Jundishapur Journal of Microbiology
Year:2019 | Volume:12 | Issue:4|Papers Count:8

Background: Klebsiella pneumoniae is an important human pathogen that causes severe diseases including urinary tract infection, pneumonia, and bacteremia. However, a rapid and sensitive detection method remains to be developed. Objectives: This study aimed to develop a rapid, real-time, and visual detection method for K. pneumoniae. This is a primary screening method to improve the diagnosis of K. pneumoniae infection and save much precious time for clinical practice. Methods: Klebsiella pneumoniae was used as an antigen to produce a monoclonal antibodies (mAb). The mAb 1E6 recognizing outer membrane protein C on the surface of K. pneumoniae was screened by an indirect enzyme-linked immunosorbent assay (ELISA), Western Blot, and mass spectrometry assay, and then conjugated with the protein A/G coated magnetic beads to generate the immune magnetic beads (IMBs). Thereafter, the IMBs were used to capture K. pneumoniae, and the complex (beads-mAb-K. pneumoniae) was used for loop-mediated isothermal amplification (LAMP) assay. Results: We developed a rapid, real-time, and visual detection method employing an immunocapture loop-mediated isothermal amplification (IC-LAMP). The sensitivity of the IC-LAMP was 4 CFU mL-1. The process of K. pneumoniae detection lasted ~ 60 minutes and had no cross-reaction to other microbial strains. Besides, the accuracy of IC-LAMP was further verified by examining 39 clinical isolates. Conclusions: Without the need for enrichment of bacteria and extraction of its genome, the IC-LAMP developed here could be used as a primary screeningmethodsupplementary to traditional detectionmethodsto improve the diagnosis of K. pneumoniae infection for clinical practice.

Background: Antimicrobial resistance is a growing healthcare system threat of huge concern worldwide. Objectives: This study aimed to report the seven-year trend of antimicrobial resistance of Acinetobacter and Pseudomonas spp. causing bloodstream infections (BSIs) in Shiraz, southern Iran, during 2010-2016. Methods: This retrospective study was conducted on the recorded blood cultures during 2010-2016. The susceptibility testing of isolates was performed by the agar diffusion test. Data were grouped into three episodes: 2010 to 2011, 2012 to 2013, and 2014 to 2016. The chi-square test was used to determine the significance of antimicrobial resistance trends. Results: The rates of resistance to antibiotics such as amikacin, cefepime, cefotaxime, ceftazidime, ciprofloxacin, and piperacillintazobactam were high within 2014-2016, with a statistically significant increasing trend over the abovementioned three periods. The resistance rates of Pseudomonas spp. to the antibiotics such as amikacin, tobramycin, gentamicin, cefepime, ceftazidime, and ciprofloxacin were high in 2014-2016 with a statistically significant increasing trend over the three periods. Conclusions: The increasing trend of antimicrobial resistance of Acinetobacter and Pseudomonas spp. to almost all the conventional antibiotics over the seven-year period of this study is alarming.

Background: Brucella spp. are Gram-positive, rod-shaped, and spore-forming bacilli. Brucella abortus and B. melitensis are the main causes of brucellosis. Objectives: The aim of the study was to establish a rapid and simple molecular method for the detection of this disease. Methods: Forty-five Brucella spp. were isolated from blood samples using the BACTEC Fluorescent 9050 system and were detected by anti-IgM or IgG Brucella specific antigen. DNA extraction was conducted on all samples. Fluorescent amplification based specific hybridization (FLASH-PCR) test was utilized to detect the 351-bp fragment from eryD gene, which was specific for Brucella spp. Results: A351-bp fragment resulted fromPCRreactionandshowedthe accuracy of designed primers. This fragmentwassuccessfully amplified in the FLASH-PCR reaction. In this study, we have positive and negative samples and a standard method. In addition, we calculated the sensitivity and specificity of this method as 100%. Conclusions: Results of the study was proved that the FLASH-PCR method was a rapid, sensitive, and safe method for the detection of Brucella genome in whole blood samples of patient harbored brucellosis and is recommended for routine usage.

Background: The genotype determination is of importance in the clinical management of hepatitis C virus (HCV) infection. Objectives: The aim of this study was to evaluate the distribution of genotypes in HCV patients in the Kahramanmaras region, Turkey, and determine if the genotype rates of Syrian refugee patients with hepatitis C are different from the population they migrated to. Methods: Blood samples sent to the microbiology laboratory between September 2014andFebruary 2018 were examined with genotyping using a real-time PCR reagent kit. Results: The results of 274 patients who were HCV genotype-assigned were obtained from the hospital electronic information system. Of 230 Turkish HCV patients, 121 (52. 8%) were identified as genotype 3, 100 (43. 3%) as genotype 1, five (2. 2%) as genotype 2, and four (1. 7%) as genotype 4. Of the 44 Syrian refugee patients, 24 (54. 5%) were identified as genotype 1, 18 (40. 9%) as genotype 4, one (2. 3%) as genotype 2, and one (2. 3%) as genotype 3. Conclusions: Predominant genotype 3 with the prevalence of 52. 8% was seen to be more frequent in the current study region than in other regions of Turkey, demonstrating the highest rate of genotype 3 infection in Turkey to date. In Syria, the predominant HCV genotype is genotype 4 with a 57% prevalence, followed by genotype 1 with 29%. In the Syrian refugees, however, the current study showed genotype 1 was more frequent than genotype 4 (54. 5 % vs. 40. 9%). This suggests that HCV genotypes may be affected by situations such as war and migration.

Background: Streptococcus mutans is considered the primary bacterial species closely associated with the etiology of dental caries in humans. Recent studies suggest an association between caries and the genetic diversity of S. mutans. Objectives: The aim of this study was to evaluate the genotypic diversity of S. mutans in schoolchildren, its stability in a one-year follow-up study, and its association with dental caries. Methods: We studied 25 schoolchildren. Representative S. mutans colonies were isolated from the dental plaque of each child, grown on mitis-salivarius-bacitracin agar, and inoculated in trypticase soy broth. We performed 16S rRNA gene PCR-RFLP analysis on S. mutans isolates. Dental caries in deciduous and permanent surfaces was scored according to the WHO criteria. After 12 months, the caries incidence, S. mutans count, and genotypic diversity were compared. We grouped samples to observe similarities using cluster analysis. A similarity coefficient of > 95% was considered for defining the genotypes. Results: At baseline, we proved the genotypic diversity of S. mutans with five different genotypes. Caries scores were higher in children with genotype A (dmfs 5. 4 vs. 3. 4). The genotypic diversity of S. mutans in the schoolchildren decreased after one year, with the predominance of genotype A (52% to 92%) that was also associated with high bacterial counts (P = 0. 0063). Conclusions: This study supports that there are changes in the genotype of S. mutans over time, and the more cariogenic genotype is more stable than the others.

Background: Staphylococcus epidermidis causes prosthetic valve endocarditis, urinary tract infection, and implant-related infections. These are difficult to treat often due to drug resistance, particularly because S. epidermidis biofilms are inherently resistant to most antibiotics. Salvia miltiorrhiza is a kind of sichuan-specific medicinal herb, and has effective ingredients, such as cryptotanshinone. Cryptotanshinone was demonstrated to have anti-microbial properties and no resistance. Objectives: The current study investigated the effects of cryptotanshinone on S. epidermidis biofilm formation, and found new agents controlling S. epidermidis biofilm formation and resistance caused by biofilm. Methods: The effects were further analyzed by crystal violet assay (CV), 2, 3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay (XTT), and scanning electron microscopy (SEM). The qRT-PCR assay was used to determine the expressions of biofilm key genes, including icaA, atlE, aap and luxS. Results: The amount treated by cryptotanshinone was reduced compared with the non-treating group, so did the metabolic activity inside the biofilm. Even the micro-structure was destroyed with cryptotanshinone. The expressions of biofilm key genes, including icaA, atlE, aap, and luxS, were down-regulated by cryptotanshinone. Conclusions: There is new insight that cryptotanshinone could inhibit immature biofilms and the down-regulations of icaA, atlE, aap, and luxS might explain this inhibitory effect.

Background: Helicobacter pylori, a Gram-negative bacteria, is the most important cause of gastric ulcer, gastric malignancies, and chronic gastritis. Clarithromycin is recognized as the most important antibiotic for the treatment. Clarithromycin resistance is related to point mutations in the 23srRNA, and the most important mutation is A2143G, A2142G. The most common cause of resistance to metronidazole is rdxA gene mutational inactivation. Objectives: This study aimed to evaluate clarithromycin and metronidazole resistance in H. pylori by phenotypic and genotypic methods. Methods: In total, 338 gastric biopsy samples were collected. The samples were cultivated on Colombia agar, consisting of various antibiotics and were incubated at 37° C under microaerophilic conditions. The biochemical tests and PCR assay were applied to identify the strains as H. pylori. The E-test was applied in the antibiogram test based on CLSI standard. The PCR-RFLP assay was performed to identify pointmutations and followed by sequencing. The PCR method was done to identify deletion of a 200-bp fragment from the rdxA gene. Results: In total, 131 (38. 7%) H. pylori strains were isolated that among them, 70 (53. 4%) and 83 (63. 4%) showed resistance to clarithromycin and metronidazole, respectively. Prevalence of A2143G, A2142G, A2142C mutations were 71. 4%, 7. 1% and 4. 3%, respectively. Seven (8. 4%) strains, included 200-bp deletion. Conclusions: The high prevalence of resistance to clarithromycin and metronidazole in H. pylori is a major concern revealed by this study which should be taken into account by physicians in selecting drug regimens. The results confirmed the necessity of phenotypic and genotypic methods of antibiotic susceptibility.

Background: Humanbocavirus (HBoV) is found worldwide and can infect the respiratory and gastrointestinal tracts of infants and children. Objectives: The aim of the present study was to characterize the complete genome of a new HBoV2A strain isolated from a patient in Korea with gastroenteritis. Methods: Viral genomic DNA was extracted from an HBoV-positive stool specimen isolated from 3-year-old female with gastroenteritis. Entire coding sequences were analyzed using a newly designed set of primers in the conserved regions in 2017. Results: The full-length genome was 5, 107 bp long. Phylogenetic analysis based on the complete genome sequence, including the three open reading frames (ORFs), indicated that CUK18 belonged to the HBoV2A genotype. The CUK18 strain showed the highest similarity with strain Nsc10-N386 isolated in Russia. Analysis of the ORF3, which encodes the viral capsid proteins VP1 and VP2, found that amino acid sequences corresponding to the three-fold-symmetry-related monomer were frequently substituted, a distinguishing feature of this specific genotype. Conclusions: Results of this study may provide valuable information for HBoV epidemiology studies and vaccine development.