Detection Of Resistance Genes Research Articles

A comparative assessment of Lactiplantibacillus plantarum isolated from chicken and humans as candidates for probiotics

Abstract. Sunardi J, Purnama ET, Sugata M, Victor H, Jan TT, Jo J. 2023. A comparative assessment of Lactiplantibacillus plantarum isolated from chicken and humans as candidates for probiotics. Biodiversitas 24: 5198-5206. Lactiplantibacillus plantarum is commonly analyzed as a potential probiotic. We hereby investigated two strains isolated from chicken crop (Lpb. plantarum F75) and human breast milk (Lpb. plantarum SU-KC1a). Ability to withstand osmotic stress (1.5%, 2.5% or 3.5% of NaCl) and phenol compounds (0.2% or 0.5%), ability to survive gastric juices for a maximum of 120 minutes and bile salt for a maximum 3 hours, as well as susceptibility to 25 antibiotic discs, were compared between both strains. Whole genomes of both strains were sequenced and analyzed in silico to determine the availability of antibiotic-resistance genes as well as the presence of mobile genetic elements and plasmid. Both strains were sensitive to increased concentrations of NaCl and phenol as well as to prolonged exposure to gastric juices. In contrast, both strains could withstand a prolonged exposure of 0.3% of bile salt. Both isolates had similar genome sizes and were susceptible to many tested antibiotics. The detected resistance genes were observed within the chromosomal genomes but no mobile genetic element nor plasmid was found. In conclusion, both strains of Lpb. plantarum displayed several characteristics of beneficial bacteria and could be used as probiotic candidates for poultry and human beings, respectively.

Antibiotics and antibiotic resistance genes in the water sources of the Wuhan stretch of the Yangtze River: Occurrence, distribution, and ecological risks

Given the ubiquitous detection of antibiotics and antibiotic resistance genes (ARGs) in waterbodies worldwide and increasing public attention to water resource safety, this study investigated the presence of antibiotics and ARGs in the water sources of the Wuhan stretch of the Yangtze River (YR) as well as potential ecological risks. In this study, 15 antibiotics and 10 ARGs in a source of drinking water were analyzed using solid-phase extraction-ultra performance liquid chromatography-mass spectrometry technology (SPE–UPLC–MS/MS) and real-time fluorescence quantitative polymerase chain reaction (qPCR). Fourteen antibiotics were detected in the samples from 18 water sources, with the highest concentration detected for tetracycline, reaching up to 1708.33 ng/L. The detection rates of norfloxacin, enrofloxacin, ofloxacin, tetracycline, and roxithromycin were 100%. The concentrations of antibiotics were highest in She Shui, followed by the Wuhan stretch of the lower reaches of the YR, whereas the lowest concentrations were found in the Wuhan stretch of the upper reaches of the YR which were approximately equal to those in the Han River (HR). Ofloxacin and roxithromycin presented a substantial threat to aquatic organisms with high sensitivity at the majority of the sampling sites. The overall abundance of ARGs was notably greater in the lower reaches of the YR compared with the upper reaches and the HR. The highest absolute abundance was observed for sulfa ARGs. Integron intl1 strongly correlated with sul1, sul2, ermB, and qnrS, and antibiotics, strongly correlated with multiple ARGs, suggesting that antibiotics and ARGs are present in water sources in Wuhan and may present a plausible hazard to both human and ecological well-being. Hence, regulating the spread and dissemination of antibiotics and ARGs in the environment is imperative. The findings of this research offer significant insights into the stewardship and safeguarding of aquatic reserves in the Wuhan stretch of the YR.

A highly selective and sensitive electrochemical sensor for tetracycline resistant genes detection based on the non-covalent interaction of graphene oxide and nucleobase

Antibiotic resistance genes (ARGs) are causing worldwide environmental problems, however, the traditional analytical methods and test equipment for them are time-consuming and expensive. The electrochemical sensor using the non-covalent bond between graphene oxide (GO) and single-stranded tet (ss-tet) was established for specific tetracycline resistance genes (tet, composed of ss-tet and complementary ss-tet (ss-tet') in water) detection, which preparation time was only 35 min and far less than most reported sensors based on covalent bond. As the result of the detection for tet, the developed sensor not only had the low detection limit of 50.0 pM (8.1 × 102 copies·mL−1), the short detection time within 42 min, but also had satisfactory stability, excellent reproducibility, and highly selectivity (RSD < 4.43 %). Besides, it also had acceptable accuracy comparing to the real-time quantitative polymerase chain reaction (RT-qPCR) and PCR array in tet detection. Noticeably, it also had been successfully applied to tetA detection in different water samples. In brief, the prepared non-covalent bond sensor is simple, rapid, and suitable for highly selective and sensitive detection of the ARGs in actual water.

Bacterial diversity and detection of resistance genes to broad-spectrum betalactams in dairy family farm soils

Bovine mastitis is a complex disease that brings great losses to the dairy producer. The microbial diversity of the soils, as well as the presence of resistance genes in the environment directly influence the maintenance of mastitis in the farm. The objective of this work was to analyze the bacterial diversity in pasture soils of a dairy family farm, detecting enterobacteria that may be involved in the etiology of bovine mastitis, and to detect genes that encode broad-spectrum betalactamases in these soils. Twelve soil samples, representative of different areas of the farm located in the municipality of Barra do Piraí, Rio de Janeiro, were collected at different times of the year. Total DNA was extracted from the samples, gene amplified by Nested-PCR and then the amplification products were separated by DGGE (Denaturing Gradient Gel Electrophoresis). With the DGGE it was possible to construct dendograms that effectively represented the bacterial diversity of these soils. Eight of the soil samples were used to amplify the genes encoding the betalactamase enzymes TEM (blaTEM gene), SHV (blaSHV gene) and CTX (blaCTXM gene). In three of the eight soil samples, the blaSHV gene was found to be present. The blaTEM and blaCTX-M genes were not detected in any of the samples. The detection of genes encoding broad-spectrum betalactamases in dairy cattle pasture soils is of concern, because the transfer of gene material between pathogenic and non-pathogenic bacteria in this environment is a reality.

Phenotypic Characterization and Resistance Genes Detection of Staphylococcus aureus Isolated from Bovine Mastitis in the Northwest of Portugal

Phenotypic Characterization and Resistance Genes Detection of Staphylococcus aureus Isolated from Bovine Mastitis in the Northwest of Portugal

Designing a Multiplex PCR for Rapid and Accurate Detection of Metallobetalactamases Resistant Genes from Acinetobacter baumaniiIsolates in Tehran City, Iran.

Acinetobacter baumannii strains harboring Meallobetalactamases (MBL) pose a significant threat in the context of nosocomial infections. The present investigation was undertaken with the objective of devising a Multiplex PCR methodology for the concurrent detection of MBL genes within A. baumannii strains prevalent in Tehran City, Iran. Between October 2020 and February 2021, 100 strains of A. baumannii were procured from burn specimens of hospitalized patients at Motahhari Hospital in Tehran. The identification of A. baumannii strains involved conventional biochemical techniques, coupled with confirmation of the presence of the bla OXA-51 gene. Antibiotic susceptibility was assessed using the Kirby-Bauer disc diffusion test. MBL-producing strains were characterized through a phenotypic approach employing the combined disk test, alongside Multiplex PCR for the simultaneous identification of bla VIM, bla IMP, bla GIM, and bla NDM genes. Statistical analyses were conducted using the chi-square test, with SPSS version 20.0 employed for data processing. Among 100 strains examined, 96.1% exhibited positivity for MBL, as determined by the combined disk test. The study revealed a predominance of extensively drug-resistant (XDR) strains, with colistin demonstrating the highest level of sensitivity. The genotypic assay unveiled that Multiplex PCR identified bla VIM, bla NDM, and bla IMP in 20 strains, bla VIM and bla NDM in 30 strains, and exclusively the bla NDM gene in 45 strains. Notably, the Multiplex PCR technique exhibited the capacity to concurrently detect MBL genes (bla VIM, bla IMP, bla GIM, bla NDM) in 2 strains. The current investigation underscores prevalence of the bla NDM gene within clinical strains of A. baumannii. Furthermore, Multiplex PCR emerges as a robust and highly sensitive technique for rapid discernment of the MBL genes within in A. baumannii strains.

A-272 Challenges in Designing an Antibiotic Gene Resistance Panel for Clinical Use

Abstract Background We faced 3 major challenges in our journey of designing a custom panel for antibiotic resistance genes (ABR). Challenge 1# Fast, robust, and affordable antimicrobial resistance testing (ABR) In general, qPCR-based methods are efficient, reliable, and fast with high specificity and sensitivity, and with the test results available within 2–3 h. We offer urinary tract infection identification by qPCR and wanted to supplement this test with detection of antibiotic resistance genes. we narrowed our choice of method to be qPCR based, Applied Biosystems™ OpenArray™ technology on the Applied Biosystems™ QuantStudio™ 12K Flex Real-Time PCR System. Challenge2# Genes to be tested. Antibiotic resistance occurs via various mechanism. Based on the literature review, we included the following gene in our custom panel 1. Aminoglycosides: aac(6′)-Ib-cr6, aac(6′)-Ib-cr, aac(6′)-Ib-cr4 2. Tetracycline: tet(M), tet(B) 3. Sulfonamides: sul1, sul2 4. Macrolide-lincosamide-streptogramin B-ketolides-oxazolidinones: erm C, erm A, erm B 5. Quinolone/(fluoroquinolones): qnrS , qnrA, aac(6′)-Ib-cr6, aac(6′)-Ib-cr, aac(6′)-Ib-cr4 6. Glycopeptides: VanA, VanB 7. Beta-lactam Class A: SHV, KPC, CTX-M 8. Beta-lactam Class B: NDM, VIM, IMP 9. Beta-lactam Class C: CMY, DHA, FOX, KPC 10. Beta-lactam Class D: OXA1, OXA-23, OXA- 48, 11. Methicillin resistance: mecA. Results Validation of the ABR panel Accuracy: 50 isolates were ordered from CDC AR isolate bank and were used to test the accuracy. The accuracy was detected to be 98% concordant. Sensitivity: The assay was sensitive to detect all urinary pathogens upto 10^3 CFU/mL. Precision: the inter and intra reproducibility was shown to be 99% concordant Specific: when tested against the CDC controls, the assays were 100% specific Challenge 3# Genotype—Phenotype correlation and prevalence of antibiotic resistance genes in USA. We compared 100 consecutive clinical specimens (urine) for genotype -phenotype co-relation. Data from the current qPCR panel and Microscan instrument was compared. It was observed for the pathogens that were phenotypically resistant to certain antibiotics, the corresponding gene association was not found in (2–50%) of antibiotic groups. This can be explained due to the limited number of genes in each drug class. Resistance to some antibiotics such as nitrofurantoin drug (100% missed) and sulphonamide drug resistance (50% missed) is induced by point mutations, which are not captured in open array genotyping qPCR assay. To overcome this gap between genotype and phenotype correlation and to understand the prevalence of antibiotic resistance genes in USA, we initiated a new project. We aim to test for 300–400 clinical samples which known resistance phenotypes and check for antibiotic resistance gene in a new expanded panel. We were successful in designing the new panel to include 88 genes (vs 30 genes in the old panel). Conclusion The current assay was approved by the CLIA authorities for clinical testing. In general, qPCR-based methods are efficient, reliable, and fast with high specificity and sensitivity, and with the test results available within 2–3 h.

Prevalence, drug resistance, molecular typing and comparative genomics analysis of MRSA strains from a tertiary A hospital in Shanxi Province, China.

Staphylococcus aureus (S. aureus) is an important zoonotic pathogen that causes a high incidence rate and mortality worldwide. This study investigated the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains in a tertiary A hospital in Shanxi Province, China, in order to determine the major epidemic clones as well as their antibiotic resistance and virulence characteristics. A total of 212 S. aureus strains were collected in this hospital, and were subjected to antimicrobial susceptibility testing, detection of virulence genes, resistance genes, and efflux pump genes. Among them, 38 MRSA strains were further subjected to detection of biofilm genes, assessment of biofilm formation ability, MLST, spa typing, SCCmec typing, and phylogenetic analysis. The majority of S. aureus strains came from the neonatology department, with secretions and purulent fluid being the main source of samples. The strains showed high resistance to penicillin (98.11%), erythromycin (64.62%) and clindamycin (59.91%), while being sensitive to vancomycin and linezolid. The detection rates of efflux pump genes and resistance genes were high, and there was a significant correlation between resistance gene types and phenotypes, with mecA showing a close correlation with oxacillin. The detection rates of virulence genes and the toxin gene profiles of MSSA and MRSA strains showed significant differences. And the detection rate of biofilm genes in MRSA strains was relatively high, with 13.16% of MRSA strains showing strong biofilm formation ability. The most common epidemic clone of MRSA was ST59-SCCmecIV-t437, followed by ST59-SCCmecV-t437. The former had a higher detection rate of resistance genes and a stronger biofilm formation ability, while the latter had a higher positive rate for pvl gene and stronger pathogenicity, making it more likely to cause systemic infections. Phylogenetic analysis showed that all MRSA strains in this study clustered into three major branches, with distinct differences in antibiotic resistance and virulence characteristics among the branches. ST59-MRSA strains from different species showed consistency and inter-species transmission, but there were differences among ST59-MRSA strains from different geographical locations. In general, most MSSA and MRSA strains exhibited multidrug resistance and carried multiple resistance genes, virulence genes, and biofilm formation genes, warranting further research to elucidate the mechanisms of drug resistance and pathogenesis.

An experimental comparison between primer and nucleotide labelling to produce RPA-amplicons used for multiplex detection of antibiotic resistance genes

Direct labelling of amplification products using isothermal amplification is currently done most frequently by incorporating previously labelled primer. Although this method is well proven and widely used, it is not a universal solution due to some weaknesses. Alternatively, labelled nucleotides could be used, whose application and functionality have been already partially demonstrated. It remains to be determined how this method performs in comparison to traditional labelling, in particular combined with isothermal amplification methods. In this work, we show a detailed analysis of the labelling efficiency under different conditions and compare the results with the traditional primer-labelling method in the context of RPA amplification. Impressively, our results showed that using Cy5-labelled dUTPs can achieve much more efficient labelling for fragments above 200 bp, while using them for smaller fragments does not bring any relevant disadvantages, but also no major benefit. Furthermore, this work successfully demonstrate for the first time a quadruplex microarray for the detection of resistance genes using RPA and direct labelling with Cy5-dUTP as a potential application scenario. The sensitivities achieved here extend to SNP discovery for the detection of the proper blaKPC variant.

A Whole-Genome Sequencing-Based Approach for the Characterization of Klebsiella pneumoniae Co-Producing KPC and OXA-48-like Carbapenemases Circulating in Sardinia, Italy.

Whole-genome sequencing (WGS) provides important information for the characterization, surveillance, and monitoring of antimicrobial resistance (AMR) determinants, particularly in cases of multi- and extensively drug-resistant microorganisms. We reported the results of a WGS analysis carried out on carbapenemases-producing Klebsiella pneumoniae, which causes hospital-acquired infections (HAIs) and is characterized by a marked resistance profile. Clinical, phenotypic, and genotypic data were collected for the AMR surveillance screening program of the University Hospital of Sassari (Italy) during 2020-2021. Genomic DNA was sequenced using the Illumina Nova Seq 6000 platform. Final assemblies were manually curated and carefully verified for the detection of antimicrobial resistance genes, porin mutations, and virulence factors. A phylogenetic analysis was performed using the maximum likelihood method. All 17 strains analyzed belonged to ST512, and most of them carried the blaKPC-31 variant blaOXA-48-like, an OmpK35 truncation, and an OmpK36 mutation. Phenotypic analysis showed a marked resistance profile to all antibiotic classes, including β-lactams, carbapenems, aminoglycosides, fluoroquinolone, sulphonamides, and novel β-lactam/β-lactamase inhibitors (BL/BLI). WGS characterization revealed the presence of several antibiotic resistance determinants and porin mutations in highly resistant K. pneumoniae strains responsible for HAIs. The detection of blaKPC-31 in our hospital wards highlights the importance of genomic surveillance in hospital settings to monitor the emergence of new clones and the need to improve control and preventive strategies to efficiently contrast AMR.

Activity of polymyxin B combinations against genetically well-characterised Klebsiella pneumoniae producing NDM-1 and OXA-48-like carbapenemases

Combination therapy can enhance the activity of available antibiotics against multidrug-resistant Gram-negative bacteria. This study assessed the effects of polymyxin B combinations against carbapenemase-producing Klebsiella pneumoniae (K. pneumoniae). Twenty clinical K. pneumoniae strains producing NDM-1 (n=8), OXA-48-like (n=10), or both NDM-1 and OXA-48-like (n=2) carbapenemases were used. Whole-genome sequencing was applied to detect resistance genes (e.g. encoding antibiotic-degrading enzymes) and sequence alterations influencing permeability or efflux. The activity of polymyxin B in combination with aztreonam, fosfomycin, meropenem, minocycline, or rifampicin was investigated in 24-hour time-lapse microscopy experiments. Endpoint samples were spotted on plates with and without polymyxin B at 4 x MIC to assess resistance development. Finally, associations between synergy and bacterial genetic traits were explored. Synergistic and bactericidal effects were observed with polymyxin B in combination with all other antibiotics: aztreonam (11 of 20 strains), fosfomycin (16 of 20), meropenem (10 of 20), minocycline (18 of 20), and rifampicin (15 of 20). Synergy was found with polymyxin B in combination with fosfomycin, minocycline, or rifampicin against all nine polymyxin-resistant strains. Wildtype mgrB was associated with polymyxin B and aztreonam synergy (P=0.0499). An absence of arr-2 and arr-3 was associated with synergy of polymyxin B and rifampicin (P=0.0260). Emergence of populations with reduced polymyxin B susceptibility was most frequently observed with aztreonam and meropenem. Combinations of polymyxin B and minocycline or rifampicin were most active against the tested NDM-1 and OXA-48-like-producing K. pneumoniae. Biologically plausible genotype-phenotype associations were found. Such information might accelerate the search for promising combinations and guide individualised treatment.

A gold nanoparticle colorimetric probe-based biosensor for rapid detection of VIM-2 and IMP-1 metallo-beta lactamase genes

Despite actions to reduce the prevalence of antibiotic resistance, antibiotic resistance remains a major threat in the treatment and control of infections. Therefore, the development of rapid diagnostic methods plays a significant role in the detection of antibiotic resistance genes and the management of hospital infections. This study aimed to design and develop a gold nanoparticle colorimetric probe-based biosensor was developed for faster and more accurate detection of VIM-2 and IMP-1 Metallo-Beta-Lactamases (MBLs) genes in different clinical samples. After identifying 248 clinical isolates of bacteria with standard biochemical methods and evaluation of antibiotic resistance and identifying MBL-producing strains, PCR method was carried out for detecting VIM-2 and IMP-1 genes as a gold standard. Synthesis of AuNPs were done by citrate reduction method and AuNP -IMP-1 biosensor was used for detecting IMP-1 gene, after functionalization of thiol modified oligonucleotides. AuNP biosensor and IMP-1 PCR were compared in terms of detection indices. Method PCR was defined as the gold standard. Bacteria examined, 87 isolates were resistant to carbapenems. Out of the 87 carbapenem-resistant isolates, 85 (34.2%) were phenotypically positive for MBLs. Also, 7 isolates had IMP-1 gene, but none of them carried VIM-2 gene. The AuNP biosensor had 100% sensitivity, specificity, PPV and NPV, respectively. The final detection limit of IMP-1 genomic DNA (LOD) by PCR and AuNP-IMP-1 biosensor technique was 0.1 fg/μl (2.5 fg/25 reactions) and 0.001 fg/μl (0.025 fg/25 reaction), respectively. According to the promising results of Diagnostic indices, AuNP biosensor is a more efficient and accurate method for direct and indirect detection of antibiotic resistance genes in clinical samples.

SERS sensor combined with the dual DNA cycling amplification assay for the sensitive detection of antibiotic resistance gene in environmental samples

Antibiotic resistance genes (ARGs) have emerged as new environmental pollutants, and even minute quantities of them can seriously affect the environment. Thus, it is urgently required to test trace ARGs. Herein, a surface-enhanced Raman scattering (SERS) sensor based on the dual cycling amplification has been developed for the ultrasensitive detection of bla-TEM. First, the capture DNA on the surface of magnetic beads and the Hg2+ aptamer-based capture strand could achieve the capture, separation, and enrichment of the bla-TEM from the relatively complex samples. Then, the dual amplification process was finished under the catalysis of the enzymes, and the entire process was operated in one step under the isothermal condition. Finally, the enriched SERS nanoprobes were obtained via the feasible magnetic separation and provided Raman signals for the detected bla-TEM under the laser irradiation. Under the optimum experimental conditions, the sensor exhibited excellent sensitivity and good selectivity, obtaining a linear range of 0.1 zM-300 aM and a limit of detection (LOD) of 0.02 zM. It can be successfully used to measure bla-TEM in actual environmental samples, which was in good accordance with the conventional polymerase chain reaction (PCR) analysis, showing great promise for detecting trace ARGs in the environmental samples.

Comorbidity and drug resistance of smear-positive pulmonary tuberculosis patients in the yi autonomous prefecture of China: a cross-sectional study

BackgroundTuberculosis (TB) has a high morbidity and mortality rate, and its prevention and treatment focus is on impoverished areas. The Liangshan Yi Autonomous Prefecture is a typical impoverished area in western China with insufficient medical resources and high HIV positivity. However, there have been few reports of TB and drug resistance in this area.MethodsWe collected the demographic and clinical data of inpatients with sputum smear positive TB between 2015 and 2021 in an infectious disease hospital in the Liangshan Yi Autonomous Prefecture. Descriptive analyses were used for the epidemiological data. The chi-square test was used to compare categorical variables between the drug-resistant and drug-susceptible groups, and binary logistic regression was used to analyse meaningful variables.ResultsWe included 2263 patients, 79.9% of whom were Yi patients. The proportions of HIV (14.4%) and smoking (37.3%) were higher than previously reported. The incidence of extrapulmonary TB (28.5%) was high, and the infection site was different from that reported previously. When drug resistance gene detection was introduced, the proportion of drug-resistant patients became 10.9%. Patients aged 15–44 years (OR 1.817; 95% CI 1.162–2.840; P < 0.01) and 45–59 years (OR 2.175; 95% CI 1.335–3.543; P < 0.01) had significantly higher incidences of drug resistance than children and the elderly. Patients with a cough of ≥ 2 weeks had a significantly higher chance of drug resistance than those with < 2 weeks or no cough symptoms (OR 2.069; 95% CI 1.234–3.469; P < 0.01). Alcoholism (OR 1.741; 95% CI 1.107–2.736; P < 0.05) and high bacterial counts on sputum acid-fast smears (OR 1.846; 95% CI 1.115–3.058; P < 0.05) were significant in the univariate analysis.ConclusionsSputum smear-positive TB predominated in Yi men (15–44 years) with high smoking, alcoholism, and HIV rates. Extrapulmonary TB, especially abdominal TB, prevailed. Recent drug resistance testing revealed higher rates in 15–59 age group and ≥ 2 weeks cough duration. Alcohol abuse and high sputum AFB counts correlated with drug resistance. Strengthen screening and supervision to curb TB transmission and drug-resistant cases in the region.

Detection of tetracycline resistance genes and their diversity in Escherichia coli isolated from pig farm waste in Banten province, Indonesia.

Livestock waste in the form of feces and liquid represents an important reservoir of antibiotic resistance genes (ARGs). Because many ARGs can be horizontally transferred to other pathogens, livestock waste plays an essential role in the emergence and transmission of various ARGs in the environment. Therefore, this study aimed to detect and assess the diversity of tet genes in Escherichia coli isolated from pig farm waste in Banten province, Indonesia. Solid waste (feces) and wastewater were collected from 44 pig farms in Banten province. The isolation and identification of E. coli referred to the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli World Health Organization (2021) guidelines. tet genes were detected using quantitative real-time polymerase chain reaction after dividing pig farms in the province into four clusters based on their adjacent areas and characteristics. tetA, tetB, tetC, tetM, tetO, and tetX were detected in solid waste and wastewater from pig farms, whereas tetE was not detected in either sample type. tetX (100%) and tetO (75%) were the most dominant genes in solid waste, whereas wastewater samples were dominated by tetA, tetM, tetO, and tetX (prevalence of 50% each). Furthermore, eight tet gene patterns were found in pig farm waste (prevalence of 12.5% each). The results showed a high prevalence of tetO and tetX in solid waste and wastewater from pig farms in Banten province. This significant prevalence and diversity indicated the transmission of tet genes from pigs to the environment, posing a serious threat to public health.

Multiplex PCR detection of antibiotic resistance and virulence genes in multidrug-resistant Staphylococcus aureus isolated from chickens, humans, rodents, and soil in Northern Tanzania

Staphylococcus aureus (S. aureus) is a zoonotic pathogen with public health and veterinary importance. We investigated the presence of antibiotic resistance genes (ARGs) and virulence genes (VGs) in 57 multidrug-resistant (MDR) S. aureus isolated from humans (n=17), chickens (n=14), rodents (n=13), and soil (n=13) using multiplex PCR. Overall, the distribution of ARGs revealed that the tetK was found in 18/57 (31.6%), mecA in 16/57 (28.1%), tetL in 5/57 (8.9%), and ermC in 1/57 (1.8%), while ermA and tetM were not detected. For VGs, the clfB was found in 6/57 (10.5 %), coa in 8/57 (14.0%), clfA in 3/57 (5.3%), hlg in 1/57 (1.8%), ebpS in 2/57 (3.5%), fnbB in 2/57 (3.5%), luk-PV in 6/57 (10.5%) and tst in 1/57 (1.8%). Resistance genes (tetK and mecA) and virulence determinants (clfB, coa, and luk-PV) were common in all sample sources, while tst, hlg, and fnbB were specific to human, chicken, and rodent isolates, respectively. Erythromycin phenotypic resistance results correlated with the presence of ermC (r=0.42), tetL (r=0.98), and mecA (r=0.51), while tetracycline resistance correlated with tetL (r=1.00) and mecA (r=0.57) genes and methicillin resistance correlated with mecA (r=0.55) and tetL (r=0.98) genes. Positive correlations were noted between ARG (ermC) and VGs; clfA (r=0.57), hlg (r=1.00), and clfB (r=0.43), and between tetK and clfB (r=0.39); tetK and coa (r=0.36) genes. Principal component analysis (PCA) shows that tetL, ermC, and mecA contributed to tetracycline, erythromycin, and methicillin resistance, respectively. The widespread presence of resistance and virulence genes, often in combination, among MDR S. aureus in isolates from humans, chicken, rodents, and soil samples require comprehensive One-Health interventions.

A double-quenching paperclip ECL biosensing platform for ultrasensitive detection of antibiotic resistance genes (mecA) based on Ti3C2 MXene-Au NPs as a coreactant accelerator

The global spread of environmental biological pollutants, such as antibiotic-resistant bacteria and their antibiotic resistance genes (ARGs), has emerged as a critical public health concern. It is imperative to address this pressing issue due to its potential implications for public health. Herein, a DNA paperclip probe with double-quenching function of target cyclic cleavage was proposed, and an electrochemiluminescence (ECL) biosensing platform was constructed using Ti3C2 MXene in-situ reduction growth of Au NPs (TCM-Au) as a coreactant accelerator, and applied to the sensitive detection of ARGs. Thanks to the excellent catalytic performance, large surface area and Au–S affinity of TCM-Au, the ECL performance of CdS QDs have been significantly improved. By cleverly utilizing the negative charge of the paperclip nucleic acid probe and its modification group, double-quenching of the ECL signal was achieved. This innovative approach, combined with target cyclic amplification, facilitated specific and sensitive detection of the mecA gene. This biosensing platform manifested highly selective and sensitive determination of mecA genes in the range of 10 fM to 100 nM and a low detection limit of 2.7 fM. The credible detectability and anti-interference were demonstrated in Yangtze river and Aeration tank outlet, indicating its promising application toward pollution monitoring of ARGs.

Detection of (CTX-M) resistance genes in Extended Spectrum Beta-lactamases bacterial isolates of Escherichia coli and Klebsiella pneumonia and the antibacterial effect of ethanolic extract of Neem plant (Azadirachta indica) against these bacteria.

Detection of (CTX-M) resistance genes in Extended Spectrum Beta-lactamases bacterial isolates of Escherichia coli and Klebsiella pneumonia and the antibacterial effect of ethanolic extract of Neem plant (Azadirachta indica) against these bacteria.

Ultrasensitive rapid detection of antibiotic resistance genes by electrochemical ratiometric genosensor based on 2D monolayer Ti3C2@AuNPs

As an important emerging pollutant, antibiotic resistance genes (ARGs) monitoring is crucial to protect the ecological environment and public health, but its rapid and accurate detection is still a major challenge. In this study, a new single-labeled dual-signal output ratiometric electrochemical genosensor (E-DNA) was developed for the rapid and highly sensitive detection of ARGs using a synergistic signal amplification strategy of T3C2@Au nanoparticles (T3C2@AuNPs) and isothermal strand displacement polymerase reaction (ISDPR). Specially, two-dimensional monolayer T3C2 nanosheets loaded with uniformly gold nanoparticles were prepared and used as the sensing platform of the E-DNA sensor. Benefiting from excellent conductivity and large specific surface area of Ti3C2@AuNPs, the probe immobilization capacity of the E-DNA sensor is doubled, and electrochemical response signals of the E-DNA sensor were significantly improved. The proposed single-labeled dual-signal output ratiometric sensing strategy exhibits three to six times higher sensitivity for the sul2 gene than the single-signal sensing strategy, which significantly reduces cost meanwhile retaining the advantages of high sensitivity and reliability offered by conventional dual-labeled ratiometric sensors. Coupled with ISDPR amplification technology, the E-DNA sensor has a wider linear range from 10 fM to 10 nM and a limit of detection as low as 2.04 fM (S/N=3). More importantly, the E-DNA sensor demonstrates excellent specificity, good stability and reproducibility for target ARGs detection in real water samples. The proposed new sensing strategy provides a highly sensitive and versatile tool for the rapid and accurate quantitative analysis of various ARGs in environmental water samples.

The Mass Spectrometry Identification, Antimicrobial Genes Detection, and Proteomics Analysis of Stutzerimonas stutzeri Strain Was Isolated from Industrial Wastewater

A large amount of organic matter, heavy metals, and even antibiotics are present in industrial wastewater, aquaculture waters, and various types of sewage, along with abundant microorganisms. To date, only a few studies involving the resistance and proteomics of Stutzerimonas stutzeri in high-salt wastewater are available. Herein, a comprehensive assessment of a newly isolated Stutzerimonas stutzeri strain, which is present in high-salt wastewater, was performed using mass spectrometry, genetic identification, and biochemical analysis to characterize the genetic and biochemical properties. Growth experiments revealed that the Stutzerimonas stutzeri strain had a moderate growth rate in nutrient broth, and the bacterial count was not high. Further analysis highlighted an apparent susceptibility of this strain to most antibiotics but some resistance to chloramphenicol and minocycline. A resistance gene assay results showed that the gene gyrB was associated with antibiotic resistance in this Stutzerimonas stutzeri strain. Proteomic analysis revealed for the first time the co-existence of two drug-resistance-related proteins (Multidrug/solvent RND membrane fusion protein and MexE) in Stutzerimonas stutzeri. Moreover, Stutzerimonas stutzeri isolated from high-salt wastewater was subjected to drug resistance gene detection, and the total protein of Stutzerimonas stutzeri was detected by protein mass spectrometry analysis. The subcellular classification shows that the 50 proteins with the highest abundance are divided into cell inner membrane, cell outer membrane, cytoplasm, cytoplasmic side, membrane, multi-pass membrane protein, and peripheral membrane protein, among which the proportion of cytoplasmic components is the highest. Overall, this study’s findings provide a new perspective for further research on the characteristics of Stutzerimonas stutzeri in high-salt wastewater.