Repetitive Sequence-based Polymerase Chain Research Articles

Munchausen syndrome mimicking refractory subcutaneous abscess with bacteremia, diagnosed by repetitive element sequence-based polymerase chain reaction: a case report

BackgroundRapid diagnosis and appropriate treatment of Munchausen syndrome is important not only for the patient but also for health care workers because a delay in diagnosis can worsen patients’ clinical outcomes, and result in a substantial medical cost.Case presentationA young and previously healthy 24-year-old Japanese woman, a nurse, presented with complaints of refractory abscess on her left upper limb for 3 months. A physical examination on admission revealed low-grade fever and a subcutaneous abscess in her left forearm. Laboratory data suggested mild systemic inflammation and liver dysfunction, but no abnormalities of the immune system, including changes in the number of lymphocytes and neutrophils, neutrophil phagocytic capacity, and natural killer (NK) cell activity, were observed. A human immunodeficiency virus test was also negative. Multiple modalities, including positron emission tomography-computed tomography, failed to detect any cause and focus of infection except her left upper limb. Streptococcus mitis and Prevotella buccae were detected from the wound, but no microorganisms were detected in a blood culture. The cellulitis promptly resolved; however, exacerbation of the subcutaneous abscess with polymicrobial bacteremia repeatedly occurred unexpectedly. Because of this puzzling clinical course, the possibility of self-injury was finally suspected. Three syringes with needles, with a turbid liquid, were found in our patient’s bag. Enterobacter cloacae and Enterococcus faecalis were detected in the liquid, and an analysis via repetitive element sequence-based polymerase chain reaction determined that Enterococcus faecalis in the wound and syringe contents were genetically identical. She was diagnosed as having Munchausen syndrome and treated with the collaboration of a psychiatrist. She finally confessed that she had injected her own saliva and toilet water into the drip line and wound.ConclusionsThis case report is valuable in that it is the first case in which this syndrome was diagnosed by a genetic method. Munchausen syndrome should not be neglected as a possible cause of refractory and recurrent infection.

DIVERSITY OFRALSTONIA SOLANACEARUMPOPULATIONS AFFECTING TOBACCO CROPS IN NORTH CAROLINA

One hundred eighty four strains of Ralstonia solanacearum isolated in 2007 and 2008 from 11 tobacco fields in North Carolina were evaluated for genotypic diversity and aggressiveness. All strains were race 1, biovar 1, and belonged to phylotype II. Genetic diversity of the strains was assessed with the use of repetitive sequence–based polymerase chain reaction. DNA primers (REP, ERIC, and BOX) were used to generate genomic fingerprints. Both REP and BOX revealed 3 patterns: Ar, Cr, and Dr and Ab, Cb, and Db, respectively. Five patterns were identified with ERIC-PCR. Pattern Ae was found in 80% of the strains collected. Pattern Be was in 4%, pattern Ce in 13%, pattern De in 2%, and pattern Ee in 1% of the strains collected. Cluster analyses showed that the strains were 88% similar and constitute a rather homogeneous group. Aggressiveness of strains was evaluated on 3 tobacco cultivars with different levels of resistance to bacterial wilt. Overall, aggressiveness depended on the field from which the strains...

Selection of enhanced antimicrobial activity posing lactic acid bacteria characterised by (GTG)5-PCR fingerprinting.

The aim of the study was a detail evaluation of genetic diversity among the lactic acid bacteria (LAB) strains having an advantage of a starter culture in order to select genotypically diverse strains with enhanced antimicrobial effect on some harmfull and pathogenic microorganisms. Antimicrobial activity of LAB was performed by the agar well diffusion method and was examined against the reference strains and foodborne isolates of Bacillus cereus, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. Antifungal activity was tested against the foodborne isolates of Candida parapsilosis, Debaromyces hansenii, Kluyveromyces marxianus, Pichia guilliermondii, Yarowia lipolytica, Aspergillus brasiliensis, Aspergillus versicolor, Cladosporium herbarum, Penicillium chrysogenum and Scopulariopsis brevicaulis. A total 40 LAB strains representing Lactobacillus (23 strains), Lactococcus (13 strains) and Streptococcus spp. (4 strains) were characterised by repetitive sequence based polymerase chain reaction fingerprinting which generated highly discriminatory profiles, confirmed the identity and revealed high genotypic heterogeneity among the strains. Many of tested LAB demonstrated strong antimicrobial activity specialised against one or few indicator strains. Twelve LAB strains were superior in suppressing growth of the whole complex of pathogenic bacteria and fungi. These results demonstrated that separate taxonomic units offered different possibilities of selection for novel LAB strains could be used as starter cultures enhancing food preservation.

An Exploratory Study of Staphylococcus epidermidis Isolates from Environment and Patient Blood Cultures from an Intensive Care Unit

This exploratory study determined the epidemiological characteristics of Staphylococcus epidermidis from near touch sites and blood cultures in one Intensive Care Unit (ICU) between July and October 2009 in a large teaching hospital in Brazil. Five different touch sites around the environment of two different patients were sampled over four days and 24 blood cultures from 24 patients were obtained and analyzed. Isolates from near touch sites and blood cultures were compared by means of the repetitive extragenic palindromic sequence-based polymerase chain reaction (rep-PCR) and analyzed by the unweighted pair-group method with arithmetic mean (UPGMA) (BioNumerics, 6.0). Results found that ciprofloxacin- resistant Staphylococcus epidermidis on stethoscopes, bed rails ( P< . 004), and patient blood cultures indicated similarity (60-65%; IC: 85%). The similarities among near touch sites and patient isolates are unsurprising due to the commensal nature of Staphylococcus epidermidis, however it does emphasize the need for further research into environmental contamination and cleaning.

Molecular Typing ofAcinetobacter baumanniiIsolated from Chinese Intensive Care Units Before and After the 2008 Sichuan Earthquake

Objective To trace the spread of Acinetobacter baumannii in intensive care units (ICUs) before and after the 2008 Sichuan earthquake in China. Methods A baumannii strains were collected between July and October 2007 from control patients and in May 2008 from survivors of the Sichuan earthquake. The multidrug resistance proflie of A baumannii was observed. The DiversiLab semi-automated rep-PCR system was used to determine the clonal relatedness of A baumannii strains; testing for antimicrobial susceptibility was performed using automated instruments. Results A total of 28 A baumannii strains were isolated, 13 of which had been isolated from control patients; 15 strains were isolated from earthquake survivors. Regardless of patient group, 2 clonal groups were observed in these isolates. The results demonstrate that most of the A baumannii isolates recovered from the earthquake survivors were similar to those obtained from the control patients. This suggests that A baumannii species, particularly those of clonal groups A and B (most of which are multidrug-resistant [MDR]), have been well established in our ICU; that earthquake survivors probably acquired A baumannii in the hospital setting; and that A baumannii recovered from earthquake survivors may bring different genetic profiles into contact with local isolates. Conclusions Our findings highlight the need for stronger infectioncontrol measures and emphasize the importance of rapid strain typing for monitoring pathogens such as A baumannii that are frequently associated with nosocomial infections. * MDR : multidrug-resistant ICU : intensive care unit rep-PCR : repetitive extragenetic palindromic sequence-based polymerase chain reaction CLSI : Clinical and Laboratory Standards Institute (CLSI)

Detection of Strain Variation in Isolates of Rhodococcus Equi from an Affected Foal Using Repetitive Sequence–Based Polymerase Chain Reaction

Rhodococcus equi is an important pathogen of foals aged 1-6 months. Evidence exists that foals are exposed to a wide diversity of R. equi strains in their environment. However, limited data are available regarding the extent to which genotypic variation exists among isolates infecting individual foals. Therefore, electrophoresis of repetitive sequence-based polymerase chain reaction (rep-PCR) amplicons in an automated microfluidics chip format was used to genotype 9 virulent R. equi isolates obtained from distinct anatomic locations in a single foal. Four of the isolates were obtained from different regions of the lung, and 5 were from abscessed intra-abdominal lymph nodes (LNs). Six distinct genotypes were identified among the 9 isolates. None of the pulmonary isolates was identical; however, a pulmonary isolate was found to be identical to an isolate recovered from a small intestinal LN, and another pulmonary isolate was identical to an isolate from a mesenteric LN. These results indicate that foals can be infected with multiple strains of virulent R. equi. Furthermore, identical strains can be found in multiple, remote anatomic locations in an infected foal, and this can occur for >1 strain in the same foal. The automated system used in the current study provided a rapid, reproducible, and discriminating method for typing R. equi isolates.

Repetitive sequence based polymerase chain reaction to differentiate close bacteria strains in acidic sites

To study the diversity of bacteria strains newly isolated from several acid mine drainage(AMD) sites in China, repetitive sequence based polymerase chain reaction (rep-PCR), a well established technology for diversity analysis of closely related bacteria strains, was conducted on 30 strains of bacteria Leptospirillum ferriphilium, 8 strains of bacteria Acidithiobacillus ferrooxidans, as well as the Acidithiobacillus ferrooxidans type strain ATCC (American Type Culture Collection) 23270. The results showed that, using ERIC and BOX primer sets, rep-PCR produced highly discriminatory banding patterns. Phylogenetic analysis based on ERIC-PCR banding types was made and the results indicated that rep-PCR could be used as a rapid and highly discriminatory screening technique in studying bacterial diversity, especially in differentiating bacteria within one species in AMD.

Interinstitutional and Intrainstitutional Transmission of a Strain ofAcinetobacter baumanniiDetected by Molecular Analysis Comparison of Pulsed-Field Gel Electrophoresis and Repetitive Sequence–Based Polymerase Chain Reaction

Pulsed-field gel electrophoresis and repetitive sequence-based polymerase chain reaction provided comparable strain discrimination with minor discordance in typing Acinetobacter baumannii clinical isolates from patients at our hospital and affiliated institutions. Typing revealed a cluster strain with intrainstitutional and interinstitutional spread during the study period. A long-term acute care facility may have been the reservoir.

Rep-PCR helps to distinguish different alloplasmic cytoplasmic male sterile lines of Brassica juncea

Cytoplasmic male sterility (CMS) in higher plants is encoded by the mitochondrial genome. Several evidences support the view that organelle genomes of eukaryotes are derived from ancestral prokaryotic symbioants. Hence, it is expected that techniques suitable for distinguishing prokaryotes will be useful for distinguishing organelle genomes. Repetitive sequence based polymerase chain reaction (rep-PCR) technique is widely employed to identify different bacterial strains. We, therefore, tested the utility of rep-PCR for distinguishing different CMS lines of Brassica juncea. Using just three sets of rep-PCR primers, all the six Brassica lines could be unambiguously identified. Cloning and sequencing of a unique 1.5 kb amplicon obtained with Box1AR primer and template DNA of CMS catholica ( W), indicated that the amplicon is indeed derived from mitochondrial genome. Further, based on sequence information of the 1.5 kb clone, a sequence characterized amplified region (SCAR) primer capable of distinguishing CMS catholica ( W) from other lines was identified. The advantages of rep-PCR over other methods for distinguishing CMS lines are discussed.

Molecular genotyping of methicillin-resistant Staphylococcus aureus strains in a teaching hospital in Turkey.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of nosocomial infections in our hospital. Therefore, we aimed to characterize MRSA isolates phenotypically from patients with nosocomial infections at Cumhuriyet University Hospital between December, 1999, and June, 2001, in Sivas by analysis of antibiotic patterns and genotypically using pulsed-field gel electrophoresis (PFGE) and repetitive element sequence-based polymerase chain reaction (rep-PCR). Forty-three nosocomial isolates were collected from various wards. All isolates were resistant to penicillin, tetracycline, oxacillin, and gentamicin. By rep-PCR and by separation of SmaI fragments of genomic DNA using PFGE, one major type (eight subtypes with PFGE) was identified among the strains. This clone was found to be different than some clones such as Iberian, Brazilian, and a major clone that was found in another Turkish University Hospital in Ankara. According to our results, there is a major MRSA clone with a potential to spread in our hospital. Infection control measures should be directed toward restricting the further spread of this clone. Therefore, in accordance with these findings, a surveillance culturing program should be established.

Multidrug-Resistant Salmonella Typhimurium DT104 in Poultry

Salmonella Typhimurium isolates from feed ingredients or poultry sources isolated during 1995 to 1997 from different geographical locations within Minnesota were examined for the presence of Salmonella Typhimurium definitive type 104 (DT104). Antibiotic susceptibility studies indicated that 15 of 50 isolates of Salmonella Typhimurium had an antibiotic resistance pattern (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) that is usually observed with multidrug-resistant Salmonella Typhimurium DT104. Of the 15 isolates showing the antibiotic resistance pattern, 8 isolates were phage type 104, 3 isolates were typed as phage type 104 complex, and the remaining 4 isolates belonged to phage types 193, 81, and 126. DT104 was recovered from both feed ingredients and poultry samples. Of the seven feed ingredient–associated Salmonella Typhimurium isolates, four were DT104, whereas only 7 of 43 poultry-associated Salmonella Typhimurium isolates were DT104. A repetitive sequence–based polymerase chain reaction (rep-PCR) of 50 isolates of Salmonella Typhimurium representing 13 phage types identified seven distinct fingerprint profiles. No correlation between phage type and rep-PCR type was noticed. Eleven Salmonella Typhimurium isolates belonging to DT104 and its complex were grouped into two closely related rep-PCR types.