Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis

Schmelcher, Mathias; Powell, Anne M.; Camp, Mary J.; Pohl, Calvin S.; Donovan, David M.

doi:10.1007/s00253-015-6579-0

Cited by 94 publications

(74 citation statements)

References 69 publications

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“…3). Such inconsistencies on a quantitative level between different methods used for analyzing synergy have been reported previously (37,38) and underline the necessity to characterize synergistic interactions in more than one assay format (39). Similarly, it is important to test PGHs and combinations thereof against multiple bacterial strains.…”

Section: Discussionmentioning

confidence: 99%

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Carolin

Dätwyler

Meile

et al. 2017

Appl Environ Microbiol

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Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of Ͼ170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of Ͼ9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by Ͼ3.5 log units within 3 h. IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.KEYWORDS antibiotic resistance, antimicrobial agents, bovine mastitis, peptidoglycan hydrolases

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Section: Discussionmentioning

confidence: 99%

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Carolin

Dätwyler

Meile

et al. 2017

Appl Environ Microbiol

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“…Synergism between lysins and antibiotics has been shown previously in the growth inhibition of facultative anaerobic bacteria (33,55). However, no synergism has been shown in the growth inhibition of strict anaerobic bacteria, possibly due to the challenge of maintaining the growth of anaerobic bacteria in minimal media.…”

Section: Discussionmentioning

confidence: 95%

Using a Novel Lysin To Help Control Clostridium difficile Infections

Wang

Euler

Delaune

et al. 2015

Antimicrob Agents Chemother

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c 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, PlyCD 1-174 , 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 PlyCD 1-174 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 PlyCD 1-174 functioned in the presence of intestinal contents, significantly decreasing colonizing C. difficile compared to controls. Together, these data suggest that PlyCD 1-174 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.

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“…These combined PGHs also showed high activity against the Staphylococcus epidermidis and Staphylococcus warneri strains tested in this study, which had shown high tolerance toward the individual PGHs (Table 2). Inconsistencies on a quantitative level between different methods used for analyzing synergy (e.g., the classical broth microdilution checkerboard assay and the 2-dimensional spot assay, which yielded ⌺FIC and ⌺FBC values for the Lyso:CHAP combination of 0.460 Ϯ 0.036 and 0.376 Ϯ 0.029, respectively) have been reported previously (37,38) and underline the necessity of characterizing synergistic interactions in more than one assay format (39). Similarly, it is important to test PGHs and combinations thereof against multiple bacterial strains.…”

Section: Screening For Endolysins With Activity In Milkmentioning

confidence: 99%

Corrected and Republished from: Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Carolin

Dätwyler

Meile

et al. 2018

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have used a microtiter plate-based protocol to screen a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk. Eight suitable PGH constructs were identified by this approach, and their efficacies against S. aureus in heat-treated milk were compared by time-kill assays. The two most active enzymes (lysostaphin and CHAPK_CWT-LST) reduced S. aureus numbers in milk to undetectable levels within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The PGH combination completely eradicated S. aureus from milk: no more bacteria were detected within 24 h after the addition of the enzymes, corresponding to a reduction of >9 log units from the level in the control. Efficacy was also retained at different inoculum levels (3 log versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas lysostaphin retained its activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci, and S. aureus in particular, are a major cause of bovine mastitis, an inflammation of the mammary gland in cows that is associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell walls and are refractory to resistance development; thus, they have promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk among a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and thus support their further characterization in animal models.

show abstract

Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis

Cited by 94 publications

References 69 publications

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Using a Novel Lysin To Help Control Clostridium difficile Infections

Corrected and Republished from: Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

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