DNA Restriction Endonuclease Patterns Research Articles

Development of antimicrobial resistance in Staphylococcus lugdunensis during treatment—report of a case of bacterial arthritis, vertebral osteomyelitis and infective endocarditis

Staphylococcus lugdunensis is a recently described coagulasenegative staphylococcus (CNS) that has been associated with a wide range of human infections [1,2]. The most frequent type of infection reported is infective endocarditis which may involve prosthetic as well as native valves [3]. In this paper we present a patient with bacterial arthritis in both knee joints (one with and one without an arthroplasty), vertebral osteomyelitis and infective endocarditis of nativemitral and aortic valves. The patient was a 79-year-old male, non-smoking, former lumberjack with a history of psoriasis and hypertension. He had seropositive rheumatoid arthritis with general joint aiection. His rheumatoid arthritis was treated with corticosteroids, methotrexate and non-steroidal anti-in£ammatory drugs. He underwent total replacement of the left knee in November 1992 because of severe osteoarthritis. The right knee did not show any signs of osteoarthritis until the end of 1994. He was admitted in April 1995 due to cellulitis of the right elbow and an infected ulcer on the left foot. Blood cultures showed growth of CNS, later veri¢ed to be S. lugdunensis. After initial intravenous treatment with benzylpenicillin and dicloxacillin for 2 days, the patient was treated with oral clindamycin for 2 weeks and the clinical course was uneventful. On follow-up at the beginning of May, the patient was well and the C-reactive protein (CRP) serum concentration was 21mg/L. Nine days later he was readmitted due to bacterial arthritis of the right knee. Cultures were positive for CNS (later shown to be S. lugdunensis) in both blood and synovial £uid. This time, treatment comprised intravenous cloxacillin for 6 days, then oral £ucloxacillin 1g three times a day for 6weeks, and then £ucloxacillin 0.5 g three times a day for 7months, in accordancewith the susceptibility test. A systolic heart murmur, not previously recognized, led to a transthoracic echocardiography (TTE) being performed in August 1995. The TTE showed mild-to-moderate aortic and mitral valve regurgitation. No vegetations were seen. The patient was followed with repeated determinations of CRP and ESRvalues (CRP 10^20mg/L and ESR 20^30mm/1h) for several months, pending total replacement of the right knee because of osteonecrosis.This was performed in November 1995. Antibiotic prophylaxis was given as intravenous cloxacillin 2 g three times a day for 7 days, and the patient was discharged with oral £ucloxacillin 0.5 g three times a day, whichwas continued until January1996. In June 1996 the patient was admitted once more but now presented symptoms of swelling, pain, eiusion and warmth of the left prosthetic knee. Arthrocenthesis was performed and the patient was given oral £ucloxacillin. The symptoms recurred over the following year.The CRP levels were 40^80 mg/L and the ESRwas 50^100mm/1h when the symptoms were present. X-ray investigation of the left knee from this period showed signs of osteitis. Altogether, six cultures of synovial £uid were taken from the left knee during this period, and they all showed growth of CNS, which in February 1997 was typed as S. lugdunensis. Initially, the patient was treated with oral £ucloxacillin, but repeated susceptibility tests showed that the isolate had developed resistance to £ucloxacillin in December1996 and the treatment was changed to cipro£oxacin (Table1). Later, the isolated strain also showed a signi¢cantly increasedMIC for cipro£oxacin, and the antibiotic treatment was changed to clindamycin. In June 1997 the patient presented with a history of lower back pain and arthralgia of the hips, and at this time the CRP was 175mg/L. The antibiotic regimen was changed to oral rifampicin plus cipro£oxacin.The patient improved, the CRP level decreased, and the patient remained well until the end of August 1997, when he was readmitted due to fever, back and hip pains. A magnetic resonance imaging (MRI) scan showed signs of vertebral osteomyelitis, andTTE showed calci¢cation of the aortic and the mitral valves but no signs of endocarditic vegetations. Mild-to-moderate mitral and aortic valve regurgitation was present, but no signs of cardiac decompensation. Blood cultures showed growth of S. lugdunensis, and the strain had now developed resistance to rifampicin. Corticosteroids CONCISE COMMUNICATIONS

Characterization of Staphylococcus intermedius from healthy dogs and cases of superficial pyoderma by DNA restriction endonuclease patterns

In order to study the possible clonal relation of Staphylococcus (S.) intermedius from canine superficial pyoderma and from healthy carriers, isolates from pustular swabs and from vaginal, nasal and normal skin swabs were typed using macrorestriction analysis with Sma I and pulsed-field gel electrophoresis. From the size of the resulting fragments the size of the chromosomes of S. intermedius could be determined to be roughly 1500 ± 200 kb on the average. The fingerprints were very heterogeneous though characteristically distinct from patterns of (human) S. aureus as published by others. Strains from superficial pyoderma were not found to be more similar to each other than strains from healthy carriers. Therefore it was concluded that strains from this type of skin infection probably did not belong to a certain subpopulation of S. intermedius, which might have indicated a higher virulence of these strains.

In vivoR–plasmid transfer in a patient with a mixed infection of shigella dysentery

Transfer of shigella R-plasmids in vivo has seldom been demonstrated. Strains of Shigella dysenteriae type 1 and Shigella flexneri type 5b were isolated from a Bulgarian traveller who visited Vietnam and developed dysentery, which was treated with trimethoprim/sulfamethoxazole (TMP/SMZ) for a short time. Both species of shigellae are unusual in Bulgaria where strains of S. sonnei predominate. Both shigella strains were multiresistant to the same antimicrobial agents. Each strain contained a 48-kilobase plasmid that conferred the entire resistance phenotype to a susceptible Escherichia coli. Restriction endonuclease patterns of plasmid DNA from the respective strains were identical. Transmissible plasmids of the same resistance phenotypes and restriction patterns were isolated from the patient's colonic E. coli. Transconjugants hybridized to a dihydrofolate reductase type I-DNA probe. These studies support the hypothesis that R-plasmid transfer may occur between non-pathogenic, faecal strains and pathogenic shigellae, a process that may have been facilitated by inadequate treatment with TMP/SMZ at the onset of the illness.

Isolation and Characterization of Nuclear Polyhedrosis Viruses from the Beet Armyworm Spodoptera exigua (Lepidoptera: Noctuidae) Found in Shiga, Japan

Eleven larvae of the beet armyworm, Spodoptera exigua, showing symptoms of nuclear polyhedrosis virus (NPV) infection were collected in Shiga Prefecture, Japan. NPVs isolated from each larva were divided into two groups. Group 1 isolates produced cuboidal polyhedra, while group 2 isolates produced icosahedral polyhedra. The restriction endonuclease patterns of viral DNA within the same group were identical. The restriction endonuclease patterns of group 1 were similar to those of Autographa californica NPV (AcNPV), whereas the patterns f group 2 were similar to S. exigua NPV isolated in California. SDS-polyacrylamide gel electrophoresis analysis of polyhedra also indicated that SeNPVs differed between the two groups. Group 1 SeNPVs could infect the cell lines SF-21 and TN-368 and group 2 SeNPVs could not. Furthermore, group 1 SeNPVs were able to infect Spodoptera litura, Plutella xylostella, and S. exigua larvae but not Bombyx mori larvae. Group 2 SeNPVs could infect S. exigua larvae, but not S. litura, P. xylostella, or B. mori larvae. The LD50 of SeNPV #5 (group 1) and SeNPV #1 (group 2) were 58 and 4.8 polyhedra per larva, respectively. These results indicated that the SeNPVs isolated in Shiga Prefecture were composed of two genetically and phenotypically distinct groups.

Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from Bombyx mori NPV

We have isolated hybrid baculoviruses of Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica NPV (AcNPV) capable of replicating in both BmN (not susceptible to AcNPV) and SF-21 (not susceptible to BmNPV) cells (A. Kondo and S. Maeda, J. Virol. 65:3625-3632, 1991). Repeated backcross infection of one of these recombinant isolates with AcNPV generated eh-AcNPV, a virus with restriction endonuclease patterns of genomic DNA nearly identical to those of AcNPV but capable of replicating in both BmN and SF-21 cells, i.e., host range expanded. Expanded host range viruses were also isolated following cotransfection of AcNPV DNA with eh-AcNPV DNA cleaved with either HindIII or PstI. Subsequent cotransfection of AcNPV DNA with plasmids from an eh-AcNPV DNA fragment library identified an 11-kbp HindIII fragment that could expand the host range of AcNPV. Subcloning and cotransfection analyses localized a 572-bp SacI-HindIII fragment within this 11-kbp fragment which could alone expand the host range of AcNPV. Mapping and nucleotide sequencing analysis revealed that this fragment was identical to the corresponding 572-bp fragment (BmScH) of BmNPV. Furthermore, this fragment originated from the coding region of the putative DNA helicase gene. Cotransfection of AcNPV DNA with BmScH also generated a host range-expanded virus, eh2-AcNPV. These results indicated that the expanded host range characteristics of eh2-AcNPV were solely the result of recombination within the coding region of the putative DNA helicase gene.

Spontaneous insertion of an IS1-like element into the virF gene is responsible for avirulence in opaque colonial variants of Shigella flexneri 2a.

Colonial variation of Shigella flexneri serotype 2a from the translucent (2457T) to the opaque form (2457O) occurs spontaneously once in 10(4) cell divisions, with concomitant loss of ipa gene expression and virulence. The appearance of 2457O was associated with the insertional inactivation of virF, an invasion plasmid-encoded positive regulator of ipa gene expression. Plasmid pWR110, a Tn5-tagged invasion plasmid that restores the invasive phenotype to plasmid-cured Shigella derivatives, was conjugally transferred into 2457O. Synthesis of the invasion-associated IpaB and IpaC polypeptides, normally present on the surface of virulent shigellae, and the invasive phenotype were restored in 2457O(pWR110) transconjugants. Plasmid DNA restriction endonuclease patterns of 2457T and 2457O, along with hybridization analysis, showed that a SalI fragment carrying the virF gene in 2457O had increased in size relative to its counterpart in 2457T. Analysis of virF DNA sequences amplified by the polymerase chain reaction revealed that the virF sequence from 2457O was 780 bp larger than that amplified from 2457T. Moreover, the virF sequence amplified from 2457O hybridized to an IS1 DNA probe whereas the amplified 2457T virF sequence did not. DNA sequence analysis mapped the insertion element, designated IS1SFO, within an A.T-rich region of the virF open reading frame and identified a 9-bp virF target sequence that was duplicated at the insertion site of IS1SFO. The DNA sequence of IS1SFO was greater than 99% homologous to IS1F. Plasmid pWR600, carrying a 1,260-bp HpaII fragment encoding a wild-type virF gene, was able to restore the virulent phenotype and translucent colonial morphology to nine independently isolated 2457O hosts.

Restriction Endonuclease Analysis of Marek's Disease Virus DNA: Differentiation of Viral Strains and Determination of Passage History

The restriction endonuclease (RE) patterns of DNA from serotype 1 Marek's disease viruses (MDVs) are unique to serotype 1 viruses and can also be used to differentiate between low and high cell-culture-passaged viruses. We compared the RE patterns of DNA from seven serotype 2 and 3 MDVs before and after serial in vitro passage. Passage of four serotype 2 strains resulted in a variety of changes in the RE pattern. Individual strains within a serotype exhibited unique restriction patterns that allowed individual isolates to be differentiated. In a similar manner, the serotype 3 virus strains displayed RE pattern variations that were unique to each strain, as well as differences between low and high cell-culture passage. Our findings, together with earlier reports, suggest that the RE patterns of MDV DNA provide a simple and accurate method to: 1) differentiate between the three MDV serotypes, 2) differentiate between virus strains within a serotype, and 3) determine whether the viruses have been passaged extensively in cell culture.

Restriction endonuclease patterns of herpes simplex virus DNA: Subtyping of HSV-1 and HSV-2 strains from genital and nongenital lesions

Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) isolates from genital and nongenital infections were submitted to restriction endonuclease analysis for possible genomic changes in relation with the adaptation of the virus to a new site on the body. HSV-1 and HSV-2 strains were successfully divided into two subgroups using the Hinc II restriction enzyme. No correlation was found, however, between the proposed genomic subtypes H1A, H1B, H2A and H2B, and the genital or nongenital origin of the HSV strains.

Chloroplast DNA polymorphism in fertile and male-sterile cytoplasms of sorghum (Sorghum bicolor (L.) Moench)

Restriction endonuclease patterns of chloroplast DNA (cpDNA) were consistently distinguishable between fertile and male-sterile cytoplasms of sorghum [Sorghum bicolor (L.) Moench], whereas no differences in restriction patterns of cpDNA among male-sterile (A1) lines, including six isocytoplasmic strains, were revealed in this study. It is suggested that chloroplast DNA may contribute to the male sterility of A1 lines used currently in hybrid sorghum production.

Restriction site variation, length polymorphism and changes in gene order in the mitochondrial DNA of the yeast Kluyveromyces lactics

The purpose of this work was to compare mitochondrial DNA restriction endonuclease patterns in strains of the yeast Kluyveromyces lactis, from different sources, to see how conserved is the organization of this organellar genome. The mitochondrial DNA of five independently-isolated strains and one of unknown origin were compared. Strains NRRL Y-1205, NRRL Y-8279 and NRRL Y-1140 gave identical patterns. Strain NRRL Y-1564 showed an insertion, with respect to the other three, of approximately 1250 bp. Strain W600B had also an insertion with extra restriction sites for EcoRI, HpaI, HaeIII, HincII and XbaI. On the other hand, strain Y-123 showed a restriction pattern quite different from the others. Sequences putatively encoding apocytochrome b, ATPase subunit 9 and ribosomal RNA large subunit, were localized on the physical maps of three strains. Results demonstrated that the order of these three genes shows a common feature in strains W600B and WM37 (auxotroph of Y-1140) but a different distribution in WM27 (auxotroph derived from Y-123). All these facts explain the extensive intraspecific polymorphism observed in the mtDNA of this yeast.

Chloroplast DNA variation in diploid and polyploid species of Bromus (Poaceae) subgenera Festucaria and Ceratochloa

Chloroplast DNA (cpDNA) restriction endonuclease patterns are used to examine phylogenetic relationships between Bromus subgenera Festucaria and Ceratochloa. Festucaria is considered monophyletic based on the L genome, while Ceratochloa encompasses two species complexes: the B. catharticus complex, which evolved by combining three different genomes, and the B. carinatus complex, which is thought to have originated from hybridization between polyploid species of B. catharticus and diploid members of Festucaria. All species of subgenus Ceratochloa (hexaploids and octoploids) were identical in chloroplast DNA sequences. Similarly, polyploid species of subgenus Festucaria, except for B. auleticus, were identical in cpDNA sequences. In contrast, diploid species of subgenus Festucaria showed various degrees of nucleotide sequence divergence. Species of subgenus Ceratochloa appeared monophyletic and phylogenetically closely related to the diploid B. anomalus and B. auleticus of subgenus Festucaria. The remaining diploid and polyploid species of subgenus Festucaria appeared in a distinct grouping. The study suggests that the B. catharticus complex must have been the maternal parent in the proposed hybrid origin of B. carinatus complex. Although there is no direct evidence for the paternal parent of the latter complex, the cpDNA study shows the complex to be phylogenetically very related to the diploid B. anomalus of subgenus Festucaria.

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Extrachromosomal DNA analysis and restriction endunuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Rapid in situ generation of DNA restriction endonuclease patterns for Neisseria gonorrhoeae

Restriction endonuclease (RE) digestion patterns of 26 isolates of Neisseria gonorrhoeae representing different serovars of serogroups WI, WII, and WIII were generated by agarose pellet entrapment and in situ digestion with HinfI and BglII. The method was fast, simple, and reproducible, and stable RE patterns were produced from subsequent in vitro passages. The cost of culture materials was reduced considerably, and no toxic or flammable solvents needed to be used. Excellent resolution of DNA fragments of higher molecular weights was obtained as a result of minimal mechanical shearing of the DNA. The REs HinfI and BglII were discriminative in the fragment length ranges of 2 to 6.5 and 2.5 to 21.5 kilobases, respectively. On the basis of densitometric scanning of electrophoretograms generated by HinfI digestion, the 26 isolates representing 12 serovars were divided into seven groups. BglII was found to be more discriminative; 15 RE patterns were established among the 26 isolates. Patterns generated by both REs showed that there was no correlation between a particular RE pattern and a serovar, since strains with identical RE patterns were from different serovars. With the exception of two strains (D3 and D14), which demonstrated positive correlation when both enzymes were used, all strains with identical serovar patterns had different RE patterns.

Mitochondrial DNA Restriction Endonuclease Patterns of Sorghums at Various Ploidy Levels

Mitochondrial DNA from Sorghum versicolor (2n=10), Sorghum bicolor race bicolor (2n=20), and Sorghum halepense (2n=40) was digested with an endonuclease

Characteristics and comparative study of fiveRhizobium meliloti bacteriophages

FiveRhizobium meliloti bacteriophages isolated from soil or lysogenic bacteria and belonging to Bradley's group B or theSiphoviridae family of tailed phaes were studied. They are of identical morphology, showing isometric heads and long, noncontractile tails with transverse bars. They are temperate and closely related by host range, DNA restriction endonuclease patterns and homology, DNA mass, serological properties, adsorption velocity, and latent period. However, the phages can be divided into three groups on the basis of burst size and frequency of lysogenization.

PM2 bacteriophage plaque morphology mutants exhibit differences in DNA length and restriction endonuclease patterns

A large plaque (LP) and a small plaque (SP) variant of PM2 bacteriophage were isolated from a mixture of the two plaque variants and were grown separately in the appropriate host bacterium,Alteromonas espejiana. They have remained pure for approximately one year from the original isolation. Restriction endonuclease analyses revealed differences in theHaeIII restriction profile between the two variants.HaeIII fragment 1 of the SP DNA was found to be smaller than the corresponding fragment from the LP variant DNA, whereas fragment 7 from the SP DNA was slightly larger than the same fragment from LP DNA. Electron microscopy of heteroduplexes formed between the DNAs from the two variants revealed that the deletion in fragment 1 mapped very close to the junction betweenHaeIII fragments 1 and 13 on the physical map of PM2 DNA. The difference in DNA length between the two variants results from addition or deletion mutations.

Occurrence and transfection of a Giardia virus.

The presence of virus-derived RNA was investigated in 38 axenically growing Giardia isolates from different geographic areas. The RNA virus was demonstrated in Giardia strains from humans in the U.S.A., England and the majority of strains from Poland. Two strains isolated respectively from a cat and a cavia also contained it. Giardia strains from humans in Belgium and Israel did not contain this RNA virus. Transfection of the RNA virus was accomplished from English and Polish strains, as well as from the cat isolate to isolates lacking it. Differences were observed both in sensitivity of Giardia strains to transfection and in infectivity of the RNA virus from different Giardia strains. Transfection could be carried out with sonicated Giardia extract as well as with filter sterilized medium in which Giardia strains containing RNA virus had grown. The RNA virus did not replicate in Giardia-free medium. No correlation could be demonstrated between the presence of the RNA virus in Giardia isolates and their in vitro resistance to some antiprotozoal drugs, nor with the fact that the strain originated from symptomatic or asymptomatic carriers. The presence of the RNA virus in Giardia trophozoites did not influence the isoenzyme patterns or restriction endonuclease patterns of repetitive DNA. A correlation may exist with the length of time since the isolation in axenic culture of the strain.

Multivariate analysis of Neisseria DNA restriction endonuclease patterns.

Chromosomal DNA was extracted from eleven Neisseria meningitidis and seven Neisseria gonorrhoeae isolates and cleaved with the restriction enzyme HindIII. The DNA fragments were separated according to their size, using a 4% polyacrylamide gel. The band patterns obtained were digitized and statistically analysed by the SIMCA method. To develop the models for N. meningitidis (class 1) and N. gonorrhoeae (class 2), all eleven meningococci and seven gonococci, were used. All strains were classified correctly and showed an extremely good class separation.

Numerical analysis of Leptospira DNA-restriction endonuclease patterns

Fifteen test strains, representing 5 leptospire serogroups were studied by DNA-restriction endonuclease (Eco RI) analysis. The resulting patterns for each strain were scanned by a densitometer and the absorbance values were recorded along the pattern at regular intervals. The absorbance profiles were compared using the correlation coefficient and the similarity matrix was clustered by the UPGMA algorithm. The results were displayed as a simplified dendrogram. The numerical analysis of DNA-restriction endonuclease patterns can be used successfully in the identification of Leptospira strains.