Swab Testing to Optimize Pneumonia treatment with empiric Vancomycin (STOP-Vanc): study protocol for a randomized controlled trial.

Background: Vancomycin, an antibiotic with activity against Methicillin-resistant Staphylococcus aureus (MRSA), is frequently included in empiric treatment for community-acquired pneumonia (CAP) despite the fact that MRSA is rarely implicated in CAP. Conducting polymerase chain reaction (PCR) testing on nasal swabs to identify the presence of MRSA colonization has been proposed as an antimicrobial stewardship intervention to reduce the use of vancomycin. Observational studies have shown reductions in vancomycin use after implementation of MRSA colonization testing, and this approach has been adopted by CAP guidelines. However, the ability of this intervention to safely reduce vancomycin use has yet to be tested in a randomized controlled trial. Methods: STOP-Vanc is a pragmatic, prospective, single center, non-blinded randomized trial. Adult patients with suspicion for CAP who are receiving vancomycin and admitted to the Medical Intensive Care Unit at Vanderbilt University Medical Center will be screened for eligibility. Eligible patients will be enrolled and randomized in a 1:1 ratio to either receive MRSA nasal swab PCR testing in addition to usual care (intervention group), or usual care alone (control group). PCR testing results will be transmitted through the electronic health record to the treating clinicians. Primary providers of intervention group patients with negative swab results will also receive a page providing clinical guidance recommending discontinuation of vancomycin. The primary outcome will be vancomycin-free hours alive, defined as the number of hours alive and free of the use of vancomycin within the first seven days following trial enrollment estimated using a proportional odds ratio model. Secondary outcomes include 30-day all-cause mortality and time alive off vancomycin. Discussion: STOP-Vanc will provide the first randomized controlled trial data regarding the use of MRSA nasal swab PCR testing to guide antibiotic de-escalation. This study will provide important information regarding the effect of MRSA PCR testing and antimicrobial stewardship guidance on clinical outcomes in an intensive care unit setting. Trial registration: This trial was registered on ClinicalTrials.gov on February 22, 2024. (ClinicalTrials.gov identifier: NCT06272994).

Coordinated adaptation of Staphylococcus aureus to calprotectin-dependent metal sequestration.

The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as "nutritional immunity." Staphylococcus aureus growth is inhibited by calprotectin in vitro, and calprotectin is localized in vivo to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of S. aureus and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of S. aureus during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in clpP, which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of S. aureus to the presence of calprotectin. ClpP contributes to pathogenesis in vivo in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for S. aureus to outcompete the host for metals. IMPORTANCE: Staphylococcus aureus is a leading cause of skin and soft tissue infections, bloodstream infections, and endocarditis. Antibiotic treatment failures during S. aureus infections are increasingly prevalent, highlighting the need for novel antimicrobial agents. Metal chelator-based therapeutics have tremendous potential as antimicrobials due to the strict requirement for nutrient metals exhibited by bacterial pathogens. The high-affinity transition metal-binding properties of calprotectin represents a potential therapeutic strategy that functions through metal chelation. Our studies provide a foundation to define mechanisms by which S. aureus combats nutritional immunity and may be useful for the development of novel therapeutics to counter the ability of S. aureus to survive in a metal-limited environment.

A multi-species outbreak of VIM-producing carbapenem-resistant bacteria in a burn unit and subsequent investigation of rapid development of cefiderocol resistance.

Carbapenem resistance due to metallo-ß-lactamases (MBLs) such as the Verona integron-encoded metallo-ß-lactamase (VIM) is particularly problematic due to the limited treatment options. We describe a case series of bacterial infections in a tertiary care hospital due to multi-species acquisition of a VIM gene along with our experience using novel ß-lactam antibiotics and antibiotic combinations to treat these infections. Four patients were treated with the combination of ceftazidime-avibactam and aztreonam, with no resistance to the combination detected. However, cefiderocol-resistant Klebsiella pneumoniae isolates were detected in two out of the five patients who received cefiderocol within 3 weeks of having started the antibiotic. Strain pairs of sequential susceptible and resistant isolates from both patients were analyzed using whole-genome sequencing. This analysis revealed that the pairs of isolates independently acquired point mutations in both the cirA and fiu genes, which encode siderophore receptors. These point mutations were remade in a laboratory strain of K. pneumoniae and resulted in a significant increase in the MIC of cefiderocol, even in the absence of a beta-lactamase enzyme or a penicillin-binding protein 3 (PBP3) mutation. While newer ß-lactam antibiotics remain an exciting addition to the antibiotic armamentarium, their use must be accompanied by diligent monitoring for the rapid development of resistance.

What's Hot This Year in Infectious Diseases Clinical Science.

The field of infectious diseases saw numerous exciting advances in 2023. Trials of new antibiotics and treatment regimens sought to address rising rates of antimicrobial resistance. Other studies focused on the most appropriate use of currently available treatments, balancing the dual goals of providing effective treatment and impactful antimicrobial stewardship. Improvements in disease prevention were made through trials of both new vaccines and new chemoprophylaxis approaches. Concerning trends this year included increasing rates of invasive group A streptococcal infections, medical tourism-associated cases of fungal meningitis, and the return of locally acquired malaria to the United States. This review covers some of these notable trials and clinical developments in infectious diseases in the past year.

Restriction of Arginine Induces Antibiotic Tolerance in Staphylococcus aureus.

Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment, particularly when they are caused by Methicillin-Resistant Staphylococcus aureus (MRSA). Antibiotic tolerance, the ability for bacteria to persist despite normally lethal doses of antibiotics, is responsible for most antibiotic treatment failure in MRSA infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-MRSA antibiotics (vancomycin, ceftaroline, delafloxacin, and linezolid) were examined using both quantitative proteomics and transposon sequencing. These screens indicated that arginine metabolism is involved in antibiotic tolerance within a biofilm and led to the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance under conditions in which the parental strain is susceptible to antibiotics. Arginine restriction was found to induce antibiotic tolerance via inhibition of protein synthesis. Finally, although S. aureus fitness in a mouse skin infection model is decreased in an argH mutant, its ability to survive in vivo during antibiotic treatment with vancomycin is enhanced, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

Imaging mass spectrometry reveals complex lipid distributions across Staphylococcus aureus biofilm layers.

Introduction: Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. Methods: S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. Results: Implementation of TIMS led to a âˆ¼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) - CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously. Conclusions: High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies.

Host Polyunsaturated Fatty Acids Potentiate Aminoglycoside Killing of Staphylococcus aureus.

Aminoglycoside antibiotics rely on the proton motive force to enter the bacterial cell, and facultative anaerobes like Staphylococcus aureus can shift energy generation from respiration to fermentation, becoming tolerant of aminoglycosides. Following this metabolic shift, high concentrations of aminoglycosides are required to eradicate S. aureus infections, which endangers the host due to the toxicity of aminoglycosides. Membrane-disrupting molecules prevent aminoglycoside tolerance in S. aureus by facilitating passive entry of the drug through the membrane. Polyunsaturated fatty acids (PUFAs) increase membrane permeability when incorporated into S. aureus. Here, we report that the abundant host-derived PUFA arachidonic acid increases the susceptibility of S. aureus to aminoglycosides, decreasing the aminoglycoside concentration needed to kill S. aureus. We demonstrate that PUFAs and aminoglycosides synergize to kill multiple strains of S. aureus, including both methicillin-resistant and -susceptible S. aureus. We also present data showing that PUFAs and aminoglycosides effectively kill S. aureus small colony variants, strains that are particularly recalcitrant to killing by many antibiotics. We conclude that cotreatment with PUFAs, which are molecules with low host toxicity, and aminoglycosides decreases the aminoglycoside concentration necessary to kill S. aureus, lowering the toxic side effects to the host associated with prolonged aminoglycoside exposure. IMPORTANCE Staphylococcus aureus infects every niche of the human host, and these infections are the leading cause of Gram-positive sepsis. Aminoglycoside antibiotics are inexpensive, stable, and effective against many bacterial infections. However, S. aureus can shift its metabolism to become tolerant of aminoglycosides, requiring increased concentrations and/or longer courses of treatment, which can cause severe host toxicity. Here, we report that polyunsaturated fatty acids (PUFAs), which have low host toxicity, disrupt the S. aureus membrane, making the pathogen susceptible to aminoglycosides. Additionally, cotreatment with aminoglycosides is effective at killing S. aureus small colony variants, strains that are difficult to treat with antibiotics. Taken together, the data presented herein show the promise of PUFA cotreatment to increase the efficacy of aminoglycosides against S. aureus infections and decrease the risk to the human host of antibiotic-induced toxicity.

Identification and Antibiotic Susceptibility Patterns of Clinical Blood Culture Isolates Not Identified by a Rapid Microarray Diagnostic System.

The use of molecular-based diagnostic testing, such as the Luminex Verigene system, to rapidly identify the most common bacterial isolates from blood cultures is an important tool that reduces the duration of inappropriate antibiotics and decreases mortality. However, 5 to 15% of microorganisms recovered from blood culture are unable to be identified by the Verigene Gram-negative (BC-GN) or Gram-positive (BC-GP) assays. In this retrospective, observational study, we evaluate the identities and antimicrobial susceptibility patterns of 229 isolates that were not identified by either the Verigene BC-GN or BC-GP assay. The results presented here suggest that important, clinically relevant information about antimicrobial susceptibility patterns can still be inferred even when isolates are not identified by Verigene. We also examined changes in antibiotic use for patients with "unidentified" Verigene results at our institution and found that this subgroup represents an opportunity to optimize empirical antibiotic therapy. IMPORTANCE Rapid diagnostic testing to identify bloodstream pathogens has arisen as an important tool both to ensure adequate antimicrobial therapy is given early and to aid in antimicrobial stewardship by allowing for more rapid deescalation of inappropriate antimicrobial therapy. However, there is a paucity of data regarding the significance of isolates that are not able to be identified by rapid diagnostic testing. In this study, we report the identification to the species level and antimicrobial susceptibilities among isolates that were not identified by one such rapid diagnostic platform, the Verigene system. This study provides important insight into how a strong understanding of the strengths and limitations of a given rapid diagnostic platform, coupled with insight into local antibiotic susceptibility patterns, can allow for more nuanced and thoughtful empirical antibiotic selection.

The Arginine Deiminase Pathway Impacts Antibiotic Tolerance during Biofilm-Mediated Streptococcus pyogenes Infections.

Bacterial biofilms are responsible for a variety of serious human infections and are notoriously difficult to treat due to their recalcitrance to antibiotics. Further work is necessary to elicit a full understanding of the mechanism of this antibiotic tolerance. The arginine deiminase (ADI) pathway is responsible for bacterial pH maintenance and is highly expressed during biofilm growth in multiple bacterial species. Using the group A Streptococcus (GAS) as a model human pathogen, the ADI pathway was demonstrated to contribute to biofilm growth. The inability of antibiotics to reduce GAS populations when in a biofilm was demonstrated by in vitro studies and a novel animal model of nasopharyngeal infection. However, disruption of the ADI pathway returned GAS biofilms to planktonic levels of antibiotic sensitivity, suggesting the ADI pathway is influential in biofilm-related antibiotic treatment failure and provides a new strategic target for the treatment of biofilm infections in GAS and potentially numerous other bacterial species.IMPORTANCE Biofilm-mediated bacterial infections are a major threat to human health because of their recalcitrance to antibiotic treatment. Through the study of Streptococcus pyogenes, a significant human pathogen that is known to form antibiotic-tolerant biofilms, we demonstrated the role that a bacterial pathway known for responding to acid stress plays in biofilm growth and antibiotic tolerance. This not only provides some insight into antibiotic treatment failure in S. pyogenes infections but also, given the widespread nature of this pathway, provides a potentially broad target for antibiofilm therapies. This discovery has the potential to impact the treatment of many different types of recalcitrant biofilm infections.

Clearance of Staphylococcus aureus from In Vivo Models of Chronic Infection by Immunization Requires Both Planktonic and Biofilm Antigens.

Staphylococcus aureus is a causative agent of chronic biofilm-associated infections that are recalcitrant to resolution by the immune system or antibiotics. To combat these infections, an antistaphylococcal, biofilm-specific quadrivalent vaccine against an osteomyelitis model in rabbits has previously been developed and shown to be effective at eliminating biofilm-embedded bacterial populations. However, the addition of antibiotics was required to eradicate remaining planktonic populations. In this study, a planktonic upregulated antigen was combined with the quadrivalent vaccine to remove the need for antibiotic therapy. Immunization with this pentavalent vaccine followed by intraperitoneal challenge of BALB/c mice with S. aureus resulted in 16.7% and 91.7% mortality in pentavalent vaccine and control groups, respectively (P < 0.001). Complete bacterial elimination was found in 66.7% of the pentavalent cohort, while only 8.3% of the control animals cleared the infection (P < 0.05). Further protective efficacy was observed in immunized rabbits following intramedullary challenge with S. aureus, where 62.5% of the pentavalent cohort completely cleared the infection, versus none of the control animals (P < 0.05). Passive immunization of BALB/c mice with serum IgG against the vaccine antigens prior to intraperitoneal challenge with S. aureus prevented mortality in 100% of mice and eliminated bacteria in 33.3% of the challenged mice. These results demonstrate that targeting both the planktonic and biofilm stages with the pentavalent vaccine or the IgG elicited by immunization can effectively protect against S. aureus infection.

Temporal proteomic profiling reveals changes that support Burkholderia biofilms.

Melioidosis associated with opportunistic pathogen Burkholderia pseudomallei imparts a huge medical burden in Southeast Asia and Australia. At present there is no available human vaccine that protects against B. pseudomallei infection and antibiotic treatments are limited particularly for drug-resistant strains and bacteria in biofilm forms. Biofilm forming bacteria exhibit phenotypic features drastically different to their planktonic states, often exhibiting a diminished response to antimicrobial therapies. Our earlier work on global profiling of bacterial biofilms using transcriptomics and proteomics revealed transcript-decoupled protein abundance in bacterial biofilms. Here we employed reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to deduce temporal proteomic differences in planktonic and biofilm forms of Burkholderia thailandensis, which is weakly surrogate model of pathogenic B. pseudomallei as sharing a key element in genomic similarity. The proteomic analysis of B. thailandensis in biofilm versus planktonic states revealed that proteome changes support biofilm survival through decreased abundance of metabolic proteins while increased abundance of stress-related proteins. Interestingly, the protein abundance including for the transcription protein TEX, outer periplasmic TolB protein, and the exopolyphosphatase reveal adaption in bacterial biofilms that facilitate antibiotic tolerance through a non-specific mechanism. The present proteomics study of B. thailandensis biofilms provides a global snapshot of protein abundance differences and antimicrobial sensitivities in planktonic and sessile bacteria.

An unusual case of Nocardia cyriacigeorgica presenting with spinal abscesses in a renal transplant recipient and a review of the literature.

Nocardia species represent a well-recognized yet uncommon cause of opportunistic infections in humans. It most frequently presents as a pulmonary infection with or without central nervous system involvement. It is a very rare cause of spinal abscesses, with only 26 cases reported in the literature. Here we report a 49-year-old man with a history of renal transplantation who presented with low back pain and was diagnosed with epidural and paraspinal abscesses due to Nocardia cyriacigeorgica that was successfully treated with antimicrobial therapy alone. In addition to the case reported here, we also conducted a systematic review of the existing literature regarding spinal abscesses due to Nocardia species and examined the success of the various treatments utilized.

Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection.

The Group A Streptococcus remains a significant human pathogen causing a wide array of disease ranging from self-limiting to life-threatening invasive infections. Epithelium (skin or throat) colonization with progression to the subepithelial tissues is the common step in all GAS infections. Here, we used transposon-sequencing (Tn-seq) to define the GAS 5448 genetic requirements for in vivo fitness in subepithelial tissue. A near-saturation transposon library of the M1T1 GAS 5448 strain was injected subcutaneously into mice, producing suppurative inflammation at 24 h that progressed to prominent abscesses with tissue necrosis at 48 h. The library composition was monitored en masse by Tn-seq and ratios of mutant abundance comparing the output (12, 24 and 48 h) versus input (T0) mutant pools were calculated for each gene. We identified a total of 273 subcutaneous fitness (scf) genes with 147 genes (55 of unknown function) critical for the M1T1 GAS 5448 fitness in vivo; and 126 genes (53 of unknown function) potentially linked to in vivo fitness advantage. Selected scf genes were validated in competitive subcutaneous infection with parental 5448. Two uncharacterized genes, scfA and scfB, encoding putative membrane-associated proteins and conserved among Gram-positive pathogens, were further characterized. Defined scfAB mutants in GAS were outcompeted by wild type 5448 in vivo, attenuated for lesion formation in the soft tissue infection model and dissemination to the bloodstream. We hypothesize that scfAB play an integral role in enhancing adaptation and fitness of GAS during localized skin infection, and potentially in propagation to other deeper host environments.

The mythos of laudable pus along with an explanation for its origin.

The presence of pus is one of the most easily recognizable signs of an infection. However, for several centuries suppuration, known as 'laudable pus,' was believed to be a sign of a healthy, healing wound. This historical misconception can be explained by the difference in the presentation of a necrotizing soft tissue infection versus other more common skin and soft tissue infections. Chronic wound infections, due to pyogenic bacteria, typically produce large amounts of thick, whitish-yellow pus. On the other hand, necrotizing soft tissue infections, despite their severe mortality and morbidity, are devoid of pus in the traditional sense. What the ancient medical observers recognized was the fact that pus is not characteristic of this subset of incredibly severe infections. This is an important distinction to remember when evaluating an infection, even today.

Global Analysis and Comparison of the Transcriptomes and Proteomes of Group A Streptococcus Biofilms.

To gain a better understanding of the genes and proteins involved in group A Streptococcus (GAS; Streptococcus pyogenes) biofilm growth, we analyzed the transcriptome, cellular proteome, and cell wall proteome from biofilms at different stages and compared them to those of plankton-stage GAS. Using high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we found distinct expression profiles in the transcriptome and proteome. A total of 46 genes and 41 proteins showed expression across the majority of biofilm time points that was consistently higher or consistently lower than that seen across the majority of planktonic time points. However, there was little overlap between the genes and proteins on these two lists. In line with other studies comparing transcriptomic and proteomic data, the overall correlation between the two data sets was modest. Furthermore, correlation was poorest for biofilm samples. This suggests a high degree of regulation of protein expression by nontranscriptional mechanisms. This report illustrates the benefits and weaknesses of two different approaches to global expression profiling, and it also demonstrates the advantage of using proteomics in conjunction with transcriptomics to gain a more complete picture of global expression within biofilms. In addition, this report provides the fullest characterization of expression patterns in GAS biofilms currently available. IMPORTANCE Prokaryotes are thought to regulate their proteomes largely at the level of transcription. However, the results from this first set of global transcriptomic and proteomic analyses of paired microbial samples presented here show that this assumption is false for the majority of genes and their products in S. pyogenes. In addition, the tenuousness of the link between transcription and translation becomes even more pronounced when microbes exist in a biofilm or a stationary planktonic state. Since the transcriptome level does not usually equal the proteome level, the validity attributed to gene expression studies as well as proteomic studies in microbial analyses must be brought into question. Therefore, the results attained by either approach, whether RNA-seq or shotgun proteomics, must be taken in context and evaluated with particular care since they are by no means interchangeable.

Type IV pili promote early biofilm formation by Clostridium difficile.

Increasing morbidity and mortality from Clostridium difficile infection (CDI) present an enormous challenge to healthcare systems. Clostridium difficile express type IV pili (T4P), but their function remains unclear. Many chronic and recurrent bacterial infections result from biofilms, surface-associated bacterial communities embedded in an extracellular matrix. CDI may be biofilm mediated; T4P are important for biofilm formation in a number of organisms. We evaluate the role of T4P in C. difficile biofilm formation using RNA sequencing, mutagenesis and complementation of the gene encoding the major pilin pilA1, and microscopy. RNA sequencing demonstrates that, in comparison to other growth phenotypes, C. difficile growing in a biofilm has a distinct RNA expression profile, with significant differences in T4P gene expression. Microscopy of T4P-expressing and T4P-deficient strains suggests that T4P play an important role in early biofilm formation. A non-piliated pilA1 mutant forms an initial biofilm of significantly reduced mass and thickness in comparison to the wild type. Complementation of the pilA1 mutant strain leads to formation of a biofilm which resembles the wild-type biofilm. These findings suggest that T4P play an important role in early biofilm formation. Novel strategies for confronting biofilm infections are emerging; our data suggest that similar strategies should be investigated in CDI.

Systemic Staphylococcus aureus infection mediated by Candida albicans hyphal invasion of mucosal tissue.

Candida albicans and Staphylococcus aureus are often co-isolated in cases of biofilm-associated infections. C. albicans can cause systemic disease through morphological switch from the rounded yeast to the invasive hyphal form. Alternatively, systemic S. aureus infections arise from seeding through breaks in host epithelial layers although many patients have no documented portal of entry. We describe a novel strategy by which S. aureus is able to invade host tissue and disseminate via adherence to the invasive hyphal elements of Candida albicans. In vitro and ex vivo findings demonstrate a specific binding of the staphylococci to the candida hyphal elements. The C. albicans cell wall adhesin Als3p binds to multiple staphylococcal adhesins. Furthermore, Als3p is required for C. albicans to transport S. aureus into the tissue and cause a disseminated infection in an oral co-colonization model. These findings suggest that C. albicans can facilitate the invasion of S. aureus across mucosal barriers, leading to systemic infection in co-colonized patients.

In vivo expression of Streptococcus pyogenes immunogenic proteins during tibial foreign body infection.

Group A streptococcus (GAS) is an important human pathogen that causes a number of diseases with a wide range of severities. While all known strains of GAS are still sensitive to penicillin, there have been reports of antibiotic treatment failure in as many as 20% to 40% of cases. Biofilm formation has been implicated as a possible cause for these failures. A biofilm is a microbially derived, sessile community where cells grow attached to a surface or as a bacterial conglomerate and surrounded by a complex extracellular matrix. While the ability of group A streptococcus to form biofilms in the laboratory has been shown, there is a lack of understanding of the role of GAS biofilms during an infection. We hypothesized that during infections, GAS exhibits a biofilm phenotype, complete with unique protein expression. To test this hypothesis, a rabbit model of GAS osteomyelitis was developed. A rabbit was inoculated with GAS using an infected indwelling device. Following the infection, blood and tissue samples were collected. Histological samples of the infected tibia were prepared, and the formation of a biofilm in vivo was visualized using peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) and confocal microscopy. In addition, Western blotting with convalescent rabbit serum detected cell wall proteins expressed in vitro under biofilm and planktonic growth conditions. Immunogenic proteins were then identified using matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These identities, along with the in vivo results, support the hypothesis that GAS forms biofilms during an infection. This unique phenotype should be taken into consideration when designing a vaccine or any other treatment for group A streptococcus infections.

Serine/threonine protein kinase Stk is required for virulence, stress response, and penicillin tolerance in Streptococcus pyogenes.

Genes encoding one or more Ser/Thr protein kinases have been identified recently in many bacteria, including one (stk) in the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). We report that in GAS, stk is required to produce disease in a murine myositis model of infection. Using microarray and quantitative reverse transcription-PCR (qRT-PCR) studies, we found that Stk activates genes for virulence factors, osmoregulation, metabolism of α-glucans, and fatty acid biosynthesis, as well as genes affecting cell wall synthesis. Confirming these transcription studies, we determined that the stk deletion mutant is more sensitive to osmotic stress and to penicillin than the wild type. We discuss several possible Stk phosphorylation targets that might explain Stk regulation of expression of specific operons and the possible role of Stk in resuscitation from quiescence.

The histone-like protein Hlp is essential for growth of Streptococcus pyogenes: comparison of genetic approaches to study essential genes.

Selection of possible targets for vaccine and drug development requires an understanding of the physiology of bacterial pathogens, for which the ability to manipulate expression of essential genes is critical. For Streptococcus pyogenes (the group A streptococcus [GAS]), an important human pathogen, the lack of genetic tools for such studies has seriously hampered research. To address this problem, we characterized variants of the inducible Ptet cassette, in both sense and antisense contexts, as tools to regulate transcription from GAS genes. We found that although the three-operator Ptet construct [Ptet(O)3] had low uninduced expression, its induction level was low, while the two-operator construct [Ptet(O)2] was inducible to a high level but showed significant constitutive expression. Use of Ptet(O)3 in the chromosome allowed us to demonstrate previously that RNases J1 and J2 are required for growth of GAS. Here we report that the uninduced level from the chromosomally inserted Ptet(O)2 construct was too high for us to observe differential growth. For the highly expressed histone-like protein (Hlp) of GAS, neither chromosomal insertion of Ptet(O)2 or Ptet(O)3 nor their use on a high-copy-number plasmid to produce antisense RNA specific to hlp resulted in adequate differential expression. However, by replacing the ribosome binding site of hlp with an engineered riboswitch to control translation of Hlp, we demonstrated for the first time that this protein is essential for GAS growth.