Genomic and Biochemical Characterization of Acinetobacter Podophage Petty Reveals a Novel Lysis Mechanism and Tail-Associated Depolymerase Activity.

The increased prevalence of drug-resistant, nosocomial Acinetobacter infections, particularly from pathogenic members of the Acinetobacter calcoaceticus-baumannii complex, necessitates the exploration of novel treatments such as phage therapy. In the present study, we characterized phage Petty, a novel podophage that infects multidrug-resistant Acinetobacter nosocomialis and Acinetobacter baumannii Genome analysis reveals that phage Petty is a 40,431-bp ϕKMV-like phage, with a coding density of 92.2% and a G+C content of 42.3%. Interestingly, the lysis cassette encodes a class I holin and a single-subunit endolysin, but it lacks canonical spanins to disrupt the outer membrane. Analysis of other ϕKMV-like genomes revealed that spaninless lysis cassettes are a feature of phages infecting Acinetobacter within this subfamily of bacteriophages. The observed halo surrounding Petty's large clear plaques indicated the presence of a phage-encoded depolymerase capable of degrading capsular exopolysaccharides (EPS). The product of gene 39, a putative tail fiber, was hypothesized to possess depolymerase activity based on weak homology to previously reported phage tail fibers. The 101.4-kDa protein gene product 39 (gp39) was cloned and expressed, and its activity against Acinetobacter EPS in solution was determined. The enzyme degraded purified EPS from its host strain A. nosocomialis AU0783, reducing its viscosity, and generated reducing ends in solution, indicative of hydrolase activity. Given that the accessibility to cells within a biofilm is enhanced by degradation of EPS, phages with depolymerases may have enhanced diagnostic and therapeutic potential against drug-resistant Acinetobacter strains.IMPORTANCE Bacteriophage therapy is being revisited as a treatment for difficult-to-treat infections. This is especially true for Acinetobacter infections, which are notorious for being resistant to antimicrobials. Thus, sufficient data need to be generated with regard to phages with therapeutic potential, if they are to be successfully employed clinically. In this report, we describe the isolation and characterization of phage Petty, a novel lytic podophage, and its depolymerase. To our knowledge, it is the first phage reported to be able to infect both A. baumannii and A. nosocomialis The lytic phage has potential as an alternative therapeutic agent, and the depolymerase could be used for modulating EPS both during infections and in biofilms on medical equipment, as well as for capsular typing. We also highlight the lack of predicted canonical spanins in the phage genome and confirm that, unlike the rounding of lambda lysogens lacking functional spanin genes, A. nosocomialis cells infected with phage Petty lyse by bursting. This suggests that phages like Petty employ a different mechanism to disrupt the outer membrane of Acinetobacter hosts during lysis.

Genomic and functional analyses of Rhodococcus equi phages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7.

The isolation and results of genomic and functional analyses of Rhodococcus equi phages ReqiPepy6, ReqiDocB7, ReqiPine5, and ReqiPoco6 (hereafter referred to as Pepy6, DocB7, Pine5, and Poco6, respectively) are reported. Two phages, Pepy6 and Poco6, more than 75% identical, exhibited genome organization and protein sequence likeness to Lactococcus lactis phage 1706 and clostridial prophage elements. An unusually high fraction, 27%, of Pepy6 and Poco6 proteins were predicted to possess at least one transmembrane domain, a value much higher than the average of 8.5% transmembrane domain-containing proteins determined from a data set of 36,324 phage protein entries. Genome organization and protein sequence comparisons place phage Pine5 as the first nonmycobacteriophage member of the large Rosebush cluster. DocB7, which had the broadest host range among the four isolates, was not closely related to any phage or prophage in the database, and only 23 of 105 predicted encoded proteins could be assigned a functional annotation. Because of the relationship of Rhodococcus to Mycobacterium, it was anticipated that these phages should exhibit some of the features characteristic of mycobacteriophages. Traits that were identified as shared by the Rhodococcus phages and mycobacteriophages include the prevalent long-tailed morphology and the presence of genes encoding LysB-like mycolate-hydrolyzing lysis proteins. Application of DocB7 lysates to soils amended with a host strain of R. equi reduced recoverable bacterial CFU, suggesting that phage may be useful in limiting R. equi load in the environment while foals are susceptible to infection.

Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens.

AIMS: Determining the effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broilers. METHODS AND RESULTS: 16S rRNA gene-based polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) profiling, DNA sequencing and real-time quantitative PCR techniques were used. The richness of both ileal and caecal microbiota increased with chicken age. The microbiota from those birds of the same age demonstrated relatively similar PCR-DGGE profiles and tended to form closely related clusters in the relatedness analyses. Dietary treatment with bacitracin (50 mg kg(-1)) and access to range did not change the richness but altered the composition of the microbiota. The impact of bacitracin was particularly obvious in 3-day-old chicks. Lactobacilli were abundant in the caecal microbiota of 3-day-old chicks regardless of the dietary treatment with bacitracin. The access to range enriched Bifidobacterium in both the ileum and caeca. CONCLUSIONS: Bacitracin, bird age and access to range all influenced bacterial microbiota in the ileum and caeca of broilers, with bird age having the greatest apparent effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing useful information for the development of antibiotic replacement therapy for poultry production.

Efficacy and pharmacokinetics of bacteriophage therapy in treatment of subclinical Staphylococcus aureus mastitis in lactating dairy cattle.

Bovine mastitis is an inflammation of the udder caused by microbial infection. Mastitis caused by Staphylococcus aureus is a major concern to the dairy industry due to its resistance to antibiotic treatment and its propensity to recur chronically. Growing concerns surrounding antibiotic resistance have spurred research into alternative treatment methods. The ability of lytic S. aureus bacteriophage K to eliminate bovine S. aureus intramammary infection during lactation was evaluated in a placebo-controlled, multisite trial. Twenty-four lactating Holstein cows with preexisting subclinical S. aureus mastitis were treated. Treatment consisted of 10-ml intramammary infusions of either 1.25 x 10(11) PFU of phage K or saline, administered once per day for 5 days. The cure rate was established by the assessment of four serial samples collected following treatment. The cure rate was 3 of 18 quarters (16.7%) in the phage-treated group, while none of the 20 saline-treated quarters were cured. This difference was not statistically significant. The effects of phage intramammary infusion on the bovine mammary gland were also studied. In healthy lactating cows, a single infusion of either filter-sterilized broth lysate or a CsCl gradient-purified phage preparation elicited a large increase in the milk somatic cell count. This response was not observed when phage was infused into quarters which were already infected with S. aureus. Phage-infused healthy quarters continued to shed viable bacteriophage into the milk for up to 36 h postinfusion. The phage concentration in the milk suggested that there was significant degradation or inactivation of the infused phage within the gland.

Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K.

AIMS: To understand the potential use of bacteriophage K to treat bovine Staphylococcus aureus mastitis, we studied the role of whey proteins in the inhibition of the phage-pathogen interaction in vitro. METHODS AND RESULTS: The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in raw bovine whey and serum. Incubation of S. aureus with phage in whey showed that the bacteria are more resistant to phage lysis when grown in whey and also bovine serum. Whey collected from 23 animals showed a wide variation in the level of phage-binding inhibition. The role of the protein component of milk whey in this inhibition was established; treatment of the whey by heat, proteases and ultrafiltration removed the inhibitory activity. Brief exposure of S. aureus cells to whey, followed by resuspension in broth, also reduced phage binding. Microscopy showed the adhesion of extracellular material to the S. aureus cell surface following exposure to whey. Chromatographic fractionation of the whey demonstrated that the inhibitory proteins were present in the high molecular weight fraction. CONCLUSIONS: The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phage attachment and thereby hindered lysis. The inhibitory whey proteins are of high molecular weight in their native form and may sterically block phage attachment sites on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have implications for any future use of phage therapy in the treatment of mastitis, and other diseases, caused by S. aureus. This pathogen is predicted to be much more resistant to phage treatment in vivo than would be expected from in vitro broth culture experiments.

Molecular typing and distribution of Staphylococcus aureus isolates in Eastern Canadian dairy herds.

Macrorestriction analysis of SmaI-digested chromosomal DNA, using pulsed field gel electrophoresis (PFGE) was performed to type and estimate genetic relationships among 288 Staphylococcus aureus isolates recovered from 58 Eastern Canadian dairy herds. In addition, a subset of the collection was phage typed and evaluated for sensitivity to 10 antimicrobial compounds. Of 288 isolates recovered, 29 distinct PFGE types were identified. Based on estimates of genetic relationships, the PFGE types were assigned to six lineage groups, designated A through F. Of all of the isolates, ca. 93% were assigned to lineage groups A, D, or F. In 58.6% of herds, only a single PFGE type was recovered, while the remainder had two to four types. Of the 212 isolates evaluated for antimicrobial resistance, 24.5% were resistant to one or more antimicrobials. Resistance to penicillin (9.9%) was most common, followed by resistance to sulfadimethoxine (7.5%). Isolates resistant to multiple antibiotics were rare. A total of 63% of isolates responded to phages from groups 1 and 3, and 32.8% could not be typed with any of the phage strains used. The other 4.1% belonged to a variety of phage types. Most of the PFGE lineage group A and F isolates corresponded to phage groups 3 and 1, respectively, and most group D isolates were not typeable. PFGE typing had better discriminatory power than phage typing in defining the relatedness of the S. aureus isolates. Distribution of PFGE types and phage types was independent across regions and within herds.

Bacteriophages of Erwinia amylovora.

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and PODOVIRIDAE:

Effect of discontinuance of type A behavioral counseling on type A behavior and cardiac recurrence rate of post myocardial infarction patients.

Three hundred post infarction participants who had received type A behavioral counseling in the Recurrent Coronary Prevention Project, (RCPP) agreed to be followed for 1 additional year after stopping 4.5 years of continuous type A behavioral counseling. One hundred fourteen participants who had served for 4.5 years as controls in the RCPP Study, thus receiving no type A behavioral counseling, volunteered to receive such counseling for 1 year. Eleven of the 300 previously type A counseled RCPP participants were lost to follow-up at the end of the additional year. The remaining 289 subjects at risk were found to have maintained their previously reduced intensity of type A behavior. Their previously observed relatively low cardiac recurrence rate during the additional year also did not significantly change. Ten of the 114 previous control RCPP participants were lost to follow-up at the end of the additional year. The remaining 104 subjects who had received a year's type A behavioral counseling showed a significantly reduced intensity of type A behavior and a similar significant decrease in both the cardiac mortality and morbidity rate. These results suggest that the decline in the intensity of type A behavior and also in the cardiac recurrence rate previously observed in post infarction subjects exposed to type A behavioral counseling persists for at least 1 year after cessation of such counseling.

Alteration of type A behavior and its effect on cardiac recurrences in post myocardial infarction patients: summary results of the recurrent coronary prevention project.

One thousand thirteen post myocardial infarction patients were observed for 4.5 years to determine whether their type A (coronary-prone) behavior could be altered and the effect such alteration might have on the subsequent cardiac morbidity and mortality rates of these individuals. Eight hundred sixty-two of these individuals were randomly assigned either to a control section of 270 participants who received group cardiac counseling or an experimental section of 592 participants who received both group cardiac counseling and type A behavioral counseling. The remaining 151 patients, serving as a "comparison group," did not receive group counseling of any kind. Using the "Intention-to-Treat" principle, we observed markedly reduced type A behavior at the end of 4.5 years in 35.1% of participants given cardiac and type A behavior counseling compared with 9.8% of participants given only cardiac counseling. The cumulative 4.5-year cardiac recurrence rate was 12.9% in the 592 participants in the experimental group that received type A counseling. This recurrence rate was significantly less (p less than 0.005) than either the recurrence rate (21.2%) observed in the 270 participants in the control group or the recurrence rate (28.2%) in those of the comparison group not receiving any special treatment. After the first year, a significant difference in number of cardiac deaths between the experimental and control participants was observed during the remaining 3.5 years of the study. Overall, the results of this study demonstrate for the first time, within a controlled experimental design, that altering type A behavior reduces cardiac morbidity and mortality in post infarction patients.

Reduction in type A behavior in healthy middle-aged American military officers.

One hundred eighteen senior officer-students of the U.S. Army War College who were healthy but exhibited type A behavior volunteered to be randomly selected and enrolled into (1) a section of 62 officers who received group type A behavior counseling for 9 months and (2) a control section of 56 officers who received no counseling of any kind. Marked or profound reduction in type A behavior at the end of 9 months was observed in 41.9% of the 62 participants who initially were enrolled to receive type A counseling; marked or profound reduction in type A behavior, however, was observed in only 8.9% of the 56 initially enrolled control subjects. No adverse effects on the military leadership qualities of type A counseled participants were observed by their classmates. Serum total cholesterol and plasma high-density lipoprotein (HDL) cholesterol measurements were obtained monthly. The serum cholesterol of the total cohort of subjects rose significantly during a month of considerable emotional tension and stress. Those subjects who underwent a profound reduction in the intensity of their type A behavior pattern also exhibited a significantly lower serum cholesterol value as the study continued than those subjects who exhibited no change in their type A behavior. No significant changes in plasma HDL cholesterol concentrations were observed in the total cohort during the above-mentioned period of stress, nor were any differences in this particular measurement noted between the type A counseled and the control participants.

Alteration of type A behavior and reduction in cardiac recurrences in postmyocardial infarction patients.

Eight hundred sixty-two postmyocardial infarction patients volunteered to be randomly selected and enrolled into: (1) a control section of 270 patients, who received group cardiologic counseling; and (2) an experimental section of 592 patients, who received group type A behavior counseling in addition to group cardiologic counseling. Reduction in type A behavior at the end of 3 years was observed in 43.8% of the 592 participants, who initially were enrolled to receive group cardiologic and type A behavioral counseling. This degree of behavioral reduction was significantly greater than that observed in participants who initially were enrolled to receive only group cardiologic counseling. The 3-year cumulative cardiac recurrence rate was 7.2% in participants who initially were enrolled to receive group cardiologic and type A behavioral counseling. This was significantly less (p less than 0.005) than that (13%) observed in participants who initially were enrolled to receive only cardiologic counseling. This difference in recurrence rates was due to a lesser incidence of nonfatal infarctions in the patients who had been enrolled in the section receiving type A behavioral as well as cardiologic counseling. These data suggest that type A behavior can be altered in a sizable fraction of postinfarction patients and that such alteration is associated with a significantly reduced rate of nonfatal myocardial infarctions.

Can the type A behavior pattern be altered after myocardial infarction? A second year report from the recurrent coronary prevention project.

The feasibility of altering a Type A style of life was investigated in 1012 nonsmoking predominantly male postinfarction volunteers in the San Francisco Bay area. A total of 862 were randomly allocated into an experimental section receiving a combination of Type A and cardiac counseling, or a control section receiving cardiac counseling alone. The remaining 150 formed a nonrandom but statistically equivalent comparison section. Assessments of change in Type A behavior were made by the participant, his or her spouse, a work colleague, and an independent rater of a videotaped structured interview. Psychometric analyses indicated that these instruments were valid and reliable measures of Type A behavior. After 24 months, participants receiving Type A/cardiac counseling exhibited a significantly greater reduction in Type A behavior than the other two sections, and had a lower cardiovascular recurrence rate than the comparison section only. No differences among the three sections were observed in total cholesterol or resting blood pressure. The results suggest that Type A behavior can be altered by group counseling in postinfarction volunteers and that such alteration is superior to no group counseling at all in the secondary prevention of coronary heart disease.

Feasibility of altering type A behavior pattern after myocardial infarction. Recurrent Coronary Prevention Project Study: methods, baseline results and preliminary findings.

We studied 1035 consecutive postinfarction patients to determine the feasibility of altering type A behavior and the effect such alteration might have on subsequent rates of infarction and cardiovascular death. Approximately 300 subjects were enrolled in small groups and primarily received cardiologic counseling on the usually accepted coronary risk factors. Six hundred subjects received, in addition to cardiologic counseling, advice and instructions designed to diminish the intensity of their type A behavior. The remaining subjects, serving as controls, received no counseling, but were examined and interviewed annually, as were those who dropped out of counseling groups. More than 98% of the 1035 subjects exhibited moderate-to-severe type A behavior during a videotaped structured interview. After the first year of this 5-year study, the rates of infarction and cardiovascular death were lower (p less than 0.01 and p less than 0.05, respectively) among subjects who received both cardiologic and behavioral counseling than among the control subjects. The rate of nonfatal infarction was lower (p less than 0.05) among subjects who received behavioral counseling than among those who received only cardiologic counseling or those who dropped out of either counseling group. The circumstances that most often preceded recurrent infarction or cardiovascular death were emotional crisis, excess physical activity, ingestion of a single fatty meal or a combination of these phenomena.

The recurrent coronary prevention project: initial findings.

A behavioural treatment programme, designed to modify type A behaviour, has been compared with cardiological measures (e.g. diet, exercise and adherence to medication), and with 'no treatment', for its ability to prevent reinfarction in patients who had suffered a myocardial infarct at least six months previously. Provisional results show fewer reinfarctions and greater modification of type A behaviour in the group receiving behavioural treatment. In the third year, to date, the difference favouring behavioural over cardiological measures was statistically significant (P less than 0.01), and other factors seem unlikely to be responsible. The five-year study still has two years to run.