Adeline R. Porter's research while affiliated with National Institute of Allergy and Infectious Diseases and other places

Klebsiella pneumoniae is an important cause of healthcare-associated infections worldwide. This opportunistic pathogen, known as classical K. pneumoniae (cKp), typically causes infections in individuals with comorbidities. cKp is often multidrug resistant, and treatment options are limited. By comparison, hypervirulent K. pneumoniae (hvKp) can cause infections in healthy individuals outside of the healthcare setting. Notably, there has been emergence of strains containing both multidrug resistance and hypervirulence genotypes (MDR hvKp). Whether these strains can circumvent killing by components of the innate immune system remains incompletely determined. Here, we compared the ability of selected hvKp (ST23 and ST86) and MDR hvKp (ST11 and ST147) clinical isolates to survive in human blood and serum and tested phagocytic killing of the microbes by human neutrophils. On average, the hvKp isolates tested had greater survival in blood and serum compared with MDR hvKp isolates. Compared with MDR hvKp isolates, the hvKp isolates had less surface-bound serum complement following culture in normal serum. Consistent with these findings, the percentage of neutrophils with phagocytosed hvKp isolates was limited (<5%), whereas >67% of neutrophils contained ingested MDR hvKp. Phagocytosis of the MDR hvKp isolates was accompanied by significant bacterial killing ( P < 0.05). The inability of neutrophils to ingest and kill these hvKp isolates was, in part, overcome by addition of rabbit antiserum specific for these clinical isolates. The results provide insight into host defense against emerging MDR hvKp and are an initial step toward assessing the potential of a vaccine or immunotherapy approach for treatment of infections. IMPORTANCE Klebsiella pneumoniae strains with a combination of multidrug resistance and hypervirulence genotypes (MDR hvKp) have emerged as a cause of human infections. The ability of these microbes to avoid killing by the innate immune system remains to be tested fully. To that end, we compared the ability of a global collection of hvKp and MDR hvKp clinical isolates to survive in human blood and resist phagocytic killing by human neutrophils. The two MDR hvKp clinical isolates tested (ST11 and ST147) were killed in human blood and by human neutrophils in vitro , whereas phagocytic killing of hvKp clinical isolates (ST23 and ST86) required specific antisera. Although the data were varied and often isolate specific, they are an important first step toward gaining an enhanced understanding of host defense against MDR hvKp.

The extent to which commensal bacteria are altered by exposure to subinhibitory concentrations of antibiotics (outside resistance) remains incompletely determined. To gain a better understanding of this phenomenon, we tested the ability of selected antibiotics (at subinhibitory concentrations) to alter survival of ST258 clinical isolates in normal human serum.

Animal models of S. aureus infection are important for evaluating bacterial pathogenesis and host immune responses. These animal infection models are often used as an initial step in the testing of vaccine antigens and new therapeutics.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

S. aureus strain USA300 has the ability to cause rapid lysis of human neutrophils after phagocytosis. Although this phenomenon likely contributes to the success of USA300 as a human pathogen, our knowledge of the mechanism remains incomplete.

Severe infections caused by multidrug-resistant Klebsiella pneumoniae sequence type 258 (ST258) highlight the need for new therapeutics with activity against this pathogen. Bacteriophage (phage) therapy is an alternative treatment approach for multidrug-resistant bacterial infections that has shown efficacy in experimental animal models and promise in clinical case reports. In this study, we assessed microbiologic, histopathologic, and survival outcomes following systemic administration of phage in ST258-infected mice. We found that prompt treatment with two phages, either individually or in combination, rescued mice with K. pneumoniae ST258 bacteremia. Among the three treatment groups, mice that received combination phage therapy demonstrated the greatest increase in survival and the lowest frequency of phage resistance among bacteria recovered from mouse blood and tissue. Our findings support the utility of phage therapy as an approach for refractory ST258 infections and underscore the potential of this treatment modality to be enhanced through strategic phage selection. IMPORTANCE Infections caused by multidrug-resistant K. pneumoniae pose a serious threat to at-risk patients and present a therapeutic challenge for clinicians. Bacteriophage (phage) therapy is an alternative treatment approach that has been associated with positive clinical outcomes when administered experimentally to patients with refractory bacterial infections. Inasmuch as these experimental treatments are prepared for individual patients and authorized for compassionate use only, they lack the rigor of a clinical trial and therefore cannot provide proof of efficacy. Here, we demonstrate that administration of viable phage provides effective treatment for multidrug-resistant K. pneumoniae (sequence type 258 [ST258]) bacteremia in a murine infection model. Moreover, we compare outcomes among three distinct phage treatment groups and identify potential correlates of therapeutic phage efficacy. These findings constitute an important first step toward optimizing and assessing phage therapy’s potential for the treatment of severe ST258 infection in humans.

Abstract A050 Introduction. Streptococcus pyogenes is a strict human pathogen causing more than 700 million infections annually worldwide. Strains of serotype M28 are among the five more abundant types causing invasive infections and pharyngitis in adults and children and have a propensity to cause puerperal sepsis and neonatal disease. A one-nucleotide indel in an intergenic homopolymeric tract (HT) located between genes Spy1336/R28, encoding the R28 virulence factor, and Spy1337 alters virulence in a mouse model of infection. Here we have analyzed size variation in this HT and determined the extent of heterogeneity in the number of tandemly-repeated 79-amino acid domains (TRs) in the coding region of Spy1336/R28 in large samples of strains recovered from humans with invasive infections [Eraso et al. PLoS ONE 15(3): e0229064, 2020]. Methods We used whole genome sequence data from 2,101 strains to analyze the number of T residues in the HTSpy1336/R28-Spy1337 and PCR analysis of the tandem repeats (TR) internal to Spy1336/R28 to analyze the number of TRs in approximately 500 strains. In addition, we analyzed transcriptome profiles (RNA-seq), made isogenic mutant strains and performed virulence assays. Results Both repeat sequence elements are highly polymorphic in natural populations of M28 strains. Variation in the HT 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 the 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 (NHP) model of necrotizing myositis. Conclusions Spy1336/R28 is very heterogeneous. Size variation in its upstream HT changes transcript levels of Spy1336/R28 and Spy1337, the transcriptome in vitro, and virulence in a mouse model of necrotizing myositis. There are three transcript states, depending on whether the HT contains 9Ts, 10Ts, or 11Ts. TR heterogeneity of Spy1336/R28 results in size variation of the R28 virulence factor. M28 strains devoid of either gene are less virulent in an NHP model of necrotizing myositis, consistent with Spy1337 being a positive regulator of Spy1336/R28 expression.

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 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.

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.