Multiple Antibiotic Resistant Bacteria Research Articles

Investigation and detection of multiple antibiotic-resistant pathogenic bacteria in municipal wastewater of Dhaka city

BackgroundWater pollution in densely populated urban areas, mainly from municipal wastewater, poses a significant threat. Pathogenic bacteria, such as Vibrio spp. and fecal coliform, endanger public health and the environment. Additionally, antibiotic-resistant bacteria in wastewater complicate treatment and heighten public health concerns.MethodsThe study sampled municipal wastewater from ten Dhaka neighborhoods, selecting treatment plants, sewage outlets, and various collection points using meticulous techniques for representative samples. Bacteriological and biochemical analyses were conducted using standardized methods. Antimicrobial susceptibility testing (AST) was performed with the disk diffusion method against 13 widely used antibiotics.ResultsAll sampled areas exhibited positive results for Vibrio spp., fecal coliform, E. coli, and Salmonella spp. Varying bacterial concentrations were observed, with the highest concentration of TVC, total vibrio spp., and total fecal coliform, total E. coli count, and total Salmonella spp. were found in Uttara (1.9 × 104 CFU/ml), Bangshal (1.8 × 102 CFU/ml), and Lalbag (2.1 × 103 CFU/ml), Mirpur (3.70 × 102 CFU/ml), and Lalbag (6 × 102 CFU/ml) respectively. AST results revealed significant resistance among all bacterial species to various antibiotics. Specifically, Vibrio spp. showed 100% resistance to cefuroxime, fecal coliform exhibited 90% resistance to cephradine, E. coli demonstrated 60% resistance to cephradine, and Salmonella spp. displayed 90% resistance to ampicillin.ConclusionThe study highlights the existence of multiple antibiotic-resistant bacteria in Dhaka's wastewater. Addressing antibiotic resistance is essential to manage the risks of multiple antibiotic-resistant infections and maintain antibiotic effectiveness. These implications are critical for various stakeholders, including public health officials, policymakers, environmentalists, and urban planners.

Multiple antibiotic resistance and herbicide catabolic profiles of bacteria isolated from Lake Villarrica surface sediments (Chile)

Antibiotics and herbicides are contaminants of emerging concern in aquatic environments. Lake Villarrica is a relevant freshwater body in Chile and was recently designated a ‘saturated nutrient zone’. Here, we investigated the occurrence of multiple antibiotic resistance (MAR) and herbicide catabolic profiles among bacteria present in the surface sediments of Lake Villarrica. The occurrence of antibiotic-resistant genes (ARGs; blaTEM, catA and tetM) and herbicide-catabolic genes (HCGs; phnJ and atzA) was investigated by qPCR. Subsequently, the presence of culturable bacteria with multiple resistance to amoxicillin (AMX), chloramphenicol (CHL) and oxytetracycline (OXT) was studied. Forty-six culturable MAR (AMX + CHL + OXT) strains were isolated and characterized with respect to their resistance to 11 antibiotics by using a disc diffusion assay and testing their ability to use herbicides as a nutrient source. qPCR analyses revealed that ARGs and HCGs were present in all sediment samples (101 to 103 gene copies g−1), with significant (P ≤ 0.05) higher values in sites near Villarrica city and cattle pastures. The plate method was used to recover MAR isolates from sediment (103–106 CFU g−1), and most of the 46 isolates also showed resistance to oxacillin (100%), cefotaxime (83%), erythromycin (96%) and vancomycin (93%). Additionally, 54 and 57% of the MAR isolates were able to grow on agar supplemented (50 mg L−1) with atrazine and glyphosate as nutrient sources, respectively. Most of the MAR isolates were taxonomically close to Pseudomonas (76.1%) and Pantoea (17.4%), particularly those isolated from urbanized sites (Pucón city). This study shows the presence of MAR bacteria with herbicide catabolic activity in sediments, which is valuable for conservation strategies and risk assessments of Lake Villarrica. However, major integrative studies on sediments as reservoirs or on the fate of MAR strains and traces of antibiotics and herbicides as a result of anthropic pressure are still needed.

Understanding the spread of antibiotic resistance in vegetables cultivated with sewage sludge: implications for food safety and human health

The conventional practice of using sewage treatment plant (STP) derived sludge as a fertilizer poses significant negative impacts on agroecosystems. Since sludge has diverse contaminants, including heavy metals (HMs), antibiotics (ABs) and antibiotic resistance genes (ARGs), its application in the agricultural fields contaminates the food and hence causes human health risks via the food chain. The transfer of ABs and ARGs from sludge to soil and then to plants can influence the development of antibiotic resistance (AR) in plant endophytes, and leads to variations in their characteristics. In a pot experiment, vegetable carrot (Daucus carota) and spinach (Spinacia oleracea) were amended with sludge samples from three sewage treatment plants (STPs) with varying treatment capacities and both above and below-ground parts of the plants were analysed for the presence of specific ABs (amoxicillin, azithromycin, chloramphenicol, ciprofloxacin, tetracycline), ARGs (blaCTX-M, blaGES, blaNDM, ermF, qnrS, Sul1), and mobile genetic elements (MGEs) (intl1, IS26). Among the characterized culturable endophytic bacteria (EB), 22 exhibited resistance to various antibiotics (highest against ampicillin, ciprofloxacin, chloramphenicol) and heavy metals (highest against lead, nickel, and chromium). Most importantly, seven multiple antibiotic-resistant endophytic bacteria (MAREB) exhibited resistance to all tested heavy metals (HMs). Additionally, all MAREB tested positive for biofilm production, and a notable proportion (72.72%) of these endophytes displayed mobility, with strong auto-aggregation ranging from 16.67 to 92.61%. The biofilm formation dynamics among these MAREB exhibited a Gaussian distribution pattern, increasing with higher antibiotic concentrations. Notably, five MAREB demonstrated survival at clarithromycin concentrations up to 150 µg ml−1. The study revealed the presence of ABs (µg kg−1) and ARGs (copies kg−1) in all parts of both vegetables, ranging from 2.87 to 314.88 and 1 × 105 to 3.2 × 1010, respectively. MAREB displayed various advantageous features to support plant growth under different stress conditions. Moreover, 51.09% of the identified EBs were reported as both plant and human-associated pathogens, and 9.09% were solely human pathogens. Transfer factor (TF), translocation factor (TLF), and bioconcentration factor (BCF) values were correlated with higher ABs and ARGs abundance in the root and shoot compartments of both vegetables. The risk assessment for ABs and ARGs highlighted children are particularly vulnerable to prolonged adverse health risks from consuming these vegetables. Therefore, this research is imperative for understanding the co-selection mechanisms, the need for improvement of the existing treatment systems in contaminants removal, and the evaluation of the presence of ABs and ARGs in sludge before its application in agricultural fields.

Care practices of contact lens solutions and microbial contamination among wearers in Ghana

PurposeThis study sought to assess contact lens solutions care practices, and their microbial contamination among contact lens wearers in Ghana and to profile their antibiotic susceptibility pattern. MethodsThe study employed a biphasic approach which involved a cross-sectional design that investigated participants’ habits related to care for the solutions with a two-part questionnaire and a microbiological analysis of samples of contact lens care solutions of the participants for microbial contamination. A snowball sampling method provided access to 32 different contact lens wearers in four care facilities in Ghana. In most cases, the participants had no pre-existing familial relationship with each other or with the care facilities. ResultsOut of 32 samples of contact lens solutions, 30 were tested for microbial contamination. A total of 23 (76.67 %) samples of contact lens solution were found to be contaminated with Enterobacter sp. (34.80 %), Pseudomonas sp. (21.70 %), Bacilli sp. (21.70 %), Klebsiella sp. (17.20 %), and Escherichia coli (4.60 %). The duration of solution storage in the open bottle and nonadherence to manufacturer instructions for solution storage showed a statistically significant association with microbial contamination (p ≤ 0.05). ConclusionContact lens care solutions have been found to harbour multiple antibiotic-resistant bacteria that are potentially pathogenic to the corneal surface. The contamination is associated with some unhealthy solution-care practices among wearers.

Spatial Distribution and Seasonal Variation of Antibiotic-Resistant Bacteria in an Urban River in Northeast China

As the largest freshwater river flowing through Harbin, the Songhua River is a standby water source. It is very important to know the species and distribution of antibiotic-resistant bacteria (ARB) in the river. In this study, five antibiotics were selected to screen and identify ARB in spring and autumn. The results showed that the concentration of cefotaxime-resistant bacteria was the highest, and the maximum concentration at S6 in spring was up to 1.40 × 104 CFU/mL. In spring and autumn, bacteria resistant to three antibiotics were screened at S1 of the Songhua River, and bacteria resistant to five antibiotics were screened at S6. No multiple antibiotic-resistant bacteria (MARB) were screened in the other four sites in autumn, while MARB were screened in the other three samples except S2 in spring. In all sample areas in spring and autumn, the probability of screening MARB at S1 and S6 was the highest, reaching 100%. The identification results of 16S rDNA polymerase chain reaction (PCR) products of ARB showed that a total of 51 ARB strains from 15 bacterial genera were screened in the Songhua River, of which 20 ARB strains were from Pseudomonas. Among the 15 bacterial genera, bacteria from 8 bacterial genera have pathogenicity. The results of this study revealed the concentration, spatial distribution, and seasonal variation of culturable ARB in the Songhua River, providing data support for the remediation of antibiotic resistance gene (ARG) pollution in the river.

Molecular characterization of antibiotic-resistant bacteria associated with maggots obtained from chicken droppings

BackgroundThe use of maggots, developed from chicken droppings as alternative fish meal might serve as a reservoir and vehicle for the widespread of single and multiple antibiotic resistant bacteria (MARB). This study aimed at investigating antibiotic resistant bacteria from chicken droppings and maggots developed from them, and the associated resistance genes and mobile genetic elements in their genomes. Freshly collected poultry droppings obtained from three commercial poultry farms, in Akure, Nigeria and maggots developed from their composting were plated on Luria–Bertani (LB) agar plates that had been incorporated with antibiotics. The emerged MARB were presumptively identified by conventional cultural microbiological methods and then authenticated using 16S rRNA gene sequence analysis. The antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in the genome of MARB were detected by standard molecular tools.ResultsPotential bacterial pathogens isolated include Escherichia coli, Bacillus cereus, Salmonella enterica, Providencia stuartii, P. rettgeri, Pseudomonas aeruginosa, Staphylococcus aureus, Alcaligenes faecalis, Paelnalcaligenes hominis and Micrococcus luteus. Seventeen ARGs were widely distributed among the 19 ARB/MARB. Tetracycline resistance gene was widely distributed among the ARB/MARB with prevalence of 62%, while macrolides and beta lactam resistance gene had 17% and 13%, respectively. At least six MARB isolates showed the presence of two or more class 1 integron, while five isolates was detected to carry two or more class 2 integron among the 19 MARB.ConclusionsThe results highlight a much higher risk of ARGs spreading through maggots derived from chicken droppings to humans and environmental microbiota.

Microbiological Assessment and Antibiotic Susceptibility of Orogodo River Exposed to Abattoir Effluent in Delta State, Nigeria

The content of abattoir effluent and its receiving water body, Orogodo River in Agbor, Delta State. Total heterotrophic and coliform counts were assessed using standard microbiological techniques. The viable heterotrophic bacteria count for the rainy season ranges from 7.7x105-4.6x104cfu/ml. Total coliform counts 2.6x105 -3.4x104 MPN/ml respectively. A total of 11 bacteria were isolated, antibiotic susceptibility pattern of bacteria was determined using the disk diffusion method. Five out of the bacteria isolates were resistant to at least four antibiotics. Out of all the antibiotics used 90 isolates were susceptible to erythromycin, 5% were resistant, also, 85 were susceptible to ofloxacin with 5% resistance and 10% were intermediate. Ampicillin had the highest resistance of 40%. The Molecular identification of multiple antibiotic-resistant bacteria was carried out using a 16S rRNA gene sequence. The identified bacteria include Shigella flexneri, Enterobacter hormaechei, Proteus vulgarus, Morganella morganii, and Pseudomonas oryzae. Data were statistically analyzed to determine the level of significance. The microbial population of bacteria isolated was found to be higher in the abattoir wastewater sample 7.7x105 and the point of entry sites 4.0x105 when compared to the downstream and upstream sample stations. The high microbial load and presence of multiple antibiotic-resistant bacteria in the river water is alarming and can lead to public health risks when such water is consumed or used for cleaning meat during processing. There is a need for proper management of abattoir waste, An on-site pretreatment strategy (collection of waste, treatment, processing, anaerobic digestion, and safe disposal) is highly recommended and should be enforced by relevant authority to safeguard the environment and the populace that depend on the Orogodo river water.

In vitro Bioactivity of Halophytic Plant Heliotropium curassavicum L. against Selected Pathogens

Heliotropium curassavicum L. was screened for antimicrobial activity against some plant and human pathogens. Plant parts of H. curassavicum, were collected from mangrove habitats of Chollangi, near Kakinada, Plant parts are dried and extracts were obtained successfully with hexane, chloroform, methanol and water, by using Soxhlet extraction apparatus. The antimicrobial activity of the plant extracts on the various test organisms, including multiple antibiotic resistant bacteria were investigated. Antimicrobial activity of the extracts was determined by the Well Diffusion Method. The results concluded that the leaf and stem extracts of H. curassavicum possess antibacterial, antifungal activities. There is a possibility of developing this plant as a source of antibacterial and antifungal agent and further investigations are necessary to identify the bioactive principles.

Proteomic analysis of anti-MRSA activity of caerin 1.1/1.9 in a murine skin infection model and their in vitro anti-biofilm effects against Acinetobacter baumannii.

Caerin 1 is a family of host defense peptides with antimicrobial properties originally isolated from the Australian tree frog. Both caerin 1.1 and caerin 1.9 have been demonstrated to inhibit infections caused by multiple antibiotic-resistant bacteria, both in vitro and in vivo. In this current study, we conducted a comparison of the minimum inhibitory concentrations of caerin 1.1 and caerin 1.9 (referred to as caerin 1.1/1.9) with commonly used antibiotics against various bacteria, including Staphylococcus aureus, Copper-Green Pseudomonas aeruginosa, Acinetobacter baumannii, and Streptococcus haemolyticus. Our findings reveal that caerin 1.1/1.9 not only prevent the formation of biofilms by A. baumannii but also exhibit a therapeutic effect on established biofilms. Furthermore, our study demonstrates that caerin 1.1/1.9 significantly inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) strains in a murine skin infection model. A quantitative proteomic analysis conducted as part of our study indicates that caerin 1.1/1.9 primarily activate oxidative phosphorylation pathways, along with several pathways associated with tissue repair and growth. These findings were observed in comparison to untreated tissues that were infected with MRSA in mice. In summary, our research outcomes suggest that caerin 1.1/1.9 exhibit the potential to serve as promising drug candidates for treating complicated antibiotic-resistant bacterial infections in humans. IMPORTANCE Caerin 1.1 and caerin 1.9, natural antimicrobial peptides derived from tree frogs, have demonstrated the ability to inhibit the growth of antibiotic-resistant bacteria, comparable to certain widely used antibiotics. Additionally, these peptides exhibit the capacity to prevent or treat biofilms formed by bacteria in conjunction with bodily components. The mechanisms underlying their antibacterial effects were investigated through a mouse model of bacterial skin infection, utilizing proteomic analysis as a technological approach.

Antimicrobial Screening of the Solvent Extracts of Halophytic Plant Suaeda maritima (L.) Demort. Against Selected Pathogens

The antimicrobial activity of leaf and stem extracts of halophytic plant Suaeda maritima (L.) Demort. against, some plant and human pathogens. Plant parts of S. maritima were collected from the mangrove habitats of Pandi and Pora regions of Godavari estuary, near Amalapuram, Andhra Pradesh, India. Plant parts are dried and extracts were obtained successfully with hexane, chloroform, methanol and water, using Soxhlet extraction apparatus. The antimicrobial activity of the plant extracts on various test organisms including multiple antibiotic resistant bacteria were investigated by Well Diffusion Method under in vitro conditions. The plant extracts have shown promising antimicrobial activity against all tested organisms. Among the four extracts, water extract of S. maritima showed appreciable antimicrobial activity against all bacterial and fungal strains. It reveals that halophytic plant has antimicrobial compounds which can act against microorganisms and they can also be used in the treatment of infectious diseases caused by resistant pathogenic microorganisms.

Microbiological investigation of bacteria in raw eggs obtained from Obafemi Awolowo University, Nigeria

This study aimed to investigate the bacterial load and antibiotic susceptibility of bacteria isolated from uncooked eggs, with the goal of assessing potential health risks associated with their consumption. Six uncooked egg samples were collected, including samples from the eggshell, egg yolk, egg albumen, and their mixture. Bacterial enumeration was performed using the serial dilution and pour plate method on nutrient agar and MacConkey agar. Bacterial isolates were identified based on morphological and biochemical characteristics using Bergey's Manual of Determinative Bacteriology. Antibiotic susceptibility testing was conducted using the disc diffusion method on Mueller Hinton agar. The mean total bacterial load in the egg samples ranged from 3.6 x 10^4 to 5.0 x 104 CFU/ml, with coliforms ranging from 2.5 x 103 to 3.0 x 104 colony-forming unit per ml. A total of 48 isolates were obtained and identified, consisting of 13 Gram-negative and 35 Gram-positive bacteria. The identified bacteria exhibited varied susceptibility and resistance patterns to antibiotics. Gram-negative bacteria showed 100% susceptibility to gentamycin, ofloxacin, pefloxacin, and ciprofloxacin, except for Neisseria denitrificans, while demonstrating 100% resistance to nitrofurantoin, augmentin, and ceftriaxone. Most Gram-positive bacteria were susceptible to Pefloxacin and Streptomycin. Some isolates displayed resistance to multiple antibiotic classes, with Micrococcus varians and Bacillus laterosporus showing resistance to 6 and 7 different classes of antibiotics, respectively. The study findings emphasize the potential health risks associated with consuming uncooked eggs due to the presence of multiple antibiotic-resistant bacteria. These findings highlight the importance of proper cooking and handling practices for eggs to minimize the risk of foodborne infections caused by antibiotic-resistant bacteria. It underscores the need for awareness and implementation of appropriate food safety measures to safeguard public health.

Antibacterial activities of Vernonia amygdalina (Del.) stem bark extracts on multiple antibiotic-resistant bacteria isolated from wound samples

Antibacterial activities of Vernonia amygdalina (Del.) stem bark extracts on multiple antibiotic-resistant bacteria isolated from wound samples

Antibacterial activities of Polyalthia longifolia leaf extracts on multiple antibiotic-resistant bacteria isolated from hospital fomites in Akure, Nigeria

Abstract. Babatunde OJ, Ogundare AO, Adebolu TT. 2023. Antibacterial activities of Polyalthia longifolia leaf extracts on multiple antibiotic-resistant bacteria isolated from hospital fomites in Akure, Nigeria. Nusantara Bioscience 15: 149-160. Polyalthia longifolia (Sonn.), an ornamental plant, is said to be therapeutic while searching for new medications to treat infections caused by multiple Antibiotic-Resistant Bacteria (MAR). This plant's leaves were investigated for prospective antibacterial activity against MAR isolated from fomites in selected hospitals in Akure and their pharmacological properties. Standard microbiological methods were used to isolate and identify bacteria from fomites. Disc diffusion was performed to test their sensitivity to conventional antibiotics and P. longifolia leaf extracts made with ethanol and water. Ciprotab® was used as the control during the antibacterial assay. Therefore, GC-MS analysis was carried out using standard methods to identify the chemicals in the plant leaf extracts. Staphylococcus aureus (29.17%), Streptococcus pyogenes (20.83%), Pseudomonas aeruginosa (14.28%), Escherichia coli (14.28%), Salmonella typhi (12.5%) and Klebsiella pneumoniae (8.33%) were the bacterial species isolated from the fomites sampled in this study. Crude ethanol leaf extract of P. longifolia (100mg/mL) inhibited the growth of these organisms with the greatest effect on P. aeruginosa with a value of 23.83±0.44 mm, which is superior to that medicated by the control antibiotic (ciprofloxacin). The GC-MS analysis of the purified leaf extracts of P. longifolia revealed the presence of bioactive compounds such as n-hexadecanoic acid and phytol, among others. The study revealed that the leaf extracts of P. longifolia can inhibit the growth of the isolated MAR from fomites with an added cidal effect, and the inhibition increase with the increase in concentration and exposure time.

Impact of soil supplemented with pig manure on the abundance of antibiotic resistant bacteria and their associated genes.

This study was conducted to evaluate the abundance of antibiotic resistant bacteria and their resistance genes from agriculture soil supplemented with pig manure. Uncultivable soil sample was supplemented with pig manure samples under microcosm experimental conditions and plated on Luria-Bertani (LB) agar incorporated with commercial antibiotics. The supplementation of soil with 15% pig manure resulted in the highest increase in the population of antibiotic resistant bacteria (ARB)/multiple antibiotic resistant bacteria (MARB). Seven genera that included Pseudomonas, Escherichia, Providencia, Salmonella, Bacillus, Alcaligenes and Paenalcaligenes were the cultivable ARB identified. A total of ten antibiotic resistant bacteria genes (ARGs) frequently used in clinical or veterinary settings and two mobile genetic elements (MGEs) (Class 1 and Class 2 integrons) were detected. Eight heavy metal, copper, cadmium, chromium, manganese, lead, zinc, iron, and cobalt were found in all of the manure samples at different concentrations. Tetracycline resistance genes were widely distributed with prevalence of 50%, while aminoglycoside and quinolone-resistance gene had 16% and 13%, respectively. Eighteen ARB isolates carried more than two ARGs in their genome. Class 1 integron was detected among all the 18 ARB with prevalence of 90-100%, while Class 2 integron was detected among 11 ARB. The two classes of integron were found among 10 ARB. Undoubtedly, pig manure collected from farms in Akure metropolis are rich in ARB and their abundance might play a vital role in the dissemination of resistance genes among clinically-relevant pathogens.

Multiple Antibiotic-Resistant Bacteria Resistant to Electrochemical Disinfection with Variation of Key Antibiotic Resistance Genes

Electrochemical disinfection (ED) is effective in removal of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) during wastewater treatment. However, the inner relationship of antibiotic-resistant phenotypes with their genotypes of ARB is still unclear in the ED process. This study explored the disinfection mechanism at the microcosmic level using four Escherichia coli (E. coli) strains with different antibiotic-resistant phenotypes. Results showed that bacteria with multiple antibiotic resistance tended to have stronger viability in disinfection compared with those resistant to fewer antibiotics. Interestingly, antibiotic-resistant phenotypes changed little in the disinfection process, while genotypes closely responded to bacterial resistance of different E. coli. In detail, beta-lactamase resistance genes played a leading role in the cross-resistance between antibiotics and electrochemical oxidation. They were proved to reduce oxidative destruction by increasing the strength of the cell wall and membrane. In addition, diaminopyrimidine and macrolide-lincosamide-streptogramin B resistance genes are also closely related to the bacterial resistance to ED. Combined with the quantitative determination of ARGs, the abundance of IntI1 and aminoglycoside resistance genes can be regarded as indicators for the risk of cross-resistance to antibiotics and electrochemical oxidation. This study provided important references for in depth understanding and accurate control of antibiotic resistance with advanced oxidation processes.

Bioactive compounds from high altitude lake Arthrospira platensis HANL01: Antioxidant property, thermal stability and antibacterial assessment against multiple antibiotics resistant bacteria

Growing concerns about antibiotic resistance in bacteria and the pursuit of novel compounds and drug candidates are significant strategies for its preparedness. The study aimed to explore Arthrospira sp. isolated from high-altitude lake water for antioxidant potential, thermal stability, and bactericidal action against various bacteria that are resistant to antibiotics. % DPPH free radical scavenging activity was recorded in the range of 15.23 ± 0.38 to 89.21 ± 0.14. Column-purified MeOH fraction and TLC fraction showed significant antibacterial potential. Crude extracts were found stable at 25 °C even after 63 days of storage. Based on the GC–MS profiling, pentadecanoic acid was reported as the primary constituent, as the spectra of this bioactive compounds (11.13 %) were considered as the highest peaks with the highest impact (9.447). The study's findings suggested that high-altitude lake A. platensis is an excellent source of antioxidant and antibacterial substances and might be employed for further drug development research, particularly against multiple antibiotic-resistant bacteria.

Isolation 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 × 105-1.2 × 109 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, thechloramphenicol acetyltransferase gene catB10, andthe 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.

Isolation 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 × 105-1.2 × 109 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.

Fate of antibiotic resistance genes and bacteria in a coupled water-processing system with wastewater treatment plants and constructed wetlands in coastal eco-industrial parks

In coastal eco-industrial zones, wastewater treatment plants (WWTPs) and constructed wetlands (CWs) can alleviate the challenge of water shortage and the negative effect of sewage discharge, while the problems of antibiotic resistance genes (ARGs) have not attracted enough attention. In this research, the Wafergen SmartChip system was adopted to investigate the ARG profiles in a coupled system combined WWTPs and CWs in a coastal industrial park. Potential risks of antibiotic resistance in chemical industrial wastewater were confirmed due to the higher abundance of target ARGs (> 107 copies/mL). General decline with partial enrichment in absolute and relative abundance of ARGs from the WWTPs to CWs revealed the effective removal of ARGs in the coupled system, while the fate of different ARG types varied greatly. Aminoglycoside and sulfonamide ARGs were detected with higher abundance (up to 5.34 ×107 and 3.61 ×107 copies/mL), especially aac(6′)-Ib and sul1. Denitrification, secondary sedimentation, and acid hydrolysis contributed to the removal of aminoglycoside, sulfonamide, β-lactamase, chloramphenicol, and multidrug ARGs. Catalytic ozonation contributed to the removal of tetracycline and MLSB ARGs. Subsurface CWs worked effectively for the removal of sulfonamide, tetracycline, and multidrug ARGs, especially tetX, cphA, tetG, and strB. Close correlations between ARGs and MGEs emphasized the vital roles of anthropogenic pollutants and horizontal gene transfer on the diffusion of ARGs. Actinobacteria, Bacteroidota, and Cyanobacteria were dominant in the CWs, while Proteobacteria, Firmicutes, and Planctomycetota were prevalent in the WWTPs. Redundancy analysis and variance partitioning analysis indicated that transposase and water quality posed greater influences on the distribution of ARGs. Co-occurrence network revealed that potential multiple antibiotic resistant pathogenic bacteria decreased in the CWs. The coupled system has a limited effect on the reduction of ARGs and potential ARG hosts, providing a comprehensive insight into the fate of ARGs in conventional water-processing systems.

Promising antibacterial activities of anethole and green-synthesized magnetite nanoparticles against multiple antibiotic-resistant bacteria.

The emergence of antibiotic resistance is considered a major threat, and this problem is exacerbated due to the excessive use of antibiotics. Therefore, it is necessary to find new antimicrobials that are effective against pathogenic bacteria resistant to existing antimicrobials. This study presents a novel antimicrobial approach for the bio-control of multi-antibiotic-resistant (MAR) bacteria. Ten antibiotic discs were used to evaluate the bacterial sensitivity. Five bacterial strains showed resistance to all the tested antibiotic groups. The following MAR bacterial strains were selected, and their identification was confirmed by 16S rRNA with an accession number on the NCBI: Shigella flexneri MZ350855.1; Klebsiella aerogenes MZ352107.1; Serratia marcescens MZ618709.1; Yersinia enterocolitica MZ673567.1; and Achromobacter denitrificans OK560350.1. Star anise extract (SAE) gives the highest inhibitory efficiency against isolated MAR bacteria between three local plant extracts. SAE was used to synthesize magnetite nanoparticles (MNPs) using a simple and eco-friendly green biosynthesis technique. Synthesized MNPs were characterized by X-ray diffraction, ultraviolet-visible spectroscopy, Fourier transform infrared, and transmission electron microscopy techniques. The effect of synthesized magnetic nanoparticles for wastewater treatment in bacterial portion demonstrated remarkable bactericidal performance against total bacterial count with a percentage reduction of 84% using 0.05 g, and the efficiency of decreasing heavy metals was estimated.