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.     

Recipient capacity of clinical strains of staphylococci belonging to different phage groups]

The recipient capacity of the strains of Staph. epidermidis and Staph. areus belonging to different phage groups, as well as the possibility of epidemic distribution of the erythromycin resistance marker among the clinical staphyloccal strains on using the defective phage obtained from strain 8325 P IIde was studied. The defective phage P IIde may be the source of epidemic distribution of the drug resistance among the competent strains of Staph. aureus. All erythromycin sensitive strains of Staph. aureus lysed by the phages of groups I and III proved to be competent recipients of the erythromycin resistance marker. The strains of Staph. aureus of phage group II and phage type 80/81, as well as the strains of Staph. epidermidis were not competent recipients under our experimental conditions. It was not possible to transfer the high level of erythromycin resistance (1000 gamma/ml) on transduction to the strains of phage group I with a relatively low level of resistance to this antibiotic (20-50 gamma/ml.     

Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus

Bacteriophage-resistant strains of Staphylococcus aureus H were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Cell walls isolated from about half of these resistant strains were incapable of inactivating phages and were shown to lack N-acetyl-d-glucosamine (GlcNAc) in their cell wall teichoic acid. Apart from the lack of GlcNAc, two of these mutant strains were deficient in cell wall phosphorus and ester-linked d-alanine. These two strains were also found to be resistant to both phage K and a host-range mutant isolated from the parent phage. These two phages could lyse the other phage-resistant mutants which lacked GlcNAc in their teichoic acid. Cell walls from the remaining phage-resistant mutant strains did inactivate phages and were found to have normal cell wall teichoic acid. Although GlcNAc in teichoic acid was required for phage inactivation, no difference in phage inactivation ability was detected with cell walls isolated from strains of S. aureus having exclusively alpha- or exclusively beta-linked GlcNAc in their cell wall teichoic acid.     

Mupirocin resistance in staphylococci: development and transfer of isoleucyl-tRNA synthetase-mediated resistance in vitro

Mupirocin resistance could be transferred from highly resistant clinical isolates of Staphylococcus aureus to highly sensitive recipients of Staph. aureus, Staph. epidermidis and Staph. haemolyticus. Transconjugants of the latter two organisms could transfer this resistance into mupirocin-sensitive Staph. aureus. Moderately resistant strains did not transfer this resistance to sensitive recipients, nor did strains with high-level mupirocin resistance developed by serial transfer or habituation. The inhibitory effects of mupirocin on crude isoleucyl-tRNA synthetases (IRS) isolated from mupirocin-sensitive and -resistant strains of Staph. aureus have been determined. Drug concentrations needed to produce 50% inhibition, I50 values, were very low against IRS from a highly sensitive strain, somewhat higher against IRS from moderately resistant strains, much higher against enzyme from strains trained in vitro to high-level resistance, and considerably higher still against IRS extracted from clinical isolates possessing high-level mupirocin resistance and from the transconjugates of such strains resulting from crosses with mupirocin-sensitive strains. It is concluded that high-level resistance in clinical isolates is plasmid-mediated involving a second, mupirocin-resistant IRS whereas in moderately resistant strains, and in strains trained in vitro to high-level resistance, chromosomal mutations are likely to be responsible for decreasing IRS sensitivity.     

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.     

Interactive Growth of Staphylococcus aureus Strains with a Poultry Skin Microflora in a Diffusion Apparatus

The growth of different biotypes of Staphylococcus aureus strains isolated from poultry was studied using the NBS Ecologen in which the interacting mixed culture was derived from the microfiora of hen skin and separated from the Staph. aureus culture by a membrane of 0.4 μm pore size. Inhibition of growth of the Staph. aureus cultures occurred with strains from each biotype. Marked inhibition of growth was always accompanied by the production of large numbers (>10⁹/ml) of plaque forming units (phage). In the mixed culture chamber poultry phage group C strains became the predominant Staph. aureus type. The phages produced by the mixed culture showed a wide spectrum of lytic activity for the propagating strains of the human, bovine and poultry phage sets.     

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph. aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50 degrees C there was no marked variation in coagulase production by the surviving strains but at 55 and 62.5 degrees C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62.5 degrees C resulted in loss of enterotoxin production by all the survivors except S6 and 234, the surviving cells of which still produced enterotoxin B after heat treatment at 55 degrees C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.     

Microbiological and molecular assessment of bacteriophage ISP for the control of Staphylococcus aureus

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the 'Twort-like viruses'. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes.     

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph, aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50°C there was no marked variation in coagulase production by the surviving strains but at 55 and 62–5dE C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62mD5dE C resulted in loss of enterotoxin production by all the survivors except S₆ and 234, the surviving cells of which still prodused enterotoxin B after heat treatment at 55dE C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.     

Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K

AIMS: To evaluate differences in biofilm or planktonic bacteria susceptibility to be killed by the polyvalent antistaphylococcus bacteriophage K. METHODS AND RESULTS: In this study, the ability of phage K to infect and kill several clinical isolates of Staphylococcus epidermidis was tested. Strains were grown in suspension or as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Most strains (10/11) were susceptible to phage K, and phage K was also effective in reducing biofilm biomass after 24 h of challenging. Biofilm cells were killed at a lower rate than the log-phase planktonic bacteria but at similar rate as stationary phase planktonic bacteria. CONCLUSIONS: Staphylococcus epidermidis biofilms and stationary growth phase planktonic bacteria are more resistant to phage K lysis than the exponential phase planktonic bacteria. SIGNIFICANCE OF STUDY: This study shows the differences in Staph. epidermidis susceptibility to be killed by bacteriophage K, when grown in biofilm or planktonic phenotypes.     

Plasmids in Staphylococcus hyicus

Fifty-one strains of Staphylococcus hyicus and six of Staph, chromogenes were collected from porcine and bovine sources in England and Belgium. Antibiotic resistance and plasmid profiles were established. Plasmids associated with resistance to tetracycline, erythromycin and streptomycin were identified in many strains; these plasmids were about the same size as those mediating similar resistances in Staph. aureus of human origin. Many small cryptic plasmids were also seen.     

Plasmids in Staphylococcus hyicus

Fifty-one strains of Staphylococcus hyicus and six of Staph. chromogenes were collected from porcine and bovine sources in England and Belgium. Antibiotic resistance and plasmid profiles were established. Plasmids associated with resistance to tetracycline, erythromycin and streptomycin were identified in many strains; these plasmids were about the same size as those mediating similar resistances in Staph. aureus of human origin. Many small cryptic plasmids were also seen.     

The Molecular and Genetic Basis of Repeatable Coevolution between Escherichia coli and Bacteriophage T3 in a Laboratory Microcosm

The objective of this study was to determine the genomic changes that underlie coevolution between Escherichia coli B and bacteriophage T3 when grown together in a laboratory microcosm. We also sought to evaluate the repeatability of their evolution by studying replicate coevolution experiments inoculated with the same ancestral strains. We performed the coevolution experiments by growing Escherichia coli B and the lytic bacteriophage T3 in seven parallel continuous culture devices (chemostats) for 30 days. In each of the chemostats, we observed three rounds of coevolution. First, bacteria evolved resistance to infection by the ancestral phage. Then, a new phage type evolved that was capable of infecting the resistant bacteria as well as the sensitive bacterial ancestor. Finally, we observed second-order resistant bacteria evolve that were resistant to infection by both phage types. To identify the genetic changes underlying coevolution, we isolated first- and second-order resistant bacteria as well as a host-range mutant phage from each chemostat and sequenced their genomes. We found that first-order resistant bacteria consistently evolved resistance to phage via mutations in the gene, waaG, which codes for a glucosyltransferase required for assembly of the bacterial lipopolysaccharide (LPS). Phage also showed repeatable evolution, with each chemostat producing host-range mutant phage with mutations in the phage tail fiber gene T3p48 which binds to the bacterial LPS during adsorption. Two second-order resistant bacteria evolved via mutations in different genes involved in the phage interaction. Although a wide range of mutations occurred in the bacterial waaG gene, mutations in the phage tail fiber were restricted to a single codon, and several phage showed convergent evolution at the nucleotide level. These results are consistent with previous studies in other systems that have documented repeatable evolution in bacteria at the level of pathways or genes and repeatable evolution in viruses at the nucleotide level. Our data are also consistent with the expectation that adaptation via loss-of-function mutations is less constrained than adaptation via gain-of-function mutations.     

An investigation of the thermal inactivation of Staphylococcus aureus and the potential for increased thermotolerance as a result of chilled storage

AIMS: The aims of this study were; (i) to provide thermal inactivation data for Staphylococcus aureus; (ii) to examine the kinetics, including decimal reduction times (D-value) and rate constants (k), that describe the thermal inactivation of Staph. aureus and to compare two different methods of calculating D-values and (iii) to determine whether or not chilled storage would toughen these microorganisms resulting in increased thermotolerance. METHODS AND RESULTS: Isolates of Staph. aureus recovered from domestic refrigerators were grown in shaken culture for 8 h at 37 degrees C, recovered and washed by centrifugation and combined to form a cocktail of five strains. Samples from this cocktail were (a) heat treated at 50, 55 and 60 degrees C or (b) held under simulated domestic refrigeration conditions for 72 h and then heat treated as above. The numbers of Staph. aureus in heat treated and chill held, heat treated samples were enumerated by direct selective plating onto Baird Parker Agar (BPA) and recovery plating on Tryptone Soya Agar (TSA) subsequently overlaid with BPA. D-values were obtained using two different methods both of which may be used when the thermal inactivation follows first order kinetics. In the first method D-values are obtained by plotting the Log(10) of the surviving cells against time and using the equation D = -1/slope. The second method uses the rate constant (k) which is obtained from the slope of a plot of ln N/N(0)vs time and D is obtained using the equation D = 2.303 k(-1). D(50), D(55) and D(60) values ranged from 94.3 to 127.9 min, 13 to 21.7 min and 4.8 to 6.5 min. Prechilling did not enhance thermal resistance. The method of calculation did not affect the D-values obtained because the thermal inactivation of Staph. aureus in this study followed first order kinetics with r(2) values of 0.91-0.99. CONCLUSIONS: The thermal inactivation of Staph. aureus in tryptone soya broth (TSB) follows first order kinetics and in general chilling of these bacteria does not increase the resistance to thermal destruction during subsequent thermal processes such as cooking. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides much needed data on the thermal resistance of Staph. aureus and validates chilling as a food storage activity which does not cause toughening of the microorganisms to subsequent cooking. However, the data generated strongly suggests that Staph. aureus is more thermotolerant than Listeria monocytogenes and should be used as the target microorganism in designing mild thermal treatments for food, in which case the current recommendations for pasteurization (70 degrees C for 2 min, minimum) should be revised.     

Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B.

The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.     

Comparative antibiotic sensitivity of different species of staphylococci isolated from humans]

A total of 206 strains of various staphylococcal species isolated from various sources were studied with respect to their sensitivity to 18 antibiotics. The number of strains poly-resistant to the antibiotics was almost the same among Staph. aureus and Staph. epidermidis, i. e. 54.8 and 51.3 per cent respectively. The coagulase-negative and mannitol-negative variants of Staph. aureus and Staph. epidermidis possessing high biological activity (10-14 properties) were resistant to more antibiotics as compared to the low active strains.     

Periodicity in the rise and fall of the numbers of the principal Staphylococcus aureus phage groups

The results of the phage typing of 5, 168 Staph. aureus strains isolated in a surgical hospital between 1959 and 1977 are analyzed for each year of this period. The wave of increase in the number of staphylococci belonging to phage group II which began, as discovered in this study, in 1965 and still showed no tendency towards decrease in 1977, as well as the periodicity of rises and falls in the number of staphylococci belonging to phage groups I and III are discussed and compared with the data contained in the literature. The authors come to the conclusion that Staph. aureus is subject to wave-like rises and falls in the number of strains belonging to the main phage groups of the species, and among them the strains belonging to phage groups I and III seem to be inversely related in respect of rises and falls in number, such changes occurring periodically at an interval of 10-12 years, while in the strains belonging to phage group II changes in number occur at a slower rate. The constant account of the percentage of phage groups I, II and III is recommended to ensure rational antibiotic therapy.     

Effect of detergents on the chloramphenicol inactivation process by resistent bacteria

The effect of detergents, i. e. cationic, anionic, nonionic and polyelectrolytes of the cationic type on the efficacy of chloramphenicol against resistant strains of E. coli and Staph. aureus was studied. It was found that the detergent effect on inactivation of chloramphenicol by the bacterial resistant strains was inconsistent. The cationic detergents and in particular chlorhexidine had the most pronounced inhibitory effect. In subbacteriostatic concentrations they significantly suppressed inactivation of chloramphenicol in the cells of E. coli and Staph. aureus. The anionic detergents and polyelectrolytes of the cationic type in the above concentrations were effective only with respect to Staph. aureus. It is noted that the detergents increased the activity of chloramphenicol against E. coli and Staph. aureus.     

Prevalence and antimicrobial susceptibility of staphylococci isolated from the skin surface of clinically normal cats

Samples were collected from 148 adult cats, processed for isolation of Staphylococcus species and tested for susceptibility to penicillin G, gentamicin, oxacillin, amoxycillin, ampicillin, cephalexin and rifampin. Methicillin resistance was also determined. Ninety-eight isolates were obtained (66% of samples). Coagulase-negative species were most common, and the most frequently isolated species (37 samples) was Staph. felis . Other coagulasenegative species, such as Staph. simulans , Staph. epidermidis and Staph. Saprophyticus were also isolated. Coagulase-positive species were obtained from 40 cats; the most frequent was Staph. intermedius (26 samples), followed by Staph. aureus (14 samples). Resistance to antibiotics was frequently observed, with 58·2% of the isolates showing resistance to at least one drug. Resistance to Penicillin G was observed in 49 of the 98 isolates (50%), 22 samples were resistant to oxacillin (22·4%) and 12 to rifampin (12·2%). Resistance to amoxycillin and ampicillin was very similar to that observed to Penicillin G. Gentamicin was the most active antimicrobial agent. Three MRSA (methicillin-resistant Staphylococcus aureus ) were isolated, which represents 21·4% of the isolates of that species. Nineteen MRS (methicillin resistant staphylococci) were also observed, distributed among Staph. intermedius (eight), Staph. simulans (six) and Staph. felis (five) isolates. The role of these micro-organisms on the skin of cats is discussed.     

Enterotoxigenicity of Staphylococcus aureus strains isolated from poultry: raw poultry carcases as a potential food-poisoning hazard

Staphylococcus aureus strains were isolated from end-of-lay poultry carcases obtained from a plant at two different stages of processing before and after storage at different temperatures. These strains were supplemented with Staph. aureus strains isolated from poultry from a wide range of sources and biotyped, phage typed, and tested for production of enterotoxins A-E. The isolates were found to consist of poultry and human specific strains and each of these groups contained strains able to produce enterotoxin. Poultry strains produced only enterotoxin D whereas human strains produced enterotoxins A, C and D. The hen carcases used in storage experiments were found to be naturally contaminated with enterotoxin D producing staphylococci. No enterotoxin D could be detected on any of the carcases even after storage at temperatures which allowed multiplication of the organisms to occur (final Staph. aureus counts ranged from 10² to 10⁷/16 cm² of breast skin).