Cross-Genus "Boot-Up" of Synthetic Bacteriophage in Staphylococcus aureus by Using a New and Efficient DNA Transformation Method.

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but these only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but new methods are needed for the efficient transformation of large DNA molecules into S. aureus to "boot-up" (i.e., rescue) infectious phages. We presented a new, efficient, and reproducible DNA transformation method, NEST (non-electroporation Staphylococcus transformation), for S. aureus to boot-up purified phage genomic DNA (at least 150 kb in length) and whole yeast-assembled synthetic phage genomes. This method was a powerful new tool for the transformation of DNA in S. aureus and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. IMPORTANCE The continued emergence of antibiotic-resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional nonmodel Siphophage from a constructed synthetic genome. This method will facilitate the engineering of Staphylococcus and Enterococcus phages for therapeutic applications and the engineering of Staphylococcus strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications.

Immune Response to Therapeutic Staphylococcal Bacteriophages in Mammals: Kinetics of Induction, Immunogenic Structural Proteins, Natural and Induced Antibodies.

Bacteriophages are able to affect the human immune system. Phage-specific antibodies are considered as major factors shaping phage pharmacokinetics and bioavailability. So far, general knowledge of phage antigenicity nevertheless remains extremely limited. Here we present comparative studies of immunogenicity in two therapeutic bacteriophages, A3R and 676Z, active against Staphylococcus aureus, routinely applied in patients at the Phage Therapy Unit, Poland. Comparison of the overall ability of whole phages to induce specific antibodies in a murine model revealed typical kinetics of IgM and IgG induction by these two phages. In further studies we identified the location of four phage proteins in the virions, with the focus on the external capsid head (Mcp) or tail sheath (TmpH) or an unidentified precise location (ORF059 and ORF096), and we confirmed their role as structural proteins of these viruses. Next, we compared the immune response elicited by these proteins after phage administration in mice. Similar to that in T4 phage, Mcp was the major element of the capsid that induced specific antibodies. Studies of protein-specific sera revealed that antibodies specific to ORF096 were able to neutralize antibacterial activity of the phages. In humans (population level), none of the studied proteins plays a particular role in the induction of specific antibodies; thus none potentially affects in a particular way the effectiveness of A3R and 676Z. Also in patients subjected to phage therapy, we did not observe increased specific immune responses to the investigated proteins.

The Indirect Impact of the SARS-CoV-2 Pandemic on Invasive Group a Streptococcus, Streptococcus Pneumoniae and Staphylococcus Aureus Infections in Houston Area Children.

Masking and social distancing have been adopted to mitigate the severe acute respiratory syndrome coronavirus 2 pandemic. We evaluated the indirect impact of severe acute respiratory syndrome coronavirus 2 prevention strategies on invasive Staphylococcus aureus, Streptococcus pneumoniae (pneumococcus) and Group A Streptococcus in Houston area children. We observed a decline in invasive pneumococcal disease and invasive Group A Streptococcus temporally associated with social distancing/masking/school closures.

Overview of the risks of Staphylococcus aureus infections and their control by bacteriophages and bacteriophage-encoded products.

Staphylococcus aureus is the leading cause of secondary infections in hospitals and a challenging pathogen in food industries. Decades after it was first reported, ß-lactam-resistant S. aureus remains a subject of intense research owing to the ever-increasing issue of drug resistance. S. aureus bacteriophages (phages) or their encoded products are considered an alternative to antibiotics as they have been shown to be effective in treating some S. aureus-associated infections. In this review, we present a concise collection of the literature on the pathogenic potential of S. aureus and examine the prospects of using S. aureus phages and their encoded products as antimicrobials.

Genomic epidemiology of methicillin-resistant and -susceptible Staphylococcus aureus from bloodstream infections.

BACKGROUND: Bloodstream infections due to Staphylococcus aureus cause significant patient morbidity and mortality worldwide. Of major concern is the emergence and spread of methicillin-resistant S. aureus (MRSA) in bloodstream infections, which are associated with therapeutic failure and increased mortality. METHODS: We generated high quality draft genomes from 323 S. aureus blood culture isolates from patients diagnosed with bloodstream infection at the Dartmouth-Hitchcock Medical Center, New Hampshire, USA in 2010-2018. RESULTS: In silico detection of antimicrobial resistance genes revealed that 133/323 isolates (41.18%) carry horizontally acquired genes conferring resistance to at least three antimicrobial classes, with resistance determinants for aminoglycosides, beta-lactams and macrolides being the most prevalent. The most common resistance genes were blaZ and mecA, which were found in 262/323 (81.11%) and 104/323 (32.20%) isolates, respectively. Majority of the MRSA (102/105 isolates or 97.14%) identified using in vitro screening were related to two clonal complexes (CC) 5 and 8. The two CCs emerged in the New Hampshire population at separate times. We estimated that the time to the most recent common ancestor of CC5 was 1973 (95% highest posterior density (HPD) intervals: 1966-1979) and 1946 for CC8 (95% HPD intervals: 1924-1959). The effective population size of CC8 increased until the late 1960s when it started to level off until late 2000s. The levelling off of CC8 in 1968 coincided with the acquisition of SCCmec Type IV in majority of the strains. The plateau in CC8 also coincided with the acceleration in the population growth of CC5 carrying SCCmec Type II in the early 1970s, which eventually leveled off in the early 1990s. Lastly, we found evidence for frequent recombination in the two clones during their recent clonal expansion, which has likely contributed to their success in the population. CONCLUSIONS: We conclude that the S. aureus population was shaped mainly by the clonal expansion, recombination and co-dominance of two major MRSA clones in the last five decades in New Hampshire, USA. These results have important implications on the development of effective and robust strategies for intervention, control and treatment of life-threatening bloodstream infections.

Evaluating the potential efficacy and limitations of a phage for joint antibiotic and phage therapy of Staphylococcus aureus infections.

In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYOSa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYOSa Because of the mode of action of PYOSa, S. aureus is unlikely to generate classical receptor-site mutants resistant to PYOSa; none were observed in the 13 clinical isolates tested. PYOSa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYOSa and antibiotics raise issues that must be addressed before PYOSa is employed clinically. Despite the maintenance of the phage, PYOSa does not clear populations of S. aureus Due to the ascent of a phenotyically diverse array of small-colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise-resurrection dynamics of PYOSa and S. aureus Critically for phage therapy, our experimental results suggest that treatment with PYOSa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.

Dysregulated Host Responses Underlie 2009 Pandemic Influenza-Methicillin Resistant Staphylococcus aureus Coinfection Pathogenesis at the Alveolar-Capillary Barrier.

Influenza viruses are a continual public health concern resulting in 3-5 million severe infections annually despite intense vaccination campaigns and messaging. Secondary bacterial infections, including Staphylococcus aureus, result in increased morbidity and mortality during seasonal epidemics and pandemics. While coinfections can result in deleterious pathologic consequences, including alveolar-capillary barrier disruption, the underlying mechanisms are poorly understood. We have characterized host- and pathogen-centric mechanisms contributing to influenza-bacterial coinfections in a primary cell coculture model of the alveolar-capillary barrier. Using 2009 pandemic influenza (pH1N1) and methicillin-resistant S. aureus (MRSA), we demonstrate that coinfection resulted in dysregulated barrier function. Preinfection with pH1N1 resulted in modulation of adhesion- and invasion-associated MRSA virulence factors during lag phase bacterial replication. Host response modulation in coinfected alveolar epithelial cells were primarily related to TLR- and inflammatory response-mediated cell signaling events. While less extensive in cocultured endothelial cells, coinfection resulted in changes to cellular stress response- and TLR-related signaling events. Analysis of cytokine expression suggested that cytokine secretion might play an important role in coinfection pathogenesis. Taken together, we demonstrate that coinfection pathogenesis is related to complex host- and pathogen-mediated events impacting both epithelial and endothelial cell regulation at the alveolar-capillary barrier.

Contribution of Podoviridae and Myoviridae bacteriophages to the effectiveness of anti-staphylococcal therapeutic cocktails.

Bacteriophage therapy is considered one of the most promising therapeutic approaches against multi-drug resistant bacterial infections. Infections caused by Staphylococcus aureus are very efficiently controlled with therapeutic bacteriophage cocktails, containing a number of individual phages infecting a majority of known pathogenic S. aureus strains. We assessed the contribution of individual bacteriophages comprising a therapeutic bacteriophage cocktail against S. aureus in order to optimize its composition. Two lytic bacteriophages vB_SauM-515A1 (Myoviridae) and vB_SauP-436A (Podoviridae) were isolated from the commercial therapeutic cocktail produced by Microgen (Russia). Host ranges of the phages were established on the panel of 75 S. aureus strains. Phage vB_SauM-515A1 lysed 85.3% and vB_SauP-436A lysed 68.0% of the strains, however, vB_SauP-436A was active against four strains resistant to vB_SauM-515A1, as well as to the therapeutic cocktail per se. Suboptimal results of the therapeutic cocktail application were due to extremely low vB_SauP-436A1 content in this composition. Optimization of the phage titers led to an increase in overall cocktail efficiency. Thus, one of the effective ways to optimize the phage cocktails design was demonstrated and realized by using bacteriophages of different families and lytic spectra.

Phage-Mediated Molecular Detection (PMMD): A Novel Rapid Method for Phage-Specific Bacterial Detection.

Bacterial infections pose a challenge to human health and burden the health care system, especially with the spread of antibiotic-resistant populations. To provide effective treatment and improved prognosis, effective diagnostic methods are of great importance. Here we present phage-mediated molecular detection (PMMD) as a novel molecular method for the detection and assessment of bacterial antibiotic resistance. This technique consists of a brief incubation, of approximately ten minutes, of the biological sample with a natural bacteriophage (phage) targeting the bacteria of interest. This is followed by total RNA extraction and RT-PCR. We applied this approach to Staphylococcus aureus (SA), a major causative agent of human bacterial infections. PMMD demonstrated a high sensitivity, rapid implementation, and specificity dependent on the phage host range. Moreover, due to the dependence of the signal on the physiological state of the bacteria, PMMD can discriminate methicillin-sensitive from methicillin-resistant SA (MSSA vs. MRSA). Finally, we extended this method to the detection and antibiotic sensitivity determination of other bacteria by proving PMMD efficacy for Bacillusanthracis.

Therapeutic potential of bacteriophages for staphylococcal infections and selected methods for in vitro susceptibility testing of staphylococci.

AIM: Staphylococcus aureus strains are the cause of frightening hospital and community infections, especially when they are resistant to antimicrobials, have important pathogenicity factors, or have biofilm production ability. Looking for novel therapeutic options which would be effective against such strains is one of the highest priorities of medicine and medical research. The study aim was to describe the occurrence of S. aureus strains and proportion of methicillin resistant strains (MRSA) detected in laboratories of the Microbiological Institute, Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno in 2011-2018. Selected strains of S. aureus were tested for biofilm production ability and susceptibility to antimicrobials and Stafal®, a phage therapeutic agent. A prerequisite was to develop a simple routine method suitable for phage susceptibility testing of bacteria. MATERIAL AND METHODS: Altogether 867 clinical isolates of S. aureus and 132 strains of other species of the genus Staphylococcus (isolated in 2011-2017) were tested for susceptibility to the phage therapy preparation Stafal® using the double-layer agar method. All strains of S. aureus were tested for biofilm production ability by the modified Christensen method with the use of titration microplates and for susceptibility to antistaphylococcal antibiotics by the disk diffusion test. For 95 S. aureus strains, the outcome of the double-layer agar method (DAM) was compared with that of our newly designed method (ODM) based on optical density decrease of the bacterial suspension. RESULTS: During the study period, the laboratories of the Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno detected 2900 strains of S. aureus per year on average. The proportion of MRSA among S. aureus isolates from blood culture and venous catheters ranged between 8.8-15.2 %. S. aureus strains recovered from venous catheters and blood culture were confirmed as stronger biofilm producers than those from other clinical specimens. MRSA strains showed higher biofilm production than methicillin susceptible strains (MSSA). As many as 90.4 % of S. aureus strains tested susceptible to the Stafal® preparation. Even a higher proportion, i.e. 99.0 %, of MRSA strains were Stafal® susceptible. No relationship was found between Stafal® susceptibility and biofilm production ability. Although Stafal® targets primarily S. aureus, some susceptibility (26.5 %) was also found for other staphylococcal species. A novel simple method designed for routine testing of susceptibility to phage therapy preparations based on optical density decrease was comparably sensitive and reliable as the commonly used double-layer agar method (DAM) and, in addition to being easy and rapid to perform, after prolonged suspension culture and at higher measurement frequency, it has an extra advantage of providing the possibility for monitoring also phage action dynamics. CONCLUSIONS: The proportion of MRSA strains detected in this study is comparable to that reported for the whole Czech Republic, and the biofilm production data are consistent with scientific evidence. The host range of the Stafal® preparation is relatively wide and covers most strains of S. aureus and some coagulase negative staphylococci. The highest efficiency of Stafal® (99.4 %) was observed against MRSA strains with multiple types of antibiotic resistance. In vitro testing of 867 strains of S. aureus and 132 other staphylococcal species has shown the phage therapy preparation Stafal® to be a suitable candidate therapeutic option for the treatment of staphylococcal infections, especially in case of failure of conventional antibiotic therapy. Moreover, a simple method for routine phage susceptibility testing of clinical bacterial isolates has been designed, which is an essential tool to be used in phage therapy.

Temperate Phages of Staphylococcus aureus.

Most Staphylococcus aureus isolates carry multiple bacteriophages in their genome, which provide the pathogen with traits important for niche adaptation. Such temperate S. aureus phages often encode a variety of accessory factors that influence virulence, immune evasion and host preference of the bacterial lysogen. Moreover, transducing phages are primary vehicles for horizontal gene transfer. Wall teichoic acid (WTA) acts as a common phage receptor for staphylococcal phages and structural variations of WTA govern phage-host specificity thereby shaping gene transfer across clonal lineages and even species. Thus, bacteriophages are central for the success of S. aureus as a human pathogen.

Whole genome sequencing revealed new molecular characteristics in multidrug resistant staphylococci recovered from high frequency touched surfaces in London.

The rise of antibiotic resistance (AMR) is one of the most important public health threats worldwide.Today, increasing attention is being paid to multidrug resistant staphylococci isolated from healthcare and non-healthcare environments as the treatment of these bacteria has become increasingly difficult. In this study, we compared staphylococci isolates recovered from high frequency touched surfaces from public areas in the community and hospitals in East and West London. 281 out of 600 (46.83%) staphylococci isolates recovered were multidrug resistant, of which 49 (8.17%) were mecA positive. There was significantly higher proportion of multidrug resistant staphylococci (P = 0.0002) in East London (56.7%) compared to West London (49.96%). The most common species identified as multidrug resistant were S. epidermidis, S. haemolyticus and S. hominis, whereas penicillin, fusidic acid and erythromycin were the most frequent antibiotics the isolates were resistant to. Whole genome sequenced of mecA positive isolates revealed that S. sciuri isolates carried the mecA1 gene, which has only 84.43% homology with mecA. In addition, other frequently identified resistance genes included blaZ, qacA/B and dfrC. We have also identified a diverse range of SCCmec types, many of which were untypable due to carrying a novel combination of ccr genes or multiple ccr complexes.

Direct interactions with influenza promote bacterial adherence during respiratory infections.

Epidemiological observations and animal models have long shown synergy between influenza virus infections and bacterial infections. Influenza virus infection leads to an increase in both the susceptibility to secondary bacterial infections and the severity of the bacterial infections, primarily pneumonias caused by Streptococcus pneumoniae or Staphylococcus aureus. We show that, in addition to the widely described immune modulation and tissue-remodelling mechanisms of bacterial-viral synergy, the virus interacts directly with the bacterial surface. Similar to the recent observation of direct interactions between enteric bacteria and enteric viruses, we observed a direct interaction between influenza virus on the surface of Gram-positive, S. pneumoniae and S. aureus, and Gram-negative, Moraxella catarrhalis and non-typeable Haemophilus influenzae, bacterial colonizers and pathogens in the respiratory tract. Pre-incubation of influenza virus with bacteria, followed by the removal of unbound virus, increased bacterial adherence to respiratory epithelial cells in culture. This result was recapitulated in vivo, with higher bacterial burdens in murine tissues when infected with pneumococci pre-incubated with influenza virus versus control bacteria without virus. These observations support an additional mechanism of bacteria-influenza virus synergy at the earliest steps of pathogenesis.

Association between the accessory gene regulator (agr) locus and the presence of superantigen genes in clinical isolates of methicillin-resistant Staphylococcus aureus.

OBJECTIVE: Methicillin-resistant Staphylococcus aureus cause to a variety of hard to cure infections. MRSA isolates also, produce an arsenal of virulence factors contribute to severe infections. The aim of this study was to find out the relationship between agr locus and presence of S. aureus superantigens (SAgs). RESULTS: Clinical isolates in two groups from two different states of Iran were collected. Antibiotic resistance patterns, agr typing, and virulence factor genes prevalence were identified and relationship between them was analyzed using SPSS software version16. Most of the samples were collected from wound 39 isolates in Group 1 and 61 isolates in Group 2. Frequency of MRSA strains was 38.1% in Group 1 and 52.1% in Group 2. Also, the most common resistance among both groups was to penicillin. agr positive isolates were detected in 132 isolates of Group 1 and 104 isolates of Group 2. In Conclusion, a significant relationship between the SAgs frequency and agr locus in both groups has been indicated. The production of superantigens in S. aureus plays an important role in the classification of agr locus, and this locus can affect differently in methicillin-resistant strains.

Characterization of Host and Bacterial Contributions to Lung Barrier Dysfunction Following Co-infection with 2009 Pandemic Influenza and Methicillin Resistant Staphylococcus aureus.

Influenza viruses are a threat to global public health resulting in ~500,000 deaths each year. Despite an intensive vaccination program, influenza infections remain a recurrent, yet unsolved public health problem. Secondary bacterial infections frequently complicate influenza infections during seasonal outbreaks and pandemics, resulting in increased morbidity and mortality. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is frequently associated with these co-infections, including the 2009 influenza pandemic. Damage to alveolar epithelium is a major contributor to severe influenza-bacterial co-infections and can result in gas exchange abnormalities, fluid leakage, and respiratory insufficiency. These deleterious manifestations likely involve both pathogen- and host-mediated mechanisms. However, there is a paucity of information regarding the mechanisms (pathogen- and/or host-mediated) underlying influenza-bacterial co-infection pathogenesis. To address this, we characterized the contributions of viral-, bacterial-, and host-mediated factors to the altered structure and function of alveolar epithelial cells during co-infection with a focus on the 2009 pandemic influenza (pdm2009) and MRSA. Here, we characterized pdm2009 and MRSA replication kinetics, temporal host kinome responses, modulation of MRSA virulence factors, and disruption of alveolar barrier integrity in response to pdm2009-MRSA co-infection. Our results suggest that alveolar barrier disruption during co-infection is mediated primarily through host response dysregulation, resulting in loss of alveolar barrier integrity.

Use of a Silkworm Larva Model in Phage Therapy Experiments.

Antibiotic-resistant bacteria can cause intractable infections in humans and animals, with damaging effects to health care and economics. Phage therapy is considered a possible alternative to chemotherapy for treating infections, but still requires laborious in vivo experiments before its introduction into society and its further development. Recently, silkworm larvae have been recognized as highly convenient and useful model animals, and an alternative to higher animals. We describe the procedure for experimental phage therapy to treat Staphylococcus aureus infections in silkworm larvae.

Slime-producing staphylococci as causal agents of subclinical mastitis in sheep.

Hitherto, research work in slime production from staphylococcal strains of mastitis origin has focused in laboratory properties of these organisms. Objective of present work was to study subclinical mastitis in sheep, caused specifically by slime-producing staphylococci: to investigate its frequency and to identify potential factors playing a role therein. Slime production was evaluated in 708 staphylococcal isolates recovered from cases of subclinical mastitis in a field study in 2198 ewes performed in an extensive countrywide field investigation across Greece. Isolates were studied by means of microbiological and molecular methods. Of these strains, 262 were characterised as slime-producing, 227 as weak slime-producing and 219 as non slime-producing. Most frequently detected genes were eno and icaB; Staphylococcus aureus possessed more genes than coagulase-negative strains; greater number of genes was detected in slime-producing than in weak slime-producing or non-slime-producing strains. Subclinical mastitis caused specifically by slime-producing staphylococci was detected in 337 ewes: prevalence in population sampled was 0.153. A multivariable mixed-effects model revealed that milking mode (highest prevalence in hand-milked flocks) and flock management system (highest prevalence in semi-intensive flocks) were the two factors associated with increased prevalence of mastitis in flocks. The results confirmed the significance of slime producing staphylococcal strains of importance in the aetiology of subclinical mastitis of sheep. Hand-milking was identified as the most important factor predisposing to that infection.

Molecular prophage typing of Staphylococcus aureus isolates from bovine mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.

In Vitro Models to Study Influenza Virus and Staphylococcus aureus Super-Infection on a Molecular Level.

Investigation of pathogen-host interactions on a molecular level requires sophisticated in vitro infection procedures, especially in the presence of different pathogens.Super-infections of influenza viruses (IV) and bacteria, with increasing incidence of Staphylococcus aureus (S. aureus) cases, are a long-known phenomenon and represent a major complication in IV-infected patients. Although several in vivo studies have improved our knowledge about pathogenesis and immune responses of super-infections that result in increased morbidity and mortality, the consequences of the direct interplay of viruses and bacteria on a molecular level in affected cells that may contribute to the deadly synergism of these pathogens are so far poorly characterized. Here we describe different infection schemes to study IV and S. aureus coinfections of distinct cell populations in vitro. Depending on the focus of interest, regulation of cell responses such as signalling mechanisms or pro- and anti-inflammatory cytokine expression, or consequences for the viral or bacterial life cycle, can be analyzed. The described infection procedures could be used as guidelines and adapted to super-infection settings of other viral and bacterial pathogens.