Phage types of Staphylococcus aureus strains and their antibiotic resistance in carriers of medical students population

The phage types of 78 S. aureus strains isolated from nose swabs obtained from a medical students in 2005 -2006 was determined and antibiotic resistance of the phage types was analysed. 680 students were tested in order to obtain the strains and 11.5% of them were carriers of S. aureus. Phage typing was performed using basic set of23 phages and 3 additional phages: 88, 89 and 187. Drug resistance was determined by the disc-diffusion method. The most frequent in studied population were the group III (21.8%) and strains lysed by phages belonging to varied groups (21.8%). Highly different phage patterns were observed among strains belonging to each of the group. Strains belonging to the group III as the strains lysed by phages from varied groups were most frequently resistant only to penicillin (52,9% respectively). Resistance to penicillin was also most often observed in the strains belonging to another groups and phage types. Usefulness of the additional phages 88,89 and 187 was in the investigations as no more than 51% of strains was lysed by this phages.     

Phage types of Staphylococcus aureus isolated in Poland in 1999-2004

The aim of study was to determine how population of Staphylococcus aureus strains from hospital environment forms itself with respect to phage types and frequency of their occurrence recently. 1157 S. aureus isolated from various clinical materials between 1999 and 2004 have been investigated. The basic set of phages and the three additional ones were used for typing strains according to Blair and Williams method. The results proved that population of S. aureus strains isolated from hospital environment differentiates with respect to phage types, like in the past. Strains belonging to phage group II dominates among them, but a considerable increase in number of phage group III have been remarked lately. A differential value of an individual phages from basic set remains on their usual level for years.     

The Distribution of Staphylococcus aureus in a Poultry Processing Plant

A set of phages previously isolated from poultry strains of Staphylococcus aureus was used to type such isolates from poultry before, during and after processing in a poultry plant. Certain poultry phage types were found to be associated with the live birds rather than the processed carcases. Strains lysed by phages from this group may represent a specific 'poultry'biotype. A site of cross-contamination within the plant was discovered.     

Phage types and plasmid profiles of plasmid DNA strains of Salmonella enterica subsp. enterica ser. Enteritidis (S. Enteritidis) isolated from food poisoning outbreaks in 2001

Salmonella Enteritidis strains are the most often isolated Salmonella serovar in Poland. In the present study, phage typing, antibiotic resistance testing and plasmid profile analysis, have been applied to characterise 41 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 41 S. Enteritidis strains coming from Poland. All 41 strains were typable and 5 different phage types were observed. Among 41 strains tested, both PT6 and PT21 were recognized in the 15 strains (36.6%). Nine strains (22%) belonged to phage type 8. The others PTs were represented by small amount of strains (PT1var and PT4). Among all tested isolates only 4 different plasmid profiles were observed. Of the 41 strains investigated, 16 (39%) contained the 57 kb plasmid alone. The remaining 25 strains (61%) except 57 kb plasmid, possessed additional DNA particles. The probable phage type conversion of PT21 to PT1var strain, possibly connected with smaller DNA particle presence, was observed. This hypothesis needs confirmation. The real S. Enteritidis epidemiological situation in Poland should be known after introducing of systematic, annual research program.     

Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log10 CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.     

Bacteriophage Types and O Antigen Groups of Escherichia coli from Urine

Urinary strains of Escherichia coli from seven geographical regions were typed serologically for O-specific antigens and with phages capable of lysing the majority of urinary isolated. The O antigen groups 4, 6, 75, 1, 50, 7, and 25 were the common ones found. Of the 454 cultures tested, 66.1% were phage typable and 65.2% were serotypable with the 48 antisera employed. Also, 71.6% of the cultures for which an O group could be determined were phage typable. Furthermore, of those seven O-antigen groups implicated in urinary tract infection, 80.2% exhibited a phage pattern. Various phage types were found within an O-antigen group, and, although one phage type associated a high percentage of the time with one O-antigen group, no correlation was observed between other O-antigen groups and phage types. Studies with bacteriuric patients by phage typing showed the presence of two strains of E. coli within an O-antigen group. Serogrouping and phage typing of fecal isolates of E. coli revealed the presence of some O-antigen groups and phage types also found as predominant types among urinary isolates. Phage typability correlated highly with hemolysis of human erythrocytes. Elevated temperatures of incubation and a chemical curing agent were used to enhance typability of cultures refractory to the typing phages. Phage typing, due to its rapidity, ease, and ability to distinguish strains of E. coli within an O-antigenic group, is suggested as a possible method by which a better insight into the epidemiology of urinary tract infections may be obtained.     

Bacteriophage types of methicillin--resistant Staphylococcus aureus strains isolated in 2005-2006 in the Gdańsk area and their resistance to other antibiotics

The phage type of 38 methicillin--resistant Staphylococcus aureus strains isolated from clinical samples in 5 hospitals in the Gdańsk area in 2005-2007 was determined and antibiotic resistance of obtained phage types was analysed. Phage typing was performed using set of 10 phages as following: MR8, MR12, MR25, 30, 33, 38, M3, M5, 622, 56B. Drug resistance was determined by the disc--diffusion method. There were 12 phage types observed. The most frequent (28.9%) was 56B type which was present in 3 hospitals and in one hospital was found to be predominated. The studied strains were most frequent (23.7%) resistant to erythromycin, clindamycin, ciprofloxacin, trimethoprim/sulfamethoxazole, tetracycline and gentamicin. This resistance pattern predominated in strains belonging to 56B type.     

Bacteriophage Typing as an Epidemiological Tool for Urinary Escherichia coli

Phage typing was used to identify strains of Escherichia coli isolated from urinary and nonurinary sources. When eight phages isolated in Pennsylvania were used to type 717 cultures from Missouri, 50.3% of 624 urinary isolates and 34.4% of 93 nonurinary isolates were typable. Strains from nonurinary sources were not found commonly in urine. When five additional phages isolated in Missouri were added to the original set of eight phages, 80.4% of 331 urinary isolates were typable. When this set of phages was used to type 552 urinary cultures isolated in California, Minnesota, Ohio, Pennsylvania, Virginia, and West Virginia, 82.0% of the cultures were typable. Some common phage types were found in high incidence among cultures from the different regions. No correlation was found between phage type and the pattern of resistance to antibiotics. Phage typing data were presented also on the number of strains in individual urine specimens and the recurrences of strains in patients with chronic bacteriuria. Of 97 fecal isolates, 75.2% of the cultures were typable, and the most common phage type was observed in high incidence among the urinary isolates from this region. When 75 cultures from nine other genera of enteric bacteria were typed, only the shigellae were lysed. In view of the information obtained by phage typing and the ease and speed with which it can be done, it is suggested that phage typing be considered a new tool in epidemiological studies of urinary tract infections by E. coli.     

Phage typing of indigenous soybean-rhizobia and relationship of a phage group strains for their asymbiotic and symbiotic nitrogen fixation

A total of 354 indigenous bradyrhizobia were isolated from soybean nodules collected from five major crop grown regions. Host-specific 12 phages, each active on particular strains were selected. Factors, which influence the interaction between the host and phage, were examined. Four different types of plaques were detected. Nearly 17% of isolates were found resistant to all phages. Phage sensitivity patterns revealed a total of 32 distinct phage genotype groups. Different set of phage combinations expressed variation in specificity for parasitizing against particular group of rhizobia. Distributions of isolates in each phage types differed markedly between regions. Interestingly, nine strains belonging to phage group 16 exhibited high ex planta nitrogenase activity in culture. However, no correlation could be established between high ex planta nitrogenase activity and their symbiotic effectiveness with soybean cultivars. Soybean cv. JS335 showed relatively superior performance than Bragg and Lee with indigenous bradyrhizobial strains. Phage typing revealed the existence of large genetic diversity among native rhizobia and selection of the superior bradyrhizobial strains can also be possible for a given soil-climate-cultivar complex.     

Use of WARD et al's scheme for bacteriophage typing of Salmonella enteritidis strains isolated in Poland

The typing phages set of Ward et al. was used to type a total of 517 strains of Salmonella Enteritidis isolated in Poland in 1986-1995. According to the Ward et al. scheme, 56.5% of the strains tested were assigned to 14 different phage types. Phage types 8, 4, 1 and 4a were placed first, second, third and fourth, respectively. They were dominated both in the outbreak isolates and in the isolates from the other sources. Ten phage types were represented by single strains. Other strains reacted with phages without showing any of the designated phage type (37.1%) or were untypable (6.4%). The Ward et al. scheme seems to demonstrate not enough high degree of strain discrimination for Salmonella Enteritidis isolates in Poland. It seems that the Ward et al. scheme is not enough useful for the epidemiological investigations of Salmonella Enteritidis in Poland.     

Bacteriophage Typing of Proteus mirabilis, Proteus vulgaris, and Proteus morganii

A bacteriphage typing scheme for differentiating Proteus isolated from clinical specimens was developed. Twenty-one distinct patterns of lysis were seen when 15 bacteriophages isolated on 8 Proteus mirabilis, 1 P. vulgaris, and 1 P. morganii were used to type 162 of 189 (85.7%) P. mirabilis and P. vulgaris isolates. Seven phages isolated on 3 P. morganii were used to type 13 of 19 (68.4%) P. morganii isolates. Overall, 84.1% of the 208 isolates were lysed by at least 1 phage at routine test dilution (RTD) or 1,000 x RTD. Fifty isolates, retyped several weeks after the initial testing, showed no changes in lytic patterns. The phages retained their titers after storage at 4 C for several months. A computer analysis of the data showed that there was no relationship between the source of the isolate and bacteriophage type. This bacteriophage typing system may provide epidemiological information on strains involved in human infections.     

Bacteriophage types of Salmonella enteritidis occurring in Poland in 1986-1995

The Lalko phages collection was used to phage type a total of 517 Salmonella Enteritidis strains isolated from food-poisoning outbreaks (312 strains) and other common sources (205 strains) in Poland, during the years 1986-1995. Above 99.0% of all strains tested were recognized as belonging to definitive phage type. Phage types 1, 6 and 7 were predominant. The strains of type 1 and 7 were most numerous. Of the 517 examined strains 312 were isolated from 46 food-poisoning outbreaks. Most of them came from the one phage type outbreaks; 8 mixed outbreaks were noted. The greatest number of the food-poisoning outbreaks was caused by Salmonella Enteritidis phage types 1, 6 and 7. Phage type 16 was isolated from persons for the first time.     

Staphylococcal and other Bacterial Species Associated with Intramammary Infections in Danish Dairy Herds

Four thousand six hundred forty– five quarter milk samples from 1179 cows from 20 commercial dairy herds were examined in order to determine the prevalence of bacterial species. A total of 859 isolates from 839 (18.1%) culture positive samples could be assigned to 34 different species and subspecies. Diagnostics of staphylococcal species was based on conventional procedures able to differentiate between all 36 species and subspecies presently acknowledged. Staphylococcus aureus was found in 10.2% of the samples and was the most common species isolated. Streptococcus dysgalactiae (1.6%) and Streptococcus uberis (1.4%) were the second and third most common species isolated. Seventeen different coagulase negative staphylococcal species (CNS) were found in 4.1% of the samples. The most frequently isolated CNS were S. epidermidis (1.3%), S. chromogenes (1.0%) and S. simulans (0.7%). Isolates of S. aureus were phage typed, and isolates of S. epidermidis were investigated by phage typing, antibiogram typing, and biotyping. A total of 378 (79.9%) isolates of S. aureus could be typed by phages, assigning them to 18 different phage types. However, 6 phage types accounted for 92.1% of the typable isolates. One to 2 phage types predominated within each herd. Eleven (18%) isolates of S. epidermidis could be typed by phages, assigning the isolates to 3 different types. Biotyping of S. epidermidis produced a total of 8 different types, the most common accounting for 29.5% of the isolates. A total of 6 different antibiogram types were observed among all isolates of S. epidermidis. Resistance towards penicillin (36.1%), tetracycline (9.8%) and streptomycin (9.8%), were recorded in the isolates of S. epidermidis. However, 35 (57.4%) of the isolates were susceptible to all 12 antibiotics tested.     

Characterization of bacteriophage nucleic acids obtained from Clostridium botulinum types C and D

Nontoxigenic strains of Clostridium botulinum types C and D are converted to toxigenic strains by infection with specific Tox+ bacteriophages. The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, and d-1873, and one nonconverting phage, c-n71, and treated with nucleases. The nucleic acids isolated were not digested by RNase A, but were digested by DNase I and exonuclease III, indicating that they were double-stranded DNA. On the basis of the restriction endonuclease digestion patterns on 0.8% agarose gel electrophoresis, the length of c-st, c-n71, c-468, and c-d6f phage DNAs was estimated to be about 110 kilobase pairs and that of c-203 and d-1873 was about 150 kilobase pairs. The digestion patterns of c-st, c-468, and c-n71 phage DNAs by PstI and HindIII were very similar. High homology was observed in the dot hybridization test. For other phages and nucleases, a good similarity was not observed. Only a little similarity was observed between c-203 and c-d6f phages. The existence of the structural genes for the toxin in both c-st and c-n71 phages was confirmed by the hybridization test with these phage DNAs and the oligonucleotide probe which represented the DNA sequence predicted for the N-terminal amino acids (2 to 17) of C. botulinum type C toxin. The loss of the converting ability of c-n71 phage may be caused not by the deletion of the tox+ gene but rather by the base mutation in c-st phage DNA.     

Characterization of bacteriophage nucleic acids obtained from Clostridium botulinum types C and D.

Nontoxigenic strains of Clostridium botulinum types C and D are converted to toxigenic strains by infection with specific Tox+ bacteriophages. The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, and d-1873, and one nonconverting phage, c-n71, and treated with nucleases. The nucleic acids isolated were not digested by RNase A, but were digested by DNase I and exonuclease III, indicating that they were double-stranded DNA. On the basis of the restriction endonuclease digestion patterns on 0.8% agarose gel electrophoresis, the length of c-st, c-n71, c-468, and c-d6f phage DNAs was estimated to be about 110 kilobase pairs and that of c-203 and d-1873 was about 150 kilobase pairs. The digestion patterns of c-st, c-468, and c-n71 phage DNAs by PstI and HindIII were very similar. High homology was observed in the dot hybridization test. For other phages and nucleases, a good similarity was not observed. Only a little similarity was observed between c-203 and c-d6f phages. The existence of the structural genes for the toxin in both c-st and c-n71 phages was confirmed by the hybridization test with these phage DNAs and the oligonucleotide probe which represented the DNA sequence predicted for the N-terminal amino acids (2 to 17) of C. botulinum type C toxin. The loss of the converting ability of c-n71 phage may be caused not by the deletion of the tox+ gene but rather by the base mutation in c-st phage DNA.     

Epidemiological analysis of a methicillin-resistant Staphylococcus aureus outbreak using restriction fragment length polymorphisms of genomic DNA.

The genomic DNA of 58 isolates of methicillin-resistant Staphylococcus aureus (MRSA) obtained during an infection outbreak at two major Canberra hospitals was analysed for restriction fragment length polymorphism (RFLP) by digestion with the endonuclease SmaI and resolution of the fragments by pulsed-field gel electrophoresis. Based on the fraction of common fragments generated by the endonuclease, DNA similarities among the isolates were estimated. Distance matrix analysis showed that the MRSA isolates could be divided into two major clusters (RFLP types I and II) and one minor one (type 46). A fourth group of miscellaneous isolates was found to be heterogeneous in terms of DNA sequence similarity. The epidemiological data indicated that RFLP type I was most common in the intensive care units in the two hospitals, with particular subtypes of RFLP type I concentrated in individual units. RFLP type II and the miscellaneous group were more generally distributed. Type 46 isolates appear to be related to a group which was present in epidemics in Melbourne hospitals in the early 1980s. Using the standard phage set, the RFLP type I group was largely untypable. However, type II isolates were all phage typable, with a shared susceptibility to phages 29/85/95/90; type 46 isolates had a shared susceptibility to phages 85/90. The miscellaneous isolates were of variable phage types.     

Phage types, plasmid profiles and chromosomal restriction profiles of Salmonella enterica subsp. enterica ser. enteritidis (S. enteritidis) isolated in Poland in 1999-2000]

Salmonella Enteritidis strains are the most often isolated Salmonella serovars in Poland. In the present study, phage typing, plasmid profile analysis, and PFGE have been applied to characterize 140 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 140 S. Enteritidis strains coming from Poland. All 140 strains were typable and six different phage types were observed. A total of 125 (89%) of 140 isolates examined belonged to PT 4. The others PTs were represented by small amount of strains (PT1-2, PT6-6, PT7-1, PT8-4 and PT21-2 strains). Among all tested isolates six different plasmid profiles were observed. Of the 140 examined strains, 128 (91.4%) contained the 57 kb plasmid alone. After XbaI digestion four distinct pulse field chromosomal restriction profiles among studied S. Enteritidis were observed. XbaI and SpeI chromosomal restriction profiles of S. Enteritidis PT4 were identical with reference strain profiles. Our findings confirmed earlier suggestions that the increase of human salmonellosis cases in Poland was caused by S. Enteritidis PT4 and was due to consumption of contaminated food. This study confirmed the importance of using PFGE in combination with phage typing, plasmid typing and antibiotic resistance testing for studying the epidemiology of S. Enteritidis.     

Capsular serotype of Staphylococcus aureus in the era of community-acquired MRSA

Capsular polysaccharide (CP) plays an important role in the pathogenicity and immunogenicity of Staphylococcus aureus, yet the common serotypes of S. aureus isolated from US pediatric patients have not been reported. We investigated capsular serotype as well as methicillin susceptibility, presence of Panton-Valentine leukocidin (PVL), and clonal relatedness of pediatric S. aureus isolates. Clinical isolates were tested for methicillin susceptibility, presence of mecA, lukS-PV and lukF-PV, cap5 and cap8 genes by PCR, and for capsular or surface polysaccharide expression (CP5, CP8, or 336 polysaccharide) by agglutination. Genetic relatedness was determined by pulsed-field gel electrophoresis. All S. aureus isolates encoded cap5 or cap8. Sixty-nine percent of 2004-2005 isolates were methicillin-susceptible (MSSA) and most expressed a detectable capsule. The majority of MRSA isolates (82%) were unencapsulated, exposing an expressed cell wall techoic acid antigen 336. Pulsed-field type USA300 were MRSA, PVL-positive, unencapsulated strains that were associated with deep skin infections and recurrent disease. Over half (58%) of all isolates from invasive pediatric dermatologic infections were USA300. All pediatric isolates contained either capsule type 5 or capsule type 8 genes, and roughly half of the S. aureus clinical disease isolates from our population were diverse MSSA-encapsulated strains. The majority of the remaining pediatric clinical disease isolates were unencapsulated serotype 336 strains of the PVL(+) USA300 community-associated-MRSA clone.     

Molecular ecology of Streptococcus thermophilus bacteriophage infections in a cheese factory.

A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control.