Restriction and modification of phage 47 and lambda by R factors

S. panama 47 (antibiotic-sensitive, phage pattern A) was infected with R factors from a number of field strains of Enterobacteriaceae (Salmonella, Escherichia coli, Citrobacter, Enterobacter, Klebsiella andProteus) isolated from human and animal sources. These R factors could be grouped into 11 types i.e. R1 R11 on the basis of induced changes in the phage type of the recipient.R8 and R11 renderS. panama resistant to the phages A H:S. panama 47 (R3) and 47 (R6) adsorb the phages A F, but there is no phage multiplication: phages G and H are considered to be restricted and modified in these strains. The R factors R5 and R7 also exert restriction and modification on a number of the typing phages A H. The nature of the changes in phage pattern brought about by R4, R9 and R10 is not understood. R2 does not exert restriction (i.e. no change in phage pattern). The R factors were also investigated for the fi (fertility inhibition) and spp (restriction of phage ) markers.The R factors R3 R11 readily segregate, in the sense that the restriction and modification loci, and occasionally the Resistance Transfer Factor as a whole was frequently lost after R transfer. These 9 types of R factors were encountered infrequently in the present material.Resistance to tetracycline inS.panama is nearly always due to R factors of type R1. In other members of Enterobacteriaceae, notably inE.coli, R1 is less frequently found than R2.     

Resistance Transfer Factors in sensitive strains ofS. panama

Two natural strains ofS.panama with an incomplete Resistance Factor (R factor) are described:S.panama I carries a Resistance Transfer Factor (RTF) exerting restriction on phageS.panama 47, but no resistance determinants; andS.panama 219 has a tetracycline-resistance determinant but no RTF.A number of drug-sensitive strains ofS.panama belonging to various phage types, were found to carry a factor which is able to mobilise the tetracycline-resistance determinant ofS.panama 219 and also of one strain ofE.coli and to transfer them toS.panama andE.coli K 12. This factor is not identical with the F factor, it is not a bacteriocinogenic factor and can therefore be considered as an RTF. These RTFs exert no restriction on (spp) and phage 47, and some of them are fi whilst others are fi.     

Characteristics of five bacteriophages of yellow-pigmented enterobacteria

Four bacteriophages (C2, C2F, E3, and E16P) belonging to morphological group C3 and one belonging to morphological group A3 (E16B) were purified by deuterium oxide gradient centrifugation and cesium chloride gradient centrifugation. Morphological group C3 phages had a densityd=1.534–1.541 and group A3 phage (E16B) had a densityd=1.492 in CsCl. Phages of morphological group C3 isolated onEnterobacter sakazakii (C2, C2F) and onErwinia herbicola (E3, E16P) were compared withSalmonella newport phage 7-11 with respect to host-range, genome size, antigenic relatedness, and ultraviolet and heat susceptibility. Phages C2 and C2F could multiply inEnterobacter cloacae, E. sakazakii, Erwinia herbicola, E. rhapontici, andLevinea malonatica; whereas phages E3, E16P, and 7-11 could multiply on these same species and onEscherichia coli and severalSalmonella serotypes. Molecular weights of phage DNAs were determined to be 58×10⁶ (C2), 60×10⁶ (7-11), 67×10⁶ (E3), and 39×10⁶ (E16B).All studied phages of morphological group C3 (includingSalmonella newport phage 7-11) were neutralized by anti-phage C2 serum. Despite differences in neutralization kinetics and in ultraviolet and heat sensitivities, these phages of morphological group C3 constitute one phage species. Phage E16B (morphological group A3) had a host-range limited toEnterobacter cloacae, Erwinia herbicola, andE. rhapontici; it was antigenically unrelated to the preceding phage group C3, and showed ultraviolet and heat susceptibility close to that of coliphage T4.     

Characterization of a Temperate Phage and Four Bacteriocins Produced by Nonpathogenic Clostridium Species

We previously reported the isolation of a temperate phage (named KT) and several bacteriocins (named clostocins) from strains of nonpathogenic Clostridium species. Later, the induction and some properties of the phage and four clostocins (A, B, C and D) were examined.

The phage was induced by UV light and mitomycin C. The phage had a polygonal head (about 85mμ in diameter) and a tail with contractile sheath (about 100mμ in length). Some other properties of the phage were also studied; plaque morphology, stability in salt solution, inactivation by UV light, pH stability, thermal inactivation, host-range and lysis of infected culture.

Clostocins A and D were partially induced by UV light and mitomycin C, whereas that of B and C were not. All clostocins failed to pass through a dialysis membrane, and were insensitive to UV light and to ribo- and deoxyribonuclease. They were destroyed by some proteolytic enzymes, but differences in degree of their susceptibility were observed among them. Clostocins A and D were very thermo-stable, whereas B and C were relatively thermo-labile. Clostocins A and D acted on some strains in the genus Clostridium, whereas B and C did on many strains in the family Bacillaceae.

There was no demonstrable serological relationship between phage KT and clostocin A, although they seemed to adsorb on the same bacterial receptor.     

Phage-typing ofSalmonella panama according to the method of Craigie and Yen

A phage-typing scheme forSalmonella panama has been established. This was done according to the method of Craigie and Yen by means of phage preparations which are all derived from a single phage. So far, eight phage types and a restgroup Z could be demonstrated by means of phage 47 and seven adaptations derived from it.     

Differentiation of methicillin-resistant strains of Staphylococcus aureus by prophage specificity]

By inducing with mitomycin C the following phages were isolated from all the tested 32 methicillin resistant strains of S. aureus: the serogroup B phage was isolated from 2 strains, the serogroup B and F phages were isolated from 5 strains and the serogroup F phage was isolated from 25 strains. The phages were divided into 5 groups by the antiphage immunity. In group 1 of the phages 4 additional phages were specified. By the specificity of the prophages in the cultures all the strains were divided into 5 groups. Group 1 of the cultures was divided into 5 subgroups (A, B, C, D and E).     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

MRSA的7种新SCCmec型别及其抗药特性

为探明海口地区耐甲氧西林金黄色葡萄球菌(MRSA)的耐药性和携带的葡萄球菌染色体mec盒(SCCmec)型别,对收集的1174株金黄色葡萄球菌用PBP2a检测法确证为MRSA有686株,用多重PCR对58株进行SCCmec分型测定,并用K-B琼脂扩散法和E-test法测定其对临床常用7类抗生素的代表性药物耐药性。结果在17株中又发现了7种新的SCCmec型别,其结构特点为:New3含A、F、H、M4个位点,New4型含F、H、M3个位点,New5含D、B、M3个位点,New6型含A、B、M3个位点,New7型含H、E、C、M4个位点,New8型含A、M两个位点,New9型含A、C、M3个位点;它们均与报道型别的结构特点存在明显差异;且携带新型的MRSA菌株,其分布特点及抗药性也与已报道的菌株存在差异:多分自门诊病人,且耐药性高,抗药谱较广,值得引起高度重视和关注。     

MRSA的7种新SCCmec型别及其抗药特性

为探明海口地区耐甲氧西林金黄色葡萄球菌(MRSA)的耐药性和携带的葡萄球菌染色体mec盒(SCCmec)型别,对收集的1174株金黄色葡萄球菌用PBP2a检测法确证为MRSA有686株,用多重PCR对58株进行SCCmec分型测定,并用K-B琼脂扩散法和E-test法测定其对临床常用7类抗生素的代表性药物耐药性。结果在17株中又发现了7种新的SCCmec型别,其结构特点为:New3含A、F、H、M4个位点,New4型含F、H、M3个位点,New5含D、B、M3个位点,New6型含A、B、M3个位点,New7型含H、E、C、M4个位点,New8型含A、M两个位点,New9型含A、C、M3个位点;它们均与报道型别的结构特点存在明显差异;且携带新型的MRSA菌株,其分布特点及抗药性也与已报道的菌株存在差异:多分自门诊病人,且耐药性高,抗药谱较广,值得引起高度重视和关注。     

Incidence of resistance to ampicillin,chloramphenicol, kanamycin,tetracycline and trimethoprim of Salmonella strains isolated in The Netherlands during 1975–1980

From 1975–1980, about 130 000 Salmonella strains isolated from various sources were tested for resistance to ampicillin, chloramphenicol, kanamycin, tetracycline and trimethoprim. Following the ban on incorporation of tetracycline in animal feeds for nutritive purposes, tetracycline resistance in S. typhimurium and S. panama strains of porcine origin dropped from about 90% in 1974 for both species, to about 34% and 1%, respectively, in 1980. The incidence of resistance in human strains concurrently decreased from about 80% in 1974 to 25% and 1%, respectively, in 1980.The build-up of multiple resistance in bovine S. dublin and S. typhimurium strains, already started in 1973–1974, has continued. Recently, phage type 193 S. typhimurium strains have become predominant and they are invariably resistant to ampicillin, chloramphenicol, tetracycline, kanamycin, neomycin, streptomycin, sulphonamide and trimethoprim. Up to now, type 193 strains were hardly encountered in human patients, but the number of human isolates is slowly increasing.A fairly large number of multiply resistant strains belonging to S. oranienburg, S. schwarzengrund, S. typhimurium and, recently, S. krefeld have been isolated from adoptive children from the Far East.     

Localization of prophages of serological group B and F on restriction fragments defined in the restriction map of Staphylococcus aureus NCTC 8325

Abstract Several Staphylococcus aureus strains were lysogenized by the phages of serological group B (phages φ53, φ85) as well as by some of serological group F (phages φ77, φ84) and macrorestriction fragment patterns of genomic DNA were estimated in the lysogenized, non-lysogenic and delysogenized (cured of prophages) strains. It was shown that the integration of phage DNA into chromosome of S. aureus leads to specific changes in restriction fragment pattern in all the lysogenized strains. These changes correlate well with the Sma I restriction map of S. aureus NCTC 8325 since they concern the restriction fragments defined in this map. Phages φ53 and φ85 integrate into Sma I fragment B. On the other hand, phages φ77 and φ84 integrate into Smal fragment E of the S. aureus restriction map. The prophages of strain NCTC 8511 have their integration sites, as follows: the phage designated by us φM integrates in fragment A, whereas the integration site for phage φJ lies in fragment E. Phage φM was estimated to be genetically related to phages of serological group A and phage φJ to those of serological group F. Evidence was given that lysogenization of S. aureus strains by at least four prophages does not cast any doubt upon the estimation of their genetic relatedness based on their similarity in restriction pattern.     

Characterization and rapid identification of phylogroup G in Escherichia coli,a lineage with high virulence and antibiotic resistance potential

The phylogeny of the Escherichia coli species, with the identification of seven phylogroups (A, B1, B2, C, D, E and F), is linked to the lifestyle of the strains. With the accumulation of whole genome sequence data, it became clear that some strains belong to a group intermediate between the F and B2 phylogroups, designated as phylogroup G. Here, we studied the complete sequences of 112 strains representative of the G phylogroup diversity and showed that it is composed of one main sequence type complex (STc)117 and four other STcs (STc657, STc454, STc738 and STc174). STc117, which phylogeny is characterized by very short internal branches, exhibits extensive O diversity, but little H-type and fimH allele diversity, whereas the other STcs are characterized by a main O, H and fimH type. STc117 strains possess many traits associated with extra-intestinal virulence, are virulent in a mouse sepsis model and exhibit multi-drug resistance such as CTX-M production. Epidemiologic data on 4,524 Australian and French strains suggest that STc117 is a poultry-associated lineage that can also establish in humans and cause extra-intestinal diseases. We propose an easy identification method that will help to trace this potentially virulent and resistant phylogroup in epidemiologic studies.     

Biological characteristics of Staphylococcus aureus isolated from nude mice

The characteristics of 50 strains of Staphylococcus aureus isolated from BALB/c nude mice (nu/nu, nu/+) with or without subcutaneous abscesses [13] were examined. All the 50 strains belonged to biotype B according to the classification by Hájek and Marsálek. All of them were phage typable, showing a single phage pattern of 52A/79/47/53/77/83A/85. The coagulase type was classified as VII. All of the 50 strains were highly sensitive to penicillin, methylphenylisoxazolyl penicillin, erythromycin, spiramycin, lincomycin, chloramphenicol, tetracycline, kanamycin, gentamicin and cephaloridine, but were resistant to sulfisoxazole. Two S. aureus strains isolated from the nostril and finger of one person working in the mouse colony were identified as the same biotype as the murine strains but different in phage type, coagulase type and drug resistance pattern.     

Gene Organization and Sequence Determination of the Two Botulinum Neurotoxin Gene Clusters in Clostridium botulinum Type A(B) Strain NCTC 2916

The gene organization and nucleotide sequence of the type A and B BoNT-gene clusters in Clostridium botulinum strain NCTC 2916 were studied. The aim was to clarify the organization of genes within C. botulinum type A strains possessing an unexpressed BoNT/B gene. The BoNT/A-gene cluster includes genes encoding BoNT, NTNH and a part of P-47 (the gene for this protein was reported in strains of C. botulinum types E and F). Clustered with the silent BoNT/B gene were genes encoding NTNH, P-21 and HA-33. Sequencing analysis of the NTNHs revealed the presence of 471 amino acids identical in the type B and A gene clusters. This gene organization contrasts markedly with the purported organization in strain NCTC 2916 described by Henderson et al. (FEMS Microbiol. Lett. 140, 151–158). In type A(B) strain NCTC 2916, the neurotoxin gene is of type BoNT/A1 within a gene cluster that has identical organization to that found in BoNT/A2 type strains; these observations may be significant in establishing the origin of the BoNT-gene cluster. Received: 28 July 1997 / Accepted: 15 October 1997     

Morphological study of five bacteriophages of yellow-pigmented enterobacteria

Two bacteriophages isolated onEnterobacter cloacae (C2, C2F) and three isolated onErwinia herbicola (E3, E16P, E16B) were purified by D₂O gradient centrifugation. Phage-containing fractions were negatively stained and examined by electron microscopy. Phages C2, C2F, E3, and E16P showed an elongated head 153×51 nm and a short noncontractile tail 12 nm long terminated by at least two short fibers. These phages correspond to the rate taxonomic group C3. Big capsomeres composing the phage head were evidenced when phage suspensions in D₂O were stained. Phage E16B showed an elongated head 97×40.5 nm, and a contractile tail 89 nm long. This phage corresponds to the extremely rate group A3.     

The cell wall receptor for bacteriophage Mu G(−) in Erwinia and Escherichia coli C

Abstract Adsorption of bacteriophage Mu with its invertible DNA segment in the G(−) orientation requires a terminal glucose residue for binding to the core lipopolysaccharide (LPS) of Gram-negative bacteria. Analysis of a Mu-resistant mutant shows that the receptor for Mu G(−) in Erwinia B374 is a Glc-β1,6-Glc disaccharide. A spontaneously occurring host-range mutant, Mu G(−)h101, grows on Escherichia coli C. The loss of the terminal β1,3-linked glucose from the LPS of E. coli C leads to resistance to the phage Mu. These mutants are also resistant to phage P1 and D108 which have largely homologous G segments. This shows that Mu G(+) and G(−) phage particles differ with respect to their cell-wall receptors in the type of glycosidic linkage of a terminal glucose residue: α1, 2 for G(+) and β1,6 for G(−).     

SEROLOGY AND TRANSDUCTION IN STAPHYLOCOCCAL PHAGE.

Dowell, C. E. (The University of Texas, Dallas) and E. D. Rosenblum. Serology and transduction in staphylococcal phage. J. Bacteriol. 84:1071-1075. 1962.-A triply lysogenic strain of Staphylococcus aureus was shown to carry a serological group B phage capable of transduction. Three typing phages (53, 80, 42D), either belonging to serological group B or having a close association with it, were also shown to have transducing ability. A rapid screening method was used to isolate two new transducing phages, both of which belonged to serological group B. Propagating strain 42B/47C was found to carry a transducing phage that was neutralized by both group B and group F antisera. Nine other phages belonging to serological groups other than group B did not have generalized transducing ability, nor did three group B typing phages that were atypical in their calcium requirement. It was postulated that transducing ability is associated with staphylococcal phages of serological group B and with related phages of group F.     

A sub-division ofSalmonella typhimurium into phage types based on the method of craigie and yen; Phages adaptable to species of the B and D group ofSalmonella; Phase adsorption as diagnostic aid

Summary and conclusions Phages were isolated which were adsorbed bySalmonella strains of groups B and D. These phages were adaptable to strains of species from these groups. The behaviour of some adaptations of these phages on strains ofS. paratyphi B has been described. A typing scheme forS. typhimurium has been established. So far, this consists of 32 types which have all been demonstrated with adaptations of a single phage.     

Radiation inactivation ofSalmonella panama andEscherichia coli K 12 present on deep-frozen broiler carcasses

Summary Low doses of ionizing radiation have been used to extend the shelf life of refrigerated poultry carcasses and to reduce the numbers of Salmonellae present. This report gives results of experiments on irradiation of deep-frozen poultry carcasses which were, before freezing, artificially contaminated withSalmonella panama and with a nalidixic acid-resistantEscherichia coli K 12. The D-values (decimal reduction) obtained with the inoculated carcasses were compared with D-values obtained with carcasses which were slaughtered in the normal way.The D-values forS.panama and forE.coli K 12 were 64.9 krad and 55.9 krad in the dripwater. Under commercial conditions approximately 100 krad were required for one decimal reduction of the Enterobacteriaceae present.The D-values estimated on the skin were higher forS.panama than forE. coli K 12 (128.6 krad vs 57.6 krad). If it is assumed that 1 positive carcass in 10,000 is allowed, the deep-frozen carcasses should be irradiated with doses of at least 700 krad to be sure of the absence of the testedS. panama strain.     

Genetic interrelationships of proteolyticClostridium botulinum types A,B, and F and other members of theClostridium botulinum complex as revealed by small-subunit rRNA gene sequences

The phylogenetic interrelationships of members of theClostridium botulinum complex of species was investigated by direct sequencing of their 16S rRNA genes. Comparative analysis of the 16S rRNA sequences demonstrated the presence of four phylogenetically distinct lineages corresponding to: i) proteolyticC. botulinum types A, B, and F, andC. sporogenes, ii) saccharolytic types B, E and F, iii) types C and D andC. novyi type A, and iv) type G andC. subterminale. The phylogenetic groupings obtained from the 16S rRNA were in complete agreement with the four divisions recognised within the species complex on the basis of phenotypic criteria.