Rapid diagnosis of acute respiratory infections by multiplex endpoint PCR technology

2021 ◽  
Vol 1 (3) ◽  
Author(s):  
Alexandru Giubelean ◽  
Aurelian Udristioiu

Introduction: The multiplex endpoint PCR technology offers a number of potential advantages, results are available in a matter of hours rather than days, the extreme sensibility facilitates detection of even minutes the amounts of pathogen DNA in clinical samples and the test is not significantly affected by prior administration of antibiotics. Aim: The aim of this work was to rapidly identify the antibiotic resistance the monitoring of pathogen growth at the patients admitted in Hospitalization Intensive Care Unit, with the diagnosis of Community Acquired Pneumonia, (CAP). Method: The Analyzer Unyvero™ Pneumonia Application was used in detection of pneumonia associated pathogens and their antibiotic resistance genes using the Pneumonia Unyvero™ System, following PCR pathogen species with sequencing of the amplified microbial DNA. Results: The main pathogens of community acquired pneumonia from the cohort study,36 cases, (20 males in mean age 35-66 years and 16 females in mean age 40-55 mean years), were Streptococcus pneumonia, (16 cases), Staphilococcus aureus, (10 cases), Klebsiella pneumonia (5 cases) and other important agents were “atypical”, such as Haemophilus Influenzae, Chlamidophilapneumonie and Moraxelacataralis. A case with Acinetobacter baumani and Proteus Sp. was also widely resistance to mefA gene / ermB gene as all cases of analyzed. The more frequency of genes resistant (29 cases) are ermA gene / ermC / ermB for Staphilococcus aureus and the gene tem+shv / gene / ctx-M with the Chromosomal mutation (7 cases), as gyrA83_87 Ecoli / Pseu for Klebsiella pneumonia agents. Also, most resistance antibiotics were Makrolides, (29 cases and Lincosamides (6 cases) and these cases have had the chromosomial integrates. The most resistance microbe, Pseudomonas aeruginosa (1 case), has been registered as multi drugs resistance [MDR]*. Conclusion: The Unyvero™ results have been available 2 days before the primary microbiology report and 3 days before the final confirmation results, obtained by microbiology culture. The Unyvero Analyzer only provides rapid data to support the therapeutic decision of currant medic.

2016 ◽  
Vol 54 (202) ◽  
pp. 72-78 ◽  
Author(s):  
Ehsan Estabraghi ◽  
Taghi Zahraei Salehi ◽  
Kumarss Amini ◽  
Mahmoud Jamshidian

Introduction: Infections caused by Gram negative bacteria, producing extended-spectrum β-lactamase, including Klebsiella pneumoniae are increasing all over the world with high morbidity and mortality. The aim of the present study was determined antimicrobial profile susceptibility and the prevalence of antibiotic resistance genes by multiplex PCR. Methods: In the present study, we obtained one-hundred isolates of K. pneumoniae from different clinical samples. The antibiotic susceptibility testing was done in thirteen antibiotic and, therefore, M-PCRs were conducted using the DNA amplification for detection of ESBLs (blaTEM, blaCTX-M, blaSHV) and int (I, II, III) genes. Results: The results of resistance to amoxicillin/clavulanate, ciprofloxacin, amikacin, trimethoprim-sulfamethoxazole, cefotaxime, ampicillin, aztreonam, imipenem, gentamicin, ceftazidime, Cefepime, ceftriaxone and levofloxacin were obtained 37%, 37%, 93%, 84%, 52%, 87%, 59%, 8%, 24%, 67%, 52%, 43% and 26%, respectively. The frequency of the extended-spectrum β-lactamase K. pneumoniae was obtained 37%. The prevalence of resistance genes of ESBLs in the M-PCR method showed that the blaTEM, blaCTX and blaSHV were 38%, 24% and 19%, respectively, however, only 8 (8%) out of 100 isolates were found to have positive outcomes for the existence of class 1 integrons and there were no detected class 2 or class 3 integrons. Conclusions: Our results recommend the likely co-carriage of some ESBLs genes and antibiotic resistance integrons on the same plasmids harboring the MDR genes. Keywords: fKlebsiella pneumonia, integrons, drug resistance. | PubMed


2021 ◽  
Author(s):  
Chen Zhao ◽  
Chenyu Li ◽  
Xiaoming Wang ◽  
Zhuosong Cao ◽  
Chao Gao ◽  
...  

Abstract Background: Antibiotic resistance genes (ARGs) have become an important public health problem. In this study, we used metagenomic sequencing to analyze the composition of ARGs in certain original habitats of northeast China, comprising three different rivers and riverbank soils of the Heilongjiang River, Tumen River, and Yalu River. Results: Twenty types of ARG were detected in every water sample. The major ARGs were multidrug resistance genes, at approximately 0.5 copies/16s rRNA, accounting for 57.5% of the total ARG abundance. The abundance of multidrug, bacitracin, beta-lactam, macrolide‑lincosamide‑streptogramin, sulfonamide, fosmidomycin, and polymyxin resistance genes covered 96.9% of the total ARG abundance. No significant ecological boundary of ARG diversity was observed. The compositions of the resistance genes in the three rivers were very similar to each other, and 92.1% of ARG subtypes were shared by all water samples. Except for vancomycin resistance genes, almost all ARGs in riverbank soils were detected in the river water. About 31.05% ARGs were carried by Pseudomonas. Opportunistic pathogenic bacteria carrying resistance genes were mainly related to diarrhea and respiratory infections. Multidrug and beta-lactam resistance genes correlated positively with mobile genetic elements (MGEs), indicating a potential risk of diffusion.Conclusions: The composition of ARGs in three different rivers was similar, indicating that climate played an important role in ARG occurrence. ARG subtypes in river water were almost completely the same as those in riverbank soil. ARGs had no significant geographical distribution characteristics. Many ARGs were carried by human pathogenic bacteria related to human diarrhea and respiratory infections, such as Pseudomonas aeruginosa and Aeromonas caviae. In general, our results provide a valuable dataset of river water ARG distribution in northeast China. The related ecological geography distribution characteristics should be further explored.


Author(s):  
Somaye Delfani ◽  
Faranak Rezaei ◽  
Setareh Soroush ◽  
Pegah Shakib

Background: Methicillin-resistant coagulase-negative staphylococci is responsible for hospital and community-acquired infections. Objective: This study aimed to investigate the antibiotic-resistance patterns, antibiotic-resistance genes, namely, ermA, ermB, ermC, blaZ, msrA, tetK, tetM, mup, and vanA, biofilm formation, and prevalence of different SCCmec types among the Staphylococcus cohniistrains isolated from clinical samples in Tehran, Iran. Methods: In this study,S. cohniiisolates were screened from the clinical samples from March 2012 to February 2013 in Tehran, Iran.Antimicrobial susceptibility test and inducible clindamycin resistance were evaluated by disc diffusion method, andresistance genes were examined using Polymerase Chain Reaction (PCR) assays. Then, biofilm formation assay was analyzed by Microtiter-plate test to detect the icaA and icaDgenes. The SCCmec and the Arginine Catabolite Mobile Element (ACME) typing were performed using the PCRmethod. Results: FromtwentyS. cohnii, all isolates were resistant to cefoxitin. 95% of the S. cohnii was defined as multidrug resistance (MDR)strains. The ermB, ermC, and vanA genes were not detected in any isolates; however, the blaZ gene had the highest frequency.95% of the S. cohnii isolates produced biofilm. Also, 4 SCCmec types, including V, IV, III+ (C2), VIII+ (AB1), were identified. Therefore, the majority of SCCmec were untypable. Based on the ACME typing, arcA and opp3 genes were positive in 13 (65%) and 1 (5%) isolates, respectively. Conclusion: Due to the high antimicrobial resistance and the spread of untypableSCCmecamong the isolates studied, the control and treatment of methicillin-resistantS. cohnii in hospitals and public health centers is a significant concern.


2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S301-S301
Author(s):  
Jessica L Snyder ◽  
Brendan Manning ◽  
Robert Shivers ◽  
Daniel Gamero ◽  
Heidi Giese ◽  
...  

Abstract Background Antibiotic-resistant bacteria are spread through selective pressure from the use of broad-spectrum empirical therapies, mobile genetic elements that pass resistance genes between species, and the inability to rapidly and appropriately respond to their presence. Resistance gene identification is often performed with post culture molecular diagnostic tests. The T2Resistance Panel, which detects methicillin resistance genes mecA/C; vancomycin resistance genes vanA/B; carbapenemases blaKPC, blaOXA-48,blaNDM, blaVIM, and blaIMP; AmpC β-lactamases blaCMY and blaDHA; and extended-spectrum β-lactamases blaCTX-M directly from patient blood samples, is based on T2 magnetic resonance (T2MR), an FDA-cleared technology with demonstrated high sensitivity and specificity for culture-independent bacterial and fungal species identification. Here we report the clinical performance of T2MR detection of resistance genes directly from patient blood samples. Methods Patients with a clinical diagnosis of sepsis and an order for blood culture (BC) were enrolled in the study at two sites. BCs were managed using standard procedures and MALDI-TOF for species identification. Resistance testing with the T2MR assay was performed on a direct patient draw and compared with diagnostic test results from concurrent BC specimen and BC specimen taken at other points in time. The potential impact on therapy was evaluated through patient chart review. Results T2MR detected the same resistance genes as detected by post culture diagnostics in 100% of samples from concurrent blood draws. Discordant results occurred when T2MR was taken ≥48 hours after BC for patients on antimicrobial therapy. The average time to positive result was 5.9 hours with T2MR vs. 30.6 hours with post-culture molecular testing. Conclusion The T2Resistance Panel detected antibiotic resistance genes in clinical samples and displayed agreement with post culture genetic testing. T2MR results were achieved faster than culture-dependent diagnostic testing results and may allow for an earlier change from empiric to directed therapy. The use of culture-independent diagnostics like T2MR could enable a quicker response to antibiotic-resistant organisms for individual patients and developing outbreaks. Disclosures All authors: No reported disclosures.


2020 ◽  
Author(s):  
Raymond Mudzana ◽  
Rooyen T Mavenyengwa ◽  
Muchaneta Gudza-Mugabe

Abstract Background: Streptococcus agalacticae (Group B Streptococcus, GBS) is one of the most important causative agents of serious infections among neonates. This study was carried out to identify antibiotic resistance and virulence genes associated with GBS isolated from pregnant women.Methods: A total of 43 GBS isolates were obtained from 420 vaginal samples collected from HIV positive and negative women who were 13-35 weeks pregnant attending Antenatal Care at Chitungwiza and Harare Central Hospitals in Zimbabwe. Identification tests of GBS isolates was done using standard bacteriological methods and molecular identification testing. Antibiotic susceptibility testing was done using the modified Kirby-Bauer method and E-test strips. The boiling method was used to extract DNA and Polymerase Chain Reaction (PCR) was used to screen for 13 genes. Data was fed into SPSS 24.0.Results: Nine distinct virulence gene profiles were identified and hly-scpB-bca-rib 37.2% (16/43) was common. The virulence genes identified were namely hly 97.8% (42/43), scpB 90.1% (39/43), bca 86.0% (37/43), rib 69.8% (30/43) and bac 11.6% (5/43). High resistance to tetracycline 97.7% (42/43) was reported followed by 72.1% (31/43) cefazolin, 69.8% (30/43) penicillin G, 58.1% (25/43) ampicillin, 55.8% (24/43) clindamycin, 46.5% (20/43) ceftriaxone, 34.9% (15/43) chloramphenicol, and 30.2% (13/43) for both erythromycin and vancomycin using disk diffusion. Antibiotic resistance genes among the resistant and intermediate-resistant isolates showed high frequencies for tetM 97.6% (41/42) and low frequencies for ermB 34.5% (10/29), ermTR 10.3% (3/29), mefA 3.4% (1/29), tetO 2.4% (1/42) and linB 0% (0/35). The atr housekeeping gene yielded 100% (43/43) positive results, whilst the mobile genetic element IS1548 yielded 9.3% (4/43).Conclusion: The study showed high prevalence of hly, scpB, bca and rib virulence genes in S. agalactiae strains isolated from pregnant women. Tetracycline resistance was predominantly caused by the tetM gene, whilst macrolide resistance was predominantly due to the presence of erm methylase, with the ermB gene being more prevalent. Multi-drug resistance coupled with the recovery of resistant isolates to antimicrobial agents such as penicillins indicates the importance of GBS surveillance and susceptibility tests. It was also observed that in vitro phenotypic resistance is not always accurately predicted by resistance genotypes.


2018 ◽  
Author(s):  
Prasanth Manohar ◽  
Thamaraiselvan Shanthini ◽  
Reethu Ann Philip ◽  
Subramani Ramkumar ◽  
Manali Kale ◽  
...  

AbstractTo evaluate the presence of biofilm-specific antibiotic-resistant genes, PA0756-0757, PA5033 and PA2070 in Pseudomonas aeruginosa isolated from clinical samples in Tamil Nadu. For this cross-sectional study, 24 clinical isolates (included pus, urine, wound, and blood) were collected from two diagnostic centers in Chennai from May 2015 to February 2016. Biofilm formation was assessed using microtiter dish biofilm formation assay and minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) were determined for planktonic and biofilm cells (MBC assay). Further, PCR amplification of biofilm-specific antibiotic resistance genes PA0756-0757, PA5033 and PA2070 were performed. Biofilm formation was found to be moderate/strong in 16 strains. MBC for planktonic cells showed that 4, 7, 10 and 14 strains were susceptible to gentamicin, ciprofloxacin, meropenem and colistin respectively. In MBC assay for biofilm cells (MBC-B), all the 16 biofilm producing strains were resistant to ciprofloxacin and gentamicin whereas nine and four were resistant to meropenem, and colistin respectively. The biofilm-specific antibiotic-resistant genes PA0756-0757 was found in 10 strains, 6 strains with PA5033 and 9 strains with PA2070 that were found to be resistant phenotypically. This study highlighted the importance of biofilm-specific antibiotic resistance genes PA0756-0757, PA5033, and PA2070 in biofilm-forming P. aeruginosa.


2021 ◽  
Vol 13 (20) ◽  
pp. 11131
Author(s):  
Osama S. Ali ◽  
Walaa G. Hozayen ◽  
Abdulwahab S. Almutairi ◽  
Sherif A. Edris ◽  
Aala A. Abulfaraj ◽  
...  

Wastewater treatment plants (WWTPs) are recognized as hotspots for the dissemination of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARBs) in the environment. Our study utilized a high-throughput sequencing-based metagenomic analysis approach to compare the ARG abundance profiles of the raw sewage, treated effluent and activated sludge samples from a full-scale WWTP in Egypt. In addition, the difference in microbial community composition due to the treatment process was assessed. As a result, 578 ARG subtypes (resistance genes) belonging to 18 ARG types (antibiotic resistance classes) were identified. ARGs encoding for resistance against multidrug, aminoglycoside, bacitracin, beta-lactam, sulfonamide, and tetracycline antibiotics were the most abundant types. The total removal efficiency percentage of ARGs in the WWTP was found to be 98% however, the ARG persistence results indicated that around 68% of the ARGs in the influent could be found in the treated effluent. This finding suggests that the treated wastewater poses a potential risk for the ARG dissemination in bacterial communities of the receiving water bodies via horizontal gene transfer (HGT). The community composition at phylum level showed that Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria were the most abundant phyla in all datasets. Although the relative abundance of several pathogenic bacteria in the influent declined to less than 1% in the effluent, the taxonomic assignments at species level for the effluent and sludge metagenomes demonstrated that clinically important pathogens such as Escherichia coli, Klebsiella pneumonia, and Aeromonas caviae were present. Overall, the results of this study would hopefully enhance our knowledge about the abundance profiles of ARGs and their fate in different wastewater treatment compartments that have never been examined before.


2021 ◽  
Vol 10 (1) ◽  
pp. 40-48
Author(s):  
O.C. Adekunle ◽  
A. Mustapha ◽  
G. Odewale ◽  
R.O. Ojedele

Introduction: Pseudomonas aeruginosa (P. aeruginosa) is a frequent nosocomial pathogen that causes severe diseases in many clinical and community settings. The objectives were to investigate the occurrence of multiple antibiotic resistant P. aeruginosa strains among clinical samples and to detect the presence of antibiotic resistance genes in the DNA molecules of the strains.Methods: Clinical specimens were collected aseptically from various human anatomical sites in five selected health institutions within Kwara State, Nigeria. Multiple drug resistance patterns of isolated micro-organisms to different antibiotics were determined using the Bauer Kirby disc diffusion technique. The DNA samples of the multiple resistant P. aeruginosa strains were extracted and subjected to Polymerase Chain Reaction (PCR) for resistance gene determination.Results: A total of 145 isolates were identified as P. aeruginosa from the clinical samples. Absolute resistance to ceftazidime, gentamicin and ceftriaxone was observed while low resistance to ciprofloxacin, piperacillin and imipenem was documented. The prevalence of bla VIM , ,bla CTX-M and blaTEM were 34.4 %, 46.7 % and 16.7 % respectively.Conclusion: This study has shown that there is a high occurrence of metallo â-lactamase- producing and antibiotic-resistant strains of P. aeruginosa in clinical specimens from the studied area. Keywords: Metallo â-lactamase enzyme, P. aeruginosa, clinical samples, antibiotic-resistance genes


2020 ◽  
Author(s):  
Raymond Mudzana ◽  
Rooyen T Mavenyengwa ◽  
Muchaneta Gudza-Mugabe

Abstract Background: Streptococcus agalacticae is one of the most important causative agents of serious infections among neonates. Group B Streptococcus (GBS) virulence factors are important in the development of vaccines, whilst antibiotic resistance genes are necessary in understanding the resistance mechanisms used by these pathogens. This study was carried out to identify the virulence genes and antibiotic resistance genes associated with GBS isolated from pregnant women.Methods: A total of 43 GBS isolates were obtained from vaginal samples that were collected from all HIV positive and HIV negative women who were 13-35 weeks pregnant attending Antenatal Care at both Chitungwiza and Harare Central Hospitals in Zimbabwe. Identification tests of GBS isolates was done using standard bacteriological methods including molecular tests. Antibiotic susceptibility testing using 3 antibiotics was done using the modified Kirby-Bauer method. The boiling method was used to extract DNA and Polymerase Chain Reaction (PCR) was used to screen for 13 genes in the isolates. Data was fed into SPSS 24.0 and the Spearman rank correlation test used to determine any correlation among genes.Results: Nine distinct virulence gene profiles were identified. The profiles hly-scpB-bca-rib 37.2% (16/43) and hly-scpB-bca 18.6% (8/43) were common among GBS isolates. The following virulence gene frequencies were obtained namely hly 97.8% (42/43), scpB 90.1% (39/43), bca 86.0% (37/43), rib 69.8% (30/43) and bac 11.6% (5/43). Antibiotic resistance genes showed high frequencies for tetM 97.6% (41/42) and low frequencies for ermB 34.5% (10/29), ermTR 10.3% (3/29), mefA 3.4% (1/29), tetO 2.4% (1/42) and linB 0% (0/35). The atr housekeeping gene amplification yielded 100% (43/43) positive results, whilst the mobile genetic element IS1548 yielded a low 9.3% (4/43).Conclusion: The study showed a high prevalence of multiple virulence genes hly, scpB, bca and rib in S. agalactiae strains isolated from pregnant women. Tetracycline resistance was found to be predominantly caused by the tetM gene, whilst macrolide resistance was predominantly due to the presence of erm methylase, with the ermB gene being more prevalent. It was also observed that in vitro phenotypic resistance is not always accurately predicted by resistance genotypes.


2010 ◽  
Vol 5 (07) ◽  
pp. 502-510 ◽  
Author(s):  
Priscila Dauros ◽  
Helia Bello ◽  
Mariana Domínguez ◽  
Juan C. Hormazábal ◽  
Gerardo González

Introduction: Vibrio (V.) parahaemolyticus has endemically established in Chilean sea shores, causing outbreaks every year, with an important number of cases. In order to know the genetic relationship, genotype dominance and antibiotic resistance of isolates obtained from two outbreaks, this study characterized 110 strains isolated from environmental and clinical samples in years 2005 and 2007 in Chile. Methodology: Genotyping was performed by determination of PFGE profiles, and pandemic group and integrons were screened by PCR. Antimicrobial susceptibility was studied by the disk diffusion method. Results: High antibiotic susceptibility frequency was found, mainly among 2007 isolates, except to ampicillin, cephalothin, cefoxitin, cefpodoxime, amikacin, streptomycin and kanamycin. Strains belonging to the pandemic group in clinical isolates account for 88% in 2005, decreasing to 66% in 2007 and among environmental isolates were detected in 20% of the strains from 2005, rising to 36% in 2007. In 2005, nine different PFGE profiles were identified, with 78% of the strains corresponding to a single clone. In 2007, sixteen different PFGE profiles were detected, with 61% of the strains included into a sole clone. The same clone was prevalent in both years. None of class 1, 2, 3 and SXT integrases genes was detected; however, the superintegron integrase gene (intIA) was present in almost all strains. Conclusions: These results suggest the persistence and dominance of a unique PFGE clone of V. parahaemolyticus during 2005 and 2007, and the absence of genetic elements that capture antibiotic resistance genes described in other species of Vibrio.


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