Genotyping of Epidemic Methicillin-Resistant Staphylococcus aureus Phage Type 15 Isolates by Fluorescent Amplified-Fragment Length Polymorphism Analysis

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for its ability to identify and subtype isolates of an epidemic methicillin-resistant phage type of Staphylococcus aureus, EMRSA-15. These isolates were also characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) of the coagulase gene and pulsed-field gel electrophoresis (PFGE). For FAFLP, DNA was double digested with restriction enzymes ApaI plus TaqI or EcoRI plus MseI. Site-specific adaptors were ligated to one or the other set of restriction fragments, and PCR amplification was carried out with adaptor-specific primers. Amplified fragments separated on an ABI 377 automated sequencer and analyzed with Genescan version 2.1 software generated FAFLP profiles for all the isolates. The presence or absence of fragments was scored, similarity coefficients were calculated, and UPGMA (unweighted pair group method using arithmatic averages) cluster analysis was performed. Either enzyme-primer combination readily differentiated EMRSA-15 from other methicillin-resistant S. aureus (MRSA) isolates and also revealed heterogeneity within the phage type. The discriminatory power of FAFLP was high. By combining both enzyme-primer data sets, 24 isolates were divided into 11 profiles. PCR-RFLP did not discriminate among these EMRSA-15 isolates. PFGE could discriminate well between isolates but was not as reproducible as FAFLP. All S. aureus and MRSA isolates in this study were typeable by FAFLP, which was easy to perform, robust, and reproducible, with evident potential to subtype MRSA for purposes of hospital infection control.     

Predictive modelling of fluorescent AFLP: a new approach to the molecular epidemiology of E. coli

Amplified fragment length polymorphism (AFLP) permits simultaneous sampling of multiple loci distributed throughout a genome, using restriction site/adaptor-specific primers under stringent conditions. Fluorescent detection instrumentation further refines this methodology, permitting internal size standards and accurate, reproducible sizing of amplified fragments. We have evaluated the potential of fluorescent AFLP (FAFLP) as a potentially definitive genotyping method for bacteria, by comparing MseI/EcoRI fragments derived experimentally from the Escherichia coli K12 MG1655 genome with those predicted by analysis of its published sequence. In silico, MseI/EcoRI digestion of this sequence produced 1200 fragments from 36 and 2151 base pairs (bp) in size. Fragment subsets which would be amplified by seven different selective (1-2 bases added to the 3' end of the core primer sequence) primer combinations were modelled. Depending on the primer pair, three to 54 fragments (range 70-400 bp) were predicted, while all seven primer pair combinations together generated 121 predicted fragments. When genomic DNA of strain MG1655 was subjected to experimental FAFLP with these seven primers, 111 correctly sized fragments were observed (+/- 1 bp) out of the 121 predicted (92% accuracy). Twenty-five unpredicted fragments were obtained; an average of four per primer pair. The size and number of fragments in FAFLP, and their gel distribution, were dictated by the choice of restriction endonucleases and the degree of primer selectivity. Our data show that FAFLP is accurate, discriminatory, reproducible and capable of standardisation. Under agreed conditions, this method shows considerable promise as a generally applicable standardised bacterial genotyping method. The fragments predicted in silico to result from amplification of MseI/EcoRI-digested DNA with the seven primer pairs described are here used to define a prototypic FAFLP analysis of E. coli.     

Molecular cloning and restriction endonuclease mapping of the murine cytomegalovirus genome (Smith strain)

We have cloned EcoRI and HindIII fragments of the Smith strain of murine cytomegalovirus (MCMV) in the plasmid vector pACYC184. These cloned fragments were used to establish a restriction endonuclease map of the genome with respect to the EcoRI and HindIII sites. The map was constructed on the basis of data derived from cross-hybridizations of EcoRI and HindIII cloned fragments, double-digestions of the cloned fragments with EcoRI and HindIII, and hybridization of cloned HindIII fragments to Southern blots of MCMV DNA cleaved with EcoRI. From our mapping data, we have determined that the length of the MCMV genome is approximately 240 kbp. The genome does not appear to undergo inversions and lacks detectable repeated sequences. One HindIII cloned fragment was obtained which contained both HindIII termini. The existence of this fragment may be related to the mode of replication of the MCMV genome.     

Chromosomal localization of a highly repeatedEcoRI DNA fragment inMegoura viciae (Homoptera,Aphididae) by nick translation and fluorescencein situ hybridization

To investigate the genome of the aphidMegoura viciae at molecular level, we have studied total DNA by agarose gel electrophoresis after cleavage with different restriction endonucleases.EcoRI digestion produced a highly repeated DNA fragment, about 600 bp long. The contribution of thisEcoRI element to the total genome ofM. viciae was estimated at about 6% by means of densitometric scanning of agarose gel photographs. The chromosomal localization of this fragment, investigated by fluorescentin situ hybridization (FISH), constantly showed one large and two narrower fluorescent bands located on the X chromosome, all corresponding to C-positive heterochromatic areas. These results are in full accordance with the data obtained byin situ nick translation experiments carried out afterEcoRI digestion, and clearly demonstrate that a substantial amount ofM. viciae heterochromatin consists ofEcoRI fragments which are mainly located on the X chromosome. Using theEcoRI restriction fragment as a molecular probe may prove to be a practical tool for the investigation of taxonomic and evolutionary relationships in this group of insects.accepted for publication by J. S. (Pat) Heslop-Harrison     

Experimental versus in silico fluorescent amplified fragment length polymorphism analysis of Mycobacterium tuberculosis: improved typing with an extended fragment range

Whole-genome fingerprinting fluorescent amplified fragment length polymorphism (FAFLP) data were compared with in silico data for the sequenced strains of Mycobacterium tuberculosis (H37Rv and CDC1551). For this G+C-rich genome, many predicted fragments were not detected experimentally. For H37Rv, only 108 (66%) of the 163 predicted EcoRI-MseI fragments between 100 and 500 bp were visualized in vitro. FAFLP was also used to identify polymorphism in 10 clinical isolates of M. tuberculosis characterized previously by IS6110 typing, examining fragments of up to 1,000 bp in size rather than up to 500 bp as was done previously. Five isolates had unique IS6110 profiles and were not known to be epidemiologically related, two isolates were the same single-band IS6110 type but were not known to be epidemiologically related, and the remaining three isolates were epidemiologically related with identical IS6110 profiles. Analysis of fragments in the 500- to 1,000-bp range using nonselective primers differentiated better between strains than analysis of fragments in the 50- to 500-bp range using a set of four selective primers. Seventeen polymorphic fragments were identified between 500 and 1,000 bp in size compared with nine polymorphic fragments between 50 and 500 bp. Using the 500- to 1,000-bp analysis, a level of discrimination similar to that of IS6110 typing was achieved which, unlike the IS6110 typing, was able to differentiate the two M. tuberculosis strains, each of which had only a single copy of IS6110.     

Molecular cloning of African swine fever virus DNA

African swine fever virus DNA (about 170 kbp) was cleaved with the restriction endonuclease EcoRI and most of the resulting 31 fragments were cloned in either the phage vector λWES.λB or the plasmid pBR325. Three fragments were not cloned in those vectors, the largest fragment EcoRI-A (21.2 kbp) and the two crosslinked terminal fragments, EcoRI-K′ and D′. Endonuclease SalI cut fragment EcoRI-A into three pieces which were cloned in plasmid pBR322. The two terminal EcoRI fragments were cloned after removal of the crosslinks with nuclease S1 and addition of EcoRI linkers to the fragment ends. The complete library of the cloned fragments accounted for about 98% of ASF virus genome, the missing sequences being those removed by the nuclease S1 in the process of cloning the terminal fragments.     

Fluorescent amplified-fragment length polymorphism subtyping of the Salmonella enterica serovar enteritidis phage type 4 clone complex

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis, a high-resolution PCR-based genome fingerprinting method, was used to subtype Salmonella enterica serovar Enteritidis phage type 4. This single phage type is responsible for the majority of salmonellosis in Europe. Twenty strains isolated from nine outbreaks, five isolates from sporadic cases of human infection, four strains of poultry origin, and one laboratory-derived strain were comparatively studied by pulsed-field gel electrophoresis (PFGE) and FAFLP analysis. Following macrorestriction with XbaI, PFGE classified 73% of PT4 strains as a single type. FAFLP analysis was carried out with the primer pair EcoRI+0 and MseI+C, by simultaneously sampling 170 to 190 loci throughout the PT4 genome. Twenty-three FAFLP profiles, with 1 to 61 amplified-fragment differences, were found among the 30 strains. The index of discriminatory power of FAFLP analysis was 0.98, compared to 0.47 for PFGE. FAFLP analysis assigned genotypes to each PT4 outbreak, as well as sporadic PT4 infections, a significant development for the epidemiology and control of this zoonotic enteric pathogen.     

Isolation and characterization of cloned fragments of bacteriophage P1 DNA.

Fragments of PI DNA generated by endo·R·EcoRI and endo·R·BamHI were mixed with appropriate cloning vectors (ColElAp^r, pBR313, pBR322) ligated in vitro and used to transform a nonsuppressing strain of Escherichia coli. In this way clones of 9 of the 26 EcoRI fragments and 5 of the 14 BamHI fragments were obtained. Marker rescue tests were used to ascertain which regions of the P1 genome were contained on the various DNA fragments. These cloned fragments are useful as probes for specific regions of the P1 genome.     

Partial characterization of herpes simplex virus type 2 (HSV-2)-specific poly(A)+ RNA by hybridization to EcoRI-generated HSV-2 DNA fragments.

Polyadenylated RNA appearing in polyribosomes from Herpes simplex virus type 2 (HSV-2)-infected cells has been isolated at different stages of infection. The amounts of virus-specific RNA hybridizing to individual EcoRI fragments of HSV-2 DNA have been detected. Templates coding for “early” virus-specific RNA are widely dispersed within the HSV-2 genome in the short and long segment since each of the EcoRI fragments showed hybridization with “early” isolated poly(A)+ RNA. The virus-specific fraction of labeled poly(A)+ RNA which hybridized to each of the EcoRI fragments increased with 1-β-d-arabinofuranosylcytosine-treated (Ara-C) poly(A)+ RNA as well as with “late” poly(A)+ RNA. Poly(A)+ RNA from infected, Ara-C-treated cells contained virus-specific RNA which hybridized to each of the EcoRI fragments. We found that the poly(A)+ RNA, specific for one EcoRI fragment, varied with the stage of infection, comparing it to other fragment-specific poly(A)+ RNAs.     

Fluorescent amplified-fragment length polymorphism genotyping of Neisseria meningitidis identifies clones associated with invasive disease

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.     

High-Resolution Genotyping of Streptococcus pyogenes Serotype M1 Isolates by Fluorescent Amplified-Fragment Length Polymorphism Analysis

We have used fluorescent amplified-fragment length polymorphism (FAFLP) analysis to subtype clinical isolates of Streptococcus pyogenes serotype M1. Established typing methods define most M1 isolates as members of a clone that has a worldwide distribution and that is strongly associated with invasive diseases. FAFLP analysis simultaneously sampled 90 to 120 loci throughout the M1 genome. Its discriminatory power, precision, and reproducibility were compared with those of other molecular typing methods. Irrespective of disease symptomatology or geographic origin, the majority of the clinical M1 isolates shared a single ribotype, pulsed-field gel electrophoresis macrorestriction profile, and emm1 gene sequence. Nonetheless, among these isolates, FAFLP analysis could differentiate 17 distinct profiles, including seven multi-isolate groups. The FAFLP profiles of M1 isolates reproducibly exhibited between 1 and more than 20 amplified fragment differences. The high discriminatory power of genotyping by FAFLP analysis revealed genetic microheterogeneity and differentiated otherwise "identical" M1 isolates as members of a clone complex.     

Activation of mouse retrovirus by herpes simplex virus type 1 cloned DNA fragments

Cloned DNA fragments from herpes simplex virus (HSV) type 1 (strain Patton) were tested for activation of endogenous mouse retrovirus in BALB/3T3 cells. Activation within the L region of HSV-1 DNA was observed with the ～3.4-kilobase pair (kbp) BamHI fragment which contains the virus thymidine kinase (TK) gene, and the ～5.3-kbp EcoRI L fragment. Activation by the TK-containing BamHI fragment was abrogated by digestion with EcoRI. Activation within the S region of HSV-1 DNA was observed with the ～15.2-kbp EcoRI H ragment and the ～8.4-kbp Eco RI/HindIII H/G fragment. Assaying for retrovirus activation serves as an additional parameter for mapping biological functions within the HSV genome.     

Identification,mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus

The thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) was identified by biochemical transformation of 3T3 TK negative (TK⁻) to 3T3 TK positive (TK⁺) cells using specific viral DNA sequences. DNA fragments of the viral genome used in this study were obtained from a defined gene library of FLDV genome containing the complete viral DNA sequences. The selection of the converted cells was carried out under the condition of the HAT selection procedure. The results of these experiments revealed that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611 to 0.718 map units) is able to transform 3T3 TK⁻ to 3T3 TK⁺ cells. Additional experiments using the subclones of EcoRI DNA fragment C revealed that DNA sequences of 4.1 kbp size between the coordinates 0.669 to 0.718 of the FLDV genome possessed the ability for biochemical transformation, indicating that the TK gene locus is located in this particular region.     

Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome

Summary To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.     

Structure of the cauliflower mosaic virus genome. II. Variation in DNA structure and sequence between isolates

The DNA genomes of four different cauliflower mosaic virus (CaMV) isolates (Cabbage B-Davis, Cabbage B-John Innes, CM4–184, and Australian) have been mapped using the restriction endonucleases, EcoRI, SalGI and BamHI, and S₁ nuclease. The differences between maps of the isolates indicate that one region of the CaMV genome is subject to considerable change while the rest is more highly conserved. EcoRI digestion patterns of the DNA from five other CaMV isolates correspond to two of the described isolates, Cabbage B-Davis and Cabbage B-John Innes. Minor DNA fragments (in less than molar amounts) appearing in restriction patterns of CaMV DNAs could be attributed to: (i) the presence of linear DNA molecules which appear to arise by double-strand breaks at specific points in the circular genome, (ii) heterogeneity within some of the viral ioslates, indicated by the appearance of a faint, underlying DNA restriction pattern characteristic of another isolate type, and (iii) the occurrence of a few “difficult” restriction sites in the genomes of some isolates.     

Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32EcoRI CIV DNA fragments), was used for transfection ofChoristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring theEcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535–0.582 m.u.), M (7.25 kbp, 0.310–0.345 m.u.), O (6.5 kbp, 0.196–0.228 m.u.), Q (5.9 kbp, 0.603–0.631 m.u.), and Y (2.0 kbp, 0.381–0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of theEcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within theEcoRI DNA fragments C and H at the genome coordinates 0.924–0.930 and 0.535–0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of theEcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661–675 and 677–691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of theEcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535–0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within theEcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661–691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within theEcoRI CIV DNA fragment Y.     

Different restriction profiles of χ prophage in Serratia marcescens K and HY

Temperate phage χ originated from the defectively lysogenic Serratia marcescens strain K, from where it was liberated after uv irradiation with low efficiency. The phage is usually indicated on strain HY that can be easily lysogenized by it and rather efficiently uv-induced. Comparing the Eco RI restriction profiles of the χ prophage in HY revealed a DNA rearrangement, by which the precursor structure in K is converted into the non-defective form. Apparently the fragment containing the pac sequence is concerned since a phage DNA probe prepared from the assumed initiation fragments of the first particles of the packaging series gave two signals instead of one with genomic K DNA. Since several independent new χ isolates showed the same Eco RI restriction pattern as the original phage of ELLMAUER and KAPLAN (1959), the generation of χ is a reproducible event.     

Prolonged and large outbreak of invasive group A Streptococcus disease within a nursing home: repeated intrafacility transmission of a single strain

Objectives

Multiple invasive group A Streptococcus (GAS) infections were reported to public health by a skilled nursing facility (facility A) in Illinois between May 2014 and August 2016. Cases continued despite interventions including antibiotic prophylaxis for all residents and staff. Two other geographically close facilities reported contemporaneous outbreaks of GAS. We investigated potential reasons for ongoing transmission.

Restriction endonuclease Hin dIII cleavage site map of bacteriophage P22.

The 14 HindIII cleavage sites on P22 DNA have been mapped. HindIII cleavage sites were located relative to EcoRI sites by determining the molecular weights and map order of fragments produced by HindIII, or HindIII and EcoRI digestion. Molecular weights were estimated from the electrophoretic mobility of fragments. The HindIII fragment order was established by HindIII cleavage of segments of the P22 genome obtained as isolated EcoRI fragments or as overlapping genetic substitutions in bacteriophage λ chromosomes. The resulting HindIII/EcoRI cleavage site map defines physical markers in all regions of the P22 genome and defines the locations of a number of P22 genes on this physical map of the P22 chromosome. Three HindIII sites and two HpaI sites have been mapped in immI, one of two P22 gene clusters controlling lysogeny. Two of these HindIII sites lie within the structural gene ant specifying one of the regulatory proteins of the immI region. Assignment of the ant gene to specific HindIII fragments utilized the insertion element Tnl, which was shown to contain no HindIII cleavage sites.     

Identification of the P1 compatibility and plasmid maintenance locus by a mini P1/ac+-plasmid.

pIH1972 is a 23.6-Mdal lac⁺ plasmid derived from P1dlw. This plasmid is stably maintained and it exhibits absolute incompatibility with P1 in RecA bacteria. A cluster of P1-EcoRI DNA fragments 15, 17, 18, 21, and 23 and part of fragments 14 and 5 are identified in the PI DNA region carried by pIH1972. It is suggested that P1 compatibility and plasmid maintenance genes are carried on the identified 10% segment of the P1 genome.