Assessment of Physico-Chemical Properties of Sewage Sludge and Unlocking Its Prospect as a Powerhouse of Antibiotic-Resistant Bacteria

With their large, diverse microbial communities chronically exposed to sub-inhibitory antibiotic concentrations, wastewater treatment works (WWTW) have been deemed hotspots for the emergence and dissemination of antimicrobial resistance, with growing concern about the transmission of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) into receiving surface waters. This study explored (1) the prevalence of ARG and ARB in local WWTW, (2) the effect of sub-inhibitory antimicrobial exposure on ARG copy numbers in pure cultures from WWTW, and (3) two WWTW with different treatment configurations. For each WWTW, qPCR determined the prevalence ofmcr3, sul1, sul2, andblaKPCduring the treatment process, and culture methods were used to enumerate and identify ARB. Bacterial colonies isolated from effluent samples were identified by 16S rDNA sequencing and their respective minimum inhibitory concentrations (MIC) were determined. These were compared to the MICs of whole community samples from the influent, return activated sludge, and effluent of each WWTW. Resistance genes were quantified in 11 isolated cultures before and after exposure to sub-MIC concentrations of target antibiotics. The numbers of ARG and ARB in both WWTW effluents were notably reduced compared to the influent.Sul1andsul2gene copies increased in cultures enriched in sub-MIC concentrations of sulfamethoxazole, whileblaKPCdecreased after exposure to amoxicillin. It was concluded, within the parameters of this study, that WWTW assist in reducing ARG and ARB, but that sub-inhibitory exposure to antimicrobials has a varied effect on ARG copy number in pure cultures.

<p>With their large, diverse microbial communities chronically exposed to sub-inhibitory antibiotic concentrations, wastewater treatment works (WWTW) have been deemed hotspots for the emergence and dissemination of antimicrobial resistance, with growing concern about the transmission of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) into receiving surface waters. This study explored (1) the prevalence of ARG and ARB in local WWTW, (2) the effect of sub-inhibitory antimicrobial exposure on ARG copy numbers in pure cultures from WWTW, and (3) two WWTW with different treatment configurations. For each WWTW, qPCR determined the prevalence of mcr3, sul1, sul2, and bla(KPC) during the treatment process, and culture methods were used to enumerate and identify ARB. Bacterial colonies isolated from effluent samples were identified by 16S rDNA sequencing and their respective minimum inhibitory concentrations (MIC) were determined. These were compared to the MICs of whole community samples from the influent, return activated sludge, and effluent of each WWTW. Resistance genes were quantified in 11 isolated cultures before and after exposure to sub-MIC concentrations of target antibiotics. The numbers of ARG and ARB in both WWTW effluents were notably reduced compared to the influent. Sul1 and sul2 gene copies increased in cultures enriched in sub-MIC concentrations of sulfamethoxazole, while bla(KPC) decreased after exposure to amoxicillin. It was concluded, within the parameters of this study, that WWTW assist in reducing ARG and ARB, but that sub-inhibitory exposure to antimicrobials has a varied effect on ARG copy number in pure cultures.</p> <p> </p>

<p>With their large, diverse microbial communities chronically exposed to sub-inhibitory antibiotic concentrations, wastewater treatment works (WWTW) have been deemed hotspots for the emergence and dissemination of antimicrobial resistance, with growing concern about the transmission of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) into receiving surface waters. This study explored (1) the prevalence of ARG and ARB in local WWTW, (2) the effect of sub-inhibitory antimicrobial exposure on ARG copy numbers in pure cultures from WWTW, and (3) two WWTW with different treatment configurations. For each WWTW, qPCR determined the prevalence of mcr3, sul1, sul2, and bla(KPC) during the treatment process, and culture methods were used to enumerate and identify ARB. Bacterial colonies isolated from effluent samples were identified by 16S rDNA sequencing and their respective minimum inhibitory concentrations (MIC) were determined. These were compared to the MICs of whole community samples from the influent, return activated sludge, and effluent of each WWTW. Resistance genes were quantified in 11 isolated cultures before and after exposure to sub-MIC concentrations of target antibiotics. The numbers of ARG and ARB in both WWTW effluents were notably reduced compared to the influent. Sul1 and sul2 gene copies increased in cultures enriched in sub-MIC concentrations of sulfamethoxazole, while bla(KPC) decreased after exposure to amoxicillin. It was concluded, within the parameters of this study, that WWTW assist in reducing ARG and ARB, but that sub-inhibitory exposure to antimicrobials has a varied effect on ARG copy number in pure cultures.</p> <p> </p>

Objectives and Design: The use of fluoroquinolones has increased against antibiotic-resistant pathogens such as Streptococcus pneumoniae and Pseudomonas aeruginosa. The E-test (AB Biodisk, Solna, Sweden) is now commonly used for susceptibility testing of fluoroquinolones against these organisms. The purpose of the present study was to evaluate the accuracy and correlation of minimum inhibitory concentrations (MICs) determined by E-testing with a National Committee for Clinical Laboratory Standards reference standard, agar-dilution MIC testing. E-test and agar dilution MICs were compared for ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin against clinical isolates of S. pneumoniae (n = 53) and P. aeruginosa (n = 62). Main Outcome Measures: MICs were determined by use of agar dilution and E-test methods. Essential agreement was defined as MICs from both methods within ± 1 log2 dilution. Categorical agreement compared MIC interpretations: susceptible, intermediate, or resistant. Categorical disagreement between methods was reported as very major, major, or minor errors. Results: E-tests produced lower MICs than the reference method for ciprofloxacin, gatifloxacin, and moxifloxacin versus P. aeruginosa. For S. pneumoniae, E-test MICs tended to be higher for all fluoroquinolones. The best correlation between testing methods was seen with levofloxacin. Essential agreement occurred more frequently with P. aeruginosa in the lower range of MICs and with S. pneumoniae in the higher range of MICs. Categorical agreement was greater than 90% for the 460 comparisons. Two very major errors (false-susceptible) occurred for gatifloxacin versus P. aeruginosa. Conclusions: For the determination of fluoroquinolone susceptibility against S. pneumoniae and P. aeruginosa, E-testing is a simple tool for clinical use, and few very major or major errors in susceptibility interpretation occur for either organism. For determining fluoroquinolone MICs, E-testing may overestimate drug activity against P. aeruginosa and underestimate drug activity versus S. pneumoniae compared with the agar dilution method. These differences could affect appropriate antimicrobial selection, leading to suboptimal outcomes.

Molecular characteristic of mcr-1 gene in Escherichia coli from aquatic products in Guangdong, China

Several studies have investigated the effects of swimming in sewage-polluted recreational beach water, highlighting the associated health hazards. To mitigate potential pathogen transmission, it is imperative that the polluted water is released away from recreational waters and foreshores, where children tend to play. At present, domestic sewage in the Faroe Islands solely undergoes primary wastewater treatment within primary settling tanks before being discharged into the ocean. Effluents are a major anthropogenic source of antibiotic resistance genes and antibiotic resistant bacteria, which are released into the environment. The aim of this study was to investigate antibiotic resistant Gram-negative bacteria and antibiotic resistance genes in influents and effluents of wastewater subjected solely to primary treatment, along with their release into the environment during both summer and winter. Water samples were collected from influents and effluents as well as with increasing distance away from the wastewater outlet and from nearby tidepools. Samples were cultured on MacConkey agar with four different antibiotics for detection of antibiotic-resistant bacteria and antibiotic resistance genes were quantified by droplet digital PCR. All multi-drug resistant bacteria were identified using the API 20E kit. We observed an overall decrease of the abundance of Gram-negative bacteria from the effluents compared to influents, however, we observed the opposite trend in the antibiotic resistance genes. Antibiotic resistant bacteria and antibiotic resistance genes in addition to multi-drug resistant bacteria were found in the surrounding oceanic and several terrestrial tidepool samples. Of the multi-drug resistant bacteria, we found, e.g., Escherichia coli, P. aeruginosa, and A. hydrophila species, which can be pathogenic, potentially causing an infection if encountering a host. These results indicate a relatively wide pollution range of the effluents from the septic tank and treated sewage released into the environment, posing a potential hazard for both humans and wildlife.

Antibiotic contamination in agroecosystems may cause serious problems, such as the proliferation of various antibiotic resistant bacteria and the spreading of antibiotic resistance genes (ARGs) in the environment or even to human beings. However, it is unclear whether environmental antibiotics, antibiotic resistant bacteria, and ARGs can directly enter into, or occur in, the endophytic systems of plants exposed to pollutants. In this study, a hydroponic experiment exposing pakchoi (Brassica chinensis L.) to tetracycline, cephalexin, and sulfamethoxazole at 50% minimum inhibitory concentration (MIC) levels and MIC levels, respectively, was conducted to explore plant growth, antibiotic uptake, and the development of antibiotic resistance in endophytic systems. The three antibiotics promoted pakchoi growth at 50% MIC values. Target antibiotics at concentrations ranging from 6.9 to 48.1 µg·kg−1 were detected in the treated vegetables. Additionally, the rates of antibiotic-resistant endophytic bacteria to total cultivable endophytic bacteria significantly increased as the antibiotics accumulated in the plants. The detection and quantification of ARGs indicated that four types, tetX, blaCTX-M, and sul1 and sul2, which correspond to tetracycline, cephalexin, and sulfamethoxazole resistance, respectively, were present in the pakchoi endophytic system and increased with the antibiotic concentrations. The results highlight a potential risk of the development and spread of antibiotic resistance in vegetable endophytic systems.

Background: Microbes are most commonly found associated with oral diseases. Globally, researchers along with the world Health Organization (WHO) suggested that this could be due to poor oral health and encourage all individuals to practice good oral hygiene using daily oral health products. The study aimed to determine the antimicrobial effects of toothpastes, as acclaimed by the manufacturers, to selected microbial flora involved in oral infections. Methodology: The antimicrobial activity of 6 toothpastes (3 herbal and 3 chemical) was tested against 3 clinical microbial isolates; Staphylococcus aureus, Escherichia coli and Candida albicans, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each toothpaste determined for each microbial isolate using the broth dilution method. Results: Colgate exhibited the highest inhibitory activity against S. aureus with respective MIC and MBC of 125mg/ml and 125mg/ml, followed by Oral-B with MIC and MBC of 125mg/ml and 500mg/ml, Dabur herbal with MIC and MBC of 250mg/ml and 500mg/ml, and Longrich with MIC of 500mg/ml and MBC of 1000mg/ml. Colgate and CloseUp exhibited highest inhibitory activity against E. coli with respective MIC and MBC of 125mg/ml and 500mg/ml, followed by Dabur herbal with MIC of 500mg/ml but no MBC. Colgate exhibited the highest inhibitory activity against C. albicans with respective MIC and MBC of 125mg/ml and 500mg/ml, followed by CloseUp with MIC of 500mg/ml, Longrich with MIC of 500mg/ml and MBC of 1000mg/ml, and oral-B with MIC of 500mg/ml but no MBC. Longrich and Oral-B exhibited no MIC/MBC against E. coli while Gavia charcoal exhibited no MIC/MBC against all the 3 microbial isolates at the concentrations used in the assay.Conclusion: The results obtained showed that chemically formulated toothpastes (Colgate, CloseUp and Oral-B) had higher inhibitory activity on microbial isolates than herbal toothpastes (Longrich, Dabur herbal and Gavia charcoal). This supports the manufacturers claim for the chemical toothpastes used, however, the herbal toothpastes showed little or no inhibitory effects on the microbial flora.

In order to evaluate the suitability of fosfomycin in combination with other agents for the treatment of Helicobacter pylori infections, the susceptibility profiles of 65 H. pylori strains were determined against multiple antimicrobial agents and combinations thereof using the agar dilution method. For fosfomycin alone, the range of minimum inhibitory concentration (MIC) results and the MICs at which 50% and 90% of strains were inhibited were 0.5-32 microg/ml and 2 and 4 microg/ml, respectively. For the combination of fosfomycin with amoxicillin, clarithromycin or metronidazole, the means calculated for the minimum and maximum fractional inhibitory concentration index were 0.70-1.17 and 1.15-2.03, respectively, suggesting partial synergy or indifference in the majority of strains. The combination of clarithromycin and metronidazole showed synergistic activity against 14 of 28 H. pylori strains tested. The in vitro activity results suggest the combination of fosfomycin with either amoxicillin or clarithromycin may be a promising alternative for the treatment of H. pylori infection. However, the clinical efficacy of these regimens remains to be investigated.

Fate of antibiotic resistant cultivable heterotrophic bacteria and antibiotic resistance genes in wastewater treatment processes

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.

Risk control of antibiotics, antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) during sewage sludge treatment and disposal: A review

Research Note: Epidemiological cutoff values and acquired resistance mechanisms of three veterinary antibiotics against Escherichia coli from chicken respiratory tract infections

The in vitro activity of fleroxacin (Ro 23-6240) against 441 bacterial isolates was compared with those of ciprofloxacin, ofloxacin, amoxycillin, cefadroxil, cefuroxime and tobramycin. An agar dilution method was used for the determination of minimal inhibitory concentrations (MICs). Ciprofloxacin showed the highest activity against the Enterobacteriaceae, 95% of the isolates were inhibited by 0.06 mg/l, but fleroxacin and ofloxacin were also highly active (MIC 90% = 0.5 and 0.25 mg/l, respectively). Ciprofloxacin was the most active agent against Pseudomonas aeruginosa (MIC 90% = 0.12 mg/l), whereas the activities of fleroxacin and ofloxacin were more variable. Tobramycin was highly active against P. aeruginosa, 75% of the isolates were inhibited by 0.5 mg/l or less. The quinolones and tobramycin exhibited high activity against Acinetobacter calcoaceticus, the great majority of the isolates being susceptible to 0.5 mg/l or less of any agent. All the quinolones showed high activity against Staphylococcus aureus, but fleroxacin was less active against Staphylococcus epidermidis and Staphylococcus saprophyticus than were the other derivatives. The pneumococcal and streptococcal isolates were markedly less susceptible to fleroxacin than to the other quinolones tested (MIC range 4-32 mg/l). All isolates of Haemophilus influenzae and Neisseria gonorrhoeae were inhibited by the lowest concentration of the quinolones employed in the study (0.03 mg/l). Cefuroxime was also highly active against N. gonorrhoeae, whether the strains were beta-lactamase-producing or not, but was somewhat less active against H. influenzae. The quinolones displayed moderate and similar activity against Bacteroides fragilis isolates (MIC range 1-16 mg/l). The MICs of fleroxacin against gram-negative rods were generally 4-16 times higher at pH 8.8 than those obtained at pH 5.8 and 7.3. The activity against gram-positive cocci was not markedly influenced by changes in pH.

The widespread occurrence of chronic and recurrent dermatophytosis has significantly affected the quality of life for patients in India and beyond. Identifying the causative dermatophytes and understanding their antifungal susceptibility can aid clinicians in tailoring effective antifungal therapies. Patients with chronic and recurrent dermatophytosis were enrolled, and conventional fungal cultures were conducted on skin scrapings. Identified isolates underwent antifungal susceptibility testing using the Clinical and Laboratory Standards Institute broth microdilution method (CLSI M38-A2) for common systemic antifungals, determining the minimum inhibitory concentration (MIC) range and calculating MIC 50 and MIC 90. Sixty samples were tested. Tinea corporis was the most common presentation (66.6%). Trichophyton mentagrophyte species complex was the prevalent species (45, 75%), followed by Trichophyton rubrum (7, 11.7%). In Trichophyton mentagrophytes species complex, MIC range was 8-64 μg/mL for fluconazole, 0.06-0.25 μg/mL for terbinafine, and 0.125-0.5 μg/mL for griseofulvin. For Trichophyton rubrum, the MIC range was 8-64 μg/mL for fluconazole, 0.06-0.25 μg/mL for terbinafine, and 0.125-0.5 μg/mL for griseofulvin. For all species, itraconazole MIC was ≤0.125 μg/mL. Hence, itraconazole and terbinafine had the best MIC range against tested isolates in our study. Absence of genotyping of isolate and not compared the results with studies where sequence-based identification to species level was done. In vitro, resistance to itraconazole for any of the four isolated agents was not seen. Terbinafine resistance appears to be an uncommon occurrence in South India. In vitro susceptibility tests shall be regularly done to design the epidemiological cutoff values.