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Background: It is challenging to determine whether Bacillus species other than Bacillus anthracis cause infections. Pseudo and true outbreaks of Bacillus spp. have been noted. Here, we present a molecular analysis of a Bacillus spp. pseudo-outbreak caused by contaminated culture tubes containing Stuart medium. Methods: Between January and March 2015, a high percentage of Bacillus spp. was isolated from the wound samples of inpatients at the Karabuk University Hospital, and an outbreak was suspected. Environmental and staff nasal samples were cultured aerobically, and Bacillus spp. were isolated from some of them. However, the isolation of Bacillus spp. in throat cultures of outpatients suggested contamination caused by culture tubes containing Stuart medium. We examined two lots of culture tubes used in the hospital. Although the culture tubes' expiry date and storage conditions were suitable, Bacillus spp. grew in one of these lots. A total of 47 Bacillus spp. isolated during this period were identified, and the clonal relationship among the isolates was investigated by arbitrarily primed polymerase chain reaction. Results: Twenty-seven strains were identified as Bacillus megaterium and 20 as Bacillus firmus. Of the four strains isolated from the Stuart medium, two were identified as B. firmus and the other two were B. megaterium. Two B. firmus strains isolated from the Stuart medium and two B. firmus strains obtained from the coronary intensive care environmental samples were matched and clustered within the same genotype. We recalled all culture tubes containing Stuart medium. After another brand's culture tubes were distributed, no growth was observed. It was then understood that the pseudo-outbreak source was contaminated culture tubes containing Stuart medium. Conclusions: Microbiological controls of medical materials and equipment should be regularly checked to prevent outbreaks (true or pseudo). [ABSTRACT FROM AUTHOR]

Background and Objective: Onychomycosis is a fungal infection of the nails; when caused by dermatophytes, it is called as Tinea unguium. The aim was to explicate the utility of arbitrarily primed polymerase chain reaction (AP-PCR) to augment the early and accurate clinical diagnosis of dystrophic onychomycosis. Materials and Methods: The collected nail samples were divided into three portions. The first portion was explored for direct microscopic examination, the second portion was used for culturing of the nail samples on sabouraud dextrose agar media, and the third portion was used for DNA extraction followed by AP-PCR identification of dermatophytes. Results: All 48 samples with dystrophic onychomycosis were diagnosed by three methods, namely, 20% Potassium hydroxide microscopy, culture growth, and AP-PCR. With all the methods, Trichophyton rubrum was found as major causative agents for dermatophyte nail infection (60%), followed by Trichophyton mentagrophytes (23% cases). AP-PCR is a convenient method to achieve a higher dermatophyte identification rate with lesser time and shows complete concordance with conventional culture for two Trichophyton species. Conclusion: AP-PCR is a rapid, specific, and sensitive procedure for the diagnosis of nail fungal infection.

Although Corynebacterium striatum is part of the human flora, it has recently drawn attention both for its multidrug resistance and its role as an invasive infection/outbreak agent. This cross-sectional study aimed to determine the antimicrobial resistance and clonal relationships among C. striatum strains. In total, 81 C. striatum strains were identified using Phoenix-100TM (BD, Sparks, MD, USA). The antimicrobial resistance of the strains was determined using the Kirby−Bauer disk diffusion method. Clonal relatedness among the strains was performed via arbitrarily primed polymerase chain reaction (AP-PCR). All 81 C. striatum strains were resistant to penicillin, cefotaxime, ciprofloxacin, and tetracycline, but susceptible to vancomycin and linezolid. The resistance rates to gentamicin, erythromycin, and clindamycin were 34.6%, 79%, and 87.7% respectively. AP-PCR results showed no predominant clone among the C. striatum strains. Corynebacterium striatum is reportedly the cause of an increasing number of invasive infections/outbreaks. Moreover, treatment options are limited. The study showed that vancomycin, linezolid, and gentamicin can be selected for the empirical treatment of C. striatum infections. Although no single-clone outbreak was observed in our hospital, small clonal circulations were observed within some units, indicating cross-contamination. Therefore, a comprehensive infection control program is warranted in future.

Introduction The aim of this study was to evaluate the diversity and similarity of Enterococcus faecalis genotype isolates from multiple oral sites using repetitive sequence-based polymerase chain reaction and arbitrarily primed polymerase chain reaction (AP-PCR). Methods Forty-two endodontically treated teeth with apical periodontitis were selected. A total of 126 microbial samples were collected from 3 different sites (saliva, pulp chamber, and root canals, all n = 42) during the nonsurgical retreatment procedures. After growth on m-Enterococcus agar, the colonies were isolated, characterized as gram-positive catalase negative cocci, and identified using an API 20 Strep kit (bioMérieux, Marcy-l'Etoile, France). Seventy-four colonies from 10 patients were confirmed as E. faecalis by polymerase chain reaction (16S ribosomal RNA). Repetitive sequence-based polymerase chain reactions using ERIC and AP-PCR using RW3A primers were performed in all 74 colonies. Fingerprints were analyzed and separated into genotypic groups based on the Dice coefficient percentage of similarity (82% or greater) as determined by ERIC reproducibility assays involving E. faecalis controls. Results Seven different E. faecalis genotypes (GTs) (GT1 = 27%, GT2 = 17.6%, GT3 = 1.3%, GT4 = 18.9%, GT5 = 9.5%, GT6 = 14.9%, and GT7 = 10.8%) were observed in different subjects and oral sites associated with endodontic failure. Remarkably, in 4 of 5 patients, the same GTs present in the infected root canals were also isolated from either the pulp chamber or the saliva samples. In particular, GT6 was detected in all 3 oral sites of patient 37. Conclusions E. faecalis GTs isolated from saliva, the pulp chamber, and the root canal were similar using the Rep-PCR and AP-PCR methods. These findings suggest that coronal microleakage is a conceivable cause of endodontic failure. [ABSTRACT FROM AUTHOR]

Objective: Sphingomonas paudmobilis is a non-fennentative, gram negative bacillus which may came epidemics especially at intensive care units (1CU), where infections are most commonly seen. We aimed to evaluate clonal relation of S. paudmobilis isolates, obtained from both patients and Internal Medicine ICU environment. Material and Method: Three S. paudmobilis isolates were detected, within one month from patients residing in Internal Medicine ICU. In order to identify the infection source, microbiological examination of environmental samples was performed. Identification of S. paudmobilis was done with conventional methods and VITEK2 (bioMerieux, France) automated identification system. Antibiotic susceptibilities of clinical and environmental S. paudmobilis isolates were evaluated with VITEK2 system and clonal relationship of the isolates were evaluated with "Arbitrarily Primed" Polymerase Chain Reaction (AP-PCR). Results: S. paudmobilis samples were isolated from three patients and five different oxygen humidifiers during environmental sampling. Phenotypic antimicrobial susceptibility patterns of total eight isolates from both groups were found identical. However, five isolates from oxygen humidifiers showed similar band patterns on AP-PCR while two patient isolates were different from environmental samples and one of them were different from all others. Conclusion: Absence of genotypic relation between environmental and patient isolates showed us that the oxygen humidifiers were not the source of the S. paudmobilis contamination. Revealing the clonal relations via molecular epidemiologic methods, helps to detection of isolates related with outbreaks and increases the effectiveness of the infection control programs of hospitals. AP-PCR is among the primarily used molecular genotyping methods during short-term outbreaks. [ABSTRACT FROM AUTHOR]

To identify and characterize novel genetic alterations in hepatocellular carcinoma (HCC). DNA was extracted from 29 HCC and corresponding normal tissues and amplified with 59 different 10-base arbitrary primers. A 550 bp DNA fragment amplified using primer Q-9 and which was present in 19 of 29 cases (66%) was cloned, sequenced, and compared with known nucleotide sequences deposited in Genome database, and quantified by real-time PCR. DNA alterations were found on chromosomes 5q34, 6p25.2 and 8q12.1 in 11 of 29 cases (38%), 7 of 29 cases (24%), and 12 of 29 cases (41%), respectively. Multivariate analysis showed that the allelic loss on chromosome 5q34 was an independent prognostic factor for poor survival of HCC patients, with the median survival time of 19 weeks for allelic loss versus 109 weeks for no allelic loss ( P = 0.001). This study indicates that allelic loss on chromosome 5q34 may be involved in the development of HCC and could be used as a prognostic indicator in HCC patients. [ABSTRACT FROM AUTHOR]

Purpose: To detect and characterize amplified DNA sequences in cholangiocarcinoma (CCA). Patients and methods: We extracted DNA from tumor and corresponding normal tissues of 30 patients with CCA and amplified with 30 random ten-mer arbitrary primers by the arbitrarily primed polymerase chain reaction (AP-PCR) technique. Results: Our results showed gains of genomic sequences at high frequency. Using the AX-11 arbitrary primer, we determined an amplified DNA fragment occurred frequently in the tumors analyzed. The DNA fragment was isolated and identified as two sequences mapped to chromosomes 2p25.3 and 7q11.23. Specific primers were designed employing these sequences and used for detecting amplification by real-time quantitative PCR. The amplification of the DNA sequences on chromosomes 2p25.3 and 7q11.23 was detected in 10 (33%) and 6 (20%) cases, respectively. Thirteen (43%) cases showed amplification on both or one of the chromosomes. In addition, amplification of the DNA on chromosome 2p25.3 was predominantly observed in poorly differentiated tumors. Conclusions: Our findings suggest that the novel amplified DNA on chromosomal regions at 2p25.3 and 7q11.23 might be involved in the development and progression of CCA. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate the colonization profile and clonal distribution of Streptococcus mutans isolated from oral cavities that presented coronal and root caries lesions. The isolation and biochemical identification of mutans streptococci were carried out by using saliva samples, dental plaque, and tissue from the caries lesions. In order to confirm their molecular identity, S. mutans and Streptococcus sobrinus were submitted to the PCR method, using specific primers for portions of the glucosyltransferase genes (gtfB and gtfI, respectively). The AP-PCR method was used to detect the genetic polymorphism of S. mutans strains. Among the isolated and identified species, S. mutans showed a significantly greater frequency of isolation (59.2%) than the other mutans streptococci. Each of the subjects harbored two to ten genotypes of S. mutans, randomly distributed in different sites. S. mutans genotypes showed no evidence of variability in colonizing noncarious and carious surfaces within the same individual, nor evidence of etiologic differences between coronal and root caries. This study showed that no particular genotype of S. mutans is uniquely associated with the initiation and progression of caries, and that root and coronal caries can emerge in the presence of a broad spectrum of S. mutans clones. [ABSTRACT FROM AUTHOR]

We investigated mother-to-newborn infant transmission of Lactobacillus species in Japanese by the typing of isolates from the vagina of pregnant women and stool specimens from their newborn infants. All infants were born by uncomplicated vaginal delivery and were basically fed breast milk. Lactobacillus strains were fingerprinted by arbitrarily primed polymerase chain reaction (AP-PCR), using ERIC1R and ERIC2 primers. Of 86 pregnant women tested, 71 (82.6%) were positive for vaginal lactobacilli. At 5 days after birth, 24 (33.8%) of the 71 infants whose mothers were lactobacilli-positive had fecal lactobacilli, while only 1 (6.7%) of the 15 infants delivered from the vaginal lactobacilli-negative mothers was lactobacilli-positive ( P < 0.01). Lactobacillus crispatus was the most prevalent species of vaginal lactobacilli in mothers and of fecal lactobacilli in infants at 5 days of age, whereas Lactobacillus gasseri was the most common in infants at 1 month of age. Identification to the species level, followed by AP-PCR typing, demonstrated that 23.3% of the 86 infants were likely to be colonized in the intestine by the vaginal lactobacilli of their mothers, and that only 2 of the infants retained the same vaginal lactobacilli until 1 month of age. These results suggest that approximately one-fourth of infants acquire vaginal lactobacilli from their mothers at birth, and that the acquired lactobacilli do not last in the intestine of the infant long-term, but rather, are replaced by ones from milk or unknown sources after birth. AP-PCR with primers ERIC1R and ERIC2 is indicated as a potential tool for the typing of Lactobacillus strains. [ABSTRACT FROM AUTHOR]

F. nucleatum is the most commonly isolated microorganism from subgingival plaque, but the role of this microorganism in periodontal diseases remains undefined. Arbitrarily primed polymerase chain reaction (AP-PCR) was evaluated as a method for fingerprinting F. nucleatum isolates and for use in clonal analysis. Pulsed field gel electrophoresis was used to further differentiate F. nucleatum isolates, with identical AP-PCR patterns. Extremely heterogeneous AP-PCR fingerprints were observed among the 98 F. nucleatum isolates, with 36 different genotypes observed with primer Cl and 30 different genotypes detected with primer C2. Combining the results of the AP-PCR genotype analysis from Cl and C2 primer amplifications revealed that up to 7 different genotypes could be distinguished from isolates from the same oral cavity and that up to 4 different genotypes were observed within a single site. An intense amplicon at approximately 450 bp generated in AP-PCR amplification with primer C2 was associated with F. nucleatum subsp. nucleatum (ATCC 25586) and with 15 F. nucleatum isolates from diseased sites and 2 isolates from healthy sites. Pulsed field gel electrophoresis confirmed the AP-PCR genotypes and demonstrated increased discriminatory power over AP-PCR. The results indicated that AP-PCR and pulsed field gel electrophoresis provide a simple and sensitive means for differentiating oral F. nucleatum isolates and further demonstrate the heterogeneity of this species. These techniques may serve as useful tools in the clonal and epidemiological analysis of F. nucleatum isolates, which may help define the role of these microorganisms in periodontal diseases. [ABSTRACT FROM AUTHOR]

The distribution and the genetic similarity of Prevotella intermedia and Prevotella nigrescens in saliva and in subgingival samples recovered from the same subject were studied in 16 subjects with different periodontal status. The isolates (4 salivary and 4 subgingival P. intermedia/nigrescens group isolates per subject) were identified to species level by hybridization with species-specific oligonucleotide probes, and the clonal analysis was performed using arbitrarily primed polymerase chain reaction (AP-PCR) (all isolates) and ribotyping (isolates from 5 subjects). In addition, the applicability of AP-PCR in differentiating between P. intermedia and P. nigrescens species was tested using 18 P. intermedia and 20 P. nigrescens isolates from 34 subjects. P. intermedia was detected in 7 and P. nigrescens in 14 of the 16 subjects. In all subjects the same species was found both in saliva and in subgingival plaque. In 15 of the 16 subjects, similar AP-PCR types of P. intermedia and/or P. nigrescens between salivary and subgingival samples were found. The salivary and subgingival isolates that were similar by AP-PCR were indistinguishable also by ribotyping. The AP-PCR analysis revealed a P. intermedia or P. nigrescens species-specific AP-PCR product in most isolates. This study indicates that both P. intermedia and P. nigrescens were found both in salivary and in subgingival samples, and both sampling sites within the same individual were usually colonized with identical AP-PCR types of the species. Thus, in addition to a subgingival sample a salivary sample seems to be suitable for detection and clonal analysis of these species. The AP-PCR method proved to be a simple method applicable for differentiation and clonal analysis of P. interinedia and P. nigrescens. [ABSTRACT FROM AUTHOR]

Although Corynebacterium striatum is part of the human flora, it has recently drawn attention both for its multidrug resistance and its role as an invasive infection/outbreak agent. This cross-sectional study aimed to determine the antimicrobial resistance and clonal relationships among C. striatum strains. In total, 81 C. striatum strains were identified using Phoenix-100TM (BD, Sparks, MD, USA). The antimicrobial resistance of the strains was determined using the Kirby–Bauer disk diffusion method. Clonal relatedness among the strains was performed via arbitrarily primed polymerase chain reaction (AP-PCR). All 81 C. striatum strains were resistant to penicillin, cefotaxime, ciprofloxacin, and tetracycline, but susceptible to vancomycin and linezolid. The resistance rates to gentamicin, erythromycin, and clindamycin were 34.6%, 79%, and 87.7% respectively. AP-PCR results showed no predominant clone among the C. striatum strains. Corynebacterium striatum is reportedly the cause of an increasing number of invasive infections/outbreaks. Moreover, treatment options are limited. The study showed that vancomycin, linezolid, and gentamicin can be selected for the empirical treatment of C. striatum infections. Although no single-clone outbreak was observed in our hospital, small clonal circulations were observed within some units, indicating cross-contamination. Therefore, a comprehensive infection control program is warranted in future. [ABSTRACT FROM AUTHOR]