Increase in invasive Streptococcus pyogenes M1 infections with close evolutionary genetic relationship, Iceland and Scotland, 2022 to 2023.

Group A Streptococcus isolates of the recently described M1UK clade have emerged to cause human infections in several European countries and elsewhere. Full-genome sequence analysis of M1 isolates discovered a close genomic relationship between some isolates from Scotland and the majority of isolates from Iceland causing serious infections in 2022 and 2023. Phylogenetic analysis strongly suggests that an isolate from or related to Scotland was the precursor to an M1UK variant responsible for almost all recent M1 infections in Iceland.

Signals of Significantly Increased Vaccine Breakthrough, Decreased Hospitalization Rates, and Less Severe Disease in Patients with Coronavirus Disease 2019 Caused by the Omicron Variant of Severe Acute Respiratory Syndrome Coronavirus 2 in Houston, Texas.

Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to dramatically alter the landscape of the coronavirus disease 2019 (COVID-19) pandemic. The recently described variant of concern designated Omicron (B.1.1.529) has rapidly spread worldwide and is now responsible for the majority of COVID-19 cases in many countries. Because Omicron was recognized recently, many knowledge gaps exist about its epidemiology, clinical severity, and disease course. A genome sequencing study of SARS-CoV-2 in the Houston Methodist health care system identified 4468 symptomatic patients with infections caused by Omicron from late November 2021 through January 5, 2022. Omicron rapidly increased in only 3 weeks to cause 90% of all new COVID-19 cases, and at the end of the study period caused 98% of new cases. Compared with patients infected with either Alpha or Delta variants in our health care system, Omicron patients were significantly younger, had significantly increased vaccine breakthrough rates, and were significantly less likely to be hospitalized. Omicron patients required less intense respiratory support and had a shorter length of hospital stay, consistent with on average decreased disease severity. Two patients with Omicron stealth sublineage BA.2 also were identified. The data document the unusually rapid spread and increased occurrence of COVID-19 caused by the Omicron variant in metropolitan Houston, Texas, and address the lack of information about disease character among US patients.

Delta Variants of SARS-CoV-2 Cause Significantly Increased Vaccine Breakthrough COVID-19 Cases in Houston, Texas.

Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have repeatedly altered the course of the coronavirus disease 2019 (COVID-19) pandemic. Delta variants are now the focus of intense international attention because they are causing widespread COVID-19 globally and are associated with vaccine breakthrough cases. We sequenced 16,965 SARS-CoV-2 genomes from samples acquired March 15, 2021, through September 20, 2021, in the Houston Methodist hospital system. This sample represents 91% of all Methodist system COVID-19 patients during the study period. Delta variants increased rapidly from late April onward to cause 99.9% of all COVID-19 cases and spread throughout the Houston metroplex. Compared with all other variants combined, Delta caused a significantly higher rate of vaccine breakthrough cases (23.7% for Delta compared with 6.6% for all other variants combined). Importantly, significantly fewer fully vaccinated individuals required hospitalization. Vaccine breakthrough cases caused by Delta had a low median PCR cycle threshold value (a proxy for high virus load). This value was similar to the median cycle threshold value for unvaccinated patients with COVID-19 caused by Delta variants, suggesting that fully vaccinated individuals can transmit SARS-CoV-2 to others. Patients infected with Alpha and Delta variants had several significant differences. The integrated analysis indicates that vaccines used in the United States are highly effective in decreasing severe COVID-19, hospitalizations, and deaths.

Sequence Analysis of 20,453 Severe Acute Respiratory Syndrome Coronavirus 2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern.

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several variants of concern emerged globally, including the UK variant (B.1.1.7), the South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California variants of interest (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. On the basis of our extensive genome sequencing program involving 20,453 coronavirus disease 2019 patient samples collected from March 2020 to February 2021, we report identification of all six of these SARS-CoV-2 variants among Houston Methodist Hospital (Houston, TX) patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency (aggregate of 1.1%) in the population, they are geographically widespread. Houston is the first city in the United States in which active circulation of all six current variants of concern has been documented by genome sequencing. As vaccine deployment accelerates, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence, frequency, and medical impact of consequential variants and their patterns and trajectory of dissemination.

Trajectory of Growth of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants in Houston, Texas, January through May 2021, Based on 12,476 Genome Sequences.

Certain genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients. SARS-CoV-2 genome sequences from 12,476 patients in the Houston Methodist health care system diagnosed from January 1 through May 31, 2021 are reported here. Prevalence of the B.1.1.7 (Alpha) variant increased rapidly and caused 63% to 90% of new cases in the latter half of May. Eleven B.1.1.7 genomes had an E484K replacement in spike protein, a change also identified in other SARS-CoV-2 lineages. Compared with non-B.1.1.7-infected patients, individuals with B.1.1.7 had a significantly lower cycle threshold (a proxy for higher virus load) and significantly higher hospitalization rate. Other variants [eg, B.1.429 and B.1.427 (Epsilon), P.1 (Gamma), P.2 (Zeta), and R.1] also increased rapidly, although the magnitude was less than that in B.1.1.7. Twenty-two patients infected with B.1.617.1 (Kappa) or B.1.617.2 (Delta) variants had a high rate of hospitalization. Breakthrough cases (n = 207) in fully vaccinated patients were caused by a heterogeneous array of virus genotypes, including many not currently designated variants of interest or concern. In the aggregate, this study delineates the trajectory of SARS-CoV-2 variants circulating in a major metropolitan area, documents B.1.1.7 as the major cause of new cases in Houston, TX, and heralds the arrival of B.1.617 variants in the metroplex.

Treatment of Coronavirus Disease 2019 (COVID-19) Patients with Convalescent Plasma.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

Human Infections Caused by Clonally Related African Clade (Clade III) Strains of Candida auris in the Greater Houston Region.

Candida auris is a pathogen of considerable public health importance. It was first reported in 2009. Five clades, determined by genomic analysis and named by the distinct regions where they were initially identified, have been defined. We previously completed a draft genome sequence of an African clade (clade III) strain cultured from the urine of a patient hospitalized in the greater Houston metropolitan region (strain LOM). Although initially uncommon, reports of the African clade in the United States have grown to include a recent cluster in California. Here, we describe a second human C. auris infection in the Houston area. Whole-genome sequence analysis demonstrated the Houston patient isolates to be clonally related to one another but distantly related to other African clade organisms recovered in the United States or elsewhere. Infections in these patients were present on admission to the hospital and occurred several months apart. Taken together, the data demonstrate the emergence and persistence of a clonal C. auris population and highlights the importance of routine high-resolution genomic surveillance of emerging human pathogens in the clinical laboratory.

Using Machine Learning To Predict Antimicrobial MICs and Associated Genomic Features for Nontyphoidal Salmonella.

Nontyphoidal Salmonella species are the leading bacterial cause of foodborne disease in the United States. Whole-genome sequences and paired antimicrobial susceptibility data are available for Salmonella strains because of surveillance efforts from public health agencies. In this study, a collection of 5,278 nontyphoidal Salmonella genomes, collected over 15 years in the United States, was used to generate extreme gradient boosting (XGBoost)-based machine learning models for predicting MICs for 15 antibiotics. The MIC prediction models had an overall average accuracy of 95% within ±1 2-fold dilution step (confidence interval, 95% to 95%), an average very major error rate of 2.7% (confidence interval, 2.4% to 3.0%), and an average major error rate of 0.1% (confidence interval, 0.1% to 0.2%). The model predicted MICs with no a priori information about the underlying gene content or resistance phenotypes of the strains. By selecting diverse genomes for the training sets, we show that highly accurate MIC prediction models can be generated with less than 500 genomes. We also show that our approach for predicting MICs is stable over time, despite annual fluctuations in antimicrobial resistance gene content in the sampled genomes. Finally, using feature selection, we explore the important genomic regions identified by the models for predicting MICs. To date, this is one of the largest MIC modeling studies to be published. Our strategy for developing whole-genome sequence-based models for surveillance and clinical diagnostics can be readily applied to other important human pathogens.

RocA Has Serotype-Specific Gene Regulatory and Pathogenesis Activities in Serotype M28 Group A Streptococcus.

Serotype M28 group A streptococcus (GAS) is a common cause of infections such as pharyngitis ("strep throat") and necrotizing fasciitis ("flesh-eating" disease). Relatively little is known about the molecular mechanisms underpinning M28 GAS pathogenesis. Whole-genome sequencing studies of M28 GAS strains recovered from patients with invasive infections found an unexpectedly high number of missense (amino acid-changing) and nonsense (protein-truncating) polymorphisms in rocA (regulator of Cov), leading us to hypothesize that altered RocA activity contributes to M28 GAS molecular pathogenesis. To test this hypothesis, an isogenic rocA deletion mutant strain was created. Transcriptome sequencing (RNA-seq) analysis revealed that RocA inactivation significantly alters the level of transcripts for 427 and 323 genes at mid-exponential and early stationary growth phases, respectively, including genes for 41 transcription regulators and 21 virulence factors. In contrast, RocA transcriptomes from other GAS M protein serotypes are much smaller and include fewer transcription regulators. The rocA mutant strain had significantly increased secreted activity of multiple virulence factors and grew to significantly higher colony counts under acid stress in vitro RocA inactivation also significantly increased GAS virulence in a mouse model of necrotizing myositis. Our results demonstrate that RocA is an important regulator of transcription regulators and virulence factors in M28 GAS and raise the possibility that naturally occurring polymorphisms in rocA in some fashion contribute to human invasive infections caused by M28 GAS strains.

Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences.

We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD(+)-glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide.

The majority of 9,729 group A streptococcus strains causing disease secrete SpeB cysteine protease: pathogenesis implications.

Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy.

Identification of point mutations in clinical Staphylococcus aureus strains that produce small-colony variants auxotrophic for menadione.

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

PBP2a mutations causing high-level Ceftaroline resistance in clinical methicillin-resistant Staphylococcus aureus isolates.

Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.

Clinical laboratory response to a mock outbreak of invasive bacterial infections: a preparedness study.

Large hospital-based clinical laboratories must be prepared to rapidly investigate potential infectious disease outbreaks. To challenge the ability of our molecular diagnostics laboratory to use whole-genome sequencing in a potential outbreak scenario and identify impediments to these efforts, we studied 84 invasive serotype emm59 group A streptococcus (GAS) strains collected in the United States. We performed a rapid-response exercise to the mock outbreak scenario using whole-genome sequencing, genome-wide transcript analysis, and mouse virulence studies. The protocol changes installed in response to the lessons learned were tested in a second iteration. The initial investigation was completed in 9 days. Whole-genome sequencing showed that the invasive infections were caused by multiple subclones of epidemic emm59 GAS strains likely spread to the United States from Canada. The phylogenetic tree showed a strong temporal-spatial structure with diversity in mobile genetic element content, features that are useful for identifying closely related strains and possible transmission events. The genome data informed the epidemiology, identifying multiple patients who likely acquired the organisms through direct person-to-person transmission. Transcriptome analysis unexpectedly revealed significantly altered expression of genes encoding a two-component regulator and the hyaluronic acid capsule virulence factor. Mouse infection studies confirmed a high-virulence capacity of these emm59 organisms. Whole-genome sequencing, coupled with transcriptome analysis and animal virulence studies, can be rapidly performed in a clinical environment to effectively contribute to patient care decisions and public health maneuvers.

Evaluation of computerized clinical decision support system to reduce unnecessary nasal methicillin-resistant Staphylococcus aureus (MRSA) polymerase chain reaction (PCR) testing.

Our health system implemented a novel clinical decision-support system to reduce unnecessary duplicate nasal methicillin-resistant Staphylococcus aureus (MRSA) polymerase chain reaction (PCR) orders. In an 8-month period, the rate of duplicate MRSA PCR orders within 7 days declined from 4.7% (370 of 7,861) to 1.2% (120 of 9,833).

A genomic day in the life of a clinical microbiology laboratory.

Next-generation sequencing technology is available to many clinical laboratories; however, it is not yet widely used in routine microbiology practice. To demonstrate the feasibility of using whole-genome sequencing in a routine clinical microbiology workflow, we sequenced the genome of every organism isolated in our laboratory for 1 day.

Creation and Evaluation of a Clinical Informatics Match: Initial Findings.

BACKGROUND: Clinical Informatics (CI) fellowship programs utilize the Electronic Residency Application Service (ERAS) to gather applications but until recently used an American Medical Informatics Association (AMIA) member-developed, simultaneous offer-acceptance process to match fellowship applicants to programs. In 2021, program directors collaborated with the AMIA to develop a new match to improve the process. OBJECTIVE: Describe the results of the first 2 years of the match and address opportunities for improvement. METHODS: We obtained applicant data for fellowship applicants in 2021 and 2022 from the ERAS and match data for the same years from the AMIA. We analyzed our data using descriptive statistics. RESULTS: There were 159 unique applicants over the 2-year period. Applicants submitted 2,178 applications with a median of 10 per applicant (interquartile range [IQR] 3-20). One hundred and four applicants (65.4%) participated in the match and ranked a median of seven programs (2-12). Forty-two programs in 2021 and 47 programs in 2022 offered a combined total 153 positions in the match. Participating programs ranked a median of eight applicants per year (IQR 5-11). Of participating applicants, 95 (91.3%) successfully matched and of those 66 (69.5%) received their top choice. Thirty-two programs (76.2%) matched at least one candidate in 2021 and 33 programs (70.2%) matched at least one candidate in 2022. In both years, 24 programs filled all available slots (57.1% in 2021 and 51.1% in 2022). CONCLUSION: Applicants were extremely successful in the new match, which successfully addressed most of the challenges of the simultaneous offer-acceptance process identified by program directors. However, applicant attrition resulted in a quarter of programs going unmatched. Although many programs still filled slots outside the match, fellowship slots may remain unfilled while the CI practice pathway remains open.

Analysis of the Genomics and Mouse Virulence of an Emergent Clone of Streptococcus dysgalactiae Subspecies equisimilis.

Streptococcus dysgalactiae subsp. equisimilis is a bacterial pathogen that is increasingly recognized as a cause of severe human infections. Much less is known about the genomics and infection pathogenesis of S. dysgalactiae subsp. equisimilis strains compared to the closely related bacterium Streptococcus pyogenes. To address these knowledge deficits, we sequenced to closure the genomes of seven S. dysgalactiae subsp. equisimilis human isolates, including six that were emm type stG62647. Recently, for unknown reasons, strains of this emm type have emerged and caused an increasing number of severe human infections in several countries. The genomes of these seven strains vary between 2.15 and 2.21 Mbp. The core chromosomes of these six S. dysgalactiae subsp. equisimilis stG62647 strains are closely related, differing on average by only 495 single-nucleotide polymorphisms, consistent with a recent descent from a common progenitor. The largest source of genetic diversity among these seven isolates is differences in putative mobile genetic elements, both chromosomal and extrachromosomal. Consistent with the epidemiological observations of increased frequency and severity of infections, both stG62647 strains studied were significantly more virulent than a strain of emm type stC74a in a mouse model of necrotizing myositis, as assessed by bacterial CFU burden, lesion size, and survival curves. Taken together, our genomic and pathogenesis data show the strains of emm type stG62647 we studied are closely genetically related and have enhanced virulence in a mouse model of severe invasive disease. Our findings underscore the need for expanded study of the genomics and molecular pathogenesis of S. dysgalactiae subsp. equisimilis strains causing human infections. IMPORTANCE Our studies addressed a critical knowledge gap in understanding the genomics and virulence of the bacterial pathogen Streptococcus dysgalactiae subsp. equisimilis. S. dysgalactiae subsp. equisimilis strains are responsible for a recent increase in severe human infections in some countries. We determined that certain S. dysgalactiae subsp. equisimilis strains are genetically descended from a common ancestor and that these strains can cause severe infections in a mouse model of necrotizing myositis. Our findings highlight the need for expanded studies on the genomics and pathogenic mechanisms of this understudied subspecies of the Streptococcus family.

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence.

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.