Frank R. DeLeo

Klebsiella pneumoniae is an important cause of human infections and is often resistant to multiple antibiotics. Most infections occur in individuals with co-morbidities and/or those who have risk factors for infection. Although progress has been made, new therapeutics and preventive measures are needed. To that end, we developed a mouse model of severe/fatal K. pneumoniae infection (intraperitoneal inoculation that leads to bacteremia) to evaluate strain virulence and test protection conferred by a multivalent CPS-conjugate vaccine. We first compared the virulence of four clinical isolates representative of important K. pneumoniae clones globally (ST23/KL1, ST86/KL2, ST11/KL64, and ST147/KL64), each of which harbors a virulence plasmid. The ST11 and ST147 isolates are additionally multidrug resistant, including resistance to carbapenem antibiotics. Compared with ST23 and ST86 isolates, the ST11 and ST147 isolates were less virulent in the mouse infection model. Moreover, numbers of viable ST11 and ST147 organisms recovered from blood decreased over time, whereas those of ST23 and ST86 increased. As a step toward evaluating the ability of a CPS-CRM197 conjugate vaccine to protect against lethal infection, we immunized CD-1 outbred mice with a cocktail of eight selected CPS conjugates, boosted them twice, and then challenged those mice with one of the two most virulent isolates (ST23/KL1 or ST86/KL2) by intraperitoneal inoculation. Mice receiving the CPS-CRM197 conjugate vaccine were protected against lethal infection caused by KL1 and KL2 isolates, whereas all sham-immunized mice developed fatal disease within 96 h. These results underscore the potential of a standard CPS-conjugate approach for development of a multivalent K. pneumoniae vaccine.

Staphylococcus aureus is an important cause of human infections globally and ranks among the top causes of death by bacteria. In addition, the microbe is notorious for developing resistance to antibiotics. Methicillin-resistant S. aureus is endemic in healthcare facilities and the community in many regions of the world. Although our understanding of S. aureus as a human commensal organism and opportunistic pathogen remains incomplete, the use of genomics and transcriptomics approaches for S. aureus research has advanced this knowledge significantly over the past 20 years. This article reviews genomics approaches, with special emphasis on transcriptomics and single-cell sequencing, used to study S. aureus , past and present, and highlights selected discoveries made with these methods and new applications moving forward.

Klebsiella pneumoniae is primarily an opportunistic pathogen known for causing healthcare-associated infections in individuals with significant risk factors and comorbidities. These bacteria are typically multidrug-resistant (MDR), a phenotype conferred in part by the production of extended-spectrum beta-lactamases and/or carbapenemases. By comparison, so-called hypervirulent K. pneumoniae (hvKp) are defined by their ability to cause severe community-acquired infections in otherwise healthy individuals. Although hvKp lineages have historically not been MDR, there has been a recent emergence of strains with both hypervirulence and multidrug resistance phenotypes. Treatment of infections caused by MDR K. pneumoniae can be difficult, and new preventative measures are needed. As a step toward the development of a vaccine directed to prevent or moderate infections caused by these pathogens, we tested the ability of capsule polysaccharide (CPS) derived from eight selected K. pneumoniae capsule types (KLs) to elicit rabbit antibodies that recognize important KLs isolated from human infections. Seventy-one out of 84 (84.5%) contemporary K. pneumoniae clinical isolates tested were recognized by CPS-specific rabbit antisera. There was the unexpected binding of the antibodies to some isolates with KLs not included in the CPS-antigen cocktails. Notably, rabbit IgG purified from CPS-specific antisera promoted and/or enhanced human PMN bactericidal activity toward all but one of the selected clinical isolates that were not killed by PMNs outright (in the absence of specific antibody). These data provide support to the idea that a CPS-antigen cocktail could be developed to protect against the K. pneumoniae KLs that are the most frequent cause of human infections. IMPORTANCE: Klebsiella pneumoniae is among the leading causes of death by infectious agents. Many of the prominent K. pneumoniae lineages are resistant to multiple classes of antibiotics, and options for treatment are limited. New countermeasures that prevent infections are needed. Here we tested the ability of capsule polysaccharide (CPS) antigen mixtures (or cocktails) to elicit rabbit antibodies that recognize K. pneumoniae from a large collection of extended-spectrum beta-lactamase-producing clinical isolates. Importantly, these antibodies had the ability to promote opsonophagocytic killing by human PMNs. Our results provide proof-of-concept for a CPS vaccine cocktail approach that could be developed to prevent infections caused by the most important K. pneumoniae lineages.

Staphylococcus aureus ( S. aureus ) is a frequent culprit in implant-associated infections and employs many virulence factors to escape killing by the host immune system. The specific immune evasion strategies used by small aggregates of S. aureus on a surface, precursors to mature biofilm, are still relatively unknown. Time-lapse confocal microscopy was leveraged to quantify interactions between S. aureus aggregates and human neutrophils in vitro and identify specific mechanisms of resistance to neutrophil killing. Surface-associated wild-type S. aureus rapidly formed small biofilm aggregates when grown in human serum. Conversely, aggregation was inhibited when the SaeR/S two-component gene regulatory system was deleted. Wild-type aggregates began to show individual and population-level resistance to neutrophil killing upon reaching sizes of approximately 50 to 75 µm ² , whereas Δ sae clusters failed to reach these sizes and were readily cleared. Aggregation of Δ sae strains was impaired by serum complement, and this inhibition required complement proteins C3 and factor B, but not C4 or C5, suggesting that this activity primarily occurs at the level of the alternative pathway. Several complement-inhibiting genes regulated by SaeR/S were identified that collectively facilitate biofilm aggregate formation in human, but not murine serum. Finally, aggregation of two related opportunistic pathogens, Staphylococcus epidermidis and Enterococcus faecalis , was inhibited by serum. These data demonstrate a function of serum complement, the ability to inhibit bacterial aggregation, that is potently blocked by S. aureus through the production of multiple complement-interfering proteins that are regulated by the SaeR/S system.

Staphylococcus aureus is a leading cause of healthcare-associated infections globally. Vancomycin-resistant S. aureus (VRSA), those with high-level resistance [minimum inhibitory concentration (MIC) of 16–32 µg/mL vancomycin], are uncommon, whereas vancomycin-intermediate S. aureus (VISA; MIC of 4–8 µg/mL), are isolated more frequently and develop during long-term and/or repeated use of the antibiotic. VISA can be difficult to eradicate and infections may persist. Our knowledge of mechanisms that underlie the development of VISA is incomplete. We used a genomics approach to investigate the VISA phenotype in three prominent S. aureus lineages. All VISA clinical isolates tested had increased cell wall thickness compared with vancomycin-susceptible S. aureus strains. Growth rates of clonal complex (CC) 5, CC8, and CC45 clinical isolates were reduced in 2 µg/mL vancomycin compared to media alone. Culture in 2 and 4 µg/mL vancomycin sequentially for two weeks reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin in a majority of CC5, CC8, and CC45 isolates tested. We identified alleles reported previously to contribute to the VISA phenotype, but unexpectedly, these alleles were unique to each CC. A subtherapeutic concentration of vancomycin elicited changes in the VISA transcriptome—common and unique—among the three CCs tested. Multiple genes, including those encoding a glycerate kinase, an M50 family metallopeptidase, and an uncharacterized membrane protein, were upregulated among all three lineages and not reported previously as associated with VISA. Although there are lineage-specific changes in DNA sequence, our findings suggest changes in the VISA transcriptome constitute a general response to stress that confers reduced susceptibility to multiple antibiotics. IMPORTANCE: Our understanding of the mechanisms that underlie the development of vancomycin-intermediate Staphylococcus aureus (VISA) is incomplete. To provide a more comprehensive view of this process, we compared genome sequences of clonal complex (CC) 5, CC8, and CC45 VISA clinical isolates and measured changes in the transcriptomes of these isolates during culture with a subtherapeutic concentration of vancomycin. Notably, we identified differentially expressed genes that were lineage-specific or common to the lineages tested, including genes that have not been previously reported to contribute to a VISA phenotype. Changes in gene expression were accompanied by reduced growth rate, increased cell wall thickness, and reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin. Our results provide support to the idea that changes in gene expression contribute to the development of VISA among three CCs that are a prominent cause of human infections.

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 (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.

Summary: Neutrophils or polymorphonuclear neutrophils (PMNs) are an important component of innate host defense. These phagocytic leukocytes are recruited to infected tissues and kill invading microbes. There are several general characteristics of neutrophils that make them highly effective as antimicrobial cells. First, there is tremendous daily production and turnover of granulocytes in healthy adults—typically 10¹¹ per day. The vast majority (~95%) of these cells are neutrophils. In addition, neutrophils are mobilized rapidly in response to chemotactic factors and are among the first leukocytes recruited to infected tissues. Most notably, neutrophils contain and/or produce an abundance of antimicrobial molecules. Many of these antimicrobial molecules are toxic to host cells and can destroy host tissues. Thus, neutrophil activation and turnover are highly regulated processes. To that end, aged neutrophils undergo apoptosis constitutively, a process that contains antimicrobial function and proinflammatory capacity. Importantly, apoptosis facilitates nonphlogistic turnover of neutrophils and removal by macrophages. This homeostatic process is altered by interaction with microbes and their products, as well as host proinflammatory molecules. Microbial pathogens can delay neutrophil apoptosis, accelerate apoptosis following phagocytosis, or cause neutrophil cytolysis. Here, we review these processes and provide perspective on recent studies that have potential to impact this paradigm.

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.

S. aureusstrain 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.