Int Gene Research Articles

Investigation of the antimicrobial susceptibility patterns of marine cyanobacteria in Bohai Bay: Cyanobacteria may be important hosts of antibiotic resistance genes in marine environment

Marine cyanobacteria, as widely distributed and photosynthetically autotrophic bacteria in the ocean, may contribute to the global dissemination of antibiotic resistance genes (ARGs) and develop a different antimicrobial susceptibility pattern from heterotrophic bacteria and cyanobacteria from freshwater environments. However, studies on antimicrobial susceptibility and the carriage of ARGs in marine cyanobacteria are still very limited. In this study, the antibiotic resistance characteristics of cyanobacteria in nearshore waters were examined through field monitoring and laboratory investigations, which included PCR detection and ARG transformation. The results showed a positive correlation between marine cyanobacteria and some ARGs in the nearshore waters of Bohai Bay. Moreover, most screened cyanobacteria showed high minimum inhibitory concentration (MIC) values for polymyxins, tetracyclines, kanamycin, and sulfonamides, moderate MIC values for streptomycin, chloramphenicol, rifampicin, and norfloxacin, and low MIC values for roxithromycin and cephalosporins. The blaTEM, blaKPC, sul1, sul2, strA, tetA, tetB, tetC, tetM, mdfA, and intI1 genes were detected in the screened marine cyanobacteria. The highest detection rates were observed for blaTEM (93.3 %), sul1 (56.6 %), sul2 (90 %), and strA (73.3 %). The detection rate of tetA (33.3 %) was the highest among the tetracycline resistance genes, and mdfA, a multidrug-resistant pump gene with resistance to tetracycline, also showed a high detection level (23.3 %). Overall, most of the screened marine cyanobacteria were found to tolerate multiple antibiotics in seawater, and the condition of the ARGs carriage was serious. Furthermore, the screened marine Synechocystis sp. C12-2 demonstrated the ability to accept ARGs on the RP4 plasmid through natural transformation and showed reduced sensitivity to ampicillin, suggesting the possibility that some marine cyanobacteria could acquire ARGs from the environment through horizontal gene transfer. Thus, marine cyanobacteria may play an important role in the propagation of marine ARGs.

Activity of propolis from Mexico on the proliferation and virulence factors of Candida albicans

BackgroundThis research evaluated the anti-Candida albicans effect of Mexican propolis from Chihuahua.Chemical composition of the ethanolic extract of propolis was determined by GC-MS, HPLC-DAD, and HPLC-MS. The presence of anthraquinone, aromatic acid, fatty acids, flavonoids, and carbohydrates was revealed.ResultsThe anti-Candida activity of propolis was determined. The inhibitions halos were between 10.0 to 11.8 mm; 25% minimum inhibitory concentration (0.5 mg/ml) was fungistatic, and 50% minimum inhibitory concentration (1.0 mg/ml) was fungicidal.The effect of propolis on the capability of C. albicans to change its morphology was evaluated. 25% minimum inhibitory concentration inhibited to 50% of germ tube formation.Staining with calcofluor-white and propidium iodide was performed, showing that the propolis affected the integrity of the cell membrane.INT1 gene expression was evaluated by qRT-PCR. Propolis significantly inhibited the expression of the INT1 gene encodes an adhesin (Int1p).Chihuahua propolis extract inhibited the proliferation of Candida albicans, the development of the germ tube, and the synthesis of adhesin INT1.ConclusionsGiven the properties demonstrated for Chihuahua propolis, we propose that it is a candidate to be considered as an ideal antifungal agent to help treat this infection since it would not have the toxic effects of conventional antifungals.

A novel strategy for eliminating antibiotic resistance genes during fertilization of dewatered sludge by earthworms: Vermicomposting practice using Chinese herbal residues derived from Lianhua Qingwen as a bulking material

Vermicomposting is a sustainable sludge recycling technology that utilizes an eco-friendly composting using earthworms and microorganisms. However, a high abundance of antibiotic resistance genes (ARGs) remains in dewatered sludge that is not satisfactorily eliminated by vermicomposting. Chinese herbs have played a major role in curing many diseases in East Asia, leading to a large amount of Chinese herbal residues (CHRs) are difficult to dispose of. The present study investigated the feasibility of CHRs on the ARGs reduction in dewatered sludge during vermicomposting. The CHRs derived from Lianhua Qingwen were added separately to sludge with weight ratios of 0%, 10%, 30%, and 50%; sludge was then vermicomposted for 30 days. The results showed that co-vermicomposting of sludge and CHRs is a feasible strategy. The CHR treatments significantly (P < 0.05) decreased antibiotic concentration and bacterial population by 23.64%–49.68% and 42.58%–93.07%, respectively, compared to counterpart. Compared to the control, the CHR addition lowered the absolute abundances of macrolide, tetracycline, and sulfonamide ARGs by 42.69%–85.15%, 22.03%–75.24%, and 23.59%–90.66%, respectively. In addition, sludge containing 30% CHRs showed significant (P < 0.05) elimination of intⅠ-1 and tnpA-4 genes with abundance reductions of 71.40% and 52.33%, respectively, relative to the control. This study suggests that the CHRs can effectively reduce ARGs content in sludge by decreasing the bacterial population and horizontal gene transfer capacity during vermicomposting.

Nutritional analysis and characterization of carbapenemase producing-Klebsiella pneumoniae resistant genes associated with bovine mastitis infected cow's milk.

The current study was designed to analyze nutritional parameters and to characterize carbapenemase producing-Klebsiella pneumoniae isolates from bovine mastitic cow's milk. Out of 700 milk samples K. pneumoniae was identified by phenotypic and molecular techniques along with their antibiogram analysis and nutritional analysis was performed using the procedure of Association of Official Analytical Chemists. Carbapenemase-producing K. pneumoniae was detected by phenotypic CarbaNP test followed by molecular characterization of their associated resistant genes blaVIM, blaKPC, blaOXA-48, blaNDM, and blaIMP along with insertion sequence common region 1 (ISCR1) and integrons (Int1, Int2, and Int3) genes. Among nutritional parameters, fat content was observed (2.99%) followed by protein (2.78%), lactose (4.32%), and total solid (11.34%), respectively. The prevalence of K. pneumoniae among bovine mastitis was found 25.71%. Antibiogram analysis revealed that more effective antibiotics was ceftazidime (80%) followed by amikacin (72%), while highly resistant antibiotics was Fusidic acid (100%). Distribution of carbapenemase producer K. pneumoniae was found 44.4%. Among carbapenem resistant genes blaKPC was found 11.25%, blaVIM 2.75%, blaNDM 17.5%, and blaOXA-48 7.5%, while blaIMP gene was not detected. Furthermore, distribution of ISCR1 was found 40%, while integron 1 was found 61.2% followed by integron 2 (20%), and integron 3 (5%). In conclusion, the recent scenario of carbapenemase resistant K. pneumoniae isolates responsible for mastitis may affect not only the current treatment regime but also possess a serious threat to public health due to its food borne transmission and zoonotic potential.

IntI1 gene abundance from septic tanks in Thailand using validated intI1 primers.

Antimicrobial resistance is a global crisis, and wastewater treatment, including septic tanks, remains an important source of antimicrobial resistance (AMR) genes. The role of septic tanks in disseminating class 1 integron, and by extension AMR genes, in Thailand, where antibiotic use is unregulated remains understudied. We aimed to monitor gene abundance as a proxy to infer potential AMR from septic tanks in Thailand. We evaluated published intI1 primers due to the lack of consensus on optimal Q-PCR primers and the absence of standardization. Our findings confirmed septic tanks are a source of class 1 integron to the environment. We highlighted the significance of intI1 primer choice, in the context of interpretation of risk associated with AMR spread from septic tanks. We recommend the validated set (F3-R3) for optimal intI1 quantification toward the goal of achieving standardization across studies.

High throughput qPCR unveils shared antibiotic resistance genes in tropical wastewater and river water

The global challenge posed by rising antimicrobial resistance, and the adoption of a One Health approach, has led to the prioritisation of surveillance for antibiotic resistance genes (ARGs) in various environments. Herein lies an information gap, particularly in the context of Thailand, where there is scarce data on ARG prevalence across diverse environmental matrices and throughout different seasons. This study aimed to fill this void, analysing ARG prevalence by high-throughput qPCR in influent (n = 12) and effluent wastewater (n = 12) and river water (n = 12). The study reveals a substantial and largely uniform presence of ARGs across all water sample types (87 % similarity). Intriguingly, no ARGs were exclusive to specific water types, indicating an extensive circulation of resistance determinants across the aquatic environment. The genes intI1, tnpA, and intI3, part of the integrons and mobile genetic elements group, were detected in high relative abundance in both wastewater and river water samples, suggesting widespread pollution of rivers with wastewater. Additional high-prevalence ARGs across all water types included qepA, aadA2, merA, sul1, qacF/H, sul2, aadB, and ereA. More alarmingly, several ARGs (e.g., blaVIM, intI3, mcr-1, mexB, qepA, vanA, and vanB) showed higher relative abundance in effluent and river water than in influents, which suggests malfunctioning or inadequate wastewater treatment works and implicates this as a possible mechanism for environmental contamination. Nine genes (i.e., blaCTX-M, blaVIM, emrD, ermX, intI1, mphA, qepA, vanA, and vanB) were recovered in greater relative abundance during the dry season in river water samples as compared to the wet season, suggesting there are seasonal impacts on the efficacy of wastewater treatment practices and pollution patterns into receiving waters. This study highlights the urgency for more effective measures to reduce antibiotic resistance dissemination in water systems.

Investigating nitrous oxide emissions and mechanisms in kitchen waste composting with leachate reuse

Solid-liquid mixed composting is a novel approach for handling kitchen waste (KW). In this study, KW was employed as composting materials to explore the nitrous oxide (N2O) emissions and corresponding mechanisms associated with the reuse of three supplementary liquids (CK: deionized water; T1: kitchen waste composting leachate (KWCL); T2: kitchen waste leachate (KWL)) during the composting process. The analysis included N2O emissions, microbial community dynamics, and nitrogen metabolism pathways. The findings demonstrated that the addition of KWCL (T1) and KWL (T2) significantly increased the nutrient content in the compost. Notably, T1 exhibited significantly higher cumulative N2O emissions than CK and T2 (P < 0.01). Both CK and T2 demonstrated more robust microbial safety, while the addition of KWCL (T1) led to more significant alterations in the structure of bacterial and fungal communities. Denitrification was identified as the primary pathway for N2O emissions during the thermophilic period. Nitrous oxide reductase activity was significantly lower in T1 than in T2 (P < 0.05), and the abundance of the nosZ gene in T1 (3.39 %) was lower than that in CK (4.18 %) and T2 (4.51 %), contributing to higher N2O emissions. Correlation analysis revealed the vital role of bacteria in N2O production, while bacterial and fungal communities exhibited positive correlations with variables such as carbon-to-nitrogen ratio and total carbon. Environmental factors were identified as key drivers of N2O emissions (r = 0.837, P < 0.05). These findings provided valuable insights for the sustainable management and recycling of KW.

High incidence of multiple intI1 genomic gene cassettes in Aeromonas strains

Antimicrobial resistance profiles of Aeromonas have been used as an effective indicator for monitoring resistances in aquatic environments. Integron gene cassettes are a major reservoir of antibiotic resistance genes in bacteria. To date, little has been reported on the antimicrobial resistance or integrons of Aeromonas in bullfrog aquaculture. In this study, 27 isolates belonging to four Aeromonas species (A. hydrophila, A. veronii, A. caviae, and A. jandaei) were isolated from farmed diseased American bullfrogs (Rana catesbeiana). all of them were multidrug-resistant, exhibiting resistance to at least seven drugs from five antibiotic classes. Class 1 integrons (intI1s), containing nine types of gene cassette arrays encoding resistance to trimethoprim (dfrA1, dfrA12, dfrA15, dfrA17), streptomycin/spectinomycin (aadA2), fluoroquinolones/aminoglycosides (aac(6′)-Ib-cr), rifampin (arr3), proline-rich antimicrobial peptides (ptrB), and multiple antimicrobial agents involved in biofilm development (dgs), were detected in 26 (26/27, 96.3%) of the isolates. Seven patterns were identified, and six of them contained two or three gene cassettes, in 27 isolates. This work highlights unusually high frequency (21/27, 77.8%) of multiple intI1 gene cassette arrays and three novel intI1 gene cassettes or arrays (dgc, ptrB, and aac(6′)-Ib-cr-arr3) in the isolates of Aeromonas spp. from American bullfrogs.

Molecular detection of Tn7-like transposons and IntI2 integron integrase in multidrug-resistant Escherichia coli and Klebsiella spp.

Abstract Background: Transposable elements are mobile DNA sequences that can move and change their location within the genome. Objectives: The aim of the study was to investigate class II integron and Tn7-like transposons and their relationship to multidrug resistance in commensal and pathogenic Klebsiella and Escherichia coli isolates. Materials and Methods: The disk-diffusion method was used to check the antibiotic susceptibility of 26 Klebsiella isolates and 76 E. coli isolates that had been isolated and preidentified using the VITEK 2 System from healthy stool, diarrhea, and urine in (urinary tract infection) specimens. The presence of the intI2 and Tn7-like transposase genes was then examined using specific primer pairs by polymerase chain reaction. Results: A total of 76% of the total isolates harbored Tn7, of which 14% of these isolates appeared to harbor intI2 in the Tn7 in three phylotypes. The other 62% of isolates had five patterns of Tn7. At the same time, the rest of the isolates (24%) were negative for the Tn7 and intI2 genes. Overall, the E. coli and Klebsiella isolates contain different Tn7-like transposons and are associated with pathogenic bacteria (P &lt; 0.05) though some normal flora harbor the transposons. The presence of intI2 and Tn7-like transposons is associated with multidrug resistance in the studied species; intI2 in Tn7-like transposon (P &lt; 0.029) and Tn7-like transposition genes only (P &lt; 0.001). A significant association (P &lt; 0.05) was detected between intI2 in the Tn7-like transposons and resistance to amoxicillin-clavulanate, piperacillin, cefotaxime, gentamicin, azithromycin, and nitrofurantoin. Conclusions: There is a correlation between multidrug resistance and the presence of Tn7-like transposition and intI2 genes.

Impact of corrosion inhibitors on antibiotic resistance, metal resistance, and microbial communities in drinking water.

Corrosion inhibitors, including zinc orthophosphate, sodium orthophosphate, and sodium silicate, are commonly used to prevent the corrosion of drinking water infrastructure. Metals such as zinc are known stressors for antibiotic resistance selection, and phosphates can increase microbial growth in drinking water distribution systems (DWDS). Yet, the influence of corrosion inhibitor type on antimicrobial resistance in DWDS is unknown. Here, we show that sodium silicates can decrease antibiotic resistant bacteria (ARB) and antibiotic-resistance genes (ARGs), while zinc orthophosphate increases ARB and ARGs in source water microbial communities. Based on controlled bench-scale studies, zinc orthophosphate addition significantly increased the abundance of ARB resistant to ciprofloxacin, sulfonamides, trimethoprim, and vancomycin, as well as the genes sul1, qacEΔ1, an indication of resistance to quaternary ammonium compounds, and the integron-integrase gene intI1. In contrast, sodium silicate dosage at 10 mg/L resulted in decreased bacterial growth and antibiotic resistance selection compared to the other corrosion inhibitor additions. Source water collected from the drinking water treatment plant intake pipe resulted in less significant changes in ARB and ARG abundance due to corrosion inhibitor addition compared to source water collected from the pier at the recreational beach. In tandem with the antibiotic resistance shifts, significant microbial community composition changes also occurred. Overall, the corrosion inhibitor sodium silicate resulted in the least selection for antibiotic resistance, which suggests it is the preferred corrosion inhibitor option for minimizing antibiotic resistance proliferation in DWDS. However, the selection of an appropriate corrosion inhibitor must also be appropriate for the water chemistry of the system (e.g., pH, alkalinity) to minimize metal leaching first and foremost and to adhere to the lead and copper rule. IMPORTANCE Antibiotic resistance is a growing public health concern across the globe and was recently labeled the silent pandemic. Scientists aim to identify the source of antibiotic resistance and control points to mitigate the spread of antibiotic resistance. Drinking water is a direct exposure route to humans and contains antibiotic-resistant bacteria and associated resistance genes. Corrosion inhibitors are added to prevent metallic pipes in distribution systems from corroding, and the type of corrosion inhibitor selected could also have implications on antibiotic resistance. Indeed, we found that sodium silicate can minimize selection of antibiotic resistance while phosphate-based corrosion inhibitors can promote antibiotic resistance. These findings indicate that sodium silicate is a preferred corrosion inhibitor choice for mitigation of antibiotic resistance.

Circulation of enterotoxigenic Escherichia coli (ETEC) isolates expressing CS23 from the environment to clinical settings.

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of infant diarrhea in low- and middle-income countries (LMICs). Diarrheal pathogens are transmitted through environmental reservoirs; however, the bacterial clones that spread across the human-environment interface remain unexplored. We aimed to determine the relationship and clonal dissemination of ETEC between children with diarrhea and polluted water samples from a local river in La Paz, Bolivia. By using WGS and the PhenePlates phenotypic system to analyze ETEC strains, we showed that ST218 and ST410 LT+STh ETEC expressing the colonization factor (CF) CS23 were found with high frequency in both samples. The CS23 ETEC isolates were found within several STs, E. coli phylogroups, and across ETEC lineages. Comparative genomic evaluation and PhenePlate screening of globally distributed clinical ETEC strains suggest that the CS23 gene is likely carried on plasmids acquired independently of the bacterial chromosomal background. Clinical strains were more often multidrug-resistant (MDR) than environmental isolates and harbored the class 1 integron-integrase gene intI1 next to the MDR cassettes. Retrospective analysis of antibiotic resistance in ETEC revealed a high frequency of MDR in clinical isolates. The LT+STh CS23 environmental ETEC isolates, showed an increased biofilm ability at environmental temperature, equal cytotoxicity, and significantly lower adherence to human epithelial cells compared to ETEC expressing other CFs. Together, we suggest that CS23 is more prevalent in ETEC than previously estimated, and the Choqueyapu River is a reservoir for LT+STh CS23 ETEC containing strains capable of causing diarrheal cases in children. IMPORTANCE The importance of clean water cannot be overstated. It is a vital resource for maintaining health and well-being. Unfortunately, water sources contaminated with fecal discharges from animal and human origin due to a lack of wastewater management pose a significant risk to communities, as they can become a means of transmission of pathogenic bacteria like enterotoxigenic E. coli (ETEC). ETEC is frequently found in polluted water in countries with a high prevalence of diarrheal diseases, such as Bolivia. This study provides novel insights into the circulation of ETEC between diarrheal cases and polluted water sources in areas with high rates of diarrheal disease. These findings highlight the Choqueyapu River as a potential reservoir for emerging pathogens carrying antibiotic-resistance genes, making it a crucial area for monitoring and intervention. Furthermore, the results demonstrate the feasibility of a low-cost, high-throughput method for tracking bacterial pathogens in low- and middle-income countries, making it a valuable tool for One Health monitoring efforts.

Antibiotic resistance indicator genes in biofilm and planktonic microbial communities after wastewater discharge.

The spread of bacteria with antibiotic resistance genes (ARGs) in aquatic ecosystems is of growing concern as this can pose a risk of transmission to humans and animals. While the impact of wastewater treatment plant (WWTP) effluent on ARG abundance in surface waters has been studied extensively, less is known about the fate of ARGs in biofilms. The proximity and dense growth of microorganisms in combination with the accumulation of higher antibiotic concentrations in biofilms might render biofilms a reservoir for ARGs. Seasonal parameters such as water temperature, precipitation, and antibiotic concentrations should be considered as well, as they may further influence the fate of ARGs in aquatic ecosystems. Here we investigated the effect of WWTP effluent on the abundance of the sulfonamide resistance genes sul1 and sul2, and the integrase gene intI1 in biofilm and surface water compartments of a river in Germany with a gradient of anthropogenic impact using quantitative PCR. Furthermore, we analyzed the bacterial community structure in both compartments via 16S rRNA gene amplicon sequencing, following the river downstream. Additionally, conventional water parameters and sulfonamide concentrations were measured, and seasonal aspects were considered by comparing the fate of ARGs and bacterial community diversity in the surface water compartment between the summer and winter season. Our results show that biofilm compartments near the WWTP had a higher relative abundance of ARGs (up to 4.7%) than surface waters (<2.8%). Sulfonamide resistance genes were more persistent further downstream (>10 km) of the WWTP in the hot and dry summer season than in winter. This finding is likely a consequence of the higher proportion of wastewater and thus wastewater-derived microorganisms in the river during summer periods. We observed distinct bacterial communities and ARG abundance between the biofilm and surface water compartment, but even greater variations when considering seasonal and spatiotemporal parameters. This underscores the need to consider seasonal aspects when studying the fate of ARGs in aquatic ecosystems.

Phenotypic and genotypic study of antibiotic-resistant Escherichia coli isolates from a wastewater treatment plant in Zulia state, Venezuela.

Antibiotic-resistance in bacteria is a global health problem, and wastewater treatment plants can play a role in their dissemination. In this work, we used PCR and plasmid transformation to characterize antibiotic-resistance and the phylogenetic groups of Escherichia coli isolated from a treatment plant in Zulia, a state in western Venezuela. Thirty-six bacteria isolates were analyzed, of which 27 resulted resistant by disc diffusion primarily to tetracycline and sulfisoxazole but also to trimethoprim, chloramphenicol, ampicillin, and cip-rofloxacin. The tetA, sul2, floR, and blaTEM resistance genes were frequently present and, in most cases, transferable. dfrA12, tetB, sul3, sul1, and aadA2genes also were detected. The integrase gene intI1 was common in multidrug-resistant isolates. These results suggest that E. coli from the treatment plant is a reservoir of antibiotic-resistance genes, which signify a potential health threat. Additionally, the phylogroup C was predominant, which is unusual and may represent an adaptation of this group to environmental conditions or per-haps the most frequent phylogroup entering from the influent.

Low Concentrations of Antibiotics Alter Microbial Communities and Induce High Abundances of Antibiotic-Resistant Genes in Ornamental Water

Antibiotic resistance poses a significant threat to the public health domain. A favorable platform for generating and disseminating antibiotic-resistant genes (ARGs) and antibiotic-resistant bacteria (ARB) is provided by landscaped fish ponds created by urbanization. This research delved into the effects exerted by different concentrations of specific antibiotics, namely tetracycline and ciprofloxacin, on the microbial community composition present in water samples obtained from a landscape pond. Additionally, we analyzed the abundance of ARGs and the class 1 integron-integrase gene (intI1), and identified potential hosts of ARGs. The results indicated that the consistent administration of antibiotics significantly influenced the microbial community structure, resulting in variations within both bacterial communities and functionalities. Furthermore, the absolute quantities of ARGs, including tetA, tetC, qnrA, and qnrS, as well as the integrase gene intI1, exhibited augmentation in response to varying types and concentrations of antibiotics. Notably, the regular input of low concentrations of antibiotics produced higher levels of abundance of ARGs than the regular input of higher concentrations of antibiotics. The use of different types of antibiotics led to diverse host bacteria structures.

Antibiotic Resistance Mediated by Escherichia coli in Kuwait Marine Environment as Revealed through Genomic Analysis.

Antibiotic-resistance gene elements (ARGEs) such as antibiotic-resistance genes (ARGs), integrons, and plasmids are key to the spread of antimicrobial resistance (AMR) in marine environments. Kuwait's marine area is vulnerable to sewage contaminants introduced by numerous storm outlets and indiscriminate waste disposal near recreational beaches. Therefore, it has become a significant public health issue and warrants immediate investigation. Coliforms, especially Gram-negative Escherichia coli, have been regarded as significant indicators of recent fecal pollution and carriers of ARGEs. In this study, we applied a genome-based approach to identify ARGs' prevalence in E. coli isolated from mollusks and coastal water samples collected in a previous study. In addition, we investigated the plasmids and intl1 (class 1 integron) genes coupled with the ARGs, mediating their spread within the Kuwait marine area. Whole-genome sequencing (WGS) identified genes resistant to the drug classes of beta-lactams (blaCMY-150, blaCMY-42, blaCTX-M-15, blaDHA-1, blaMIR-1, blaOKP-B-15, blaOXA-1, blaOXA-48, blaTEM-1B, blaTEM-35), trimethoprim (dfrA14, dfrA15, dfrA16, dfrA1, dfrA5, dfrA7), fluroquinolone (oqxA, oqxB, qnrB38, qnrB4, qnrS1), aminoglycoside (aadA2, ant(3'')-Ia, aph(3'')-Ib, aph(3')-Ia, aph(6)-Id), fosfomycin (fosA7, fosA_6, fosA, fosB1), sulfonamide (sul1, sul2, sul3), tetracycline (tet-A, tet-B), and macrolide (mph-A). The MFS-type drug efflux gene mdf-A is also quite common in E. coli isolates (80%). The plasmid ColRNAI was also found to be prevalent in E. coli. The integron gene intI1 and gene cassettes (GC) were reported to be in 36% and 33%, respectively, of total E. coli isolates. A positive and significant (p < 0.001) correlation was observed between phenotypic AMR-intl1 (r = 0.311) and phenotypic AMR-GC (r = 0.188). These findings are useful for the surveillance of horizontal gene transfer of AMR in the marine environments of Kuwait.

Antimicrobial resistance- and pathogen patterns in the fecal microbiota of sows and their offspring in German commercial pig farms.

Reducing antibiotic use is one of the biggest challenges in pig farming, as antibiotics have been used for years to control typical problems such as newborn or post-weaning diarrhea. The pressure a one health approach has created on animal production regarding antimicrobial resistance is an opportunity to find other strategies against enterobacterial pathogens in suckling and weaned piglets. A farm-specific approach could have a good success due to the individual farm structures in Germany and other countries. In this study, non-metric multidimensional scaling, hierarchical clustering, and latent class analysis were used to determine the impact of antibiotic use on antibiotic resistance patterns and pathogen prevalence in 20 German pig farms. This may help to develop individualized health strategies. 802 fresh fecal samples were collected from sows and piglets from 20 piglet production and rearing farms at different production times (sows antepartum and postpartum, suckling piglets, weaned piglets). In addition, the use of antibiotics was recorded. DNA extracts were subjected to quantitative real-time qPCR with primers specific for antibiotic resistance genes (int1, sul1-3, dfrA1, mcr-1, blaCTX-M), and virulence factors of relevant bacteria (C. difficile, C. perfringens, Salmonella, Escherichia/Shigella/Hafnia, E. coli). Linear and logistic regression models were used to analyze the relationship between different antibiotics and the major genes contributing to the clustering of observations for the different animal groups. Clustering revealed different farm clusters for sows, suckling piglets, and weaned piglets, with the most remarkable diversity in antibiotic use among weaned piglets. Amoxicillin, lincomycin, and enrofloxacin were identified as the most probable cause of increased odds of the presence of relevant antibiotic resistance genes (mcr1, dfrA1, blaCTX-M). Still, direct effects of a specific antibiotic on its associated resistance gene were rare. Enrofloxacin and florfenicol favored the occurrence of C. difficile in sows. The E. coli fimbriae genes were less affected by antibiotic use in sows and piglets, but the F4 fimbriae gene could be associated with the integrase 1 gene in piglets. The results confirm that multidrug-resistant enterobacteria are widespread in German pig farms and give awareness of the impact of current antibiotic use while searching for alternative health strategies.

Comparison of concentration and extraction workflows for qPCR quantification of intI1 and vanA in untreated wastewater

Quantitative polymerase chain reaction (qPCR) measurement of antibiotic resistance genes (ARGs) in untreated municipal wastewater may prove useful in combating the antimicrobial resistance crisis. However, harmonizing and optimizing qPCR-based workflows is essential to facilitate comparisons across studies, and includes achieving highly-effective ARG capture through efficient concentration and extraction procedures. In the current study, combinations of sample volume, membrane types and DNA extraction kits within filtration and centrifugation-based workflows were used to quantify 16S ribosomal RNA (16S rRNA), class 1 integron-integrase gene (intI1) and an ARG encoding resistance to vancomycin (vanA) in untreated wastewater sampled from three wastewater treatment plants (WWTPs). Highly abundant 16S rRNA and intI1 were detected in 100 % of samples from all three WWTPs using both 2 and 20 mL sample volumes, while lower prevalence vanA was only detected when using the 20 mL volume. When filtering 2 mL of wastewater, workflows with 0.20-/0.40-μm polycarbonate (PC) membranes generally yielded greater concentrations of the three targets than workflows with 0.22-/0.45-μm mixed cellulose ester (MCE) membranes. The improved performance was diminished when the sample volume was increased to 20 mL. Consistently greater concentrations of 16S rRNA, intI1 and vanA were yielded by filtration-based workflows using PC membranes combined with a DNeasy PowerWater (DPW) Kit, regardless of the sample volume used, and centrifugation-based workflows with DNeasy Blood & Tissue Kit for 2-mL wastewater extractions. Within the filtration-based workflows, the DPW kit yielded more detection and quantifiable results for less abundant vanA than the DNeasy PowerSoil Pro Kit and FastDNA™ SPIN Kit for Soil. These findings indicate that the performance of qPCR-based workflows for surveillance of ARGs in wastewater varies across targets, sample volumes, concentration methods and extraction kits. Workflows must be carefully considered and validated considering the target ARGs to be monitored.

Mobile genetic elements profiling, gene flow, and antimicrobial susceptibility profiles, among Pseudomonas aeruginosa isolates, isolated from Al Muthanna hospitals' wound and burn units in Iraq

The prevalence assessment of integrons among multidrug-resistant strains of Pseudomonas aeruginosa receives much-needed attention from this study, as we achieved our desired objective by conducting a thorough analysis on one hundred swabs obtained from burn and clinical cases at the hospitals present in Al Muthanna governorate during November of the year 2021 through to March of the year 2022. By implementing various methodologies encompassing the scrutiny of growth traits and cellular composition as well as executing biochemical assays, a total of 55 isolates were determined to exhibit the existence of P. aeruginosa.When cultured in Hifluoro agar media, Pseudomonas aeruginosa produced diverse hues; particularly noticeable was its blue-green colour. It was discovered through investigation that there were no intI2 and inti3 genes present in those isolated. Findings from this research disclosed that about one-fifth, or precisely twelve out of fifty-five P. aeruginosa strains screened, had an actively expressed Integrase I gene. The association between elevated rates of resistance to multiple antimicrobial agents and the existence of integrons is worth mentioning. Furthermore, the assemblage of isolates that were efficacious in the presence of integrons demonstrated an augmented resistance towards several frequently employed antibiotics like rifampicin and ceftazidime. In conclusion, it can be stated with confidence that a considerable occurrence of integrons can be observed in Pseudomonas aeruginosa strains that display resistance to numerous pharmaceutical agents. Additionally, the discovery of the intI1 gene in a considerable proportion of isolates underscores the effectiveness of integrons in conferring resistance to a variety of antimicrobial agents. These revelations supplement our insight into antibiotic-resistant mechanisms while also underscoring the necessity for viable strategies aimed at halting and preventing bacterial drug resistance.

Assessment of the stability of antimicrobials and resistance genes during short- and long-term storage condition: accounting for uncertainties in bioanalytical workflows

Unravelling complexities in antimicrobial agent–microbe interactions in the context of antimicrobial resistance (AMR) requires robust analytical workflows accounting for all uncertainties. Temporal storage of wastewater samples under refrigerated or frozen conditions prior to chemical and biological analysis is widely used to facilitate laboratory routine but may affect stability of analytes over time. Yet, little knowledge exists regarding stability of biological and chemical determinants in environmental samples, which hampers validity of research outputs. This study examines, for the first time, the stability of 32 antimicrobials (AAs) including commonly used classes of antibiotics and their representative metabolites and variation of 5 antibiotic resistance genes (ARGs) (ermB, sul1, tetW, blaCTX-M, qnrS), as well as intI1 and 16S rRNA genes in a reference wastewater sample stored under freezing condition for up to 1 year. Ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) and quantitative PCR (qPCR) techniques were adopted to measure concentration of AAs and ARGs, respectively. Results suggested that parent compounds are less affected by freezing storage compared to the metabolites. β-Lactams, clindamycin, and N-desmethyl clindamycin are the most-affected compounds which were poorly recovered (34–67%) from the starting concentration. By contrast, sulfonamides, macrolides, quinolones, and azoles are generally stable under freezing condition. No consistent differences were observed in gene copies between fresh and frozen samples, and ermB and tetW showed the highest variabilities at 30% under freezing condition. Overall, this study adds to the current knowledge on environmental AMR monitoring and emphasises the need for standardised protocols for AMR monitoring in the wastewater samples.Graphical

Characterization of Multiple Resistance Genes of Escherichia coli Strains Isolated from Food and Clinical Samples.

Multi-drug resistance (MDR), which is formed by the development of antimicrobial resistance, which is one of the defense mechanisms in bacteria, has pushed human beings to constantly seek new antimicrobial agents in the fight against these microorganisms. Speed and precision are very important in identifying resistance in strains with MDR that reach humans in an open system through the consumption of contaminated food or water. The main aim of this study was the molecular characterization of ESBL gene variants (blaTEM, blaOXA, blaSHV, and blaCTX-M), integron genes (int1, int2, and int3), and sulphonamide (sul1, sul2, and sul3) resistance genes by PCR from E. coli isolates originated from food and clinical samples. A total of 17 sets of primers were used for phylogenetic identification, molecular detection, and resistance and integron gene characterization of phenotypically identified E. coli isolates. The 45 red meat samples were collected from local markets, which are located in four (Adıyaman, Gaziantep, Kahramanmaraş, and Hatay) different provinces, clinical isolates were obtained from urine samples of patients with UTI from Şanlıurfa Mehmet Akif İnan Training and Research Hospital. Three gene groups were screened with 3 multiplex PCR applications in 63 food and 33 clinical isolates found to be E. coli by molecular identification. In terms of the 3 gene groups screened in food samples, the highest rates were found in the blaSHV gene at 44.44%, the sul1 gene at 69.84% and the int2 gene at 73.02%; in clinical samples, it is listed as blaCTX-M gene at 15.15%, sul2 gene at 81.82% and int1 gene at 54.55%. In terms of 3 gene groups scanned, the presence of 3 or more genes, including at least one gene from each gene group, was detected in 31 isolates from food samples and 2 isolates from clinical samples. Overall, it can be said that the high frequency of MDR genes isolated from E. coli-contaminated red meat samples and clinical samples gives a clue about the overuse of antibiotics in Türkiye.