Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes. - Related Documents




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192401.0000Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes. The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 × 10(5)-1.2 × 10(9) CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of ≥0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae, and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (β-lactams), aadA, aac (6')-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, β-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the chloramphenicol acetyltransferase gene catB10, and the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.202336939224
192610.9999Whole genome sequencing revealed high occurrence of antimicrobial resistance genes in bacteria isolated from poultry manure. BACKGROUND: Global demand for food has driven expansion and intensification of livestock production, particularly in developing nations where antibiotic use is often routine. Waste from poultry production, including manure, is commonly utilized as fertilizers in agroecosystems, risking environmental contamination with potentially zoonotic bacteria and antimicrobial resistance genes (ARGs). METHODS: Here, 33 bacterial isolates were recovered from broiler (n = 17) and layer (n = 16) chicken manure by aerobic culture using Luria Bertani agar. Antimicrobial susceptibility testing (AST) was performed using disc diffusion method. MALDI-ToF and 16S rRNA sequencing were used to identify and compare a subset of antibiotic-resistant isolates (n = 13). Comparison of whole genome sequence assemblies and phenotypic assays were used to assess capacity for biofilm formation, heavy metal tolerance and virulence. RESULTS: AST by disc diffusion revealed all isolates were resistant to a minimum of three antibiotics, with resistance to ampicillin, co-trimoxazole, fluoroquinolones, tetracyclines, streptomycin, rifampicin and/or chloramphenicol detected. Stutzerimonas sp. and Acinetobacter sp. were the common genera observed in this study. Genome sequencing of each selected isolate revealed carriage of multiple ARGs capable of conferring resistance to many antimicrobials commonly employed in poultry production and human medicine, including tetracyclines, quinolones, macrolides, sulfonamide and cephalosporins. CONCLUSIONS: The high occurrence of ARGs in studied bacterial isolates confirms that poultry manure could act as a source of genetic material that could be transferred to commensal microbiota and opportunistic pathogens of humans. Understanding the complex resistome interplay between humans, animals, and the environment requires a One Health approach, with implications for agricultural settings and public health.202539880102
273420.9998High Frequency of Antibiotic Resistance Genes (ARGs) in the Lerma River Basin, Mexico. The spread of beta-lactamase-producing bacteria is of great concern and the environment has been found to be a main source of contamination. Herein, it was proposed to determine the frequency of antimicrobial-resistant-Gram-negative bacteria throughout the Lerma River basin using phenotypic and molecular methods. Resistant bacteria were isolated with chromogenic media and antimicrobial susceptibility tests were used to characterize their resistance. ARGs for beta-lactams, aminoglycosides, and quinolones were detected by PCR. Species were identified by Sanger sequencing the 16S rRNA gene and the representative genomes of MDR strains were sequenced by NGS. A high variation in the number of isolates was observed in the 20 sampled sites, while observing a low diversity among the resistant bacteria. Of the 12 identified bacterial groups, C. freundii, E. coli, and S. marcescens were more predominant. A high frequency of resistance to beta-lactams, quinolones, and aminoglycosides was evidenced, where the bla(CTX,)qnrB, qnrS y, and aac(6')lb-cr genes were the most prevalent. C. freundii showed the highest frequency of MDR strains. Whole genome sequencing revealed that S. marcescens and K. pneumoniae showed a high number of shared virulence and antimicrobial resistance genes, while E. coli showed the highest number of unique genes. The contamination of the Lerma River with MDR strains carrying various ARGs should raise awareness among environmental authorities to assess the risks and regulations regarding the optimal hygienic and sanitary conditions for this important river that supports economic activities in the different communities in Mexico.202236360888
192330.9998Emerging Issues on Antibiotic-Resistant Bacteria Colonizing Plastic Waste in Aquatic Ecosystems. Antibiotic-resistant bacteria (ARB) adhesion onto plastic substrates is a potential threat to environmental and human health. This current research investigates the prevalence of two relevant human pathogens, Staphylococcus spp. and Klebsiella spp., and their sophisticated equipment of antibiotic-resistant genes (ARGs), retrieved from plastic substrates submerged into an inland water body. The results of microbiological analysis on selective and chromogenic media revealed the presence of colonies with distinctive phenotypes, which were identified using biochemical and molecular methods. 16S rDNA sequencing and BLAST analysis confirmed the presence of Klebsiella spp., while in the case of Staphylococcus spp., 63.6% of strains were found to be members of Lysinibacillus spp., and the remaining 36.3% were identified as Exiguobacterium acetylicum. The Kirby-Bauer disc diffusion assay was performed to test the susceptibility of the isolates to nine commercially available antibiotics, while the genotypic resistant profile was determined for two genes of class 1 integrons and eighteen ARGs belonging to different classes of antibiotics. All isolated bacteria displayed a high prevalence of resistance against all tested antibiotics. These findings provide insights into the emerging risks linked to colonization by potential human opportunistic pathogens on plastic waste commonly found in aquatic ecosystems.202438667014
286440.9998Case study on the soil antibiotic resistome in an urban community garden. Urban agricultural soils can be an important reservoir of antibiotic resistance, and have great food safety and public health indications. This study investigated antibiotic-resistant bacteria and antibiotic resistance genes in urban agricultural soils using phenotypic and metagenomic tools. In total, 207 soil bacteria were recovered from 41 soil samples collected from an urban agricultural garden in Detroit, MI, USA. The most prevalent antibiotic resistance phenotype demonstrated by Gram-negative bacteria was resistance to ampicillin (94.2%), followed by chloramphenicol (80.0%), cefoxitin (79.5%), gentamicin (78.4%) and ceftriaxone (71.1%). All Gram-positive bacteria were resistant to gentamicin, kanamycin and penicillin. Genes encoding resistance to quinolones, β-lactams and tetracyclines were the most prevalent and abundant in the soil. qepA and tetA, both encoding efflux pumps, predominated in the quinolone and tetracycline resistance genes tested, respectively. Positive correlation (P<0.05) was identified among groups of antibiotic resistance genes, and between antibiotic resistance genes and metal resistance genes. The data demonstrated a diverse population of antibiotic resistance in urban agricultural soils. Phenotypic determination together with soil metagenomics proved to be a valuable tool to study the nature and extent of antibiotic resistance in the environment.201829857032
192750.9998First Molecular Characterization and Antibiogram of Bacteria Isolated From Dairy Farm Wastewater in Bangladesh. This pioneering study in Bangladesh combines phenotypic and genotypic approaches to characterize antibiotic-resistant bacteria in dairy farm wastewater, addressing a critical gap in regional antimicrobial resistance (AMR) research. Dairy farming is integral to global food production, yet the wastewater generated by these operations is a significant source of environmental and public health concerns, particularly in the context of antibiotic resistance. This study aimed to isolate and identify antibiotic-resistant bacteria from dairy farm wastewater and evaluate their antibiogram profiles to inform effective management strategies. A total of 60 wastewater samples were collected and subjected to conventional bacterial characterization, followed by molecular detection via PCR and 16S rRNA gene sequencing. The study identified Pseudomonas aeruginosa (35%), Escherichia coli (30%), Bacillus subtilis (16.67%), and Acinetobacter junii (8.33%) as the predominant bacterial species. Sequencing results demonstrated high compatibility with reference sequences, confirming the identities of the isolates. Antibiogram analysis revealed significant resistance patterns: P. aeruginosa exhibited the highest resistance to penicillin (85.71%) and amoxicillin (76.19%), while demonstrating greater sensitivity to ciprofloxacin and cotrimoxazole. E. coli showed notable resistance to penicillin (88.89%), amoxicillin, and ceftriaxone, while B. subtilis and A. junii also demonstrated high levels of resistance to multiple antibiotics. Notably, a substantial proportion of the isolates exhibited multidrug resistance (MDR), with MAR indices ranging from 0.37 to 0.75. Moreover, several antibiotic resistance genes (ARGs) including penA, bla (TEM) , bla (CTX-M) , tetA, tetB, tetC, and ermB were detected across the bacterial species, with high prevalence rates in P. aeruginosa and A. junii, suggesting the potential for horizontal gene transfer and further spread of resistance. These findings underscore the critical need for a One Health approach to mitigate the risks posed by antibiotic-resistant bacteria in dairy farm wastewater, emphasizing the critical importance of responsible antibiotic use and sustainable farming practices to protect public health and environmental integrity.202540458482
192960.9998Research Note: Detection of antibiotic-resistance genes in commercial poultry and turkey flocks from Italy. Antibiotics are routinely used in commercial poultry farms for the treatment of economically important bacterial diseases. Repeated use of antibiotics, usually administered in the feed or drinking water, may also result in the selection of resistant bacteria in animal feces, able to transfer their antimicrobial-resistance genes (ARG), residing on mobile elements, to other microorganisms, including human pathogens. In this study, single and multiplex PCR protocols were performed to detect tetracycline-, lincomycin-, chloramphenicol-, aminoglycoside-, colistin-, vancomycin-, and carbapenem-resistance genes, starting from 38 litter samples collected from 6 poultry and 2 turkey Italian flocks. The ARG were confirmed for all investigated classes of antimicrobials, except for colistin (mcr-1, mcr-2, mcr-3,mcr-4 mcr-5) and carbapenem (IMP, OXA-48, NDM, KPC), while the vanB gene was only detected for vancomycin. The highest positivity was obtained for tetracycline (tet[L], tet[M], tet[K], tetA[P]] and aminoglycoside (aadA2) ARG, confirming the predominant use of these antimicrobials in the veterinary practice and their potential to enhance the resistance patterns also in humans as a consequence of environmental contamination. On the contrary, the dissemination by poultry of ARG for critically important antimicrobials seems to be of minor concern, suggesting a negligible environmental dissemination by these genes in the Italian poultry industry. Finally, the molecular screening performed in this study using a noninvasive sampling method represents a simple and rapid tool for monitoring the ARG patterns at the farm level.202133799114
273670.9998Characterization of Bacterial Communities and Their Antibiotic Resistance Profiles in Wastewaters Obtained from Pharmaceutical Facilities in Lagos and Ogun States, Nigeria. In Nigeria, pharmaceutical wastewaters are routinely disseminated in river waters; this could be associated with public health risk to humans and animals. In this study, we characterized antibiotic resistant bacteria (ARB) and their antibiotic resistance profile as well as screening for sul1 and sul2 genes in pharmaceutical wastewater effluents. Bacterial composition of the wastewater sources was isolated on non-selective media and characterized by the polymerase chain reaction (PCR) amplification of the 16S rRNA genes, with subsequent grouping using restriction fragment length polymorphism (RFLP) and sequencing. The antibiotics sensitivity profiles were investigated using the standard disk diffusion plate method and the minimum inhibitory concentrations (MICs) of selected antibiotics on the bacterial isolates. A total of 254 bacterial strains were isolated, and majority of the isolates were identified as Acinetobacter sp., Klebsiella pneumonia, Proteus mirabilis, Enterobacter sp. and Bacillus sp. A total of 218 (85.8%) of the bacterial isolates were multidrug resistant. High MICs values were observed for all antibiotics used in the study. The result showed that 31.7%, 21.7% and 43.3% of the bacterial isolates harbored sul1, sul2, and Intl1 genes, respectively. Pharmaceuticals wastewaters are potential reservoirs of ARBs which may harbor resistance genes with possible risk to public health.201829966226
273580.9998Insight into the Antibiotic Resistance of Bacteria Isolated from Popular Aquatic Products Collected in Zhejiang, China. The present study was aimed to obtain a close insight into the distribution and diversity of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) among the aquatic products collected in Zhejiang, China. A total of 136 presumptive ARB picked up from six aquatic samples were classified into 22 genera and 49 species based on the 16S rDNA sequencing. Aeromonas spp., Shewanella spp., Acinetobacter spp., Myroides spp., Pseudomonas spp., and Citrobacter spp. accounted for 80% of the ARB. Among them, 109 isolates (80.15%) exhibited resistance to at least one antibiotic. Most isolates showed resistance to not only the originally selected drug but also to one to three other tested drugs. The diversity of ARB distributed in different aquatic products was significant. Furthermore, the resistance data obtained from genotypic tests were not entirely consistent with the results of the phenotypic evaluation. The genes qnrS, tetA, floR, and cmlA were frequently detected in their corresponding phenotypic resistant isolates. In contrast, the genes sul2, aac(6')-Ib, and bla (PSE) were less frequently found in the corresponding phenotypically resistant strains. The high diversity and detection rate of ARB and ARGs in aquaculture might be a significant threat to the food chains closely related to human health.202336929890
284990.9998Antibiotic-resistant bacteria and gut microbiome communities associated with wild-caught shrimp from the United States versus imported farm-raised retail shrimp. In the United States, farm-raised shrimp accounts for ~ 80% of the market share. Farmed shrimp are cultivated as monoculture and are susceptible to infections. The aquaculture industry is dependent on the application of antibiotics for disease prevention, resulting in the selection of antibiotic-resistant bacteria. We aimed to characterize the prevalence of antibiotic-resistant bacteria and gut microbiome communities in commercially available shrimp. Thirty-one raw and cooked shrimp samples were purchased from supermarkets in Florida and Georgia (U.S.) between March-September 2019. The samples were processed for the isolation of antibiotic-resistant bacteria, and isolates were characterized using an array of molecular and antibiotic susceptibility tests. Aerobic plate counts of the cooked samples (n = 13) varied from < 25 to 6.2 log CFU/g. Isolates obtained (n = 110) were spread across 18 genera, comprised of coliforms and opportunistic pathogens. Interestingly, isolates from cooked shrimp showed higher resistance towards chloramphenicol (18.6%) and tetracycline (20%), while those from raw shrimp exhibited low levels of resistance towards nalidixic acid (10%) and tetracycline (8.2%). Compared to wild-caught shrimp, the imported farm-raised shrimp harbored distinct gut microbiota communities and a higher prevalence of antibiotic-resistance genes in their gut. The presence of antibiotic-resistant strains in cooked shrimps calls for change in processing for their mitigation.202133558614
1941100.9998The association between antimicrobials and the antimicrobial-resistant phenotypes and resistance genes of Escherichia coli isolated from hospital wastewaters and adjacent surface waters in Sri Lanka. The presence of antimicrobials, antimicrobial-resistant bacteria (ARB), and the associated antimicrobial resistance genes (ARGs) in the environment is a global health concern. In this study, the concentrations of 25 antimicrobials, the resistance of Escherichia coli (E. coli) strains in response to the selection pressure imposed by 15 antimicrobials, and enrichment of 20 ARGs in E. coli isolated from hospital wastewaters and surface waters were investigated from 2016 to 2018. In hospital wastewaters, clarithromycin was detected at the highest concentration followed by sulfamethoxazole and sulfapyridine. Approximately 80% of the E. coli isolates were resistant, while 14% of the isolates exhibited intermediate resistance against the tested antimicrobial agents. Approximately 61% of the examined isolates were categorized as multidrug-resistant bacteria. The overall abundance of phenotypes that were resistant toward drugs was in the following order: β-lactams, tetracycline, quinolones, sulfamethoxazole/trimethoprim, aminoglycosides, and chloramphenicol. The data showed that the E. coli isolates frequently harbored bla(TEM), bla(CTX-M), tetA, qnrS, and sul2. These results indicated that personal care products were significantly associated with the presence of several resistant phenotypes and resistance genes, implying their role in co-association with multidrug resistance. Statistical analysis also indicated a disparity specific to the site, treatment, and year in the data describing the prevalence of ARB and ARGs and their release into downstream waters. This study provides novel insights into the abundance of antimicrobial, ARB and ARGs in Sri Lanka, and could further offer invaluable information that can be integrated into global antimicrobial resistance databases.202133894511
2853110.9998Antibiotic resistance and virulence genes in coliform water isolates. Widespread fecal pollution of surface water may present a major health risk and a significant pathway for dissemination of antibiotic resistance bacteria. The River Rhine is one of the longest and most important rivers in Europe and an important raw water source for drinking water production. A total of 100 coliform isolates obtained from River Rhine (Germany) were examined for their susceptibility to seven antimicrobial agents. Resistances against amoxicillin, trimethoprim/sulfamethoxazole and tetracycline were detected in 48%, 11% and 9% of isolates respectively. The antibiotic resistance could be traced back to the resistance genes bla(TEM), bla(SHV), ampC, sul1, sul2, dfrA1, tet(A) and tet(B). Whereby, the ampC gene represents a special case, because its presence is not inevitably linked to a phenotypic antibiotic resistance. Multiple antibiotics resistance was often accompanied by the occurrence of class 1 or 2 integrons. E. coli isolates belonging to phylogenetic groups A and B1 (commensal) were more predominant (57%) compared to B2 and D groups (43%) which are known to carry virulent genes. Additionally, six E. coli virulence genes were also detected. However, the prevalence of virulence genes in the E. coli isolates was low (not exceeding 4.3% per gene) and no diarrheagenic E. coli pathotypes were detected. This study demonstrates that surface water is an important reservoir of ARGs for a number of antibiotic classes such as sulfonamide, trimethoprim, beta-lactam-antibiotics and tetracycline. The occurrence of antibiotic resistance in coliform bacteria isolated from River Rhine provides evidence for the need to develop management strategies to limit the spread of antibiotic resistant bacteria in aquatic environment.201627497615
2861120.9998Antibiotic Resistance Profiles and Genomic Analysis of Endophytic Bacteria Isolates from Wild Edible Fungi in Yunnan. The use of antibiotics has led to the emergence of antibiotic resistance, posing significant challenges in the prevention, control, and treatment of microbial diseases, while threatening public health, the environment, and food safety. In this study, the antibiotic resistance phenotypes and genotypes of 56 endophytic bacteria isolates from three species of wild edible fungi in Yunnan were analyzed using the Kirby-Bauer disk diffusion method and PCR amplification. The results revealed that all isolates were sensitive to ofloxacin, but resistance was observed against 17 other antibiotics. Specifically, 55, 53, and 51 isolates exhibited resistance to amoxicillin, penicillin, and vancomycin, respectively. Antibiotic resistance gene (ARG) detection indicated that the sulfonamide sul1 gene had the highest detection rate (53.57%). Excluding the ARG that was not detected, the lowest detection rates were the sulfonamide sul2 and sul3 genes, both at 1.79%. Among six tetracycline resistance genes, only tetK and tetM were detected. For β-lactam antibiotics, blaTEM, blaVIM, and blaSHV genes were present, while blaOXA was absent. In aminoglycoside resistance genes, aadB was not detected, while detection rates for aac(3')-IIa, acrB, and aadA1 were 3.57%, 1.79%, and 37.5%, respectively. The chloramphenicol Cat gene was detected at a rate of 14.29%, whereas floR was absent. For polypeptide resistance, VanC was detected at 3.57%, with EmgrB not detected. All three quinolone genes were detected, with detection rates of 8.92% for GyrA, 39.29% for GyrB, and 37.5% for ParC. Through phylogenetic analysis, 12 isolates that are closely related to ten common foodborne pathogenic bacteria were further selected for whole-genome sequencing and assembly. Gene annotations revealed that each isolate contained more than 15 ARGs and over 30 virulence factors. Notably, the detection rate of antibiotic resistance phenotypes was higher than that of genotypes, highlighting the importance of studying phenotypic antibiotic resistance that lacks identifiable ARGs. This study enriches the research on endophytes in wild edible fungi and provides new data for microbial ecology and antibiotic resistance research. It also offers critical insights for monitoring microbial antibiotic resistance in wild edible fungi and potentially other food sources, contributing to more effective strategies for ecological protection, sustainable agricultural development, and public health security.202540005728
5545130.9998Healthy broilers disseminate antibiotic resistance in response to tetracycline input in feed concentrates. Wide varieties of antibiotics are used in poultry farms to improve the growth and also to control the infection in broiler chicken. To identify the seriousness of the same in the poultry sector, current study has been designed to analyze the presence of tetracycline in poultry feed and also the tetracycline resistance among the bacteria released through the excreta of poultry. In the study, 27 bacteria belonging to the Escherichiacoli and Klebsiellapneumoniae. were isolated from the faecal samples collected from five different farms. Antibiotic susceptibility analysis showed 77% of E. coli and 100% of the K. pneumoniae. to be resistant to tetracycline. Further, molecular screening for tetA and tetB genes showed 85.18% of isolates to have tetA and 22.22% with tetB. The presence of tetracycline in collected feed samples was also analysed quantitatively by Liquid chromatography-mass spectrometry (LC-MS). Here, three out of five feed samples were found to be positive for tetracycline. The study showed a direct correlation between the antibiotic supplemented feed and the emergence of antimicrobial resistance among the intestinal microflora. The results of the study indicate the need for strict control over antibiotic use in animal feed to limit the rapid evolution and spread of antimicrobial resistance.202033039593
1967140.9998Identification of molecular determinants of antibiotic resistance in some fish farms of Ghana. Antimicrobial resistance is a global health challenge caused by the ability of microorganisms including bacteria, fungi, protozoans and viruses to survive the effects of drugs that hitherto were effective against them. This study sought to investigate the presence of antibiotic-resistant bacteria and their corresponding molecular determinants in fish farms of the Central and Western Regions of Ghana. Management practices and antibiotic use at the fish farms were obtained through the administration of a questionnaire. Coliform and Gram-positive bacterial loads of catfish (Clarias gariepinus), tilapia (Oreochromis niloticus) intestinal microbiota, and pond water samples recovered on MacConkey Agar and Mannitol Salt Agar were determined. Bacterial isolates were identified using various biochemical assays. Antibiotic resistance profiles and possible responsible genes of bacterial isolates were determined using the disc diffusion and Polymerase Chain Reaction (PCR) methods respectively. The study revealed that none of the fish farm managers admitted using antibiotics for prevention and treatment of diseases and no major disease outbreak had ever been recorded. Bacterial loads of pond water exceeded the acceptable level of ≤100 E. coli and <10 coliforms per mL for wastewater recommended for use in fish farming. In all, 145 bacterial isolates comprising 99 Gram negative and 46 Gram-positive bacteria were stored and identified. Most isolates were resistant to at least an antibiotic. Both Gram-negative and Gram-positive bacteria were highly resistant to beta-lactam antibiotics with a corresponding high percentage detection of the bla (TEM) gene compared to other classes of antibiotics. This study has revealed the presence of various molecular determinants of antibiotic resistance including bla (TEM), cmIA, qnrS, tetB and bla (CTX-M), in multidrug-resistant bacteria at some fish farms in Ghana. There is the need to increase awareness about risks associated with the misuse and overuse of antibiotics by humans and the potential risk of spread of multi-drug resistant-bacteria in the environment.202236097488
3128150.9998Diversity and antibiotic susceptibility pattern of cultivable anaerobic bacteria from soil and sewage samples of India. Soil and sewage act as a reservoir of animal pathogens and their dissemination to animals profoundly affects the safety of our food supply. Moreover, acquisition and further spread of antibiotic resistance determinants among pathogenic bacterial populations is the most relevant problem for the treatment of infectious diseases. Bacterial strains from soil and sewage are a potential reservoir for antimicrobial resistance genes. Accurate species determination for anaerobes from environmental samples has become increasingly important with the re-emergence of anaerobic bacteremia and prevalence of multiple-drug-resistant microorganisms. Soil samples were collected from various locations of planar India and the diversity of anaerobic bacteria was determined by 16S rRNA gene sequencing. Viable counts of anaerobic bacteria on anaerobic agar and SPS agar ranged from 1.0 × 10(2)cfu/g to 8.8 × 10(7)cfu/g and nil to 3.9 × 10(6)cfu/g, respectively. Among clostrdia, Clostridium bifermentans (35.9%) was the most dominant species followed by Clostridium perfringens (25.8%). Sequencing and phylogenetic analysis of C. perfringens beta2 toxin gene (cpb2) fragment indicated specific phylogenetic affiliation with cluster Ia for 5 out of 6 strains. Antibiotic susceptibility for 30 antibiotics was tested for 74 isolates, revealing resistance for as high as 16-25 antibiotics for 35% of the strains tested. Understanding the diversity of the anaerobic bacteria from soil and sewage with respect to animal health and spread of zoonotic pathogen infections is crucial for improvements in animal and human health.201120965279
1933160.9998Antibiotic Resistance Genes Occurrence in Conventional and Antibiotic-Free Poultry Farming, Italy. Antimicrobial resistance is a complex and widespread problem threatening human and animal health. In poultry farms, a wide distribution of resistant bacteria and their relative genes is described worldwide, including in Italy. In this paper, a comparison of resistance gene distribution in litter samples, recovered from four conventional and four antibiotic-free broiler flocks, was performed to highlight any influence of farming systems on the spreading and maintenance of resistance determinants. Conventional PCR tests, targeting the resistance genes related to the most used antibiotics in poultry farming, along with some critically important antibiotics for human medicine, were applied. In conventional farms, n. 10 out of n. 30 investigated genes were present in at least one sample, the most abundant fragments being the tet genes specific for tetracyclines, followed by those for aminoglycosides and chloramphenicol. All conventional samples resulted negative for colistin, carbapenems, and vancomycin resistance genes. A similar trend was observed for antibiotic-free herds, with n. 13 out of n. 30 amplified genes, while a positivity for the mcr-1 gene, specific for colistin, was observed in one antibiotic-free flock. The statistical analysis revealed a significant difference for the tetM gene, which was found more frequently in the antibiotic-free category. The analysis carried out in this study allowed us to obtain new data about the distribution of resistance patterns in the poultry industry in relation to farming types. The PCR test is a quick and non-expensive laboratory tool for the environmental monitoring of resistance determinants identifying potential indicators of AMR dissemination.202236139170
2843170.9998High Throughput Screening of Antimicrobial Resistance Genes in Gram-Negative Seafood Bacteria. From a global view of antimicrobial resistance over different sectors, seafood and the marine environment are often considered as potential reservoirs of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and sparse results on this sector. This study aims to provide new data and insights regarding the content of resistance markers in various seafood samples and sources, and therefore the potential exposure to humans in a global One Health approach. An innovative high throughput qPCR screening was developed and validated in order to simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons, integrons, and insertion sequences in Gram-negative bacteria. Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) show the occurrence of sul-1, ant(3″)-Ia, aph(3')-Ia, strA, strB, dfrA1, qnrA, and bla(CTX-M-9) genes in Pseudomonas spp., Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates and the presence of MGEs in all bacterial species investigated. We found that the occurrence of MGE may be associated with the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE were detected in half of the seafood isolates investigated, association with ARG was only identified for twelve isolates. The results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria (ARB) and ARG decreases with increasing distance from potential sources of fecal contamination. This unique and original high throughput micro-array designed for the screening of ARG and MGE in Gram-negative bacteria could be easily implementable for monitoring antimicrobial resistance gene markers in diverse contexts.202235744743
2865180.9998Antibiotic resistance in soil and water environments. Seven locations were screened for antibiotic-resistant bacteria using a modified agar dilution technique. Isolates resistant to high levels of antibiotics were screened for r plasmids. Low-level resistance (25 micro g x ml(-1)) was widespread for ampicillin, penicillin, tetracycline, vancomycin and streptomycin but not for kanamycin. Resistant populations dropped sharply at high antibiotic levels, suggesting that intrinsic non-emergent mechanisms were responsible for the multiple drug resistance exhibited at low doses. Dairy farm manure contained significantly (P < 0.01) more (%) resistant bacteria than the other sites. Bacteria isolated from a dairy water canal, a lake by a hospital and a residential garden (fertilized by farm manure) displayed resistance frequencies of 77, 75 and 70%, respectively. Incidence of tetracycline resistance was most prevalent at 47-89% of total bacteria. Out of 200 representative isolates analyzed, Pseudomonas, Enterococcus-like bacteria, Enterobacter and Burkholderia species constituted the dominant reservoirs of resistance at high drug levels (50-170 micro g x ml(-1)). Plasmids were detected in only 29% (58) of these bacteria with tetracycline resistance accounting for 65% of the plasmid pool. Overall, resistance trends correlated to the abundance and type of bacterial species present in the habitat. Environmental reservoirs of resistance include opportunistic pathogens and constitute some public health concern.200212396530
1928190.9998Targeted Antimicrobial Resistance Gene Screening from Metagenomic DNA of Raw Milk Samples Identifies the Presence of Multiple Genes Including the mcr9. The current study has investigated the prevalence of antimicrobial resistance (AMR) genes in cow and goat raw milk samples. The misuse of antibiotics in the livestock sector has already been reported to be a major factor contributing to AMR risk. For the study, milk samples were collected from five different farms, and metagenomic DNA was extracted. Then, PCR amplification was carried out using primers specific to 15 different AMR genes. From the results obtained, the prevalence of β-lactam resistance genes, particularly blaTEM (24%), along with other genes like blaZ (12%) and blaSHV (8%), were observed in addition to the transmissible mcr9 gene (12%) conferring resistance to colistin. These findings underscore the urgent need for monitoring AMR genes and regulating antibiotic use in dairy farming to safeguard public health, as it poses a potential risk with the consumption of unpasteurized milk.202540488653