scholarly journals Comprehensive Pathogen Identification, Antibiotic Resistance, and Virulence Genes Prediction Directly From Simulated Blood Samples and Positive Blood Cultures by Nanopore Metagenomic Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Menglan Zhou ◽  
Yarong Wu ◽  
Timothy Kudinha ◽  
Peiyao Jia ◽  
Lei Wang ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Virulence Genes ◽  
Direct Sequencing ◽  
Blood Cultures ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Hybrid Assembly ◽  
Pathogen Identification ◽  
Blood Samples

Bloodstream infection is a major cause of morbidity and mortality worldwide. We explored whether MinION nanopore sequencing could accelerate diagnosis, resistance, and virulence profiling prediction in simulated blood samples and blood cultures. One milliliter of healthy blood samples each from direct spike (sample 1), anaerobic (sample 2), and aerobic (sample 3) blood cultures with initial inoculation of ∼30 CFU/ml of a clinically isolated Klebsiella pneumoniae strain was subjected to DNA extraction and nanopore sequencing. Hybrid assembly of Illumina and nanopore reads from pure colonies of the isolate (sample 4) was used as a reference for comparison. Hybrid assembly of the reference genome identified a total of 39 antibiotic resistance genes and 77 virulence genes through alignment with the CARD and VFDB databases. Nanopore correctly detected K. pneumoniae in all three blood samples. The fastest identification was achieved within 8 h from specimen to result in sample 1 without blood culture. However, direct sequencing in sample 1 only identified seven resistance genes (20.6%) but 28 genes in samples 2–4 (82.4%) compared to the reference within 2 h of sequencing time. Similarly, 11 (14.3%) and 74 (96.1%) of the virulence genes were detected in samples 1 and 2–4 within 2 h of sequencing time, respectively. Direct nanopore sequencing from positive blood cultures allowed comprehensive pathogen identification, resistance, and virulence genes prediction within 2 h, which shows its promising use in point-of-care clinical settings.

scholarly journals Combined effects of composting and antibiotic administration on cattle manure–borne antibiotic resistance genes

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Ishi Keenum ◽  
Robert K. Williams ◽  
Partha Ray ◽  
Emily D. Garner ◽  
Katharine F. Knowlton ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Land Application ◽  
Multidimensional Analysis ◽  
Small Scale ◽  
Antibiotic Administration ◽  
Metagenomic Sequencing ◽  
Combined Effects ◽  
Bacterial Microbiota

Abstract Background Research is needed to delineate the relative and combined effects of different antibiotic administration and manure management practices in either amplifying or attenuating the potential for antibiotic resistance to spread. Here, we carried out a comprehensive parallel examination of the effects of small-scale (> 55 °C × 3 days) static and turned composting of manures from dairy and beef cattle collected during standard antibiotic administration (cephapirin/pirlimycin or sulfamethazine/chlortetracycline/tylosin, respectively), versus from untreated cattle, on “resistomes” (total antibiotic resistance genes (ARGs) determined via shotgun metagenomic sequencing), bacterial microbiota, and indicator ARGs enumerated via quantitative polymerase chain reaction. To gain insight into the role of the thermophilic phase, compost was also externally heated to > 55 °C × 15 days. Results Progression of composting with time and succession of the corresponding bacterial microbiota was the overarching driver of the resistome composition (ANOSIM; R = 0.424, p = 0.001, respectively) in all composts at the small-scale. Reduction in relative abundance (16S rRNA gene normalized) of total ARGs in finished compost (day 42) versus day 0 was noted across all conditions (ANOSIM; R = 0.728, p = 0.001), except when externally heated. Sul1, intI1, beta-lactam ARGs, and plasmid-associated genes increased in all finished composts as compared with the initial condition. External heating more effectively reduced certain clinically relevant ARGs (blaOXA, blaCARB), fecal coliforms, and resistome risk scores, which take into account putative pathogen annotations. When manure was collected during antibiotic administration, taxonomic composition of the compost was distinct according to nonmetric multidimensional analysis and tet(W) decayed faster in the dairy manure with antibiotic condition and slower in the beef manure with antibiotic condition. Conclusions This comprehensive, integrated study revealed that composting had a dominant effect on corresponding resistome composition, while little difference was noted as a function of collecting manure during antibiotic administration. Reduction in total ARGs, tet(W), and resistome risk suggested that composting reduced some potential for antibiotic resistance to spread, but the increase and persistence of other indicators of antibiotic resistance were concerning. Results indicate that composting guidelines intended for pathogen reduction do not necessarily provide a comprehensive barrier to ARGs or their mobility prior to land application and additional mitigation measures should be considered.

scholarly journals Molecular characteristics and virulence gene profiles of Staphylococcus aureus isolates in Hainan, China

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xuehan Li ◽  
Tao Huang ◽  
Kai Xu ◽  
Chenglin Li ◽  
Yirong Li
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Virulence Genes ◽  
Mainland China ◽  
Geographical Location ◽  
Antibiotic Resistance Genes ◽  
Virulence Gene ◽  
Molecular Characteristics ◽  
Staphylococcal Protein A

Abstract Background There have been no reports regarding the molecular characteristics, virulence features, and antibiotic resistance profiles of Staphylococcus aureus (S. aureus) from Hainan, the southernmost province of China. Methods Two hundred twenty-seven S. aureus isolates, consisting of 76 methicillin-resistant S. aureus (MRSA) and 151 methicillin-susceptible S. aureus (MSSA), were collected in 2013–2014 and 2018–2019 in Hainan, and investigated for their molecular characteristics, virulence genes, antibiotic resistance profiles and main antibiotic resistance genes. Results Forty sequence types (STs) including three new STs (ST5489, ST5492 and ST5493), and 79 Staphylococcal protein A (spa) types were identified based on multilocus sequence typing (MLST) and spa typing, respectively. ST398 (14.1%, 32/227) was found to be the most prevalent, and the prevalence of ST398-MSSA increased significantly from 2013 to 2014 (5.5%, 5/91) to 2018–2019 (18.4%, 25/136). Seventy-six MRSA isolates were subject to staphylococcus chromosomal cassette mec (SCCmec) typing. SCCmec-IVa was the predominant SCCmec type, and specifically, ST45-SCCmec IVa, an infrequent type in mainland China, was predominant in S. aureus from Hainan. The antibiotic resistance profiles and antibiotic resistance genes of S. aureus show distinctive features in Hainan. The resistant rates of the MRSA isolates to a variety of antibiotics were significantly higher than those of the MSSA isolates. The predominant erythromycin and tetracycline resistance genes were ermC (90.1%, 100/111) and tetK (91.8%, 78/85), respectively. Eleven virulence genes, including the Panton-Valentine leukocidin (pvl) and eta, were determined, and the frequency of eta and pvl were found to be 57.3 and 47.6%. Such high prevalence has never been seen in mainland China before. Conclusion S. aureus isolates in Hainan have unique molecular characteristics, virulence gene and antibiotic resistance profiles, and main antibiotic resistance genes which may be associated with the special geographical location of Hainan and local trends in antibiotic use.

scholarly journals 655. Detection of Antibiotic Resistance Genes in Clinical Samples using T2 Magnetic Resonance

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S301-S301
Author(s):  
Jessica L Snyder ◽  
Brendan Manning ◽  
Robert Shivers ◽  
Daniel Gamero ◽  
Heidi Giese ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Magnetic Resonance ◽  
Species Identification ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Resistance Testing ◽  
Clinical Samples ◽  
Antibiotic Resistant ◽  
Blood Samples ◽  
Culture Independent

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.

scholarly journals Emerging MDR-Pseudomonas aeruginosa in fish commonly harbor oprL and toxA virulence genes and blaTEM, blaCTX-M, and tetA antibiotic-resistance genes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Abdelazeem M. Algammal ◽  
Mahmoud Mabrok ◽  
Elayaraja Sivaramasamy ◽  
Fatma M. Youssef ◽  
Mona H. Atwa ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Virulence Genes ◽  
Antibiotic Resistance Genes ◽  
Health Concern ◽  
Diffusion Method ◽  
Multi Drug Resistance ◽  
Fish Farms ◽  
M Gene ◽  
Total Prevalence

Abstract This study aimed to investigate the prevalence, antibiogram of Pseudomonasaeruginosa (P.aeruginosa), and the distribution of virulence genes (oprL,exoS, phzM, and toxA) and the antibiotic-resistance genes (blaTEM, tetA, and blaCTX-M). A total of 285 fish (165 Oreochromisniloticus and 120 Clariasgariepinus) were collected randomly from private fish farms in Ismailia Governorate, Egypt. The collected specimens were examined bacteriologically. P. aeruginosa was isolated from 90 examined fish (31.57%), and the liver was the most prominent infected organ. The antibiogram of the isolated strains was determined using a disc diffusion method, where the tested strains exhibited multi-drug resistance (MDR) to amoxicillin, cefotaxime, tetracycline, and gentamicin. The PCR results revealed that all the examined strains harbored (oprL and toxA) virulence genes, while only 22.2% were positive for the phzM gene. On the contrary, none of the tested strains were positive for the exoS gene. Concerning the distribution of the antibiotic resistance genes, the examined strains harbored blaTEM, blaCTX-M, and tetA genes with a total prevalence of 83.3%, 77.7%, and 75.6%, respectively. Experimentally infected fish with P.aeruginosa displayed high mortalities in direct proportion to the encoded virulence genes and showed similar signs of septicemia found in the naturally infected one. In conclusion, P.aeruginosa is a major pathogen of O.niloticus and C.gariepinus.oprL and toxA genes are the most predominant virulence genes associated with P.aeruginosa infection. The blaCTX-M,blaTEM, and tetA genes are the main antibiotic-resistance genes that induce resistance patterns to cefotaxime, amoxicillin, and tetracycline, highlighting MDR P.aeruginosa strains of potential public health concern.

Abundance, diversity and mobility potential of antibiotic resistance genes in pristine Tibetan Plateau soil as revealed by soil metagenomics

2020 ◽  
Vol 96 (10) ◽  
Author(s):  
Bo Li ◽  
Zeng Chen ◽  
Fan Zhang ◽  
Yongqin Liu ◽  
Tao Yan
Keyword(s):  
Antibiotic Resistance ◽  
Microbial Diversity ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Soil Samples ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Metagenomic Sequencing ◽  
Clinical Issue ◽  
Abundance And Diversity

ABSTRACT Widespread occurrence of antibiotic resistance genes (ARGs) has become an important clinical issue. Studying ARGs in pristine soil environments can help to better understand the intrinsic soil resistome. In this study, 10 soil samples were collected from a high elevation and relatively pristine Tibetan area, and metagenomic sequencing and bioinformatic analyses were conducted to investigate the microbial diversity, the abundance and diversity of ARGs and the mobility potential of ARGs as indicated by different mobile genetic elements (MGEs). A total of 48 ARG types with a relative abundance of 0.05–0.28 copies of ARG/copy of 16S rRNA genes were detected in Tibetan soil samples. The observed ARGs were mainly associated with antibiotics that included glycopeptide and rifamycin; the most abundant ARGs were vanRO and vanSO. Low abundance of MGEs and potentially plasmid-related ARGs indicated a low horizontal gene transfer risk of ARGs in the pristine soil. Pearson correlation and redundancy analyses showed that temperature and total organic carbon were the major environmental factors controlling both microbial diversity and ARG abundance and diversity.

scholarly journals Antibiotic Resistance and Molecular Epidemiological Characteristics of Streptococcus agalactiae Isolated from Pregnant Women in Guangzhou, South China

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Zhaomin Cheng ◽  
Pinghua Qu ◽  
Peifeng Ke ◽  
Xiaohan Yang ◽  
Qiang Zhou ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Pregnant Women ◽  
Resistance Genes ◽  
Streptococcus Agalactiae ◽  
Virulence Genes ◽  
Mass Peak ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Epidemiological Characteristics ◽  
Tof Ms

Streptococcus agalactiae colonization in pregnant women can cause postpartum intrauterine infections and life-threatening neonatal infections. To formulate strategies for the prevention and treatment of S. agalactiae infections, we performed a comprehensive analysis of antibiotic resistance and a molecular-based epidemiological investigation of S. agalactiae in this study. Seventy-two S. agalactiae strains, collected from pregnant women, were subjected to antibiotic susceptibility tests; then, the screened erythromycin and clindamycin nonsusceptible isolates were used for macrolides and clindamycin resistance genes detection, respectively. Detection of resistance genes, serotyping, and determination of virulence genes were performed by polymerase chain reaction. The clonal relationships among the colonized strains were evaluated by multilocus sequence typing. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) mass peak analysis was performed to discriminate the specific sequence types (STs). In our study, 69.4% and 47.2% of the strains were nonsusceptible to erythromycin and clindamycin, respectively; the multidrug resistance rate was 66.7%. All erythromycin nonsusceptible strains harbored resistance genes, whereas only 52.9% of the clindamycin nonsusceptible strains possessed the linB gene. Erythromycin resistance was mainly mediated by the ermB or mefA/E genes. Four serotypes were identified, and the most common serotype was serotype III (52.8%), followed by Ib (22.2%), Ia (18.0%), and II (4.2%). All the strains were divided into 18 STs that were assigned to nine clonal complexes. Most of the major STs were distributed into specific serotypes, including ST19/serotype III, ST17/serotype III, ST485/serotype Ia, ST862/serotype III, and ST651/serotype III. Analysis of virulence genes yielded seven clusters, of which bca-cfb-scpB-lmb (61.6%) was the predominant virulence gene cluster. Among all ST strains distributed in this region, only the ST17 strains had a mass peak at 7620 Da. The outcomes of this study are beneficial for the epidemiological comparison of colonized S. agalactiae in different regions and may be helpful for developing the strategies for the prevention of S. agalactiae infection in Guangzhou. Furthermore, our results show that MALDI-TOF MS can be used for the rapid identification of the ST17 strains.

scholarly journals Phylogenetic Analysis of Antibiotic Resistance Genes and Virulence Genes of Klebsiella species in silico

2017 ◽  
Vol 16 (1) ◽  
pp. 119-127 ◽  
Author(s):  
Nusrat Nahar ◽  
Ridwan Bin Rashid
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
In Silico ◽  
Iron Uptake ◽  
Virulence Genes ◽  
Antibiotic Resistance Genes ◽  
Genotype 1 ◽  
Klebsiella Species ◽  
Tract Infections ◽  
Klebsiella Infections

A total of twelve isolates were screened for virulence and antibiotic resistance genes associated with Klebsiella pneumoniae infections. Virulence and antibiotic resistance genes were detected by in silico PCR amplification. Iron uptake protein entB was detected in 66.67% (n=8) of the isolates while no isolate was found to harbour chelating agent irp2. Iron uptake system kfu, involved in purulent tissue infections and capsule formation, was identified in 25% (n=3) of the isolates. Regulator of mucoid phenotype A, rmpA was not found in any of the isolates. The wabG gene, responsible for urinary tract infections was found in seven K. pneumoniae strains. Five uge positive strains might play role in the pathogenicity of K. pneumoniae infections. About 83.33% of the isolates were positive for type 1 fimbriae fimH1 while no type 3 fimbriae mrkD gene was found. Complement reaction blocked by plasmid traT gene was not observed in Klebsiella species while eight isolates harboured outer membrane lipoprotein, ycfM which protects Klebsiella species from antibiotics. Antibiotic resistance genes blaTEM and blaSHV were detected in 33.33% (n=4) and 66.67% (n=8) of the isolates while 25% isolates carried both blaTEM and blaSHV genes. Genotype 1 carried fimH1 and ycfM genes while all the virulence genes studied were present in genotype 2 and 3. The blaSHV gene was detected in all the genotypes while blaTEM gene was found in only genotype 1 and 3. The findings of this study would be helpful to predict virulence gene associated with Klebsiella infections. This data also helps us to choose antibiotics for treating Klebsiella infections. By assessing the genotypic distribution of antibiotic resistance gene, correct antibiotic can be used to treat the infection. This could help reduce emergence of antibiotic resistance since it is known that incorrect choice of antibiotics contributes to antibiotic resistance.Dhaka Univ. J. Pharm. Sci. 16(1): 119-127, 2017 (June)

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