carbapenemase-producing organisms is unprecedented in the U.S. The clinical impacts from this outbreak underscore the need for improved requirements for U.S. OTC product importers.

Whole-Genome Sequencing Reveals Diversity of Carbapenem-Resistant Pseudomonas aeruginosa Collected through CDC's Emerging Infections Program, United States, 2016-2018.

The CDC's Emerging Infections Program (EIP) conducted population- and laboratory-based surveillance of US carbapenem-resistant Pseudomonas aeruginosa (CRPA) from 2016 through 2018. To characterize the pathotype, 1,019 isolates collected through this project underwent antimicrobial susceptibility testing and whole-genome sequencing. Sequenced genomes were classified using the seven-gene multilocus sequence typing (MLST) scheme and a core genome (cg)MLST scheme was used to determine phylogeny. Both chromosomal and horizontally transmitted mechanisms of carbapenem resistance were assessed. There were 336 sequence types (STs) among the 1,019 sequenced genomes, and the genomes varied by an average of 84.7% of the cgMLST alleles used. Mutations associated with dysfunction of the porin OprD were found in 888 (87.1%) of the genomes and were correlated with carbapenem resistance, and a machine learning model incorporating hundreds of genetic variations among the chromosomal mechanisms of resistance was able to classify resistant genomes. While only 7 (0.1%) isolates harbored carbapenemase genes, 66 (6.5%) had acquired non-carbapenemase ß-lactamase genes, and these were more likely to have OprD dysfunction and be resistant to all carbapenems tested. The genetic diversity demonstrates that the pathotype includes a variety of strains, and clones previously identified as high-risk make up only a minority of CRPA strains in the United States. The increased carbapenem resistance in isolates with acquired non-carbapenemase ß-lactamase genes suggests that horizontally transmitted mechanisms aside from carbapenemases themselves may be important drivers of the spread of carbapenem resistance in P. aeruginosa.

Dialysis Water Supply Faucet as Reservoir for Carbapenemase-Producing Pseudomonas aeruginosa.

During June 2017-November 2019, a total 36 patients with carbapenem-resistant Pseudomonas aeruginosa harboring Verona-integron-encoded metallo-ß-lactamase were identified in a city in western Texas, USA. A faucet contaminated with the organism, identified through environmental sampling, in a specialty care room was the likely source for infection in a subset of patients.

Epidemiology of extended-spectrum ß-lactamase-producing Enterobacterales in five US sites participating in the Emerging Infections Program, 2017.

OBJECTIVE: The incidence of infections from extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales (ESBL-E) is increasing in the United States. We describe the epidemiology of ESBL-E at 5 Emerging Infections Program (EIP) sites. METHODS: During October-December 2017, we piloted active laboratory- and population-based (New York, New Mexico, Tennessee) or sentinel (Colorado, Georgia) ESBL-E surveillance. An incident case was the first isolation from normally sterile body sites or urine of Escherichia coli or Klebsiella pneumoniae/oxytoca resistant to ≥1 extended-spectrum cephalosporin and nonresistant to all carbapenems tested at a clinical laboratory from a surveillance area resident in a 30-day period. Demographic and clinical data were obtained from medical records. The Centers for Disease Control and Prevention (CDC) performed reference antimicrobial susceptibility testing and whole-genome sequencing on a convenience sample of case isolates. RESULTS: We identified 884 incident cases. The estimated annual incidence in sites conducting population-based surveillance was 199.7 per 100,000 population. Overall, 800 isolates (96%) were from urine, and 790 (89%) were E. coli. Also, 393 cases (47%) were community-associated. Among 136 isolates (15%) tested at the CDC, 122 (90%) met the surveillance definition phenotype; 114 (93%) of 122 were shown to be ESBL producers by clavulanate testing. In total, 111 (97%) of confirmed ESBL producers harbored a blaCTX-M gene. Among ESBL-producing E. coli isolates, 52 (54%) were ST131; 44% of these cases were community associated. CONCLUSIONS: The burden of ESBL-E was high across surveillance sites, with nearly half of cases acquired in the community. EIP has implemented ongoing ESBL-E surveillance to inform prevention efforts, particularly in the community and to watch for the emergence of new ESBL-E strains.

Genomic Surveillance for SARS-CoV-2 Variants: Predominance of the Delta (B.1.617.2) and Omicron (B.1.1.529) Variants - United States, June 2021-January 2022.

Genomic surveillance is a critical tool for tracking emerging variants of SARS-CoV-2 (the virus that causes COVID-19), which can exhibit characteristics that potentially affect public health and clinical interventions, including increased transmissibility, illness severity, and capacity for immune escape. During June 2021-January 2022, CDC expanded genomic surveillance data sources to incorporate sequence data from public repositories to produce weighted estimates of variant proportions at the jurisdiction level and refined analytic methods to enhance the timeliness and accuracy of national and regional variant proportion estimates. These changes also allowed for more comprehensive variant proportion estimation at the jurisdictional level (i.e., U.S. state, district, territory, and freely associated state). The data in this report are a summary of findings of recent proportions of circulating variants that are updated weekly on CDC's COVID Data Tracker website to enable timely public health action.† The SARS-CoV-2 Delta (B.1.617.2 and AY sublineages) variant rose from 1% to >50% of viral lineages circulating nationally during 8 weeks, from May 1-June 26, 2021. Delta-associated infections remained predominant until being rapidly overtaken by infections associated with the Omicron (B.1.1.529 and BA sublineages) variant in December 2021, when Omicron increased from 1% to >50% of circulating viral lineages during a 2-week period. As of the week ending January 22, 2022, Omicron was estimated to account for 99.2% (95% CI = 99.0%-99.5%) of SARS-CoV-2 infections nationwide, and Delta for 0.7% (95% CI = 0.5%-1.0%). The dynamic landscape of SARS-CoV-2 variants in 2021, including Delta- and Omicron-driven resurgences of SARS-CoV-2 transmission across the United States, underscores the importance of robust genomic surveillance efforts to inform public health planning and practice.

GAMMA: a tool for the rapid identification, classification and annotation of translated gene matches from sequencing data.

MOTIVATION: Tools used to identify genes in microbial sequences using a reference database generally report matches as a percent identity, which can be difficult to interpret in cases with <100% sequence identity, as changes to specific amino acids can have dramatic effects on protein function, such as when they occur in substrate binding regions or enzyme active sites, which in turn can have dramatic effects on phenotypes like antimicrobial resistance or virulence. RESULTS: Here, we present GAMMA, an open-source tool for Gene Allele Mutation Microbial Assessment, which uses protein coding-level identity to make gene calls from any gene database and generates a classification (e.g. mutant, truncation) and translated annotation (e.g. Y190S mutation, truncation at residue 110) for these calls. GAMMA accurately called antimicrobial resistance genes from a large set of genomes faster than three other tools. It can also be used with any gene database, as we demonstrated by identifying virulence genes in the same genome set. Because of its speed and flexibility, GAMMA can be used to rapidly find and annotate any gene matches of interest in microbial sequencing data. AVAILABILITY AND IMPLEMENTATION: GAMMA is freely available as a Bioconda package (https://bioconda.github.io/recipes/gamma/README.html) and as a command line script (https://github.com/rastanton/GAMMA). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Extensively Drug-Resistant Carbapenemase-Producing Pseudomonas aeruginosa and Medical Tourism from the United States to Mexico, 2018-2019.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) producing the Verona integron‒encoded metallo-ß-lactamase (VIM) are highly antimicrobial drug-resistant pathogens that are uncommon in the United States. We investigated the source of VIM-CRPA among US medical tourists who underwent bariatric surgery in Tijuana, Mexico. Cases were defined as isolation of VIM-CRPA or CRPA from a patient who had an elective invasive medical procedure in Mexico during January 2018‒December 2019 and within 45 days before specimen collection. Whole-genome sequencing of isolates was performed. Thirty-eight case-patients were identified in 18 states; 31 were operated on by surgeon 1, most frequently at facility A (27/31 patients). Whole-genome sequencing identified isolates linked to surgeon 1 were closely related and distinct from isolates linked to other surgeons in Tijuana. Facility A closed in March 2019. US patients and providers should acknowledge the risk for colonization or infection after medical tourism with highly drug-resistant pathogens uncommon in the United States.

Detection and Characterization of Targeted Carbapenem-Resistant Health Care-Associated Threats: Findings from the Antibiotic Resistance Laboratory Network, 2017 to 2019.

Carbapenemase gene-positive (CP) Gram-negative bacilli are of significant clinical and public health concern. Their rapid detection and containment are critical to preventing their spread and additional infections they can cause. To this end, CDC developed the Antibiotic Resistance Laboratory Network (AR Lab Network), in which public health laboratories across all 50 states, several cities, and Puerto Rico characterize clinical isolates of carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB) and conduct colonization screens to detect the presence of mobile carbapenemase genes. In its first 3 years, the AR Lab Network tested 76,887 isolates and 31,001 rectal swab colonization screens. Targeted carbapenemase genes (blaKPC, blaNDM, blaOXA-48-like, blaVIM, or blaIMP) were detected by PCR in 35% of CRE, 2% of CRPA, and <1% of CRAB isolates and 8% of colonization screens tested, respectively. blaKPC and blaVIM were the most common genes in CP-CRE and CP-CRPA isolates, respectively, but regional differences in the frequency of carbapenemase genes detected were apparent. In CRE and CRPA isolates tested for carbapenemase production and the presence of the targeted genes, 97% had concordant results; 3% of CRE and 2% of CRPA isolates were carbapenemase production positive but PCR negative for those genes. Isolates harboring blaNDM showed the highest frequency of resistance across the carbapenems tested, and those harboring blaIMP and blaOXA-48-like genes showed the lowest frequency of carbapenem resistance. The AR Lab Network provides a national snapshot of rare and emerging carbapenemase genes, delivering data to inform public health actions to limit the spread of these antibiotic resistance threats.

Gram-Negative Bacteria Harboring Multiple Carbapenemase Genes, United States, 2012-2019.

Reports of organisms harboring multiple carbapenemase genes have increased since 2010. During October 2012-April 2019, the Centers for Disease Control and Prevention documented 151 of these isolates from 100 patients in the United States. Possible risk factors included recent history of international travel, international inpatient healthcare, and solid organ or bone marrow transplantation.

Detection of CTX-M-27 ß-Lactamase Genes on Two Distinct Plasmid Types in ST38 Escherichia coli from Three U.S. States.

Infections caused by extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli are a significant cause of morbidity and health care costs. Globally, the prevailing clonal type is ST131 in association with the blaCTX-M-15 ß-lactamase gene. However, other ESBLs, such as blaCTX-M-14 and blaCTX-M-27, can also be prevalent in some regions. We identified ST38 ESBL-producing E. coli from different regions in the United States which carry blaCTX-M-27 embedded on two distinct plasmid types, suggesting the potential emergence of new ESBL lineages.

The allometry of movement predicts the connectivity of communities.

Connectivity has long played a central role in ecological and evolutionary theory and is increasingly emphasized for conserving biodiversity. Nonetheless, connectivity assessments often focus on individual species even though understanding and preserving connectivity for entire communities is urgently needed. Here we derive and test a framework that harnesses the well-known allometric scaling of animal movement to predict community-level connectivity across protected area networks. We used a field translocation experiment involving 39 species of southern African birds to quantify movement capacity, scaled this relationship to realized dispersal distances determined from ring-and-recovery banding data, and used allometric scaling equations to quantify community-level connectivity based on multilayer network theory. The translocation experiment explained observed dispersal distances from ring-recovery data and emphasized allometric scaling of dispersal based on morphology. Our community-level networks predicted that larger-bodied species had a relatively high potential for connectivity, while small-bodied species had lower connectivity. These community networks explained substantial variation in observed bird diversity across protected areas. Our results highlight that harnessing allometric scaling can be an effective way of determining large-scale community connectivity. We argue that this trait-based framework founded on allometric scaling provides a means to predict connectivity for entire communities, which can foster empirical tests of community theory and contribute to biodiversity conservation strategies aimed at mitigating the effects of environmental change.

Development and Application of a Core Genome Multilocus Sequence Typing Scheme for the Health Care-Associated Pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that frequently causes health care-associated infections (HAIs). Due to its metabolic diversity and ability to form biofilms, this Gram-negative nonfermenting bacterium can persist in the health care environment, which can lead to prolonged HAI outbreaks. We describe the creation of a core genome multilocus sequence typing (cgMLST) scheme to provide a stable platform for the rapid comparison of P. aeruginosa isolates using whole-genome sequencing (WGS) data. We used a diverse set of 58 complete P. aeruginosa genomes to curate a set of 4,440 core genes found in each isolate, representing ∼64% of the average genome size. We then expanded the alleles for each gene using 1,991 contig-level genome sequences. The scheme was used to analyze genomes from four historical HAI outbreaks to compare the phylogenies generated using cgMLST to those of other means (traditional MLST, pulsed-field gel electrophoresis [PFGE], and single-nucleotide variant [SNV] analysis). The cgMLST scheme provides sufficient resolution for analyzing individual outbreaks, as well as the stability for comparisons across a variety of isolates encountered in surveillance studies, making it a valuable tool for the rapid analysis of P. aeruginosa genomes.

Multicenter Outbreak of Gram-Negative Bloodstream Infections in Hemodialysis Patients.

RATIONALE & OBJECTIVE: Contaminated water and other fluids are increasingly recognized to be associated with health care-associated infections. We investigated an outbreak of Gram-negative bloodstream infections at 3 outpatient hemodialysis facilities. STUDY DESIGN: Matched case-control investigations. SETTING & PARTICIPANTS: Patients who received hemodialysis at Facility A, B, or C from July 2015 to November 2016. EXPOSURES: Infection control practices, sources of water, dialyzer reuse, injection medication handling, dialysis circuit priming, water and dialysate test findings, environmental reservoirs such as wall boxes, vascular access care practices, pulsed-field gel electrophoresis, and whole-genome sequencing of bacterial isolates. OUTCOMES: Cases were defined by a positive blood culture for any Gram-negative bacteria drawn July 1, 2015 to November 30, 2016 from a patient who had received hemodialysis at Facility A, B, or C. ANALYTICAL APPROACH: Exposures in cases and controls were compared using matched univariate conditional logistic regression. RESULTS: 58 cases of Gram-negative bloodstream infection occurred; 48 (83%) required hospitalization. The predominant organisms were Serratia marcescens (n=21) and Pseudomonas aeruginosa (n=12). Compared with controls, cases had higher odds of using a central venous catheter for dialysis (matched odds ratio, 54.32; lower bound of the 95% CI, 12.19). Facility staff reported pooling and regurgitation of waste fluid at recessed wall boxes that house connections for dialysate components and the effluent drain within dialysis treatment stations. Environmental samples yielded S marcescens and P aeruginosa from wall boxes. S marcescens isolated from wall boxes and case-patients from the same facilities were closely related by pulsed-field gel electrophoresis and whole-genome sequencing. We identified opportunities for health care workers' hands to contaminate central venous catheters with contaminated fluid from the wall boxes. LIMITATIONS: Limited patient isolates for testing, on-site investigation occurred after peak of infections. CONCLUSIONS: This large outbreak was linked to wall boxes, a previously undescribed source of contaminated fluid and biofilms in the immediate patient care environment.

Identification of a Carbapenemase-Producing Hypervirulent Klebsiella pneumoniae Isolate in the United States.

We report on a carbapenemase-producing hypervirulent Klebsiella pneumoniae (CP-hvKP) isolate collected from a U.S. patient at an outpatient clinic. The isolate was identified as K. pneumoniae serotype K1 sequence type 23 and included both a hypervirulence (with rmpA, rmpA2 iroBCDN, peg-344, and iucABCD-iutA genes) and a carbapenemase-encoding (blaKPC-2) plasmid. The emergence of CP-hvKP underscores the importance of clinical awareness of this pathotype and the need for continued monitoring of CP-hvKP in the United States.

Discovery, characterization, and lead optimization of 7-azaindole non-nucleoside HIV-1 reverse transcriptase inhibitors.

A library of 585 compounds built off a 7-azaindole core was evaluated for anti-HIV-1 activity, and ten hits emerged with submicromolar potency and therapeutic index >100. Of these, three were identified as non-nucleoside reverse transcriptase (RT) inhibitors and were assayed against relevant resistant mutants. Lead compound 8 inhibited RT with submicromolar potency (IC50=0.73µM) and also maintained some activity against the clinically important RT mutants K103N and Y181C (IC50=9.2, 3.5µM) in cell-free assays. Free energy perturbation guided lead optimization resulted in the development of a compound with a two-fold increase in potency against RT (IC50=0.36µM). These data highlight the discovery of a unique scaffold with the potential to move forward as next-generation anti-HIV-1 agents.

Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094.

Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on ß-d-2'-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2'-C-methyl-GTP, is a well-characterized inhibitor of NS5B polymerase, whereas the second metabolite, 2'-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo Finally, we found that although both 2'-C-methyl-GTP and 2'-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2'-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2'-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites.

Ligand similarity guided receptor selection enhances docking accuracy and recall for non-nucleoside HIV reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTI) are allosteric inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a viral polymerase essential to infection. Despite the availability of >150 NNRTI-bound RT crystal structures, rational design of new NNRTI remains challenging because of the variability of their induced fit, hydrophobic binding patterns. Docking NNRTI yields inconsistent results that vary markedly depending on the receptor structure used, as only 27% of the >20k cross-docking calculations we performed using known NNRTI were accurate. In order to determine if a hospitable receptor for docking could be selected a priori, we evaluated more than 40 chemical descriptors for their ability to pre-select a best receptor for NNRTI cross-docking. The receptor selection was based on similarity scores between the bound- and target-ligands generated by each descriptor. The top descriptors were able to double the probability of cross-docking accuracy over random receptor selection. Additionally, recall of known NNRTI from a large library of similar decoys was increased using the same approach. The results demonstrate the utility of pre-selecting receptors when docking into difficult targets. Graphical Abstract Cross-docking accuracy increases when using chemical descriptors to determine the NNRTI with maximum similarity to the new compound and then docking into its respective receptor.

Design, synthesis and evaluation of novel anti-HCV molecules that deliver intracellularly three highly potent NS5A inhibitors.

The design and synthesis of new non-symmetrical NS5A inhibitors with sulfur containing amino acids is reported along with their ability to block HCV replication in an HCV 1b replicon system. These compounds display EC50 values in the picomolar range with a large therapeutic index (>10(6)). Moreover, cellular pharmacology studies show that our preferred compounds intracellularly deliver three potent NS5A inhibitors.

Predicting Zika virus structural biology: Challenges and opportunities for intervention.

BACKGROUND: Zika virus is an emerging crisis as infection is implicated in severe neurological disorders-Guillain-Barré syndrome and fetal microcephaly. There are currently no treatment options available for Zika virus infection. This virus is part of the flavivirus genus and closely related to Dengue Fever Virus, West Nile Virus, and Japanese Encephalitis Virus. Like other flaviviruses, the Zika virus genome encodes three structural proteins (capsid, precursor membrane, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Currently, no structural information exists on these viral proteins to facilitate vaccine design and rational drug discovery. METHODS: Structures for all Zika virus viral proteins were predicted using experimental templates available from closely related viruses using the online SwissModel server. These homology models were compared to drug targets from other viruses using Visual Molecular Dynamics Multiseq software. Sequential alignment of all Zika virus polyproteins was performed using Clustal Omega to identify mutations in specific viral proteins implicated in pathogenesis. RESULTS: The precursor membrane, envelope, and NS1 proteins are unique to Zika virus highlighting possible challenges in vaccine design. Sequential differences between Zika virus strains occur at critical positions on precursor membrane, envelope, NS2A, NS3, NS4B, and NS5 as potential loci for differential pathogenesis. Druggable pockets in Dengue Fever Virus and West Nile Virus NS3 and NS5 are retained in predicted Zika virus structures. CONCLUSIONS: Lead candidates for Zika virus can likely be established using NS3 and NS5 inhibitors from other flaviviruses, and the structures presented can provide opportunities for Zika virus intervention strategies.