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PubMed2006

[Use of molecular subtyping methods to investigate two nosocomial outbreaks due to Salmonella Livingstone in Sfax hospital, Tunisia].

OBJECTIVE: The aim of the study was to investigate two nosocomial outbreaks due to Salmonella Livingstone in a pediatric ward in Sfax hospital using molecular typing techniques. MATERIALS AND METHODS: We included 84 strains of S. Livingstone isolated from patients hospitalized in a pediatric ward between November 1999 through August 2002 in addition to one environmental sample. Three epidemiological unrelated strains of S. Livingstone were also tested. The molecular typing techniques were: plasmid analysis, enterobacterial repetitive intergenic consensus (ERIC-PCR), random amplification of polymorphic DNA (RAPD-PCR) and pulsed field gel electrophoresis (PFGE). RESULTS: The plasmid analysis and the ERIC-PCR generated a similar profile for outbreak isolates including the environmental sample while the epidemiologically unrelated strains demonstrated distinct patterns. The RAPD-PCR applied on 20 strains showed three patterns but one profile was predominating. All the strains isolate of S. Livingstone, except the veterinary strain, could not be typed by PFGE. CONCLUSION: Using the molecular typing techniques, we showed that these two outbreaks in the pediatric ward were due to the clonal spread of a single strain of S. Livingstone. The identification of the source of contamination and the improvement of hygiene conditions are required.

Bacterial Typing Techniques↗
PubMed1998

Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region.

The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9-kbp DNA fragment and its sequence was determined. An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length. The pRA2 replication region has two open reading frames (ORFs). ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end. ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively. The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1.

Consensus Sequence↗
PubMed1992

Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

Base Sequence↗
PubMed1988

Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. I. T4 endonuclease V-initiated excision repair.

The process by which DNA-interactive proteins locate specific sequences or target sites on cellular DNA within Escherichia coli is a poorly understood phenomenon. In this study, we present the first direct in vivo analysis of the interaction of a DNA repair enzyme, T4 endonuclease V, and its substrate, pyrimidine dimer-containing plasmid DNA, within UV-irradiated E. coli. A pyrimidine dimer represents a small target site within large domains of DNA. There are two possible paradigms by which endonuclease V could locate these small target sites: a processive mechanism in which the enzyme "scans" DNA for dimer sites or a distributive process in which dimers are located by random three-dimensional diffusion. In order to discriminate between these two possibilities in E. coli, an in vivo DNA repair assay was developed to study the kinetics of plasmid DNA repair and the dimer frequency (i.e. the number of dimer sites on a given plasmid molecule) in plasmid DNA as a function of time during repair. Our results demonstrate that the overall process of plasmid DNA repair initiated by T4 endonuclease V (expressed from a recombinant plasmid within repair-deficient E. coli) occurs by a processive mechanism. Furthermore, by reducing the temperature of the repair incubation, the endonuclease V-catalyzed incision step has been effectively decoupled from the subsequent steps including repair patch synthesis, ligation, and supercoiling. By this manipulation, it was determined that the overall processive mechanism is composed of two phases: a rapid processive endonuclease V-catalyzed incision reaction, followed by a slower processive mechanism, the ultimate product of which is the dimer-free supercoiled plasmid molecule.

DNA Repair↗
PubMed2000

Isolation and sequence analysis of plasmid pNO33 in the epsilon-poly-L-lysine-producing actinomycete Streptomyces albulus IFO14147.

A high-molecular-size plasmid (37-kb), named pNO33, has been isolated from Streptomyces albulus IFO14147, a producer of epsilon-poly-L-lysine which exhibited antimicrobial activity. The sequence analysis of a 4.6-kb fragment in pNO33 revealed four putative open reading frames, one of which exhibited a significant homology to the bldB gene product involved in morphogenesis and antibiotic production by S. coelicolor.

Journal Article↗
PubMed1996

Molecular characterization of Salmonella enterica subsp. enterica serovar choleraesuis field isolates and differentiation from homologous live vaccine strains suisaloral and SC-54.

Four independent molecular methods were used to characterize the Salmonella enterica subsp. enterica serovar choleraesuis live vaccine strains SC-54 and Suisaloral and to differentiate them from S. choleraesuis field isolates. Plasmid analysis revealed the presence of seven plasmid profiles. A virulence plasmid of 52-kbp was identified by hybridization with an spvB-spvC gene probe in each of the S. choleraesuis field isolates and in the Suisaloral vaccine strain, but not in the SC-54 vaccine strain. Ribotyping, performed with a gene probe that recognized 23S, 16S, and 5S rRNA genes, resulted in three closely related hybridization patterns. IS200 elements were not detected in the field isolates or in the two S. choleraesuis live vaccine strains. Macrorestriction analysis with the enzymes XbaI, SpeI, NotI, and SfiI differentiated the 29 S. choleraesuis strains included in this study into 10, 13, 8, and 13 different fragment patterns, respectively. While the Suisaloral vaccine strain showed a unique XbaI macrorestriction pattern, the fragment patterns of the SC-54 strain obtained with the different enzymes were shared by 2 to 18 S. choleraesuis field strains. A combination of plasmid analysis and macrorestriction analysis proved to be most suitable for the molecular typing of S. choleraesuis and the differentiation of both live vaccine strains from field isolates of this serovar.

Animals↗
PubMed1995

Epidemiologic investigation of an outbreak of coagulase-negative Staphylococcus primary bacteremia in a newborn intensive care unit.

Infections due to coagulase-negative Staphylococcus (CNS) are an ever-increasing nosocomial problem, particularly in the pediatric population. The authors describe a cluster of three primary bloodstream infections due to CNS in a newborn intensive care unit that occurred between November 23 and December 2, 1992. Two children died as a direct consequence of the bacteremia; at autopsy, one had a large bacteria-containing thrombus extending from the insertion site of a central catheter to the superior vena cava. The children were placed in isolation, and the nursing and medical staff were given topical nasal mupirocin. Plasmid analysis performed later disclosed three different blood isolates that also were different from any of the staff's nasal isolates. The authors concluded that molecular methods such as plasmid analysis are important tools in identifying true outbreaks and can prevent needless interventions, such as those during this cluster.

Bacteremia↗
PubMed2005

Pulsed-field gel electrophoresis, plasmid profiles and phage types for the human isolates of Salmonella enterica serovar Enteritidis obtained over 13 years in Taiwan.

AIMS: Plasmid profile, phage typing, and pulsed-field gel electrophoresis (PFGE) patterns of 124 Salmonella Enteritidis strains isolated in 1998-2002 in Taiwan were analysed and the results were compared with those of the 63 strains obtained in 1991-1997, so that molecular subtypes and epidemic strains for Salmonella Enteritidis over a 13-year period (1991-2002) could be elucidated. METHODS AND RESULTS: A total of 124 strains of Salmonella Enteritidis isolated from human in Taiwan between 1998 and 2002 were analysed by PFGE, plasmid analysis and phage typing. The results obtained were compared with those of the 63 strains obtained in 1991-1997, so that the clonal relationships for a total of 187 strains obtained over 13 years could be elucidated. For PFGE, restriction enzymes XbaI, SpeI and NotI were used for chromosomal DNA digestion. Results showed 28 PFGE pattern combinations for the 187 Salmonella strains. Of them, pattern X3S3N3 was the major subtype as 130 strains isolated from different locations during 1991-2002 showed this PFGE pattern. For all these 187 strains, the genetic similarity was higher than 80%. Plasmid analysis showed 17 distinct types, which consist of one to four plasmids and the predominant phage type of those strains was PT4 (71.6%) and PT6a (13.4%). The three methods identified different degrees of polymorphism in the following order: plasmid profile (18 types, D = 0.659) > PFGE (28 types, D = 0.512) > phage typing (13 types, D = 0.438). As PFGE patterns, phage type and plasmid profile were combined for subtyping, the 187 strains could be grouped into 46 subtypes and the discriminatory index was raised to 0.795. For these 46 subtypes, the predominant one was X3S3N3/P1/PT4, which contained 77 (41%) isolates. CONCLUSIONS: Most of the Salmonella Enteritidis strains from sporadic cases were with pattern X3S3N3. They were the prevalent and may be the epidemic strains found in Taiwan during 1991-2002. The present study suggested that the several variants were derived from a single clonal line and the genome for strains of Salmonella Enteritidis are highly conserved over a 13-year period (1991-2002). SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here are useful for epidemiolgical study of salmonellosis caused by Salmonella Enteritidis in Taiwan. Comparing the data of the present study with those obtained for strains from other countries, the major subtypes for Salmonella Enteritidis infection in the world can be elucidated.

Bacteriophage Typing↗
PubMedDate not supplied

[Dissemination of an extended spectrum beta-lactamase producing Klebsiella pneumoniae strains in a Tunisian university hospital center].

On a period of four years (january 1999-december 2002), 49 strains (non redundant) of extended spectrum beta-lactamase-producing Klebsiella pneumoniae from 43 hospitalised patients and three strains from the environment hospital were collected at Mongi Slim University Hospital Center. The objectives of our work were to investigate for clonality of strains, to clarify transmission fashions and reservoirs of the infection by reviewing clinical records and typing strains. Antibiotic susceptibility testing, plasmid analysis and Random Amplified Polymorphic DNA (RAPD) have been done. 84% of the patients were hospitalized in the intensive care and pediatric units. Urinary infections and septicaemias were the more frequent infections (74.2%). The 49 isolats have been classified in 13 antibiotypes. The plasmid analysis showed 16 patterns. The RAPD revealed 28 patterns and variation within patterns in five cases. Our results showed a diversity of the strains suggesting endemicity, possible transmission of plasmids and persistence of some clones which circulated between services. The used markers permitted the evaluation of a long-term strategy of prevention requiring a strict observance of hygiene rules and rational use of antibiotics.

Adolescent↗
PubMed1990

A nonlinear technique for the analysis of plasmid instability in micro-organisms.

A nonlinear technique to calculate parameters for segregational instability and differences in cellular growth-rate for plasmid-bearing micro-organisms growing in batch or continuous culture is presented. This method is compared with an approximate technique based upon linear regression. The accuracy and sensitivity of the results are evaluated by use of simulated data and biological data taken from experiments with Pseudomonas aeruginosa(pGSS15) and Escherichia coli(pHSG415). It is demonstrated that the nonlinear analysis gives results which are significantly more accurate and which show much better agreement with the data. Consequently, the new analysis leads to quite different conclusions with regard to the nature of the instability of the plasmid-bearing strain. This method offers an opportunity to study the genetic and physiological aspects of plasmid instability and so aid the design and optimization of cloning vectors.

Escherichia coli↗
PubMed2003

Molecular epidemiology of systemic infection caused by Enterobacter cloacae in a high-risk neonatal intensive care unit.

OBJECTIVE: To investigate the molecular epidemiology of systemic nosocomial infections caused by Enterobacter cloacae. SETTING: Neonatal intensive care unit (NICU) of a tertiary-care university hospital. PATIENTS: Forty-two high-risk neonates with systemic infections caused by E. cloacae. METHODS: From 1995 to 1997, the variables associated with death in these patients were evaluated. The molecular epidemiology of the strains responsible for the systemic infections, and 14 unrelated strains, was studied using plasmid analysis and pulsed-field gel electrophoresis (PFGE). RESULTS: The overall mortality rate for infection caused by E. cloacae was 34%, whereas the crude mortality rate during the study period was 8.12% (P < .001). Gestational age (preterm neonates) and birth weight (small for gestational age) were not associated with a higher risk of death. Insertion of a venous catheter by dissection of a peripheral vein was the only invasive procedure related to death (P = .016) in this study. A molecular analysis showed that three outbreaks, each occurring in a different year, were caused by strains with distinctive DNA profiles. Only one outbreak was identified by the infection control service, in the NICU. Plasmid analysis and PFGE showed similar ability to discriminate control strains from the E. cloacae strains isolated from the neonates. CONCLUSIONS: Systemic infections caused by E. cloacae in our NICU were associated with a high mortality rate and occurred as small, unrecognized outbreaks. These results may not be generalizable because the data were from a single center.

Bacterial Typing Techniques↗
PubMed1996

Epidemiological study of an outbreak of infection with Staphylococcus aureus resistant to lincosamides and streptogramin A in a French hospital.

A significant increase in the incidence of isolates of methicillin-resistant Staphylococcus aureus (MRSA), that were also resistant to lincosamides and streptogramin A (LSA-MRSA), was observed in a French university hospital. Twenty-seven isolates from the outbreak were characterised, including 17 isolates from a plastic surgery ward and six control strains of MRSA. The strains were examined by antibiotyping and biotyping, and by three molecular methods: plasmid analysis, ribotyping and insertion sequence (IS) typing with IS256 sequence as a probe. Antibiotyping (five antibiotypes) was discriminatory because of the uncommon resistance phenotype of the epidemic strain. Biotyping (three biotypes), DNA plasmid analysis (four profiles) and ribotyping (two profiles) were poorly sensitive, in contrast to IS-typing (12 profiles). By the latter method, a coefficient of similarity (percentage similarity) compared to the predominant IS profile was calculated. Strains with a coefficient of similarity > or = to 82% were considered as highly related to the epidemic strain, while those with a coefficient of similarity < or = to 40% were regarded as distant. Results obtained with the five markers confirmed that an outbreak of hospital infection had occurred in the plastic surgery ward, with spread of the epidemic strain throughout the hospital.

Adult↗
PubMed1999

Molecular typing of methicillin-resistant Staphylococcus aureus in a university teaching hospital.

Plasmid analysis and pulsed-field gel electrophoresis (PFGE) were used to study the epidemiologic relationship among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated at Tokyo Medical and Dental University Hospital. We found that 263 of 276 MRSA isolates had plasmids, which could be classified into 30 different patterns according to the number and plasmid molecular weight. Strains which harboured a single plasmid of approximately 13.4 Mds in molecular weight were the most numerous (55.7% of the isolates). These strains were isolated from 14 of 17 hospital wards. The largest number of strains with this plasmid pattern (33 strains) were isolated from a single ward. PFGE typing was then performed to further confirm the relationships among these 33 strains. The PFGE banding patterns of these strains were highly similar. The antibiogram profiles of these strains were also correlated with the PFGE pattern. Thus, the results suggest that these strains are epidemiologically related and spread throughout the ward. Combined plasmid analysis and PFGE were effective for discriminating the various MRSA isolates.

DNA, Bacterial↗
PubMed1993

Recurrent epidemics caused by a single strain of erythromycin-resistant Staphylococcus aureus. The importance of molecular epidemiology.

OBJECTIVE--To compare the accuracy of clinical epidemiologic findings with results of molecular epidemiologic analysis in identifying the source of recurrent epidemics of erythromycin-resistant Staphylococcus aureus (ERSA) infections in a well-baby nursery. DESIGN--Epidemic investigations with retrospective and prospective microbiological surveillance. Staphylococcus aureus isolates were evaluated by plasmid analysis and genotyping. SETTING--A well-baby nursery in a 700-bed university teaching hospital with approximately 250 deliveries per month. PATIENTS--Newborn infants who developed ERSA infections during 1990 and 1991. INTERVENTION--Traditional infection control measures, including cohorting of infected infants and hand washing, were implemented. Personnel were cultured for nasal carriage of S aureus during both epidemics. Employees carrying ERSA were removed from the nursery and treated with mupirocin. RESULTS--In the first epidemic, 15 infants were infected with ERSA. A nursing assistant who cared for most of the infants was found to be a carrier of ERSA. She was removed from the nursery and the epidemic resolved. Fifteen months after the first epidemic ended, an epidemic that involved 11 infants began. The attending physician had a facial furuncle and was found to be a carrier of ERSA. The physician was treated and the epidemic resolved. Plasmid and genotyping showed the ERSA organisms from both epidemics were the same. The employee implicated in the first epidemic did not have the epidemic strain, but the physician who attended during both epidemics did. CONCLUSIONS--Traditional epidemic investigations may lead to false conclusions that can only be recognized with molecular epidemiologic techniques. For these techniques to be useful in the control of outbreaks, plasmid analysis and/or genotyping must be readily available.

Bacterial Typing Techniques↗
PubMed1989

Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants.

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.

Conjugation, Genetic↗