Chromosomal Restriction Endonuclease Research Articles

Strain Differentiation and Taxonomic Characterisation of a Thermophilic Group of Phenol-degrading Bacilli

Thirteen new thermophilic phenol degrading strains were isolated from a number of geographically distinct thermal sources. The strains demonstrated similar growth characteristics with pH optima between 6.0–7.2, temperature optima between 60 °C–70 °C and similar substrate utilisation spectra. The comparison of chromosomal restriction endonuclease, ribotyping and total cellular protein patterns in addition to bacteriocin typing was used for strain differentiation and resulted in the formation of five different groups to which the isolates could be assigned. Eight of the isolates were assigned to group 2 based on identical patterns generated for each method applied. Additional methods such as the comparison of antibiotic MIC values, phenol tolerance, co-metabolic transformation of chlorophenols, fatty acid methyl ester (FAME) patterns and RFLP patterns revealed minor differences between the isolates of group 2. Isolate A2, of this group and A7, which displayed differences using every technique applied, were selected for taxonomic identification and were identified as B. thermoleovorans and B. thermoglucosidasius respectively. This is the first report of phenol degrading isolates belonging to these species.

Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order

We have constructed physical and genetic maps of the chromosomes of 21 Lyme disease agent spirochetes from geographically diverse locations. All have linear chromosomes whose lengths range from 935 to 955 kbp, and all contain multiple linear plasmids in the 16- to 175-kbp size range. The locations of 11 gene clusters on the chromosomes of these different isolates are indistinguishable at the resolution achieved in this study, indicating that the members of this related group of species have highly conserved chromosomal gene orders. However, chromosomal restriction endonuclease cleavage site maps are unique for nearly all isolates. The 22 chromosomal maps currently available define eight classes of Lyme disease agents. Four of these correspond to the previously proposed species Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, and Borrelia japonica. In addition, the North American isolates 21038, DN127 c19-2, 25015, and CA55 typify four additional chromosomal types that are as phylogenetically distinct as the species listed above. These findings support the idea that comparison of restriction maps is currently the most robust and definitive method for determining overall chromosomal relationships among closely related bacteria. In the course of this work, we located on the chromosome the previously unmapped outer surface protein-encoding LA7 gene and genes homologous to the Escherichia coli priA, plsC, parE, and parC genes, and we have substantially refined the locations of the recA, fla, p22A, and flgE genes.

Chromosomal restriction endonuclease analysis and ribotyping of Bacteroides fragilis

We have analysed the restriction fragment length patterns of chromosomal DNA and of ribosomal RNA (rRNA) genes in order to investigate the clonal distribution within Bacteroides fragilis isolates. Eighteen blood culture isolates from 18 patients and 4 faecal isolates from 4 subjects were examined. Chromosomal restriction endonuclease analysis (REA) was performed by separating BamHI-generated DNA fragments using polyacrylamide gel electrophoresis (PAGE). Ribotyping was accomplished by hybridizing EcoRI-treated genomic DNA subjected to conventional REA on agarose to a radiolabelled probe obtained from 16+23S rRNA of E. coli. All 22 isolates could be differentiated by their REA patterns with a varying percentage of similar fragments. Analysis of the rRNA gene patterns displayed heterogeneity, and revealed 14 ribotypes among the 18 blood culture isolates and 3 among the 4 faecal isolates. The predominant ribotype among the clinical isolates was also shared by one faecal isolate. The results suggest that no particular clones are predominantly responsible for systemic infection.

Further consideration of the clonal nature of Salmonella typhi: evaluation of molecular and clinical characteristics of strains from Indonesia and Peru.

We examined envelope protein profiles, chromosomal restriction endonuclease digest patterns, and immune responses to envelope proteins for collections of Salmonella typhi strains isolated in Peru and Indonesia. Only minor differences in envelope protein patterns were apparent among strains. Strains from 7 of 20 Indonesian patients had a distinct chromosomal digest pattern compared with patterns of Peruvian and other Indonesian strains. Strains with this pattern carried the gene for the j flagellar antigen (H1-j); differences in response to envelope proteins of j and d strains were noted on immunoblot analysis. Our data suggest that there are genotypic and phenotypic differences among S. typhi strains. The clinical importance of these differences remains to be fully evaluated; however, in this study it was not possible to show a clear correlation between strain characteristics and disease severity.

Molecular characterization of strains of enteroinvasive Escherichia coli O143, including isolates from a large outbreak in Houston, Texas.

A large diarrhea outbreak due to enteroinvasive Escherichia coli (EIEC) serogroup O143 occurring in Houston, Tex., provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. This was done by comparing the plasmid patterns and the chromosomal restriction endonuclease digestion patterns by pulsed-field gel electrophoresis (PFGE) of EIEC from the outbreak, other E. coli from the same serogoup (O143), and EIEC isolated from other patients with diarrhea. Among the isolates studied, there was marked restriction fragment length polymorphism. All 3 non-O143 EIEC isolates had very different restriction endonuclease digestion patterns, as did 5 of 5 O143 non-EIEC isolates and 6 of 15 O143 EIEC isolates. Four Houston outbreak O143 EIEC isolates had the same restriction pattern as an O143 EIEC strain isolated 2 months before in Mexico and was nearly identical to another two O143 EIEC Mexican isolates. These related strains also had the same plasmid pattern; however, the presence of only a few plasmid bands, versus the 21 to 30 chromosomal bands seen with PFGE, suggests that plasmid patterns could be a less specific way to distinguish different strains. These results demonstrate that PFGE can distinguish between different E. coli strains of the same serogroup and phenotype. This technique can also identify relatedness within O143 EIEC, and our data suggest the spread of a strain of EIEC from Mexico to Houston, where it caused a large outbreak. PFGE may be useful to study the epidemiology of EIEC.

Comparison of epidemic neisseria meningitidis strains by dna restriction endonuclease profile analysis

Meningococcal disease (MD) in Western Australia prior to 1987 was caused by endemic, predominantly serogroup B Neisseria meningitidis strains. In 1987/S3 an epidemic of serogroup A MD occurred in remote outback Aboriginal communities in Western Australia. Comparable epidemiologic features of this epidemic with overseas epidemics suggested spread of a single clone of serogroup A Neisseria meningitidis . DNA analysis of serogroup A strains isolated from MD cases admitted to Princess Margaret Hospital for Children in 1987/88 was undertaken. Chromosomal DNA extraction and restriction endonuclease profiles obtained with EcoRI and Hind III demonstrate that these serogroup A strains are clonal. This indicates that the molecular epidemiology of epidemic serogroup A MD in Western Australia is similar to recent experience in Saudi Arabia, Sudan, Chad and Ethiopia, where spread of single closely related clones has been iesponsible for pandemic waves of MD in the past decade.

Epidemiology of an endemic strain of beta-lactamase-producing Enterococcus faecalis

Three previously reported beta-lactamase-producing (Bla+) enterococci with distinct but related antibiotic resistance phenotypes, plasmid profiles, and plasmid restriction endonuclease digestion patterns were isolated at the West Haven Veterans Administration Medical Center in Connecticut (WH245, WH257, WH571) in July 1986 and March 1987. In this study, we analyzed the whole-cell DNA of these isolates by using pulsed-field gel electrophoresis of large chromosomal fragments generated by SmaI digestion. The three West Haven isolates showed very similar chromosomal restriction endonuclease digestion patterns; these patterns were distinct from those of Bla+ and Bla- enterococci from other geographic areas and from those of other West Haven enterococci, suggesting an endemic Bla+ strain at this institution. Clinical information regarding these isolates suggests that exposure to the genitourinary clinic, obstructive urinary tract disease, and frequent antibiotic therapy may have been common risk factors for acquisition of this endemic strain.

Epidemiologic typing of Enterobacter sakazakii in two neonatal nosocomial outbreaks

Two unrelated hospital outbreaks of Enterobacter sakazakii, involving meningitis, bacteremia, and colonization of neonates, were investigated. In each of these outbreaks, E. sakazakii was isolated from both patients and dried infant formula. In previous outbreaks, the source and mode of transmission of E. sakazakii in neonatal infections was not determined. In this study, we used a combination of typing methods (plasmid analysis, antibiograms, chromosomal restriction endonuclease analysis, ribotyping, and multilocus enzyme electrophoresis) to evaluate the isolates from each outbreak as to their relatedness. The typing results differed among outbreaks, but in each one, patient and formula isolates shared the same typing pattern. The only exceptions were disk antibiograms, which often varied among colonies selected from each of the isolates. Plasmid analysis, chromosomal restriction endonuclease analysis, ribotyping, and multilocus enzyme electrophoresis all were effective as epidemiological typing methods for E. sakazakii, especially when used in combination. By using this typing scheme, we have confirmed that E. sakazakii from intrinsically contaminated dried infant formula was the source of neonatal infection.

Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites

Epidemiologic evaluation of enterococci has been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared chromosomal restriction endonuclease digestion patterns of 27 isolates of Enterococcus faecalis from three different locations by using pulsed-field electrophoresis of large chromosomal fragments (14 to 1,000 kilobases). All but two isolates generated a clear, evaluable pattern with a single lysis and digestion, and the remaining two were visualized when a larger quantity of bacteria was used. All isolates from different locations generated different restriction patterns, as did most isolates within a single location; there was also evidence of spread of strains between individuals in each location. The ease with which this analysis can be performed, together with the clarity and polymorphism seen in the patterns, suggests that this technique will be very useful for epidemiological evaluations of nosocomial enterococcal infections.

Nosocomial Clostridium difficile colonisation and disease

To assess the risk of acquiring Clostridium difficile diarrhoea or colitis in patients colonised with C difficile, rectal swabs taken weekly for 9 weeks from patients with long-term (at least 7 days) hospital stays on three wards were cultured for C difficile. 60 (21%) of 282 patients were culture-positive for C difficile during their hospital stay, of whom 51 were symptom-free faecal excretors. C difficile diarrhoea developed in the other 9 patients; 2 were culture-positive for C difficile and had diarrhoea at the time of first culture, and 7 had diarrhoea or pseudomembranous colitis after 1-6 previously negative weekly rectal cultures. All patients with diarrhoea were on one ward, but symptom-free, excretors were found on all wards. HindIII chromosomal restriction endonuclease analysis (REA) of the C difficile isolates revealed 18 distinct types. All isolates from the patients with diarrhoea were one of two nearly identical REA types, B or B2. 26 of the 29 total B/B2 isolates were from patients on the same ward, which points to a nosocomial outbreak. The symptom-free excretors were not at increased risk of subsequent clinical illness.

Antibiotic and toxicant susceptibility profiles of clinical and environmental Klebsiella pneumoniae isolates

AbstractKlebsiella pneumoniae strains isolated from different clinical and environmental sources were examined for resistance to antibiotics, pentachlorophenol, and heavy metals using intracellular ATP measurements. Resistance to kanamycin, neomycin, gentamicin, and tobramycin was noted for the hospital strain but not for the environmentally derived isolates. On the other hand, strains isolated from pulp and paper mill effluents and receiving waters exhibited a higher degree of pentachlorophenol and heavy metal resistance. Chromosomal restriction endonuclease analysis (REA) digests of three environmental strains produced patterns that were different and readily distinguishable. Plasmids were detectable in these same environmental isolates; two of the three carried a 70 × 106 Da plasmid that is thought to mediate both antibiotic and heavy metal resistance.

Application of chromosomal restriction endonuclease digest analysis for use as typing method for Clostridium difficile.

The usefulness of restriction endonuclease analysis of chromosomal DNA as a typing method for Clostridium difficile was tested. Over four months all faecal samples were routinely cultured for C difficile. DNA of all isolated strains was isolated and tested with the restriction endonuclease Hind III. The patterns obtained after electrophoresis in agarose gels seemed to be strain specific. Antibiotic susceptibility profiles agreed with the results of the restriction endonuclease analysis, though they were much less discriminating. Analysis of the results indicated that restriction endonuclease analysis is a suitable typing method for C difficile, which may be very valuable in epidemiological studies where a highly discriminating typing method is needed.

Molecular epidemiology of bacterial infections: examples of methodology and of investigations of outbreaks.

Plasmid profile analysis, plasmid and chromosomal bacterial restriction endonuclease DNA analysis, and DNA hybridization are increasingly used in clinical microbiology and epidemiology. These techniques have been applied, singly and in combination, to investigations of outbreaks, including those of diarrheal diseases caused by pandemic Vibrio cholerae, enterotoxigenic and enterohemorrhagic Escherichia coli, Salmonella muenchen, and Salmonella typhimurium. The techniques have been critical in the unraveling of outbreaks of disease caused by nosocomial and community-acquired pathogens. Although there are limitations to these methods, their applications have important ramifications for basic science and public health.

Molecular epidemiology of neonatal meningitis due to Citrobacter diversus: a study of isolates from hospitals in Maryland.

Six cases of neonatal meningitis due to Citrobacter diversus were diagnosed in three Baltimore (Maryland) hospitals between 1983 and 1985. Using plasmid profiles, biotypes, serotypes, and chromosomal restriction endonuclease digests as epidemiological markers, we studied 63 isolates of C. diversus (including four isolates from cerebrospinal fluid) from these and seven other hospitals in Maryland. Within two of the three hospitals with meningitis cases, the same strain of C. diversus was isolated from case infant(s), healthy neonates, and nursery personnel. In all three hospitals, C. diversus strains different from those implicated as a cause of meningitis were also isolated. Other than the meningitis-associated strains, 15 different strains of C. diversus were isolated from infants in the hospitals studied, with several distinct clusters of asymptomatic, colonized infants identified.

Investigation of a Campylobacter jejuni outbreak by serotyping and chromosomal restriction endonuclease analysis.

Fifty Campylobacter jejuni isolates, including 29 from humans associated with an outbreak of enteritis, 20 from cattle, and 1 from a milk source, were serotyped on the basis of extractable thermostable antigens and examined by bacterial chromosomal restriction endonuclease digest analysis. Serotyping showed specific differences between the human isolates and the milk isolates, but each of these generally, although not consistently, reacted with 4 of the 42 C. jejuni typing antisera. Restriction patterns of all of the human isolates and some of the cattle isolates were indistinguishable, confirming the suspected link between the cattle and the human outbreak. The single milk isolate had a restriction pattern unlike those of the human isolates, and therefore its involvement in the transmission of infection from the cattle to the humans could not be confirmed.

Application of serotyping and chromosomal restriction endonuclease digest analysis in investigating a laboratory-acquired case of Campylobacter jejuni enteritis.

A frequently passaged laboratory strain of Campylobacter jejuni was confirmed by serotyping on the basis of thermostable antigens and by bacterial chromosomal restriction endonuclease digests to be the causative agent of enteritis in a laboratory worker.