A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression.

CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

PlyKp104, a Novel Phage Lysin for the Treatment of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Other Gram-Negative ESKAPE Pathogens.

Klebsiella pneumoniae and Pseudomonas aeruginosa are two leading causes of burn and wound infections, pneumonia, urinary tract infections, and more severe invasive diseases, which are often multidrug resistant (MDR) or extensively drug resistant. Due to this, it is critical to discover alternative antimicrobials, such as bacteriophage lysins, against these pathogens. Unfortunately, most lysins that target Gram-negative bacteria require additional modifications or outer membrane permeabilizing agents to be bactericidal. We identified four putative lysins through bioinformatic analysis of Pseudomonas and Klebsiella phage genomes in the NCBI database and then expressed and tested their intrinsic lytic activity in vitro. The most active lysin, PlyKp104, exhibited >5-log killing against K. pneumoniae, P. aeruginosa, and other Gram-negative representatives of the multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumonia, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species) without further modification. PlyKp104 displayed rapid killing and high activity over a wide pH range and in high concentrations of salt and urea. Additionally, pulmonary surfactants and low concentrations of human serum did not inhibit PlyKp104 activity in vitro. PlyKp104 also significantly reduced drug-resistant K. pneumoniae >2 logs in a murine skin infection model after one treatment of the wound, suggesting that this lysin could be used as a topical antimicrobial against K. pneumoniae and other MDR Gram-negative infections.

Strains of bacterial species induce a greatly varied acute adaptive immune response: The contribution of the accessory genome.

A fundamental question in human susceptibility to bacterial infections is to what extent variability is a function of differences in the pathogen species or in individual humans. To focus on the pathogen species, we compared in the same individual the human adaptive T and B cell immune response to multiple strains of two major human pathogens, Staphylococcus aureus and Streptococcus pyogenes. We found wide variability in the acute adaptive immune response induced by various strains of a species, with a unique combination of activation within the two arms of the adaptive response. Further, this was also accompanied by a dramatic difference in the intensity of the specific protective T helper (Th) response. Importantly, the same immune response differences induced by the individual strains were maintained across multiple healthy human donors. A comparison of isogenic phage KO strains, demonstrated that of the pangenome, prophages were the major contributor to inter-strain immune heterogeneity, as the T cell response to the remaining "core genome" was noticeably blunted. Therefore, these findings extend and modify the notion of an adaptive response to a pathogenic bacterium, by implying that the adaptive immune response signature of a bacterial species should be defined either per strain or alternatively to the species' 'core genome', common to all of its strains. Further, our results demonstrate that the acquired immune response variation is as wide among different strains within a single pathogenic species as it is among different humans, and therefore may explain in part the clinical heterogeneity observed in patients infected with the same species.

Isolation of Phage Lysins That Effectively Kill Pseudomonas aeruginosa in Mouse Models of Lung and Skin Infection.

Multidrug resistance (MDR) is rapidly increasing in prevalence among isolates of the opportunistic pathogen Pseudomonas aeruginosa, leaving few treatment options. Phage lysins are cell wall hydrolases that have a demonstrated therapeutic potential against Gram-positive pathogens; however, the outer membrane of Gram-negative bacteria prevents most lysins from reaching the peptidoglycan, making them less effective as therapeutics. Nevertheless, a few lysins from Gram-negative bacterial phage can penetrate the bacterial outer membrane with the aid of an amphipathic tail found in the molecule's termini. In this work, we took a phylogenetic approach to systematically identify those lysins from P. aeruginosa phage that would be most effective therapeutically. We isolated and performed preliminary characterization of 16 lysins and chose 2 lysins, PlyPa03 and PlyPa91, which exhibited >5-log killing activity against P. aeruginosa and other Gram-negative pathogens (particularly Klebsiella and Enterobacter). These lysins showed rapid killing kinetics and were active in the presence of high concentrations of salt and urea and under pH conditions ranging from 5.0 to 10.0. Activity was not inhibited in the presence of the pulmonary surfactant beractant (Survanta). While neither enzyme was active in 100% human serum, PlyPa91 retained activity in low serum concentrations. The lysins were effective in the treatment of a P. aeruginosa skin infection in a mouse model, and PlyPa91 protected mice in a lung infection model, making these lysins potential drug candidates for Gram-negative bacterial infections of the skin or respiratory mucosa.

Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria.

The prevalence of multidrug-resistant Pseudomonas aeruginosa has stimulated development of alternative therapeutics. Bacteriophage peptidoglycan hydrolases, termed lysins, represent an emerging antimicrobial option for targeting Gram-positive bacteria. However, lysins against Gram-negatives are generally deterred by the outer membrane and their inability to work in serum. One solution involves exploiting evolved delivery systems used by colicin-like bacteriocins (e.g., S-type pyocins of P. aeruginosa) to translocate through the outer membrane. Following surface receptor binding, colicin-like bacteriocins form Tol- or TonB-dependent translocons to actively import bactericidal domains through outer membrane protein channels. With this understanding, we developed lysocins, which are bioengineered lysin-bacteriocin fusion molecules capable of periplasmic import. In our proof-of-concept studies, components from the P. aeruginosa bacteriocin pyocin S2 (PyS2) responsible for surface receptor binding and outer membrane translocation were fused to the GN4 lysin to generate the PyS2-GN4 lysocin. PyS2-GN4 delivered the GN4 lysin to the periplasm to induce peptidoglycan cleavage and log-fold killing of P. aeruginosa with minimal endotoxin release. While displaying narrow-spectrum antipseudomonal activity in human serum, PyS2-GN4 also efficiently disrupted biofilms, outperformed standard-of-care antibiotics, exhibited no cytotoxicity toward eukaryotic cells, and protected mice from P. aeruginosa challenge in a bacteremia model. In addition to targeting P. aeruginosa, lysocins can be constructed to target other prominent Gram-negative bacterial pathogens.

Novel Engineered Peptides of a Phage Lysin as Effective Antimicrobials against Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative bacterial pathogen responsible for a range of nosocomial infections. The recent rise and spread of multidrug-resistant A. baumannii clones has fueled a search for alternative therapies, including bacteriophage endolysins with potent antibacterial activities. A common feature of these lysins is the presence of a highly positively charged C-terminal domain with a likely role in promoting outer membrane penetration. In the present study, we show that the C-terminal amino acids 108 to 138 of phage lysin PlyF307, named P307, alone were sufficient to kill A. baumannii (>3 logs). Furthermore, P307 could be engineered for improved activity, the most active derivative being P307SQ-8C (>5-log kill). Both P307 and P307SQ-8C showed high in vitro activity against A. baumannii in biofilms. Moreover, P307SQ-8C exhibited MICs comparable to those of levofloxacin and ceftazidime and acted synergistically with polymyxin B. Although the peptides were shown to kill by disrupting the bacterial cytoplasmic membrane, they did not lyse human red blood cells or B cells; however, serum was found to be inhibitory to lytic activity. In a murine model of A. baumannii skin infection, P307SQ-8C reduced the bacterial burden by ∼2 logs in 2 h. This study demonstrates the prospect of using peptide derivatives from bacteriophage lysins to treat topical infections and remove biofilms caused by Gram-negative pathogens.

Using a Novel Lysin To Help Control Clostridium difficile Infections.

As a consequence of excessive antibiotic therapies in hospitalized patients, Clostridium difficile, a Gram-positive anaerobic spore-forming intestinal pathogen, is the leading cause of hospital-acquired diarrhea and colitis. Drug treatments for these diseases are often complicated by antibiotic-resistant strains and a high frequency of treatment failures and relapse; therefore, novel nonantibiotic approaches may prove to be more effective. In this study, we recombinantly expressed a prophage lysin identified from a C. difficile strain, CD630, which we named PlyCD. PlyCD was found to have lytic activity against specific C. difficile strains. However, the recombinantly expressed catalytic domain of this protein, PlyCD1-174, displayed significantly greater lytic activity (>4-log kill) and a broader lytic spectrum against C. difficile strains while still retaining a high degree of specificity toward C. difficile versus commensal clostridia and other bacterial species. Our data also indicated that noneffective doses of vancomycin and PlyCD1-174 when combined in vitro could be significantly more bactericidal against C. difficile. In an ex vivo treatment model of mouse colon infection, we found that PlyCD1-174 functioned in the presence of intestinal contents, significantly decreasing colonizing C. difficile compared to controls. Together, these data suggest that PlyCD1-174 has potential as a novel therapeutic for clinical application against C. difficile infection, either alone or in combination with other preexisting treatments to improve their efficacy.

Novel phage lysin capable of killing the multidrug-resistant gram-negative bacterium Acinetobacter baumannii in a mouse bacteremia model.

Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killing A. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found in Acinetobacter phage.

A novel chimeric phage lysin with high in vitro and in vivo bactericidal activity against Streptococcus pneumoniae.

OBJECTIVES: Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide and new antimicrobials are urgently needed. Our aim was new chimeric phage endolysins, or lysins, with improved bactericidal activity by swapping the structural components of two pneumococcal phage lysozymes: Cpl-1 (the best lysin tested to date) and Cpl-7S. METHODS: The bactericidal effects of four new chimeric lysins were checked against several bacteria. The purified enzymes were added at different concentrations to resuspended bacteria and viable cells were measured after 1 h. Killing capacity of the most active lysin, Cpl-711, was tested in a mouse bacteraemia model, following mouse survival after injecting different amounts (25-500 µg) of enzyme. The capacity of Cpl-711 to reduce pneumococcal biofilm formation was also studied. RESULTS: The chimera Cpl-711 substantially improved the killing activity of the parental phage lysozymes, Cpl-1 and Cpl-7S, against pneumococcal bacteria, including multiresistant strains. Specifically, 5 µg/mL Cpl-711 killed ≥7.5 log of pneumococcal R6 strain. Cpl-711 also reduced pneumococcal biofilm formation and killed 4 log of the bacterial population at 1 µg/mL. Mice challenged intraperitoneally with D39_IU pneumococcal strain were protected by treatment with a single intraperitoneal injection of Cpl-711 1 h later, resulting in about 50% greater protection than with Cpl-1. CONCLUSIONS: Domain swapping among phage lysins allows the construction of new chimeric enzymes with high bactericidal activity and a different substrate range. Cpl-711, the most powerful endolysin against pneumococci, offers a promising therapeutic perspective for the treatment of multiresistant pneumococcal infections.

A highly active and negatively charged Streptococcus pyogenes lysin with a rare D-alanyl-L-alanine endopeptidase activity protects mice against streptococcal bacteremia.

Bacteriophage endolysins have shown great efficacy in killing Gram-positive bacteria. PlyC, a group C streptococcal phage lysin, represents the most efficient lysin characterized to date, with a remarkably high specificity against different streptococcal species, including the important pathogen Streptococcus pyogenes. However, PlyC is a unique lysin, in terms of both its high activity and structure (two distinct subunits). We sought to discover and characterize a phage lysin active against S. pyogenes with an endolysin architecture distinct from that of PlyC to determine if it relies on the same mechanism of action as PlyC. In this study, we identified and characterized an endolysin, termed PlyPy (phage lysin from S. pyogenes), from a prophage infecting S. pyogenes. By in silico analysis, PlyPy was found to have a molecular mass of 27.8 kDa and a pI of 4.16. It was active against a majority of group A streptococci and displayed high levels of activity as well as binding specificity against group B and C streptococci, while it was less efficient against other streptococcal species. PlyPy showed the highest activity at neutral pH in the presence of calcium and NaCl. Surprisingly, its activity was not affected by the presence of the group A-specific carbohydrate, while the activity of PlyC was partly inhibited. Additionally, PlyPy was active in vivo and could rescue mice from systemic bacteremia. Finally, we developed a novel method to determine the peptidoglycan bond cleaved by lysins and concluded that PlyPy exhibits a rare d-alanyl-l-alanine endopeptidase activity. PlyPy thus represents the first lysin characterized from Streptococcus pyogenes and has a mechanism of action distinct from that of PlyC.

Anti-CRISPR proteins trigger a burst of CRISPR-Cas9 expression that enhances phage defense.

CRISPR-Cas immune systems provide bacteria with adaptive immunity against bacteriophages, but they are often transcriptionally repressed to mitigate auto-immunity. In some cases, CRISPR-Cas expression increases in response to a phage infection, but the mechanisms of induction are largely unknown, and it is unclear whether induction occurs strongly and quickly enough to benefit the bacterial host. In S. pyogenes, Cas9 is both an immune effector and auto-repressor of CRISPR-Cas expression. Here, we show that phage-encoded anti-CRISPR proteins relieve Cas9 auto-repression and trigger a rapid increase in CRISPR-Cas levels during a single phage infective cycle. As a result, fewer cells succumb to lysis, leading to a striking survival benefit after multiple rounds of infection. CRISPR-Cas induction also reduces lysogeny, thereby limiting a route for horizontal gene transfer. Altogether, we show that Cas9 is not only a CRISPR-Cas effector and repressor but also a phage sensor that can mount an anti-anti-CRISPR transcriptional response.

Novel bacteriophage lysin with broad lytic activity protects against mixed infection by Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are among the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. As a result, alternative therapies need to be developed to combat these pathogens. We have identified a novel bacteriophage lysin (PlySs2), derived from a Streptococcus suis phage, with broad lytic activity against MRSA, vancomycin-intermediate S. aureus (VISA), Streptococcus suis, Listeria, Staphylococcus simulans, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae (group B streptococcus [GBS]), S. pyogenes, Streptococcus sanguinis, group G streptococci (GGS), group E streptococci (GES), and Streptococcus pneumoniae. PlySs2 has an N-terminal cysteine-histidine aminopeptidase (CHAP) catalytic domain and a C-terminal SH3b binding domain. It is stable at 50 °C for 30 min, 37 °C for >24 h, 4°C for 15 days, and -80 °C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 at 128 µg/ml in vitro reduced MRSA and S. pyogenes growth by 5 logs and 3 logs within 1 h, respectively, and exhibited a MIC of 16 µg/ml for MRSA. A single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of mixed MRSA and S. pyogenes infection. Serially increasing exposure of MRSA and S. pyogenes to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; as such, PlySs2 has all the characteristics to be an effective therapeutic.

PaP1, a Broad-Spectrum Lysin-Derived Cationic Peptide to Treat Polymicrobial Skin Infections.

Most skin infections, including those complicating burns, are polymicrobial involving multiple causative bacteria. Add to this the fact that many of these organisms may be antibiotic-resistant, and a simple skin lesion or burn could soon become life-threatening. Membrane-acting cationic peptides from Gram-negative bacteriophage lysins can potentially aid in addressing the urgent need for alternative therapeutics. Such peptides natively constitute an amphipathic region within the structural composition of these lysins and function to permit outer membrane permeabilization in Gram-negative bacteria when added externally. This consequently allows the lysin to access and degrade the peptidoglycan substrate, resulting in rapid hypotonic lysis and bacterial death. When separated from the lysin, some of these cationic peptides kill sensitive bacteria more effectively than the native molecule via both outer and cytoplasmic membrane disruption. In this study, we evaluated the antibacterial properties of a modified cationic peptide from the broad-acting lysin PlyPa01. The peptide, termed PaP1, exhibited potent in vitro bactericidal activity toward numerous high priority Gram-positive and Gram-negative pathogens, including all the antibiotic-resistant ESKAPE pathogens. Both planktonic and biofilm-state bacteria were sensitive to the peptide, and results from time-kill assays revealed PaP1 kills bacteria on contact. The peptide was bactericidal over a wide temperature and pH range and could withstand autoclaving without loss of activity. However, high salt concentrations and complex matrices were found to be largely inhibitory, limiting its use to topical applications. Importantly, unlike other membrane-acting antimicrobials, PaP1 lacked cytotoxicity toward human cells. Results from a murine burn wound infection model using methicillin-resistant Staphylococcus aureus or multidrug-resistant Pseudomonas aeruginosa validated the in vivo antibacterial efficacy of PaP1. In these studies, the peptide enhanced the potency of topical antibiotics used clinically for treating chronic wound infections. Despite the necessity for additional preclinical drug development, the collective data from our study support PaP1 as a potential broad-spectrum monotherapy or adjunctive therapy for the topical treatment of polymicrobial infections and provide a foundation for engineering future lysin-derived peptides with improved antibacterial properties.

Prophage integration into CRISPR loci enables evasion of antiviral immunity in Streptococcus pyogenes.

CRISPR loci are composed of short DNA repeats separated by sequences, known as spacers, that match the genomes of invaders such as phages and plasmids. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defence, phages can produce small proteins that inhibit these nucleases, termed anti-CRISPRs (Acrs). Here we demonstrate that the ΦAP1.1 temperate phage utilizes an alternative approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection, this phage expresses a small anti-CRISPR protein, AcrIIA23, that prevents Cas9 function, allowing ΦAP1.1 to integrate into the direct repeats of the CRISPR locus, neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transduction of spacers, enabling a complex modulation of the type II-A CRISPR immune response. A bioinformatic search identified prophages integrated not only in the CRISPR repeats, but also the cas genes, of diverse bacterial species, suggesting that prophage disruption of the CRISPR-cas locus is a recurrent mechanism to counteract immunity.

Gram-Negative Bacterial Lysins.

Antibiotics have had a profound impact on human society by enabling the eradication of otherwise deadly infections. Unfortunately, antibiotic use and overuse has led to the rapid spread of acquired antibiotic resistance, creating a major threat to public health. Novel therapeutic agents called bacteriophage endolysins (lysins) provide a solution to the worldwide epidemic of antibiotic resistance. Lysins are a class of enzymes produced by bacteriophages during the lytic cycle, which are capable of cleaving bonds in the bacterial cell wall, resulting in the death of the bacteria within seconds after contact. Through evolutionary selection of the phage progeny to be released and spread, these lysins target different critical components in the cell wall, making resistance to these molecules orders of magnitude less likely than conventional antibiotics. Such properties make lysins uniquely suitable for the treatment of multidrug resistant bacterial pathogens. Lysins, either naturally occurring or engineered, have the potential of being developed into fast-acting, narrow-spectrum, biofilm-disrupting antimicrobials that act synergistically with standard of care antibiotics. This review focuses on newly discovered classes of Gram-negative lysins with emphasis on prototypical enzymes that have been evaluated for efficacy against the major antibiotic resistant organisms causing nosocomial infections.

Evaluation of Topical Lysostaphin as a Novel Treatment for Instrumented Rhesus Macaques (Macaca mulatta) Infected with Methicillin-Resistant Staphylococcus aureus.

Lytic enzymes are novel antimicrobial agents that degrade bacterial cell walls, resulting in cell rupture and death. We tested one enzyme, the bacteriocin lysostaphin, for treatment of nonhuman primates (Macaca mulatta) with persistent methicillinresistant Staphylococcus aureus (MRSA) infection of their cranial implant margins. The goal of this study was to determine if topical lysostaphin, either alone or as an adjunct therapy, could eliminate MRSA. Lysostaphin had in vitro lytic activity against all 4 previously identified NHP MRSA clones, as well as against 12 MRSA isolates of the same clonal type (MLST ST3862 and spa type t4167) before and after treatment, with no resistance discovered. In an in vivo pilot study, a 2-d application of lysostaphin alone reduced MRSA in the implant margins by 3-logs during treatment of one animal; however, MRSA titers had returned to control levels by 1 wk after treatment. In the main study, all animals (n = 4) received 10 d of systemic antibiotic treatment and both the animals and their environment (cages, equipment, room) underwent 5-d of decontamination. The experimental animals (n = 2) received 5 doses of topical lysostaphin (15 mg, every other day) applied onto their implant margins. Daily cultures showed that MRSA counts decreased significantly (≤ 25 colony-forming units/mL; P < 0.05). However, sampling of the cranial implant margin 7 d after last treatment showed that MRSA counts had returned to control levels. Our study suggests that lysostaphin, coupled with other treatment modalities, can decrease MRSA infection short-term but do not completely eradicate MRSA in the long-term. This reappearance of MRSA may be due to cross-contamination or reinfection from other infected areas, an inability of the treatment to reach all colonized areas, or insufficient dosing or length of treatment. Topical lysostaphin may be more useful clinically for superficial nonimplant associated wounds in which the lytic enzyme has better access to the infected tissue.

Staphylococcus aureus Infecting and Colonizing Experimental Animals, Macaques, in a Research Animal Facility.

An outbreak of methicillin-resistant Staphylococcus aureus (MRSA) infections on the skin and soft tissues of experimental macaques in the vivarium of The Rockefeller University, New York, triggered this observational and interventional study. We screened 14 macaques in the colony (samples from head, nares, and rectum) and their housing (40 environmental surfaces) four times in 1 year, for S. aureus colonization or contamination, while implementing enhanced decolonization and decontamination procedures. A total of 114 isolates of S. aureus were recovered and characterized (antibiograms, spa typing, multilocus sequence typing, pulsed-field gel electrophoresis [PFGE], mecA, Panton-Valentine Leukocidin, and arginine catabolic mobile element). Based on these results, six strains of S. aureus were identified: two MRSA strains (t16708/ST3862/PFGE-A, t16709/ST3862/PFGE-C) and one methicillin-sensitive S. aureus (t8397/ST3884/PFGE-D) were characterized for the first time in this study; strains belonging to spa types t189 and t4167 have been identified in primates in previous studies. None of these strains was common to the neighboring New York City human community. Thus, it seems probable that the animals were already colonized upon arrival to the University. We suggest screening primates for S. aureus carriage upon arrival to University vivaria and possible implementation of extensive decolonization procedures before any surgical interventions.

Novel algorithms reveal streptococcal transcriptomes and clues about undefined genes.

Bacteria-host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence.

The Phage Lysin PlySs2 Decolonizes Streptococcus suis from Murine Intranasal Mucosa.

Streptococcus suis infects pigs worldwide and may be zoonotically transmitted to humans with a mortality rate of up to 20%. S. suis has been shown to develop in vitro resistance to the two leading drugs of choice, penicillin and gentamicin. Because of this, we have pursued an alternative therapy to treat these pathogens using bacteriophage lysins. The bacteriophage lysin PlySs2 is derived from an S. suis phage and displays potent lytic activity against most strains of that species including serotypes 2 and 9. At 64 µg/ml, PlySs2 reduced multiple serotypes of S. suis by 5 to 6-logs within 1 hour in vitro and exhibited a minimum inhibitory concentration (MIC) of 32 µg/ml for a S. suis serotype 2 strain and 64 µg/ml for a serotype 9 strain. Using a single 0.1-mg dose, the colonizing S. suis serotype 9 strain was reduced from the murine intranasal mucosa by >4 logs; a 0.1-mg dose of gentamicin reduced S. suis by <3-logs. A combination of 0.05 mg PlySs2 + 0.05 mg gentamicin reduced S. suis by >5-logs. While resistance to gentamicin was induced after systematically increasing levels of gentamicin in an S. suis culture, the same protocol resulted in no observable resistance to PlySs2. Thus, PlySs2 has both broad and high killing activity against multiple serotypes and strains of S. suis, making it a possible tool in the control and prevention of S. suis infections in pigs and humans.