Antibacterial and biofilm removal activity of a podoviridae Staphylococcus aureus bacteriophage SAP-2 and a derived recombinant cell-wall-degrading enzyme

Antibacterial and biofilm removal activity of a new podoviridae Staphylococcus aureus bacteriophage (SAP-2), which belongs to the φ29-like phage genus of the Podoviridae family, and a cell-wall-degrading enzyme (SAL-2), which is derived from bacteriophage SAP-2, have been characterized. The cell-wall-degrading enzyme SAL-2 was expressed in Escherichia coli in a soluble form using a low-temperature culture. The cell-wall-degrading enzyme SAL-2 had specific lytic activity against S. aureus, including methicillin-resistant strains, and showed a minimum inhibitory concentration of about 1 μg/ml. In addition, this enzyme showed a broader spectrum of activity within the Staphylococcus genus compared with bacteriophage SAP-2 in its ability to remove the S. aureus biofilms. Thus, the cell-wall-degrading enzyme SAL-2 can be used to prevent and treat biofilm-associated S. aureus infections either on its own or in combination with other cell-wall-degrading enzymes with anti-S. aureus activity.     

Complete genome sequence of a newly isolated lytic bacteriophage,EFAP‐1 of Enterococcus faecalis,and antibacterial activity of its endolysin EFAL‐1

Aims: In this work, we aimed to identify an effective treatment of infections caused by Enterococcus spp. strains resistant to conventional antibiotics. Methods and Results: We report the isolation and characterization of a new lytic bacteriophage, designated bacteriophage EFAP‐1, that is capable of lysing Enterococcus faecalis bacteria that exhibit resistance to multiple antibiotics. EFAP‐1 has low sequence similarity to all known bacteriophages. Transmission electron microscopy confirmed that EFAP‐1 belongs to the Siphoviridae family. A putative lytic protein of EFAP‐1, endolysin EFAL‐1, is encoded in ORF 2 and was expressed in Escherichia coli. Recombinant EFAL‐1 had broad‐spectrum lytic activity against several Gram‐positive pathogens, including Ent. faecalis and Enterococcus faecium. Conclusions: The complete genome sequence of the newly isolated enterococcal lytic phage was analysed, and it was demonstrated that its recombinant endolysin had broad lytic activity against various Gram‐positive pathogens. Significance and Impact of the Study: Bacteriophage EFAP‐1 and its lytic protein, EFAL‐1, can be utilized as potent antimicrobial agents against Enterococcus spp. strains resistant to conventional antibiotics in hospital infections and also as environmental disinfectants to control disease‐causing Enterococcus spp. in dairy farms.     

Potential of the Polyvalent Anti-Staphylococcus Bacteriophage K for Control of Antibiotic-Resistant Staphylococci from Hospitals

The increasing prevalence of antibiotic-resistant staphylococci has prompted the need for antibacterial controls other than antibiotics. In this study, a lytic bacteriophage (phage K) was assessed in vitro for its ability to inhibit emerging drug-resistant Staphylococcus aureus strains from hospitals and other species of Staphylococcus isolated from bovine infections. In in vitro inhibitory assays, phage K lysed a range of clinically isolated methicillin-resistant S. aureus (MRSA) strains, S. aureus with heterogeneous vancomycin resistance and vancomycin resistance, and teicoplanin-resistant strains. In these assays, 14 of the MRSA strains were initially only weakly sensitive to this phage. However, propagation of phage K on these less-sensitive strains resulted in all 14 being sensitive to the modified phages. The results enforce the principle that, while certain target bacteria may be relatively insensitive to lytic phage, this can be overcome by obtaining modified phage variants from passage of the phage through the insensitive strains. Model in situ hand wash studies using a phage-enriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti-Staphylococcus activity from the cream on plates and in broth.     

The polyphenol (-)-epicatechin gallate disrupts the secretion of virulence-related proteins by Staphylococcus aureus

Aim: (?)‐Epicatechin gallate (ECg) modifies the morphology, cell wall architecture and β‐lactam antibiotic susceptibility of Staphylococcus aureus. As these effects result primarily from intercalation into the bacterial cytoplasmic membrane, the capacity of ECg to modulate the secretion of two key staphylococcal virulence factors, coagulase and α‐toxin, was examined. Methods and Results: Bioassays were used to determine coagulase and haemolysin activity in culture supernatants of a number of S. aureus isolates grown in the presence and absence of ECg; α‐toxin secretion was also evaluated by immunoblotting. Growth in ECg reduced the levels of activity of both proteins in culture supernatants; the effects could only be partly explained by ECg‐mediated inhibition of bioactivity and by induction of secreted proteases. Conclusion: ECg suppresses the secretion of coagulase and α‐toxin by clinical isolates of S. aureus. Significance and Impact of the Study: The observation that secretion of key components of staphylococcal virulence can be compromised by a naturally occurring polyphenol supports the notion that ECg and related compounds may have therapeutic utility for the control of infections that are currently difficult to treat due to the propensity of methicillin‐resistant S. aureus to accumulate antibiotic resistance genes.     

Detection of Methicillin-Resistant Staphylococcus aureus Using mecA/nuc Genes and Antibiotic Susceptibility Profile of Malaysian Clinical Isolates

The aim of this study is to compare methicillin-resistant Staphylococcus aureus (MRSA) detection methods and to generate antibiogram profile of S. aureus clinical isolates from two teaching hospitals in Malaysia including three reference isolates from American Type Culture Collection (ATCC). The mecA/nuc gene PCR amplification, spot inoculation test and oxacillin disc diffusion test were applied to compare its MRSA detection abilities. No disagreement between the three methods was observed. From 29 bacterial isolates (including the ATCC strains) tested, 19 isolates were confirmed as S. aureus with 14 isolates exhibiting multidrug-resistance. All isolates are still susceptible to vancomycin as indicated by the E-test result. Current biochemical tests are comparable with the molecular detection method for MRSA used in this study while multidrug-resistance traits are present in both MRSA and MSSA clinical isolates. Presently, mupirocin seems to be the best alternative for vancomycin against multidrug-resistant S. aureus infections in Malaysia. Susceptibility profile of 19 S. aureus isolates acquired from two teaching hospitals and ATCC towards 16 selected antibiotics was analyzed and an antibiogram was generated. Findings also indicated resistance against many of the available antibiotics and thus an urgent need to search for alternative antibiotics.     

Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci

A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.     

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin‐derived protein

New anti‐infective approaches are much needed to control multi‐drug‐resistant (MDR) pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4‐ to 23.4‐fold as response to V12CBD. More significantly, V12CBD could activate macrophages through NF‐κB pathway and enhance phagocytosis against S. aureus. As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections.     

Immunogenicity and antimicrobial effectiveness of Pseudomonas aeruginosa specific bacteriophage in a human lung in vitro model

The rise of antibiotic resistant bacteria is posing a serious threat to human health. For example, resistant strains of Pseudomonas aeruginosa have resulted in untreatable and potentially lethal infections in both cystic fibrosis and immunocompromised patients. Due to the growing need for alternative treatment options, bacteriophage, or phage, therapy is gaining considerable attention. While previous studies have demonstrated the effectiveness of phage in combating persistent bacterial infections, there is currently a lack of knowledge regarding the host immunological response following phage exposure. In the present study, the bioresponses of an enhanced in vitro model were characterized following exposure to either DMS3 or PEV2, P. aeruginosa targeting phages. Results demonstrated a PEV2-dependent increase in IL-6 and TNF-α production, but no changes associated with DMS3 exposure. Additionally, following the establishment of an in vitro infection model, DMS3 was found to successfully protect mammalian lung cells from P. aeruginosa. Taken together, the biocompatibility and antibacterial effectiveness distinguish DMS3 bacteriophage as a strong candidate for phage therapy. However, as DMS3 is pilin dependent and bacterial receptor expression varies significantly, this work highlights the necessity of generating phage cocktails.

     

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

Anti-biofilm activity of Quercus infectoria G. Olivier against methicillin-resistant Staphylococcus aureus

Aims: To establish the effect of Quercus infectoria G. Olivier extract and its main constituent, tannic acid, on staphylococcal biofilm and their anti‐biofilm mechanisms. Methods and Results: Anti‐biofilm activity of the plant materials on clinical isolated of methicillin‐resistant Staphylococcus aureus and methicillin‐susceptible Staph. aureus was employed using a crystal violet‐stained microtiter plate method. The extract at minimum inhibitory concentration (MIC; 0·25 mg ml^?1) was significantly reduced the biofilm formation of the isolates (P < 0·05). The effect on staphylococcal cell surface hydrophobicity (CSH) of the test compounds was investigated as a possible mode of action of the anti‐biofilm activity. The hydrophobicity index of all the bacterial isolates increased following treatment with supra‐MIC, MIC and sub‐MIC of the extract and tannic acid. Observation of the treated bacterial cells by electron microscopy revealed that the test compounds caused clumps of partly divided cocci with thickened and slightly rough cell wall. Conclusions: The results indicated that Q. infectoria extract and tannic acid affected staphylococcal biofilm formation and their effect on bacterial CSH and cell wall may involve in the anti‐biofilm activity. Significance and Impact of the Study: This evidence highlighted the anti‐biofilm potency of the natural products and clarified their anti‐biofilm mechanisms.     

噬菌体治疗肺炎克雷伯菌感染的研究进展

随着细菌的进化以及部分抗生素的滥用，耐药细菌的感染已成为21世纪主要的公共卫生挑战之一。其中，耐药肺炎克雷伯菌(Klebsiella pneumoniae)问题尤为突出。噬菌体在治疗耐药细菌感染引起的疾病方面展现出一定的潜力及独特优势，但目前噬菌体治疗尚缺乏统一的临床指导规范。虽然临床上有少数将噬菌体用于治疗肺炎克雷伯菌感染的成功案例，但多数情况下是采用噬菌体配合抗生素疗法，噬菌体在其中的作用仍不明确。本文综合评述国内外研究数据，回顾与噬菌体治疗肺炎克雷伯菌感染相关的数个重点问题，包括噬菌体的特性以及影响其疗效的因素，旨在为肺炎克雷伯菌和其他耐药细菌的噬菌体治疗提供参考。     

Prevention of Staphylococcus aureus biofilm formation and reduction in established biofilm density using a combination of phage K and modified derivatives

Aims: To investigate the ability of a mixture of phage K and six of its modified derivatives to prevent biofilm formation by Staphylococcus aureus and also to reduce the established biofilm density. Methods and Results: The bioluminescence‐producing Staph. aureus Xen29 strain was used in the study, and incubation of this strain in static microtitre plates at 37°C for 48 h confirmed its strong biofilm‐forming capacity. Subsequently, removal of established biofilms of Staph. aureus Xen29 with the high‐titre phage combination was investigated over time periods of 24 h, 48 h and 72 h. Results suggested that these biofilms were eliminated in a time‐dependant manner, with biofilm biomass reduction significantly greater after 72 h than after 24–48 h. In addition, initial challenge of Staph. aureus Xen29 with the phage cocktail resulted in the complete inhibition of biofilm formation over a 48‐h period with no appearance of phage resistance. Conclusions: In general, our findings demonstrate the potential use of a modified phage combination for the prevention and successful treatment of Staph. aureus biofilms, which are implicated in several antibiotic‐resistant infections. Significance and Impact of the Study: This study highlights the first use of phage K for the successful removal and prevention of biofilms of Staph. aureus.     

M13 Virus based detection of bacterial infections in living hosts

We report a first method for using M13 bacteriophage as a multifunctional scaffold for optically imaging bacterial infections in vivo. We demonstrate that M13 virus conjugated with hundreds of dye molecules (M13‐Dye) can target and distinguish pathogenic infections of F‐ pili expressing and F ‐negative strains of E. coli. Further, in order to tune this M13‐Dye complex suitable for targeting other strains of bacteria, we have used a 1‐step reaction for creating an anti‐bacterial antibody ‐M13‐Dye probe. As an example, we show anti‐S. aureus ‐M13‐Dye able to target and image infections of S. aureus in living hosts, with a 3.7× increase in fluorescence over background. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Efficacy of Polyvalent Bacteriophage P-27/HP to Control Multidrug Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Associated with Human Infections

Emergence of multidrug resistant strains has created serious problem for safe eradication of Staphylococcus aureus infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages kill bacteria irrespective of their antibiotic sensitivity and thus they can be used as potent prophylactic/therapeutic agent to treat such infections. Here, we report isolation of broad host range anti-staphylococcal lytic bacteriophage P-27/HP from sewage water. This phage was able to inhibit 17 of 28 (60%) human disease associated S. aureus isolates. In vitro studies revealed its strong lytic efficacy to diminish S. aureus 27/HP population (c.f.u.) by more than 5.0 logs (P < 0.0001) (equivalent to 99.99%) in 3 h at 0.01 MOI. In vivo lytic efficacy analysis showed that a single subcutaneous injection of phage P-27/HP (10⁷ p.f.u.) was sufficient to protect S. aureus 27/HP infected (5 × 10⁸ c.f.u.) mice from bacteremia and subsequent death. A considerable decline of more than 6 logs (99.9%) in splenic S. aureus 27/HP c.f.u. count was noted at the 3 days of phage treatment. In conclusion, our results suggest that phage P-27/HP is polyvalent in nature and has high-lytic potential towards S. aureus, thus, a therapy employing this phage would be efficacious to control S. aureus infections.     

Strain Specific Phage Treatment for Staphylococcus aureus Infection Is Influenced by Host Immunity and Site of Infection

The response to multi-drug resistant bacterial infections must be a global priority. While mounting resistance threatens to create what the World Health Organization has termed a “post-antibiotic era”, the recent discovery that antibiotic use may adversely impact the microbiome adds further urgency to the need for new developmental approaches for anti-pathogen treatments. Methicillin-resistant Staphylococcus aureus (MRSA), in particular, has declared itself a serious threat within the United States and abroad. A potential solution to the problem of antibiotic resistance may not entail looking to the future for completely novel treatments, but instead looking into our history of bacteriophage therapy. This study aimed to test the efficacy, safety, and commercial viability of the use of phages to treat Staphylococcus aureus infections using the commercially available phage SATA-8505. We found that SATA-8505 effectively controls S. aureus growth and reduces bacterial viability both in vitro and in a skin infection mouse model. However, this killing effect was not observed when phage was cultured in the presence of human whole blood. SATA-8505 did not induce inflammatory responses in peripheral blood mononuclear cultures. However, phage did induce IFN gamma production in primary human keratinocyte cultures and induced inflammatory responses in our mouse models, particularly in a mouse model of chronic granulomatous disease. Our findings support the potential efficacy of phage therapy, although regulatory and market factors may limit its wider investigation and use.     

肺炎克雷伯菌噬菌体解聚酶的研究进展

肺炎克雷伯菌(Klebsiella pneumoniae)是在临床引起多种感染的常见条件致病菌之一。多重耐药肺炎克雷伯菌株的出现,给防控细菌感染带来了巨大阻力。肺炎克雷伯菌噬菌体编码的解聚酶是一种稳定性高、特异性强的生物酶,具有分解细菌胞外多糖、限制细菌生长等多种功能。解聚酶可为防控肺炎克雷伯菌感染提供新思路,在抗菌应用中具有广阔前景。本文就肺炎克雷伯菌噬菌体解聚酶的研究进展进行综述。     

Synonymous codon usage in forty staphylococcal phages identifies the factors controlling codon usage variation and the phages suitable for phage therapy

The immergence and dissemination of multidrug-resistant strains of Staphylococcus aureus in recent years have expedited the research on the discovery of novel anti-staphylococcal agents promptly. Bacteriophages have long been showing tremendous potentialities in curing the infections caused by various pathogenic bacteria including S. aureus. Thus far, only a few virulent bacteriophages, which do not carry any toxin-encoding gene but are capable of eradicating staphylococcal infections, were reported. Based on the codon usage analysis of sixteen S. aureus phages, previously three phages were suggested to be useful as the anti-staphylococcal agents. To search for additional S. aureus phages suitable for phage therapy, relative synonymous codon usage bias has been investigated in the protein-coding genes of forty new staphylococcal phages. All phages appeared to carry A and T ending codons. Several factors such as mutational pressure, translational selection and gene length seemed to be responsible for the codon usage variation in the phages. Codon usage indeed varied phage to phage. Of the phages, phages G1, Twort, 66 and Sap-2 may be extremely lytic in nature as majority of their genes possess high translational efficiency, indicating that these phages may be employed in curing staphylococcal infections.     

On the challenges of detecting whole Staphylococcus aureus cells with biosensors

Due to the increasing number of nosocomial infections and multidrug‐resistant bacterial strains, Staphylococcus aureus is now a major worldwide concern. Rapid detection and characterization of this bacterium has become an important issue for biomedical applications. Biosensors are increasingly appearing as low‐cost, easy‐to‐operate and fast alternatives for rapid detection. In this review, we will introduce the main characteristics of S. aureus and will focus on the interest of biosensors for a faster detection of whole S. aureus cells. In particular, we will review the most promising strategies in the choice of ligand for the design of selective and efficient biosensors. Their specific characteristics as well as their advantages and/or disadvantages will also be commented.     

Characterization of the staphylococcal bacteriophage lysin CHAP(K)

Aims: To develop an efficient purification strategy for the bacteriophage lysin CHAP_K. To evaluate its antibacterial spectrum_, enzymatic properties, optimal reaction conditions and lytic activity against live Staphlyococcus aureus. Methods and Results: Recombinant CHAP_K was purified to homogeneity by cation exchange chromatography, with yields of up to 10 mg from 1 l of Escherichia coli culture. The lytic spectrum of CHAP_K includes all staphylococcal species and also members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Carnobacterium. The enzyme was active from pH 6 to 11 with an optimum activity at pH 9, from 5 to 40°C, with an optimum activity at 15°C. When cell lysis by CHAP_K and lysostaphin was compared over a concentration range of 2·5–10 μg ml^?1 using live Staph. aureus for 5 min at 37°C, CHAP_K gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin. Conclusions: This study describes in detail the purification and characteristics of the novel phage‐derived enzyme CHAP_K demonstrating that it has excellent biochemical properties as an anti‐staphylococcal agent. Significance and Impact of the Study: Currently, there is a need for new antimicrobial agents due to the increasing worldwide prevalence of antibiotic resistance. Our findings demonstrate the potential for development of CHAP_K as an alternative therapeutic against pathogenic staphylococci including MRSA.