BACKGROUND: Nontuberculous mycobacteria (NTM) cause pulmonary and extrapulmonary infections. Although isolation of NTM from clinical specimens has increased nationally, few studies delineated the molecular characteristics of extrapulmonary NTM. METHODS: Extrapulmonary isolates were collected by four Emerging Infections Program sites from October 2019 to March 2020 and underwent laboratory characterization, including matrix-assisted laser desorption ionization-time of flight mass spectrometry, Sanger DNA sequencing, and whole genome sequencing. Bioinformatics analyses were employed to identify species, sequence types (STs), antimicrobial resistance (AR), and virulence genes; isolates were further characterized by phylogenetic analyses. RESULTS: Among 45 isolates, the predominant species were Mycobacterium avium (n=20, 44%), Mycobacterium chelonae (n=7, 16%), and Mycobacterium fortuitum (n=6, 13%). The collection represented 31 STs across 10 species; the most common ST was ST11 (M. avium, n=7). Mycobacterium fortuitum and Mycobacterium abscessus isolates harbored multiple genes conferring resistance to aminoglycosides, beta-lactams, and macrolides. No known AR mutations were detected in rpoB, 16S, or 23S rRNAs. Slow-growing NTM species harbored multiple virulence genes including type-VII secretion components, adhesion factors, and phospholipase C. CONCLUSION: Continued active laboratory- and population-based surveillance will further inform the prevalence of NTM species and STs, monitor emerging clones, and allow AR characterization.

Importance: Immunization with tetanus, diphtheria, and acellular pertussis (Tdap) vaccine is recommended in the United States during weeks 27 through 36 of pregnancy to prevent life-threatening infant pertussis. The optimal gestation for immunization to maximize concentrations of neonatal pertussis toxin antibodies is unknown. Objective: To determine pertussis toxin antibody concentrations in cord blood from neonates born to women immunized and unimmunized with Tdap vaccine in pregnancy and optimal gestational age for immunization to maximize concentrations of neonatal antibodies. Design, Setting, and Participants: Prospective, observational, cohort study of term neonates in Houston, Texas (December 2013-March 2014). Exposures: Tdap immunization during weeks 27 through 36 of pregnancy or no Tdap immunization. Main Outcomes and Measures: Primary outcome was geometric mean concentrations (GMCs) of pertussis toxin antibodies in cord blood of Tdap-exposed and Tdap-unexposed neonates and proportions of Tdap-exposed and Tdap-unexposed neonates with pertussis toxin antibody concentrations of 15 IU/mL or higher, 30 IU/mL or higher, and 40 IU/mL or higher, cutoffs representing quantifiable antibodies or levels that may be protective until the infant immunization series begins. Secondary outcome was the optimal gestation for immunization to achieve maximum pertussis toxin antibodies. Results: Six hundred twenty-six pregnancies (mean maternal age, 29.7 years; 41% white, 27% Hispanic, 26% black, 5% Asian, 1% other; mean gestation, 39.4 weeks) were included. Three hundred twelve women received Tdap vaccine at a mean gestation of 31.2 weeks (range, 27.3-36.4); 314 were unimmunized. GMC of neonatal cord pertussis toxin antibodies from the Tdap-exposed group was 47.3 IU/mL (95% CI, 42.1-53.2) compared with 12.9 IU/mL (95% CI, 11.7-14.3) in the Tdap-unexposed group, for a GMC ratio of 3.6 (95% CI, 3.1-4.2; P < .001). More Tdap-exposed than Tdap-unexposed neonates had pertussis toxin antibody concentrations of 15 IU/mL or higher (86% vs 37%; difference, 49% [95% CI, 42%-55%]), 30 IU/mL or higher (72% vs 17%; difference, 55% [95% CI, 49%-61%]), and 40 IU/mL or higher (59% vs 12%; difference, 47% [95% CI, 41%-54%]); P < .001 for each analysis. GMCs of pertussis toxin antibodies were highest when Tdap vaccine was administered during weeks 27 through 30 and declined thereafter, reaching a peak at week 30 (57.3 IU/mL [95% CI, 44.0-74.6]). Conclusions and Relevance: Immunization with Tdap vaccine during the third trimester of pregnancy, compared with no immunization, was associated with higher neonatal concentrations of pertussis toxin antibodies. Immunization early in the third trimester was associated with the highest concentrations.

Despite high vaccine coverage, pertussis cases in the United States (US) have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The US exclusively employs acellular vaccines and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the US retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 US surveillance isolates collected from 2010-2016 identified two that were both Prn- and Fha-deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutation to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure.

Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genome structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine potential evolution of chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. Observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigation of disease resurgence and molecular epidemiology. IMPORTANCE: Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen's evolution. The chromosome of B. pertussis includes a high number of repetitive, mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-coding genes which otherwise exhibit little nucleotide sequence diversity. By comparing complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology.