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Klebsiella pneumoniae is a serious human pathogen associated with resistance to multiple antibiotics and high mortality. K. variicola and K. quasipneumoniae are closely related organisms that are generally considered to be less-virulent opportunistic pathogens. We used a large, comprehensive, population-based strain collection and whole-genome sequencing to investigate infections caused by these organisms in our hospital system. We discovered that K. variicola and K. quasipneumoniae isolates are often misidentified as K. pneumoniae by routine clinical microbiology diagnostics and frequently cause severe life-threatening infections similar to K. pneumoniae . The presence of KPC in K. variicola and K. quasipneumoniae strains as well as NDM-1 metallo-beta-lactamase in one K. variicola strain is particularly concerning because these genes confer resistance to many different beta-lactam antibiotics. The sharing of plasmids, as well as evidence of homologous recombination, between these three species of Klebsiella is cause for additional concern. Klebsiella pneumoniae is a major threat to public health, causing significant morbidity and mortality worldwide. The emergence of highly drug-resistant strains is particularly concerning. There has been a recognition and division of Klebsiella pneumoniae into three distinct phylogenetic groups: Klebsiella pneumoniae , Klebsiella variicola , and Klebsiella quasipneumoniae . K. variicola and K. quasipneumoniae have often been described as opportunistic pathogens that have less virulence in humans than K. pneumoniae does. We recently sequenced the genomes of 1,777 extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates recovered from human infections and discovered that 28 strains were phylogenetically related to K . variicola and K. quasipneumoniae . Whole-genome sequencing of 95 additional non-ESBL-producing K. pneumoniae isolates recovered from patients found 12 K. quasipneumoniae strains. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis initially identified all patient isolates as K. pneumoniae , suggesting a potential pitfall in conventional clinical microbiology laboratory identification methods. Whole-genome sequence analysis revealed extensive sharing of core gene content and plasmid replicons among the Klebsiella species. For the first time, strains of both K. variicola and K. quasipneumoniae were found to carry the Klebsiella pneumoniae carbapenemase (KPC) gene, while another K. variicola strain was found to carry the New Delhi metallo-beta-lactamase 1 (NDM-1) gene. K. variicola and K. quasipneumoniae infections were not less virulent than K. pneumoniae infections, as assessed by in-hospital mortality and infection type. We also discovered evidence of homologous recombination in one K. variicola strain, as well as one strain from a novel Klebsiella species, which challenge the current understanding of interrelationships between clades of Klebsiella . IMPORTANCE Klebsiella pneumoniae is a serious human pathogen associated with resistance to multiple antibiotics and high mortality. K. variicola and K. quasipneumoniae are closely related organisms that are generally considered to be less-virulent opportunistic pathogens. We used a large, comprehensive, population-based strain collection and whole-genome sequencing to investigate infections caused by these organisms in our hospital system. We discovered that K. variicola and K. quasipneumoniae isolates are often misidentified as K. pneumoniae by routine clinical microbiology diagnostics and frequently cause severe life-threatening infections similar to K. pneumoniae . The presence of KPC in K. variicola and K. quasipneumoniae strains as well as NDM-1 metallo-beta-lactamase in one K. variicola strain is particularly concerning because these genes confer resistance to many different beta-lactam antibiotics. The sharing of plasmids, as well as evidence of homologous recombination, between these three species of Klebsiella is cause for additional concern.

Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts. A fundamental goal of contemporary biomedical research is to understand the molecular basis of disease pathogenesis and exploit this information to develop targeted and more-effective therapies. Necrotizing myositis caused by the bacterial pathogen Streptococcus pyogenes is a devastating human infection with a high mortality rate and few successful therapeutic options. We used dual transcriptome sequencing (RNA-seq) to analyze the transcriptomes of S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates. The in vivo bacterial transcriptome was strikingly remodeled compared to organisms grown in vitro , with significant upregulation of genes contributing to virulence and altered regulation of metabolic genes. The transcriptome of muscle tissue from infected nonhuman primates (NHPs) differed significantly from that of mock-infected animals, due in part to substantial changes in genes contributing to inflammation and host defense processes. We discovered significant positive correlations between group A streptococcus (GAS) virulence factor transcripts and genes involved in the host immune response and inflammation. We also discovered significant correlations between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness, as assessed by previously conducted genome-wide transposon-directed insertion site sequencing (TraDIS). By integrating the bacterial RNA-seq data with the fitness data generated by TraDIS, we discovered five new pathogen genes, namely, S. pyogenes 0281 ( Spy0281 [ dahA ]), ihk-irr , slr , isp , and ciaH , that contribute to necrotizing myositis and confirmed these findings using isogenic deletion-mutant strains. Taken together, our study results provide rich new information about the molecular events occurring in severe invasive infection of primate skeletal muscle that has extensive translational research implications. IMPORTANCE Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts.

Bacteria control gene expression in concert with their population density by a process called quorum sensing, which is modulated by bacterial chemical signals and environmental factors. In the human pathogen Streptococcus pyogenes , production of secreted virulence factor SpeB is controlled by a quorum-sensing pathway and environmental pH. The quorum-sensing pathway consists of a secreted leaderless peptide signal (SIP), and its cognate receptor RopB. Here, we report that the SIP quorum-sensing pathway has a pH-sensing mechanism operative through a pH-sensitive histidine switch located at the base of the SIP-binding pocket of RopB. Environmental acidification induces protonation of His144 and reorganization of hydrogen bonding networks in RopB, which facilitates SIP recognition. The convergence of two disparate signals in the SIP signaling pathway results in induction of SpeB production and increased bacterial virulence. Our findings provide a model for investigating analogous crosstalk in other microorganisms. The mechanism by which environmental pH controls the virulence of the pathogen Streptococcus pyogenes is unclear. Here, Do et al. show that changes in pH affect the activity of the virulence regulator RopB via its interaction with a quorum-sensing peptide signal.

There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves. We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein—the primary target of global vaccine efforts—are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution. IMPORTANCE There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.

Streptococcus pyogenes is a strict human pathogen responsible for more than 700 million infections annually worldwide. Strains of serotype M28 S. pyogenes are typically among the five more abundant types causing invasive infections and pharyngitis in adults and children. Type M28 strains also have an unusual propensity to cause puerperal sepsis and neonatal disease. We recently discovered that a one-nucleotide indel in an intergenic homopolymeric tract located between genes Spy1336/R28 and Spy1337 altered virulence in a mouse model of infection. In the present study, we analyzed size variation in this homopolymeric tract and determined the extent of heterogeneity in the number of tandemly-repeated 79-amino acid domains in the coding region of Spy1336/R28 in large samples of strains recovered from humans with invasive infections. Both repeat sequence elements are highly polymorphic in natural populations of M28 strains. Variation in the homopolymeric tract results in (i) changes in transcript levels of Spy1336/R28 and Spy1337 in vitro, (ii) differences in virulence in a mouse model of necrotizing myositis, and (iii) global transcriptome changes as shown by RNAseq analysis of isogenic mutant strains. Variation in the number of tandem repeats in the coding sequence of Spy1336/R28 is responsible for size variation of R28 protein in natural populations. Isogenic mutant strains in which genes encoding R28 or transcriptional regulator Spy1337 are inactivated are significantly less virulent in a nonhuman primate model of necrotizing myositis. Our findings provide impetus for additional studies addressing the role of R28 and Spy1337 variation in pathogen-host interactions.

Streptococcus pyogenes (group A streptococcus; GAS) causes 600 million cases of pharyngitis annually worldwide. There is no licensed human GAS vaccine despite a century of research. Although the human oropharynx is the primary site of GAS infection, the pathogenic genes and molecular processes used to colonize, cause disease, and persist in the upper respiratory tract are poorly understood. Using dense transposon mutant libraries made with serotype M1 and M28 GAS strains and transposon-directed insertion sequencing, we performed genome-wide screens in the nonhuman primate (NHP) oropharynx. We identified many potentially novel GAS fitness genes, including a common set of 115 genes that contribute to fitness in both genetically distinct GAS strains during experimental NHP pharyngitis. Targeted deletion of 4 identified fitness genes/operons confirmed that our newly identified targets are critical for GAS virulence during experimental pharyngitis. Our screens discovered many surface-exposed or secreted proteins — substrates for vaccine research — that potentially contribute to GAS pharyngitis, including lipoprotein HitA. Pooled human immune globulin reacted with purified HitA, suggesting that humans produce antibodies against this lipoprotein. Our findings provide new information about GAS fitness in the upper respiratory tract that may assist in translational research, including developing novel vaccines.

Streptococcus pyogenes causes 700 million human infections annually worldwide, yet, despite a century of intensive effort, there is no licensed vaccine against this bacterium. Although a number of large-scale genomic studies of bacterial pathogens have been published, the relationships among the genome, transcriptome, and virulence in large bacterial populations remain poorly understood. We sequenced the genomes of 2,101 emm28 S. pyogenes invasive strains, from which we selected 492 phylogenetically diverse strains for transcriptome analysis and 50 strains for virulence assessment. Data integration provided a novel understanding of the virulence mechanisms of this model organism. Genome-wide association study, expression quantitative trait loci analysis, machine learning, and isogenic mutant strains identified and confirmed a one-nucleotide indel in an intergenic region that significantly alters global transcript profiles and ultimately virulence. The integrative strategy that we used is generally applicable to any microbe and may lead to new therapeutics for many human pathogens. This study presents the genomes of 2,101 emm28 Streptococcus pyogenes invasive strains, of which 492 were transcriptionally profiled, and 50 were assessed for virulence. GWAS, eQTL analysis, and study of isogenic mutant strains identified an intergenic region that alters global transcript profiles and bacterial virulence.

Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon-directed insertion-site sequencing (TraDIS), we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins and exporters for toxins, quorum-sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding 5 transporters confirmed that each isogenic mutant strain was significantly (P < 0.05) impaired in causing necrotizing myositis in NHPs. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis showed that these 5 genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface exposed, suggesting that our findings have translational research implications.

Chronic airways infection with methicillin-resistant Staphylococcus aureus (MRSA) is associated with worse respiratory disease cystic fibrosis (CF) patients. Ceftaroline is a cephalosporin that inhibits the penicillin-binding protein (PBP2a) uniquely produced by MRSA. We analyzed 335 S. aureus isolates from CF sputum samples collected at three US centers between 2015–2018. Molecular relationships demonstrated that high-level resistance of preceding isolates to carbapenems were associated with subsequent isolation of ceftaroline resistant CF MRSA. In vitro evolution experiments showed that pre-exposure of CF MRSA to meropenem with further selection with ceftaroline implied mutations in mecA and additional mutations in pbp1 and pbp2 , targets of carbapenems; no effects were achieved by other β-lactams. An in vivo pneumonia mouse model showed the potential therapeutic efficacy of ceftaroline/meropenem combination against ceftaroline-resistant CF MRSA infections. Thus, the present findings highlight risk factors and potential therapeutic strategies offering an opportunity to both prevent and address antibiotic resistance in this patient population. Varela, Roch et al. find that resistance of preceding isolates to carbapenems is associated with the subsequent isolation of methicillin-resistant Staphylococcus aureus (MRSA) resistant to ceftaroline. Addition of meropenem reduces the cystic fibrosis MRSA’s resistance to ceftaroline in a pneumonia mouse model, suggesting a strategy to control antibiotics resistance.

Candida auris is an emerging pathogen with considerable public health importance. We present the draft genome sequence of a strain recently cultured from the urine of a patient hospitalized in the greater Houston metropolitan region. Two combined Oxford Nanopore sequencing runs provided sufficient data to rapidly generate a draft genome with 9 contigs covering the expected 7 chromosomes. Footnotes * https://www.ncbi.nlm.nih.gov/bioproject/PRJNA540998