Random amplified polymorphic DNA typing versus pulsed-field gel electrophoresis for epidemiological typing of vancomycin-resistant enterococci

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing with SmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the "gold standard." The discriminating power of RAPD typing, with seven primers and 11 combinations of primers, was tested on 18 strains, and only the most discriminating combination was further tested on the whole collection. We compared the epidemiological usefulness of RAPD typing of 60 clinical VRE isolates with that of SmaI PFGE typing. With primers AP4 and ERIC1R, RAPD generated 30 patterns versus the 36 patterns generated by SmaI PFGE. However, this did not hamper the epidemiologically correct clustering of 15 related strains and the detection of multiple colonization in nine patients. We conclude that this simple RAPD technique is well suited to the epidemiological typing of VRE and the monitoring of its nosocomial spread.     

Pulsed-field gel electrophoresis is more efficient than ribotyping and random amplified polymorphic DNA analysis in discrimination of Pasteurella haemolytica strains

OBJECTIVE: Factors such as size of hyphema, intraocular pressure, initial visual acuity, and use of steroids or antifibrinolytic drugs may be associated with the likelihood of rebleeding in traumatic hyphema. The association of the visual outcome with secondary hemorrhage has been questioned. DESIGN: Randomized, placebo-controlled, clinical trial. PARTICIPANTS: Two hundred and thirty-eight patients who had hyphema develop after blunt trauma. INTERVENTION: Eighty patients received oral tranexamic acid, 80 patients received placebo, and 78 patients received oral prednisolone. MAIN OUTCOME MEASURES: Secondary hemorrhage and vision at the time of discharge from the hospital were measured. RESULTS: Rebleeding occurred in 43 (18%) of the patients and was prevented significantly by oral tranexamic acid compared with the placebo (odds ratios [OR] = 0.39; 95% confidence interval [CI], 0.17, 0.89). Occurrence of secondary hemorrhage had weak associations with initial high intraocular pressure (OR = 2.7; 95% CI, 0.99, 7.3) and initial visual acuity of 6/60 or less (OR = 1.8; 95% CI, 0.9, 3.7). Secondary hemorrhage had no statistical association with age, gender, oral prednisolone, size of hyphema, and retinal damage. Visual acuity of 6/60 or less at the time of discharge was significantly associated with rebleeding (OR = 10.5; 95% CI, 3.7, 29.2), initial visual acuity of 6/60 or less (OR = 9.9; 95% CI, 2.8, 38.0), retinal damage (OR = 14.6; 95% CI, 3.8, 55.8), and male gender (OR = 6.5; 95% CI, 1.4, 31.9). Final visual acuity had no significant statistical association with age, use of oral prednisolone or tranexamic acid, and size of hyphema. CONCLUSIONS: High intraocular pressure and low vision at the time of first examination may be associated with increased chance of rebleeding. Retinal damage, secondary hemorrhage, male gender, and initial poor vision are associated with a worse visual outcome in patients with traumatic hyphema.     

Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis and ribotyping

A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates. We conclude that the close genetic similarity observed between epidemiologically unrelated and outbreak-related isolates of S. enteritidis suggests that both PFGE and ribotyping are of limited value in the epidemiological analysis of these particular isolates, possibly because of the highly clonal nature of pathogenic strains of S. enteritidis.     

Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis

The structured clinical interview for diagnosis (axis 1) according to the Diagnostic and Statistical Manual for Mental Disorders (DSM-III-R) was used to assess psychiatric morbidity in 110 infertile patients. They were divided into two groups according to whether referral to the service of psychosomatic medicine was deemed advisable by the physician in charge. Psychiatric disorders were diagnosed in 39 of 56 (69.6%) patients in the referred group and in 13 of 54 (24.1%) in the non-referred group. Psychiatric morbidity was found in 61.1% of females and 21% of males. Adjustment disorders were found in 59.6% (31/52) of all patients, in 59% (24/39) of patients among the referred group and in 61.5% (8/13) of patients among the non-referred group. Fourteen (67%) of 21 women in the referred group with adjustment disorders suffered from anxiety. In addition, 33.3% of patients in the non-referred group showed important psychological dysfunction, although DSM-III-R criteria were not met. Psychiatric morbidity was significantly associated with the number of treatment cycles and female gender in the whole study population, as well as with the type and length of infertility in the non-referred group. Psychological services in an infertility clinic help to identify at an early stage those individuals who are more likely to be vulnerable. This would enable psychological interventions to be targeted towards those in greater need.     

Investigation of a pseudo-outbreak of Nocardia asteroides infection by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA PCR

Molecular strain typing by pulsed-field gel electrophoresis and by randomly amplified polymorphic DNA analysis was used to investigate a cluster of four Nocardia asteroides isolates associated with the BACTEC 460 TB system. An instrument motor drive misalignment resulted in inadequate needle sterilization and cross-contamination of BACTEC vials. This pseudo-outbreak illustrates the importance of proper BACTEC 460 needle sterilization and maintenance and confirms the usefulness of molecular typing methods for epidemiologic investigations.     

Genotypic characterization of Salmonella enteritidis phage types by plasmid analysis, ribotyping, and pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to resolve XbaI and SpeI macrorestriction fragments from 60 defined phage type (PT) reference strains of Salmonella enteritidis. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct XbaI pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb spv-associated fragment was observed for three PT reference strains, and the profile was designated PFP X1A. The XbaI macrorestriction profiles of a further four PT reference strains were altered by the presence of plasmid-associated bands. Twenty-six SpeI-generated PFPs (plus one subtype) were observed for the same strains. No SpeI fragment corresponding to the 38-MDa serovar-specific plasmid was detected. The distribution of XbaI and SpeI profiles did not always correspond, producing a total of 32 combined PFPs for the 60 PT reference strains. This compared with a total of 18 different plasmid profiles and three PvuII ribotypes generated by the same strains. The results of this study indicate that PFGE may offer an improved level of discrimination over other genotypic typing methods for the epidemiological typing of S. enteritidis.     

Application of random amplified polymorphic DNA analysis for detection of Salmonella spp. in foods

The random amplified polymorphic DNA (RAPD) band patterns from 23 Salmonella spp. produced by use of an oligonucleotide primer (called du primer) designed on the basis of the N-terminal sequence of dulcitol 1-phosphate dehydrogenase (5'-GTGGTGACCCAGGATGGCCAGGTG-3') were different from those from 16 non-Salmonella spp. The bands at 460 and 700 bp were produced in all Salmonella strains tested. These RAPD fragments obtained from Salmonella typhimurium strongly hybridized with the corresponding RAPD bands from the other strains of Salmonella, but not with those from non-Salmonella spp. in Southern blot analysis. The RAPD bands were detected by ethidium bromide staining even when genomic DNA prepared from as few as 2.8 x 10(3) cells was used. The minimum detectable cell number in the initial inoculum of S. typhimurium was 4 x 10(-1) CFU/25 g of raw beef after the preenrichment in Enterobacteriaceae enrichment mannitol (EEM) broth for 6 h and the selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin (DMPBN) medium for 18 h at 42 degrees C. Seven raw foods inoculated with S. typhimurium at numbers from 4 x 10(-1) to 2.6 x 10(2) CFU/25 g of food were positive in both the RAPD analysis and the conventional culture method.     

Molecular epidemiological analysis of Pseudomonas aeruginosa by pulsed-field gel electrophoresis]

In order to see the nosocomial infection by Pseudomonas aeruginosa (P. aeruginosa) in the Akita University Hospital, we analyzed the serotype and genotype for 150 strains isolated from various clinical specimens from 1994 to 1996. One hundred strains chosen at random were divided into 11 serotypes by serotyping and into 50 genotypes by pulsed-field gel electrophoresis of Spe I-digested P. aeruginosa genomic DNAs. Serotype E strains, most common, gave 17 genome patterns. Fifty strains separated periodically in certain wards had further 7 genome patterns. The strains isolated from one patient with different times or with different serotypes showed the same stable genotype. Genome patterns were inconsistent with susceptibility to antimicrobial agents. Both drug-susceptible and -resistant strains were found in strains with the same genome pattern. The genome pattern was identical, even though the susceptibility was different due to isolation time or storage. This suggested that the multidrug-resistant strains of P. aeruginosa expanded in the hospital were not derived from one original strain.     

Application of physico-chemical typing methods for the epidemiological analysis of Salmonella enteritidis strains of phage type 25/17

Eighty-nine Salmonella enteritidis phage type 25/17 strains isolated from a localized outbreak in the German state Nordrhein-Westfalen (outbreak NWI) could not be further differentiated by biochemotyping and plasmid pattern analysis. They were submitted to a complex typing system consisting of modern physico-chemical analytical procedures. In lipopolysaccharide pattern analysis the strains proved to be homogeneous. In multilocus enzyme electrophoresis, outer membrane and whole cell protein pattern (WCPP) analysis, and Fourier-transform infrared (FT-IR) spectroscopy (increasing extent of differentiation in the given order) strains deviating from each basal pattern were found. The extent of correspondence in these deviations was satisfactory. Forty-six strains of the same sero- and phage type, however, obtained from different outbreaks, were additionally typed. The results obtained with them indicate that the data of the first group were not restricted to strains from outbreak NWI, but of general validity. It was found that both WCPP and FT-IR represent valuable methods for the sub-grouping of bacteria.     

Reorientation time of DNA molecules in pulsed-field gel electrophoresis

The precise determination of the influence of an electric field strength E on the resolution of DNA molecules during a pulsed-field gel electrophoresis shows that the maximal molecular size Nmax of still resolved DNA molecules is described by the equation Nmax = k tau E alpha, where k is a coefficient, tau is a pulse time, and alpha is an exponent (calculated as approximately 3/2). We assume that the best estimation of the reorientation time tau R for each DNA fragment is such a pulse time in which this DNA molecule is the largest separated one.     

Insertion possibility of 16S-23S space amplification and random amplified polymorphic DNA analysis for typing of methicillin-resistant Staphylococcus aureus strains in the context of nosocomial infections

Within the scope of the present study n = 183 MRSA isolates from the extended area of Düsseldorf and n = 93 international MRSA strains from seven different countries were typed by pulsed-field gel electrophoresis and two PCR methods (RAPD and 16S-23S-spacer amplification). The isolates could be subdivided into 30 different types by PFGE, into 21 by means of RAPD and 18 by 16S-23S-spacer amplification. PFGE had the highest discriminatory potential, however, a combined use of the three typing methods allows a more detailed differentiation even of those isolates with identical PFGE pattern. Both amplification procedures were rapid, easy in handling with reproductable results. For a temporary epidemiological analysis within 24 hours, both amplification methods could be combined. In case the investigated isolates were still suspected of showing a "clonal identity", they should be analysed by additional PFGE (lasting about four days). Although the international isolates were chosen by random selection, several MRSA strains with identical pattern could be found in different countries of the world. Some RAPD-, spacer- and PFGE pattern were constant over many years. This reflects a high genetic stability of single strains.     

Restriction fragment length polymorphism analysis and random amplified polymorphic DNA analysis of Campylobacter jejuni strains isolated from patients with Guillain-Barré syndrome

Campylobacter jejuni serotype O19 strains associated with the Guillain-Barré syndrome (GBS) and other strains were examined by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction products of the flaA genes and by random amplified polymorphic DNA (RAPD) analysis. RFLP analysis showed that regardless of LIO serotype, geographic origins, or association with GBS, the O19 isolates shared an identical digestion pattern by each of four restriction endonucleases, DdeI, MboI, MseI, and AluI. In contrast, among C. jejuni O1 or O2 strains, RFLP patterns were different even among strains of the same LIO serotype. The results of the RAPD analysis were consistent with the flaA RFLP data. These data indicate that all of the O19 strains that were tested were closely related to one another whether they were or were not associated with GBS.     

Simplified analysis of pathogenic leptospiral serovars by random amplified polymorphic DNA fingerprinting

Recent studies have suggested that platelet activating factor (PAF) plays an important role in various reproductive functions, including ovulation, implantation and parturition, and that the local concentration of PAF is modulated by PAF-acetylhydrolase (PAF-AH), a potent PAF inactivator. In this study, we investigated the possible effects of various bioactive substances, which are present at high concentrations in the human pregnant uterus, on PAF-AH secretion from decidual macrophages using a monocyte-macrophage model system, human myelocytic leukaemia cells (HL-60). By treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells were transformed to macrophage-like cells, which secreted PAF-AH into the culture medium time- and dose-dependently. After treatment with 10(-8) M TPA, the effects of various substances on the secretion of PAF-AH were examined. Among the substances examined, cortisol and TGF-beta suppressed PAF-AH secretion from TPA-stimulated HL-60 cells in a significant and dose-dependent way. Endothelin, epidermal growth factor, and brain natriuretic peptide had no significant effect on PAF-AH secretion from TPA-stimulated HL-60 cells. These results suggest that local PAF concentrations in the pregnant uterus might be regulated, at least partly, by cortisol and TGF-beta; thus these substances may play a role in the initiation of parturition via regulation of local PAF concentrations.     

Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis

To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.     

Diversity among clinical isolates of Proteus penneri detected by random amplified polymorphic DNA analysis

Previous findings indicate that wheel running can have either an aversive or an appetitive effect. That is, wheel running for 30 min induces conditioned taste aversion (CTA) in rats trained while hungry and thirsty but facilitates feeding in non-deprived rats. In Experiment 1, wheel running was also found to be effective in producing CTA in non-deprived rats. Therefore, Experiment 2 tested whether wheel running produces the aversive and appetitive effects simultaneously. During each of four training trials, two groups of non-deprived rats were given a flavored solution to drink for 10 min. Then those in the wheel group were put in running wheels for 30 min whereas those in the cage group spent 30 min in small cages. Finally, all rats were given a 60-min feeding test. After the first trial, the wheel group drank less flavored solution than the cage group during each of the remaining trials. The wheel group also ate more than the cage group on each feeding test. These results indicate that wheel running produces CTA and facilitates eating at the same time. A role for the mesolimbic dopamine reward system in these effects was considered.     

Segregation analysis of random amplified polymorphic DNA (RAPD) markers in Picea abies Karst

The reliability of arbitrarily primed amplification products was tested. The segregation analysis of 266 amplification products obtained using 17 different 10-mer oligonucleotides in 34 megagametophytes from a single tree of Picea abies was carried out. Fifty-four out of the 165 variable bands fit the 1:1 segregation ratio expected for Mendelian traits. The segregation ratio of a subset of six RAPD markers in five other individuals from the same population confirmed their genetic nature. Our results strengthen the evidence previously reported that RAPDs markers can be considered Mendelian traits useful in the detection of genetic variability among both different individuals and populations.     

Phage restriction and the presence of small plasmids in Salmonella enteritidis

Sequence variation within the variable region of the 16S rRNA at position 440 to 480 allowed the synthesis of specific PCR primers for the identification of groups within the species Photorhabdus luminescens, symbionts of entomopathogenic nematodes of the genus Heterorhabditis. For the second PCR primer the highly conserved region at 755 to 795 was used. The P. luminescens type strain specific primer could not recognize any other P. luminescens strain. The primer TEMPERATUS based on the sequence of strain DSM12190 (isolated from North West European H. megidis strain HSH2) identified all P. luminescens associated with H. megidis from North West Europe and two isolates from closely the related nematode strains from Ireland. The primer TROPICUS based on strain DSM12191 (isolated from the nematode type strain H. indica strain LN2) identified P. luminescens of tropical origin isolated from H. indica. Symbionts of H. bacteriophora could not yet be separated into well described groups with the primers used. A comparison of sequence data resulted in the identification of additional groups. The non-symbiotic P. luminescens isolates are distinct in the variable region. The group HELIOTHIDIS contains 15 P. luminescens associated with H. bacteriophora from North East America. The MARELATUS group contains symbionts of the nematode H. marelatus from the West Coast of the US. The data together with the specific symbiotic association of P. luminescens strains with different nematode species support the division of the taxon P. luminescens into different species.     

Phage types and ribotypes of Salmonella enteritidis in southern Italy

Differently from other European countries, Southern Italy was affected by a considerable increase in human infections due to Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) only after 1990. On the present investigation, two groups of S. Enteritidis strains isolated during the low-incidence period 1980-1984 and the epidemic period 1990-1993, respectively, have been submitted to phage-typing and ribotyping in order to ascertain whether the epidemic increase was determined by the spread of a foreign bacterial clone or not. Among the 150 isolates relative to the aforesaid two periods, 12 different phage types (PTs) were observed. PT4 was the most common phage type among the strains isolated in 1980-1984 (61%) as well as in those of the epidemic period 1990-1993 (72%). PT8 was the second most frequent (33%) phage type in 1980-1984. It was substituted by PT1 (19%) in the 1990-1993 period. Analysis of rDNA patterns obtained after Hinc II digestions and Escherichia coli rRNA hybridizations showed 8 different patterns, A to H. The great majority of the strains studied (140 isolates, 93%) belonged to the ribotype A, showing a similar frequency both in 1980-1984 (36 of 39, 92%) and in 19901993 (104 of 111, 94%). The predominance of PT4 and ribotype A among both preepidemic and epidemic strains is in agreement with the hypothesis that host genetic diversity decline and modern farming practices in the poultry industry have facilitated a widespread dissemination of preexisting endemic strains. This hypothesis urges to plan new strategies in preventing S. Enteritidis infections.