Recombinant of the Staphylococcal Bacteriophage Lysin CHAPk and Its Elimination against Streptococcus agalactiae Biofilms

Abstract

Bovine mastitis is the most important infectious disease, causing significant losses in the dairy industry, in which Streptococcus agalactiae is a major pathogen. In this study, lysin CHAPk, derived from bacteriophage K, was expressed heterogeneously, and its antimicrobial and anti-biofilm effects against S. agalactiae isolated from bovine mastitis were further analyzed. CHAPk was expressed in Escherichia coli BL21 (DE3), in which the purified yield of CHAPk was up to 14.6 mg/L with the purity of 95%. Time-killing kinetic curves showed that CHAPk fastly killed S. agalactiae in TSB medium and in milk within 25 min (by 3.3 log10 CFU/mL and 2.4 log10 CFU/mL, respectively). Observation of scanning electron microscope (SEM) showed cells wrinkled and ruptured after the treatment of CHAPk. CHAPk effectively inhibited early biofilms by 95% in 8 × MIC, and eradicated mature biofilms by 89.4% in 16 × MIC. Moreover, CHAPk killed 99% bacteria in mature biofilms. Confocal laser scanning microscopy (CLSM) also demonstrated the potent antimicrobial and anti-biofilm action of CHAPk. It was firstly demonstrated CHAPk had the characters of inhibition/elimination of S. agalactiae biofilms and killing the bacteria in biofilms. CHAPk has the potential to develop a new antibacterial agent for mastitis treatment of S. agalactiae infections.

Dual Antibacterial Activities and Biofilm Eradication of a Marine Peptide-N6NH2 and Its Analogs against Multidrug-Resistant Aeromonas veronii

“…Planktonic bacteria were gently rinsed twice, and the biofilms were dyed with SYTO9 and PI (LIVE/DEAD Bac Light Bacterial Viability Kit) for 15 min. After washing with PBS, the biofilms were observed by Zeiss LSM880 confocal microscope (CLSM) (Zeiss, Oberkochen, Germany) [ 48 , 51 ].…”

“…PBS was used as the negative control. The plates were treated with ultrasound for 5 min and the viable bacteria were counted on the TSA plates [ 48 , 51 ].…”

Phage Therapy in Veterinary Medicine

“…Phage endolysin expressed not only a lytic action but also a reduction of biofilm of approximately 90% and destroyed 99% of the bacteria that were present in the mature biofilm. The action of endolysin was well highlighted, thanks to the use of a confocal microscope [ 79 ].…”

Potential for Phages in the Treatment of Bacterial Sexually Transmitted Infections

“…Considered a concerning threat by CDC, clindamycin resistance prevalent, some erythromycin, azithromycin and vancomycin resistance reported [5] Considered a part of normal flora for 10-30% of women [55], the CDC estimates 27,000 severe cases of GBS infections in the US, with 49% (~13,230) being erythromycin-resistant and with 28% (~7560) being clindamycin-resistant [5]; a majority of infants colonized with GBS do not develop a GBS infection; about 60% of cases of early-onset GBS infection occur in neonates born to patients with negative GBS culture at 35-37 weeks [56] Leading cause of neonatal sepsis and meningitis [55]; asymptomatic in colonized women [57] Intravenous penicillin G (during labor); ampicillin or vancomycin may be substituted [56] Gram-positive, with ten different capsular types (Ia, Ib, II-XI) [58] Growth observed with normal laboratory conditions (37 • C and enriched media) [59,60] Temperate phages have been isolated and characterized for S. agalactiae [59,61,62]; phage lysins have successfully shown activity in vitro (lysins from S. agalactiae phages B30 [63,64], λSA2 [65] and λSA1 [66], as well as CHAP k lysin derived from S. aureus [67]) and in vivo (PlyGBS from phage NCTC11261 [68] and chimeric ClyV [69]). There can be a wide host range with streptococcal phages and phage enzymes [70,71]…”

“…Biofilms are notoriously resistant to antibiotics; however, certain phages possess the ability to effectively degrade biofilms where antibiotics fail (see Figure 1 F,G) [ 121 , 122 ]. Interestingly, CHAP k lysine—a derivative of native lysin of S. aureus —presented strong activity against S. agalactiae biofilm even more potent than vancomycin [ 67 ]. Currently, work is being performed to biologically engineer hybrid phage enzymes for both Gram-positive and Gram-negative bacteria.…”