Pulsed-field Gel Electrophoretic Research Articles

Genotypic Characterization of Clinical Isolates of Staphylococcus aureus from Pakistan.

In this study, we compared pulsed-field gel electrophoretic (PFGE), multilocus sequence typing (MLST), Staphylococcal cassette chromosome mec (SCCmec), spa typing, and virulence gene profiles of 19 Panton–Valentine leucocidin (PVL)-positive, multidrug-, and methicillin-resistant clinical Staphylococcus aureus (MRSA) isolates obtained from a hospital intensive care unit in Pakistan. The isolates exhibited 10 pulsotypes, contained eight adhesin genes (bbp, clfA, clfB, cna, fnbA, fnbB, map-eap, and spa), 10 toxin genes (hla, hlb, hld, hlg, pvl, sed, see, seg, seh, and tst), and two other virulence genes (cfb, v8) that were commonly present in all isolates. The spa-typing indicated seven known spa types (t030, t064, t138, t314, t987, t1509, and t5414) and three novel spa types. MLST analysis indicated eight ST types (ST8, ST15, ST30, ST239, ST291, ST503, ST772, and ST1413). All isolates belonged to the agr group 1. Most of the isolates possessed SCCmec type III, but some isolates had it in combination with types SCCmec IV and V. The presence of multidrug-resistant MRSA isolates in Pakistan indicates poor hygienic conditions, overuse of antibiotics, and a lack of rational antibiotic therapy that have led to the evolution and development of hypervirulent MRSA clones. The study warrants development of a robust epidemiological screening program and adoption of effective measures to stop their spread in hospitals and the community.

Genetic diversification of Neisseria meningitidis during waves of colonization and disease in the meningitis belt of sub-Saharan Africa

Although Neisseria meningitidis is a highly variable organism, most invasive disease is caused by a minority of genotypes. Hypervirulent lineages have been identified and their pandemic spread has been traced. During a longitudinal meningococcal colonization study in a district of northern Ghana clonal waves of carriage and disease were observed. Genetic diversification of genoclouds was analysed by pulsed field gel electrophoretic (PFGE) analysis of isolates from healthy carriers and from meningitis patients. Even during the limited time of persistence in the district, microevolution of the dominating genoclouds took place. Population genomic analyses are required to understand the genetic basis for the emergence of new lineages with epidemic potential, which is of crucial importance for the development of long-term global vaccination strategies against meningococcal disease.

Characterization of glycopeptide-resistant enterococcus faecium (GRE) from broilers and pigs in Denmark: genetic evidence that persistence of GRE in pig herds is associated with coselection by resistance to macrolides.

Glycopeptide-resistant enterococci (GRE) from broilers and pigs were characterized to investigate the background for the persistence of GRE in pig herds. All porcine isolates belonged to closely related pulsed-field gel electrophoretic (PFGE) types, with the ermB and vanA genes located on the same transferable genetic element. Broiler isolates belonged to different PFGE types. The persistence of GRE in Danish pig herds after the ban of glycopeptides may be explained by the genetic link between ermB and vanA and coselection by use of macrolides for treatment and growth promotion.

Genetic diversity in Helicobacter pullorum from human and poultry sources identified by an amplified fragment length polymorphism technique and pulsed-field gel electrophoresis

Helicobacter pullorum was first isolated from the faeces and carcasses of poultry and has been associated with human gastroenteritis. The aim of this study was to examine interstrain genetic diversity within H. pullorum. Two fingerprinting techniques were used: amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoretic (PFGE) analysis. The 20 strains examined were from four countries and comprised 13 human isolates and seven poultry isolates. Their identity was confirmed by a species-specific PCR assay. The human and poultry isolates had distinct genotypes and most strains showed a high degree of genetic diversity. Genotyping also indicated a clonal origin for two strains from the same poultry flock, and established a close relatedness between three chicken carcass isolates from a processing plant. It is concluded that these two genotyping techniques will provide a useful basis for future epidemiological investigations of H. pullorum in poultry, and may provide a link with its possible causal role in human gastrointestinal infections.

Interspecies recombination between enterococci: genetic and phenotypic diversity of vancomycin-resistant transconjugants.

Handwerger and colleagues demonstrated that a particular clinical isolate of Enterococcus faecium, designated GUC, and here redesignated as GUCR, can conjugatively transfer vancomycin resistance. The vancomycin resistance is encoded by a chromosomally born linked set of genes in the donor, designated the vanA cluster, to the chromosome of an E. faecalis recipient, JH2-2. Here it is reported that an earlier isolate of E. faecium from the same patient who later harbored the vancomycin-resistant E. faecium GUCR lacks the vanA gene cluster but is otherwise similar (by SmaI chromosomal fingerprint and metabolic fingerprinting) to the vancomycin-resistant GUCR. Therefore, "GUCS" is a strong suspect as the base strain for the clinical acquisition of the vanA cluster present in GUCR. Thirteen laboratory-generated vanA transconjugants derived from conjugation between GUCR and JH2-2 were subjected to further analysis, allowing a comparison between transfer in the laboratory and transfer that occurred in the clinical setting. Surprisingly, each JH2-2 transconjugant had a unique constellation of abilities to oxidize various members of a panel of potential carbon sources. This pattern was stable for each transconjugant, and it was not changed by growing the strains with or without vancomycin. Transconjugants had pulsed-field gel electrophoretic (PFGE) patterns largely consistent with that of JH2-2, the recipient in conjugation experiments. However, PFGE analysis showed that a large but variable amount of DNA, between 145 kb and 277 kb, was transferred into different transconjugants. The mechanism appears to be conjugative transposition in which new DNA is added to the pre-existing genome rather than substituting for a segment in the recipient. Mapping and hybridization studies of several transconjugants showed that each received similar, but not exactly the same, DNA fragment of at least 30 kb from the donor. Sequencing of 16S ribosomal genes was used to confirm that the recipient and donor strains used in transconjugation experiments were different species. Sequence analysis was also used to consider the possibility that rRNA operons might be mobilized in conjugation, but no evidence for the transfer of rDNA operons was found. An apparent insertion sequence in E. faecium almost identical to IS 1485 and 57% sequence identity to IS 199 of Streptococcus mutans was found in the region of DNA transferred. The results imply new consequences of conjugative transfer and interspecies recombination.

Acquisition of new capsular genes among clinical isolates of antibiotic-resistant Streptococcus pneumoniae.

Antibiotic-resistant clones of Streptococcus pneumoniae are recognizable through a combination of unique molecular, microbiological, and serological properties. In the course of surveillance of epidemic clones of S. pneumoniae, several isolates were identified that shared the clone-specific pulsed-field gel electrophoretic (PFGE) pattern and antibiotype but expressed serotypes atypical for the particular clone. A selected group of isolates belonging to the Spanish/USA clone but expressing serotypes 19, 14, or 3, instead of the expected serotype 23F, were tested using DNA probes for each of the 18 open reading frames (ORFs) of the 23F capsular locus. In no case were there any 23F-specific genes retained, with the possible exception of genes already known to be common to the capsular loci involved. Analysis of the sequence of the capsular locus of a penicillin-resistant serotype 23F isolate from Mexico showed that part of the cpsA gene of this strain, as well as genes cpsQ and cpsR, had high degrees of identity to the sequence of the homologous genes in isolates expressing serotype 19F. The capsular locus of this Mexican strain may have originated from an in vivo capsular switch event in which the original 19F locus was replaced by 23F-specific capsular genes.

In vitro genotypic variation of Campylobacter coli documented by pulsed-field gel electrophoretic DNA profiling: implications for epidemiological studies

Six isolates of Campylobacter coli from different pig herds were subcultured up to 50 times over a 6-month period and DNA samples suitable for pulsed-field gel electrophoretic (PFGE) profiling prepared at regular (1, 20, 40 and 50 passages) intervals. In 5/6 strains, changes in the banding patterns of Sma1, Sal1 and/or BamH1 digests were observed. In one such strain the differences were considered artifactual. However, significant alterations in PFGE profiles between subcultures of four strains were seen, irrespective of the restriction enzyme used. Spontaneous intramolecular genomic rearrangements were considered the most likely mechanism for the changes observed. A numerical analysis based upon the combined distribution of Sma1- and Sal1-derived fragments clustered most strain subcultures together, with the exception of those from one isolate which were divided into two clusters. The effect of spontaneous genetic change on PFGE profiles must be considered when evaluating strain relationships. Numerical techniques may aid data interpretation but results must be evaluated cautiously.

In vitro genotypic variation of Campylobacter coli documented by pulsed-field gel electrophoretic DNA profiling: implications for epidemiological studies.

Six isolates of Campylobacter coli from different pig herds were subcultured up to 50 times over a 6-month period and DNA samples suitable for pulsed-field gel electrophoretic (PFGE) profiling prepared at regular (1, 20, 40 and 50 passages) intervals. In 5/6 strains, changes in the banding patterns of Sma1, Sal1 and/or BamH1 digests were observed. In one such strain the differences were considered artifactual. However, significant alterations in PFGE profiles between subcultures of four strains were seen, irrespective of the restriction enzyme used. Spontaneous intramolecular genomic rearrangements were considered the most likely mechanism for the changes observed. A numerical analysis based upon the combined distribution of Sma1- and sal1-derived fragments clustered most strain subcultures together, with the exception of those from one isolate which were divided into two clusters. The effect of spontaneous genetic change on PFGE profiles must be considered when evaluating strain relationships. Numerical techniques may aid data interpretation but results must be evaluated cautiously.

Subtyping of Campylobacter jejuni Penner heat-stable (HS) serotype 11 isolates from human infections.

High resolution molecular subtyping was applied to Campylobacter jejuni Penner heat-stable (HS) 11 isolates from human infections and other sources. Strains were genotyped by restriction fragment length polymorphism analysis involving PCR-based flagellin gene (flaA) profiling with HinfI and DdeI, and pulsed-field gel electrophoretic (PFGE) profiling with SmaI and KpnI. Fla-genes of the strains were highly conserved as most (95%) had the same fla-profile. PFGE analysis of SmaI digests was more discriminatory with 15 profile subtypes identified, although 36% of isolates had a common profile. The study showed that strains of C. jejuni HS11, unlike those of HS1 and the HS4 complex, were relatively homogeneous at the genomic level and that high resolution molecular techniques were essential for detailed epidemiological subtyping.

Recurrent peritonitis in a patient on dialysis and prophylactic vancomycin

A 34-year-old woman with renal failure had been on peritoneal dialysis for 6 years, during which she had recurrent episodes of peritonitis. Because of the increasing frequency of her episodes, prophylactic vancomycin (30 mg/L) was put in each dialysate exchange for 3 weeks of each month (five exchanges per day) from January to June, 1997. Five consecutive peritoneal dialysates (PD) collected beween March 27 and May 28, 1997, yielded pure cultures of a meticillin-resistant Staphylocccus epidermidis with reduced susceptibility to vancomycin and teicoplanin and identical pulsed-field gel electrophoretic (PFGE) pattern.

Duration of detection of fecal excretion of Escherichia coli O157:H7 in cattle.

To better define the bovine reservoir of Escherichia coli O157:H7, cattle were tested monthly by bacteriologic culture for fecal excretion of E. coli. E. coli O157:H7 was isolated from feces of 56 cattle sampled an average of 6.98 times (2-12 samples). By broth enrichment culture, immunomagnetic separation, or both, 35 cattle had 1 positive sample, 12 had 2, 7 had 3, and 1 each had 4 and 5. Five cattle with > or = 2 positive samples were in a herd in which 5 pulsed-field gel electrophoretic (PFGE) types were simultaneously present; in 3 of these cattle, different PFGE types were detected in different samples. The duration of detected excretion E. coli O157:H7 by individual cattle in this study was <1 month in 35 (63%) of 56 cattle. Both serial and concurrent excretion of different E. coli O157:H7 strains by individual cattle was observed.

Spread of the serotype 23F multidrug-resistant Streptococcus pneumoniae clone to South Korea.

Thirty-eight antibiotic-resistant isolates of Streptococcus pneumoniae recovered in a hospital in Seoul, Korea, between February 1990 and March 1992 were analyzed for serotype, antibiotic susceptibility patterns, and chromosomal relatedness using pulsed-field gel electrophoretic (PFGE) analysis of SmaI chromosomal digests. Most of the isolates were from sputum samples, and a few strains were from otitis media and meningitis. The great majority of isolates (34 of 38, or 89%) were multiresistant, sharing virtually identical, elevated minimal inhibitory concentration (MIC) values (microgram/ml) for penicillin (1-2), chloramphenicol (12-25), tetracycline (25-30), and sulfamethoxazole/trimethoprim (> 100). Twenty of the isolates were also resistant to erythromycin, and all isolates were also considered to be resistant to ceftriaxone and cefotaxime (1-2) according to the new breakpoint definitions. The most frequent serotypes were 23F (17 of 38) and 19F(14 of 38); 2 belonged to serotype 15B and 1 of 24F. Of the remaining 4 isolates (2 serotype 3, 1 type 6B, and 1 type 9V) all were resistant to tetracycline and sulfamethoxazole/trimethoprim and with the exception of 1 of the type 3 strains, were also resistant to chloramphenicol. Eleven of the 14 serotype 19F isolates shared a relatively homogeneous PFGE pattern, which was indistinguishable from the PFGE pattern shown by most (12 of 17) of the 23F isolates. The PFGE pattern of these 19F and 23F isolates was also indistinguishable from the PFGE pattern shown by representative multiresistant capsular type 23F isolates from Croatia, Portugal, and New York City and the findings document the extensive geographic spread of this multidrug-resistant S. pneumoniae clone. The data also suggests in vivo capsular transformation of the multiresistant clone from serotype 23F to serotype 19F.

Flagellin gene profiling of Campylobacter jejuni heat-stable serotype 1 and 4 complex

Flagellin gene ( flaA) sequence polymorphisms were used to discriminate amongst 167 strains of Campylobacter jejuni serotype HS1 and the HS4 complex. Direct PCR of cell suspensions provided a rapid method for analysing DNase-negative strains, whereas purified DNA was necessary for the DNase-positive strains. Nine different PCR-RFLP patterns (genotypes) were identified by analysis with HinΛ and 12 with Ddel, giving a total of 19 combined flaA profile types. The most common combined fla types were H1D1 (35%) and H1D2 (20%) for serotype HS1, and H1D2 (23%) and H4D7 (43%) for serotype HS4. Comparison of flaA typing with other key subtyping methods for C. jejuni showed it to be less discriminatory than pulsed field gel electrophoretic (PFGE) profiling, but more so than ribotyping. Fla types provided a useful indication of strain diversity, but as some were conserved across different serotypes, ribotypes and PFGE types, the same fla type could not be used as the sole basis for grouping strains. We provide evidence for several distinct subgroups based on conserved multiple genomic criteria within the HS1 and HS4 strains, and conclude that monitoring of such subgroups could provide a novel basis for future epidemiological surveillance of C. jejuni.

A YAC-based contig of 1.5 Mb spanning the human multidrug resistance gene region and delineating the amplification unit in three human multidrug-resistant cell lines.

A contig of 21 nonchimeric yeast artificial chromosomes (YACs) has been assembled across 1.5 Mb of the multidrug resistance (MDR) gene region located at 7q21, and formatted with four previously reported probes, six newly isolated probes, and three sequence-tagged sites (STSs) from internal and end fragments of YACs. A physical map of rare cutter restriction enzyme sites across the region was also constructed by pulsed-field gel electrophoretic (PFGE) analysis of four overlapping YAC clones. The amplification unit of this region in different cell lines was then determined by Southern blot analysis on the basis of the physical map and probes. Amplified DNA was located in extrachromosomal elements in human MDR cell lines studied here, and the size of the amplification unit was determined to be discrete in one MDR amplification but variable in others.

Resolution of clonal subgroups among Neisseria gonorrhoeae IB-2 and IB-6 serovars by pulsed-field gel electrophoresis.

Analysis of macrorestriction patterns by PFGE to resolve the relatedness of clonal subgroups amongst N gonorrhoeae IB-2 and IB-6 serovar strains. Nineteen IB-2 and eight IB-6 serovar strains that differed in either auxotype or penicillin sensitivity were isolated over a two and a half-year period from patients attending several STD clinics in Sydney. During this period, a major clone, Wt/IB-2 (FS), established on epidemiological grounds, was circulating amongst homosexual males. The genetic relation of this major clone to the other strains present in the community was determined by pulsed-field gel electrophoretic (PFGE) analysis of DNA restriction fragments. Genomic DNA from the 27 isolates were prepared, digested with SpeI and BglII and the restriction patterns were analysed by contour-clamped homogeneous electric field electrophoresis (CHEF) in a CHEF DRIII equipment. Phenotypic characterisation of the 27 isolates by the combined use of auxotype, serological characterisation and penicillin sensitivity indicated the presence of subgroups within each of the two serovars. In the present study, PFGE analysis of SPeI and BglII-generated genomic DNA restriction patterns from six of the ten Wt/IB-2 (FS) correlated well with phenotypic characterisation of this major clone. Four of the ten Wt/IB-2 (FS) were found to be clonally-derived variants of this major clone as minor genome variations (less than 3 DNA fragments) were observed. Distinct clones were represented by three Wt/IB-2 (LS) isolates as the DNA fingerprints generated from these were unrelated to the major clone. Analysis of PFGE patterns of 6 Pro/IB-2 isolates showed that one was genotypically identical to the major clone, two were clonal variants and three had significantly different patterns to indicate that they were genotypically unrelated. Wt/IB-6 isolates had heterogenous PFGE patterns that were clearly unrelated to the Wt/IB-2 serovar strains. Within the IB-6 serovar, there were three isolates with the Wt/IB-6 (FS) phenotype that could be considered as clonal variants whilst the rest were genotypically distinct. PFGE analysis of macrorestriction patterns generated from SpeI- and BglII-cleavage of genomic DNA has enabled the establishment of clonal origins of strains present in the Sydney community during the period of study. The delineation of strains belonging to major A/S groups by PFGE analysis presents a clearer epidemiological picture than phenotypic characterisation alone.

Penicillin-resistant and multidrug-resistant Streptococcus pneumoniae in a pediatric hospital in Zagreb, Croatia.

Sixty-four penicillin-resistant Streptococcus pneumoniae isolates [benzylpenicillin minimal inhibitory concentrations (MICs) between 0.05 and 1.6 micrograms/ml] recovered at the Pediatric Hospital "Dr. Fran Mihaljevic" in Zagreb, Croatia between October 1990 and March 1993 were analyzed for serotype, antibiotic susceptibility patterns, and chromosomal relatedness using pulsed-field gel electrophoretic (PFGE) analysis of chromosomal DNA fragmented by digestion with the SmaI endonuclease. Hospital "Dr. Fran Mihaljevic" services the capital of Croatia and its vicinity. Most of the isolates were from nasopharyngeal carriage, but several isolates were from otitis media, sinusitis, and meningitis. Most isolates belonged to either serotype 23F (36/64) or 19F (12/64); the rest, including three 15C isolates, were in 11 additional distinct serotypes. The overwhelming majority (25/36) of the serotype 23F isolates had penicillin MIC values of 1-2 micrograms/ml and shared variants of a common PFGE pattern, closely related to the PFGE identified in multiresistant pneumococci of the same serotype with wide geographic spread to Spain, Portugal, France, and the United States. This group of bacteria was also resistant to tetracycline, chloramphenicol, and sulfamethoxazole/trimethoprim. In contrast to the relative genetic and phenotypic homogeneity of the more highly penicillin resistant isolates, pneumococci with penicillin MICs between 0.5 and 0.4 microgram/ml (29/64) were distributed in 13 different serotypes and as many as 20 distinct PFGE patterns.

Novel penicillin-resistant clones of Streptococcus pneumoniae in the Czech Republic and in Slovakia.

Seventy-two penicillin-resistant Streptococcus pneumoniae isolates collected from clinical specimens in the Czech and Slovakian Republics between 1990 and 1992 were analyzed by a variety of molecular techniques. Most of the highly resistant isolates (40/72) (penicillin MIC between 1 up to 16 micrograms/ml) were represented by two distinct pneumococcal clones, and most of these isolates (35/40) were also resistant to at least two other antibiotics (tetracycline plus chloramphenicol or erythromycin). All 17 isolates belonging to the first clone were of serotype 14, had very high penicillin MICs (8-12 micrograms/ml), shared a common, abnormal penicillin-binding protein (PBP) pattern and one of two related pulsed-field gel electrophoretic (PFGE) patterns. The 15 isolates belonging to the second clone were all of serotype 19A, had penicillin MICs between 1 and 4 micrograms/ml, shared a unique, abnormal PBP pattern, and could be divided into two subgroups on the basis of PFGE patterns, one of which was indistinguishable from the PFGE pattern of a multiresistant capsular type 19A clone of S. pneumoniae already identified earlier in Hungary. Thirty-two of the 72 pneumococcal isolates had lower penicillin MICs (0.1-0.5 microgram/ml), and these isolates differed from the more highly resistant ones in several respects: They belonged to seven different serotypes, showed large variation in PFGE patterns (20 patterns in 32 isolates) and most of them (21/32) were resistant to penicillin only. Tentative explanations for these findings, in terms of epidemiological and molecular mechanisms, are considered.

Molecular involvement of the pvt-1 locus in a gamma/delta T-cell leukemia bearing a variant t(8;14)(q24;q11) translocation.

A highly malignant human T-cell receptor (TCR) gamma/delta+ T-cell leukemia was shown to have a productive rearrangement of the TCR delta locus on one chromosome 14 and a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on the other chromosome 14. Chromosome walking coupled with pulsed-field gel electrophoretic (PFGE) analysis determined that the TCR J delta 1 gene fragment of the involved chromosome was relocated approximately 280 kb downstream of the c-myc proto-oncogene locus found on chromosome band 8q24. This rearrangement was reminiscent of the Burkitt's lymphoma variants that translocate to a region identified as the pvt-1 locus. Sequence comparison of the breakpoint junctions of interchromosomal rearrangements in T-cell leukemias involving the TCR delta-chain locus revealed novel signal-like sequence motifs, GCAGA(A/T)C and CCCA(C/G)GAC. These sequences were found on chromosome 8 at the 5' flanking site of the breakpoint junction of chromosome 8 in the TCR gamma/delta leukemic cells reported here and also on chromosome 1 in T-cell acute lymphocytic leukemia patients carrying the t(1;14)(p32;q11) rearrangement. These results suggest that (i) during early stages of gamma delta T-cell ontogeny, the region 280 kb 3' of the c-myc proto-oncogene on chromosome 8 is fragile and accessible to the lymphoid recombination machinery and (ii) rearrangements to both 8q24 and 1p32 may be governed by novel sequence motifs and be subject to common enzymatic mechanisms.

Molecular Involvement of the pvt-1 Locus in a γ/δ T-Cell Leukemia Bearing a Variant t(8;14)(q24;qll) Translocation

A highly malignant human T-cell receptor (TCR) gamma/delta+ T-cell leukemia was shown to have a productive rearrangement of the TCR delta locus on one chromosome 14 and a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on the other chromosome 14. Chromosome walking coupled with pulsed-field gel electrophoretic (PFGE) analysis determined that the TCR J delta 1 gene fragment of the involved chromosome was relocated approximately 280 kb downstream of the c-myc proto-oncogene locus found on chromosome band 8q24. This rearrangement was reminiscent of the Burkitt's lymphoma variants that translocate to a region identified as the pvt-1 locus. Sequence comparison of the breakpoint junctions of interchromosomal rearrangements in T-cell leukemias involving the TCR delta-chain locus revealed novel signal-like sequence motifs, GCAGA(A/T)C and CCCA(C/G)GAC. These sequences were found on chromosome 8 at the 5' flanking site of the breakpoint junction of chromosome 8 in the TCR gamma/delta leukemic cells reported here and also on chromosome 1 in T-cell acute lymphocytic leukemia patients carrying the t(1;14)(p32;q11) rearrangement. These results suggest that (i) during early stages of gamma delta T-cell ontogeny, the region 280 kb 3' of the c-myc proto-oncogene on chromosome 8 is fragile and accessible to the lymphoid recombination machinery and (ii) rearrangements to both 8q24 and 1p32 may be governed by novel sequence motifs and be subject to common enzymatic mechanisms.