Fate of fluoroquinolones associated with antimicrobial resistance in circular periurban agriculture.

Animal antibiotic use contributes to antimicrobial resistance (AMR) in humans. While animal manure benefits soil fertility, it also acts as hotspot for antibiotic residues, antibiotic-resistant bacteria, and their genes. Amending soils with poultry litter is recognized as "magic" among horticulture farmers and it remains a common practice globally. However, this poses a risk especially in countries where prophylactic use of antibiotics is allowed. In Argentina, fluoroquinolones are used in this way besides being listed as essential medicines and classified as "watch" by the World Health Organization. Antibiotic selective pressure can favour AMR in the environment but the fate of antibiotic residues and AMR dissemination from these practices remains poorly understood. Our research addresses this gap with a biological model tracing fluoroquinolones from poultry to soil to lettuce and tracking anthropogenic AMR with the proposed biomarker genes sul1 and intI1. Fresh poultry litter was stored for six months before application in a horticulture field experiment. The experiment included control and manured plots where lettuce was cultivated till harvest. Enrofloxacin concentration was 7.3 µg/kg in fresh poultry litter, while its metabolite ciprofloxacin was 39.22 µg/kg after storage. Although no fluoroquinolones were detected in soils, lettuce from manured plots contained enrofloxacin and ciprofloxacin at 14.97 and 9.77 µg/kg, respectively, providing evidence of fluoroquinolone bioaccumulation in plants. Abundance of sul1 and intI1 in poultry litter was not affected by storage. Manured soils showed better soil quality than controls, but sul1 gene abundance was 1.6 times higher, reaching 7.61 Log sul1/g soil. A less sensitive, but significant effect was registered for intI1. These findings show that static storage is insufficient to stop the transmission of antibiotics and AMR biomarkers from poultry to horticulture. Amending soil with industrial poultry litter contributes to pollution with these emergent contaminants and risks human antibiotic exposure through fresh vegetables.

High Carriage of tetA, sul1, sul2 and bla TEM Resistance Genes among the Multidrug-resistant Uropathogenic Escherichia coli (UPEC) Strains from Malaysian Patients.

The rapid emergence of multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) strains pose a critical challenge in urinary tract infection (UTI) treatments. However, little work elucidated the resistance mechanisms of the MDR UPEC clinical strains in Malaysia. Therefore, this study aimed to determine the antimicrobial susceptibility profiles and the prevalence of antimicrobial resistance genes among the UPEC strains. Polymerase chain reactions were conducted to detect the presence of 6 antimicrobial resistance genes among 60 UPEC strains. Meanwhile, the antimicrobial resistance profiles against 9 antimicrobials were examined through the Kirby-Bauer disk diffusion method. In this study, the MDR isolates accounted for 40.0% (24/60), with the highest prevalence of resistance towards ampicillin (43/60; 71.7%), followed by tetracycline (31/60; 51.7%), nalidixic acid (30/60; 50.0%), co-trimoxazole (20/60, 33.3%), ciprofloxacin (19/60, 31.7%), levofloxacin (16/60, 21.6%) and chloramphenicol (10/60, 16.7%). In contrast, low resistance rates were observed among minocycline (1/60; 1.7%) and imipenem (0/60; 0.0%). bla TEM was the most prevalent gene (36/60; 60.0%), followed by tetA (27/60; 45.0%), sul2 (25/60; 41.7%), sul1 (13/60; 21.7%) and tetB (8/60; 13.3%). Surprisingly, bla SHV was not detected among the UPEC isolates. The MDR, ampicillin and tetracycline-resistant isolates were significantly associated with a higher prevalence of tetA, sul1, sul2 and bla TEM. In contrast, tetB displayed no significant relationship with any of the antimicrobials tested. The patient's age and gender were not the risk factors for the carriage of the resistance genes. Our findings identified the common resistance genes carried by the antimicrobial resistant UPEC isolates and provide valuable insights into developing the best antibiotic prescription regime to treat UTIs in our local scene.

Temporal and spatial changes in the abundance of antibiotic resistance gene markers in a wastewater treatment plant.

In this study, we investigated the temporal and spatial quantitative changes in the concentration of antibiotic resistance gene (ARG) markers in a municipal wastewater treatment plant (WWTP). Four ARGs conferring resistance to different classes of antibiotics (ermB, sul1, tet[W], and blaCTXM) and a gene used as a proxy for ARG pollution (intl1) were quantified in two separate sampling campaigns covering two and half years of operation of the WWTP. First, a systematic monthly monitoring of multiple points in the inlet and the outlet revealed an absolute decrease in the concentration of all analyzed ARGs. However, the relative abundance of sul1 and intl1 genes relative to the total bacterial load (estimated using the universal marker 16S rDNA) increased in the outlet samples as compared to the inlet. To pinpoint the exact stage of removal and/or enrichment within the WWTP, a second sampling including the stages of the biological treatment was performed bimonthly. This revealed a distinct enrichment of sul1 and intl1 genes during the biological treatment phase. Moreover, the temporal and spatial variations in ARG abundance patterns within the WWTP underscored the complexity of the dynamics associated with the removal of ARGs during wastewater treatment. Understanding these dynamics is pivotal for developing efficient strategies to mitigate the dissemination of ARGs in aquatic environments. PRACTITIONER POINTS: Regular monitoring of ARG markers in WWTPs is essential to assess temporal and spatial changes, aiding in the development of effective mitigation strategies. Understanding the dynamics of ARG abundance during biological treatment is crucial for optimizing processes and minimizing dissemination in aquatic environments. Increased relative abundance of certain ARGs highlights potential enrichment during wastewater treatment, necessitating targeted interventions. Systematic monitoring of multiple points within WWTPs can provide valuable insights into the efficacy of treatment processes in reducing ARG levels over time. The complexity of ARG abundance patterns underscores the need to develop holistic approaches to tackle antibiotic resistance in wastewater systems.

Native Microalgae-Bacteria Consortia: A Sustainable Approach for Effective Urban Wastewater Bioremediation and Disinfection.

Urban wastewater is a significant by-product of human activities. Conventional urban wastewater treatment plants have limitations in their treatment, mainly concerning the low removal efficiency of conventional and emerging contaminants. Discharged wastewater also contains harmful microorganisms, posing risks to public health, especially by spreading antibiotic-resistant bacteria and genes. Therefore, this study assesses the potential of a native microalgae-bacteria system (MBS) for urban wastewater bioremediation and disinfection, targeting NH4+-N and PO43--P removal, coliform reduction, and antibiotic resistance gene mitigation. The MBS showed promising results, including a high specific growth rate (0.651 ± 0.155 d-1) and a significant average removal rate of NH4+-N and PO43--P (9.05 ± 1.24 mg L-1 d-1 and 0.79 ± 0.06 mg L-1 d-1, respectively). Microalgae-induced pH increase rapidly reduces coliforms (r > 0.9), including Escherichia coli, within 3 to 6 days. Notably, the prevalence of intI1 and the antibiotic resistance genes sul1 and blaTEM are significantly diminished, presenting the MBS as a sustainable approach for tertiary wastewater treatment to combat eutrophication and reduce waterborne disease risks and antibiotic resistance spread.

Evaluation of the presence of integrons, sul and smqnr genes and the prevalence of antibiotic resistance in Stenotrophomonas maltophilia clinical isolates.

OBJECTIVES: The objective of this investigation was to examine the mechanisms associated with antibiotic resistance in Stenotrophomonas maltophilia clinical isolates retrieved from hospitalized patients undergoing open heart surgery in a Heart Center located in Tehran, Iran. MATERIALS AND METHODS: This investigation encompassed a cross-sectional study of 60 S. maltophilia isolates, which were procured from diverse clinical specimens. Primary identification of the isolates was conducted through conventional microbiologic methods and subsequently verified by means of PCR primers. The E-test was utilized to establish the minimum inhibitory concentrations (MICs). PCR was then employed to ascertain the antibiotic resistance genes (sul1, sul2, Smqnr and intl1 - intl3). RESULTS: In this study, a total of sixty clinical isolates of S. maltophilia were collected, with the majority of them being obtained from Intensive Care Units (ICU) (n = 54; 90%). The disk diffusion method yielded results indicating that 55% of the isolates were sensitive to minocycline, whereas 30% were intermediate and 15% were found to be resistant. Additionally, the MIC results revealed that the resistant rates of the isolates towards ceftazidime, cotrimoxazole and levofloxacin were 46.7%, 1.7% and 5%, respectively. The PCR amplification of three classes of integrons genes indicated that fifteen (25%) of the isolates carried int1, while no detection for intl2 and intl3 was reported. Furthermore, the prevalence of antibiotic resistance genes (sul1, sul2, and Smqnr) was identified in 15 (25%), 6 (10%), and 28 (46.7%) isolates, respectively. CONCLUSION: The reported increasing rate of antibiotic resistance and mobile genetic elements that could extend the resistance genes to other strains in the hospital, finally it could be an alarming issue for healthcare settings that need special attention to this strain and the epidemiological study on this issue.

Antibiotic Resistance Profile of Salmonella sp. Isolates from Commercial Laying Hen Farms in Central-Western Brazil.

Microbial resistance to antibiotics poses a significant threat to both human and animal health, necessitating international efforts to mitigate this issue. This study aimed to assess the resistance profiles of Salmonella sp. isolates and identify the presence of intl1, sul1, and blaTEM resistance genes within antigenically characterized isolates, including Agona, Livingstone, Cerro, Schwarzengrund, Salmonella enterica subsp. enterica serotype O:4.5, Anatum, Enteritidis, Johannesburg, Corvallis, and Senftenberg. These isolates underwent susceptibility testing against 14 antibiotics. The highest resistance percentages were noted for sulfamethoxazole (91%), sulfonamides (51%), and ceftiofur (28.9%), while no resistance was observed for ciprofloxacin. Salmonella Johannesburg and Salmonella Corvallis showed resistance to one antibiotic, whereas other serovars were resistant to at least two. Salmonella Schwarzengrund exhibited resistance to 13 antibiotics. The intl1 gene was detected in six out of the ten serovars, and the sul1 gene in three, always co-occurring with intl1. The blaTEM gene was not identified. Our findings highlight the risk posed by the detected multiple resistances and genes to animal, human, and environmental health. The multidrug resistance, especially to third-generation cephalosporins and fluoroquinolones, highlights the need for stringent monitoring of Salmonella in laying hens. The potential of the environment, humans, eggs, and their products to act as vectors for antibiotic resistance represents a significant concern for One Health.

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

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

Corrigendum: The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.

[This corrects the article DOI: 10.3389/fmicb.2023.1058350.].

The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.

Introduction: Currently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2. Methods: Here we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP-impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ). Results: Our results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/l), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65%, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3-fold over 13 km of the river stretch, suggesting an internal gene multiplication. Discussion: We found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream from the WWTP. Therefore, limiting the release of ARG-harboring microorganisms may be more crucial for restricting the environmental spread of antimicrobial resistance than attenuating ng/L concentrations of antibiotics.

Antibiotic resistance and resistance mechanism of Corynebacterium kroppenstedtii isolated from patients with mastadenitis.

To investigate the antibiotic resistance and resistance mechanism of Corynebacterium kroppenstedtii (C. kroppenstedtii) isolated from patients with mastadenitis. Ninety C. kroppenstedtii clinical isolates were obtained from clinical specimens in 2018-2019. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed by the broth microdilution method. The resistance genes were detected using PCR and DNA sequencing. The results of antimicrobial susceptibility testing indicated that the resistance rates of C. kroppenstedtii to erythromycin and clindamycin, ciprofloxacin, tetracycline, and trimethoprim-sulfamethoxazole were 88.9%, 88.9%, 67.8%, 62.2%, and 46.6%, respectively. None of the C. kroppenstedtii isolates was resistant to rifampicin, linezolid, vancomycin, or gentamicin. The gene of erm(X) was detected in all clindamycin and erythromycin-resistant strains. The gene of sul(1) and tet(W) were detected among all trimethoprim sulfamethoxazole-resistant strains and tetracycline-resistant strains, respectively. Furthermore, 1 or 2 amino acid mutations (mainly single mutation) were observed in the gyrA gene among ciprofloxacin-resistant strains.

Abundance, diversity, and host assignment of total, intracellular, and extracellular antibiotic resistance genes in riverbed sediments.

Human health risk assessment for environmental antibiotic resistant microbes requires not only quantifying the abundance of antibiotic resistance genes (ARGs) in environmental matrices, but also understanding their hosts and genetic context. Further, differentiating ARGs in intracellular and extracellular DNA (iDNA and eDNA) fractions may help refine our understanding of ARG transferability. The objectives of this study were to understand the (O1) abundance and diversity of extracellular, intracellular, and total ARGs along a land use gradient and (O2) impact of bioinformatics pipeline on the assignment of putative hosts for the ARGs observed in the different DNA fractions. Sediment samples were collected along a land use gradient in the Raritan River, New Jersey, USA. DNA was extracted to separate eDNA and iDNA and qPCR was performed for select ARGs and the 16S rRNA gene. Shotgun metagenomic sequencing was performed on DNA extracts for the different DNA fractions. ARG hosts were assigned via two different bioinformatic pipelines: network analysis of raw reads versus assembly. Results of the two pipelines were compared to evaluate their performance in terms of number and diversity of linkages and accuracy of in silico matrix spike host assignments. No differences were observed in the 16S rRNA gene normalized sul1 concentrations between the DNA fractions. The overall microbial community structure was more similar for iDNA and total DNA compared to eDNA and generally clustered by sampling site. ARGs associated with mobile genetic elements increased in iDNA for the downstream sites. Regarding host assignment, the raw reads pipeline via network analysis identified 247 ARG hosts as compared to 53 hosts identified by assembly pipeline. Other comparisons between the pipelines were made including ARG assignment to taxa containing waterborne pathogens and practical considerations regarding processing time.

Complete genome sequencing of transposon-mediated sulfamethoxazole resistance encoded by the Sul1 gene in multidrug-resistant Nocardia farcinica SZ 1509.

OBJECTIVES: The objective of this study is to explore the molecular basis of trimethoprim-sulfamethoxazole (SXT) resistance in Nocardia, an SXT-resistant N. farcinica strain, named SZ 1509, by whole-genome sequencing. METHODS: Antimicrobial susceptibility testing of SZ 1509 was performed by broth microdilution, Etest, and disk diffusion arrays. Genome sequencing and analysis were performed to discover the SXT resistance determinant and its genetic context. Inverse PCR was conducted to confirm the circular form of the composite transposon. PCR for the sul1 gene was performed among SXT-susceptible isolates. RESULTS: SZ 1509 is resistant to many drugs, especially SXT, with a minimum inhibitory concentration (MIC) of up to 32/608 µg/mL (ratio of 1:19 for trimethoprim: sulfamethoxazole). Its assembled genome consists of one chromosome and four plasmids with a total size of 6 613 629 bp and 71.1% of GC content. The plasmid 2 was found to carry one IS6-composite transposon containing IS6100 carrying the sul1 gene, one tellurite resistance gene TerC, and several transcriptional regulators. Inverse PCR analyses showed its circular form. All 10 SXT-susceptible isolates do not contain sul1. In addition, mutations with strong associations to SXT resistance were not conclusive. CONCLUSION: This is the first study to elucidate the transposon-mediated sulfamethoxazole resistance in N. farcinica. Our results provide insights on acquired drug resistance of N. farcinica and further suggest that the prevalence and correlation of this resistance's determinants in clinical isolates should be continuously monitored to provide effective clinical management of its resultant diseases.

Analysis of Nucleotide Sequences Similarity and Protein Prediction of Some Resistance Genes in Escherichia coli Isolated from Iraqi Patients with Urinary Tract Infections.

Antibiotic resistance leads to a dramatic increase in the morbidity and mortality caused by infectious diseases. Even though estimates vary widely, the economic cost of antimicrobial-resistant bacteria is on a rise. The current aimed to identify the antimicrobial resistance of Escherichia coli (E. coli). In fact, this study focused on the recent deep-learning methods (sequencing) to investigate E. coli antibiotic resistance and their protein sequences. To evaluate antibiotic resistance, the sequencing method could be considered the method of choice. The E. coli was identified by either specific biochemical tests or polymerase chain reaction (PCR) using the 16S rRNA gene. The results of aadA1 gene sequences demonstrated 10 nucleic acid substitutions throughout, as compared to the reference NCBI database (MG385063). Out of the 10 nucleic acid substitutions, 9 missense effects were observed. While the dfrA1 gene sequences illustrated 20 nucleic acid substitutions throughout, compared to the reference NCBI database (KY706080), out of the 20 nucleic acid substitutions, 8 missense effects were observed. Furthermore, the sul1 gene sequences displayed 20 nucleic acid substitutions throughout, in comparison with the reference NCBI database (CP069561), and out of the 20 nucleic acid substitutions, 12 missense effects were detected. The cat1 gene sequences showed 14 nucleic acid substitutions throughout, compared to the reference NCBI database (NC017660), and out of the 14 nucleic acid substitutions, 8 missense effects were observed. The precise point (Missense) mutation in four genes (aadA1, dfrA1, sul1, and cat1) in the expected sequence is interpreted to be the target site of a site-specific recombination mechanism that led to antibiotics resistance in E. coli isolates.

Development of a harmonized method for antimicrobial susceptibility testing of Bordetella avium using broth microdilution and detection of resistance genes.

AIMS: In response to a request from the Clinical and Laboratory Standards Institute (CLSI), the objective of this study was to develop a harmonized method for broth microdilution susceptibility testing of Bordetella (B.) avium, the major causative agent of infectious coryza in poultry. METHODS AND RESULTS: To find a suitable test medium, growth curves with four epidemiologically unrelated B. avium isolates were created in cation-adjusted Mueller-Hinton broth (CAMHB), CAMHB + 2.5% lysed horse blood and veterinary fastidious medium. All isolates showed good growth in CAMHB, therefore MIC values were determined using this medium and the homogeneity of the values was determined. An essential MIC agreement of 99.7% was calculated. Testing of a larger strain collection (n = 49) for their susceptibility to 24 antimicrobials confirmed the suitability of the tested method and revealed some isolates with elevated MICs of florfenicol (n = 1), streptomycin (n = 2), tetracyclines (n = 5), and trimethoprim/sulfamethoxazole (n = 6). PCR assays detected the resistance genes aadA1, dfrB1, floR, sul1, sul2 and tet(A). CONCLUSIONS: The method used enables easy reading and a good reproducibility of MIC values for B. avium. SIGNIFICANCE AND IMPACT OF STUDY: Application of the tested method allows harmonized resistance testing of B. avium and identification of isolates with elevated MIC values.

Emergence of phenotypic and genotypic resistance in the intestinal microbiota of rainbow trout (Oncorhynchus mykiss) exposed long-term to sub-inhibitory concentrations of sulfamethoxazole.

Natural waters are contaminated globally with pharmaceuticals including many antibiotics. In this study, we assessed the acquisition of antimicrobial resistance in the culturable intestinal microbiota of rainbow trout (Oncorhynchus mykiss) exposed for 6 months to sub-inhibitory concentrations of sulfamethoxazole (SMX), one of the most prevalent antibiotics in natural waters. SMX was tested at three concentrations: 3000 µg/L, a concentration that had no observed effect (NOEC) on the in vitro growth of fish intestinal microbiota; 3 µg/L, a theoretical predicted no effect concentration (PNEC) for long-term studies in natural environments; and 0.3 µg/L, a concentration detected in many surveys of surface waters from various countries including the USA. In two independent experiments, the emergence of phenotypic resistance and an increased prevalence of bacteria carrying a sulfonamide-resistance gene (sul1) were observed in SMX-exposed fish. The emergence of phenotypic resistance to1000 mg/L SMX was significant in fish exposed to 3 µg/L SMX and was in large part independent of sul resistance genes. The prevalence of bacteria carrying the sul1 resistance gene increased significantly in the culturable intestinal microbiota of SMX-exposed fish, but the sul1-positive population was in large part susceptible to 1000 mg/L SMX, suggesting that the gene confers a lower resistance level or a growth advantage. The increased prevalence of sul1 bacteria was observed in all groups of SMX-exposed fish. Overall, this study suggests that fish exposed long-term to waters contaminated with low levels of antibiotics serve as reservoir of antimicrobial resistant genes and of resistant bacteria, a potential threat to public health.

Prevalence of trimethoprim/sulfamethoxazole resistance genes among Stenotrophomonas maltophilia clinical isolates in Egypt.

Stenotrophomonas maltophilia is an important multidrug resistant nosocomial pathogen. Trimethoprim/sulfamethoxazole (TMP/SMX) is considered the drug of choice for treatment of S. maltophilia infections, thus emerging resistance to TMP/SMX poses a serious threat. In the present study we aimed to investigate the frequency of TMP/SMX resistance genes (sul1, sul2, dfrA), and to evaluate their relatedness with integron 1 (int1), and insertion sequence common regions (ISCR) among 100 S. maltophilia from different clinical isolates in Egypt. Isolates were identified biochemically and confirmed by VITEK2. Detection of sul1, sul2, and dfrA genes, int1 and ISCR elements was performed by PCR. Among the 16 TMP/SMX resistant isolates, sul1 gene was detected in all of them, and it was associated with int1 gene presence in all resistant isolates. The sul2 gene was detected in 6 out of 16 resistant isolates (37.5%), and only 2 of the 16 resistant isolates (12.5%) harboured dfrA gene. ISCR was detected in 10 of the resistant isolates (62.5%) and in 4 of them it was associated with the presence of sul2 gene. Among the 84 TMP/SMX sensitive isolates, sul1 gene was detected in 15 (17.8%), int1 in 16 (19%) and ISCR in 6 (7.1%). None of the susceptible isolates had sul2 or dfrA genes. These findings point out an increasing frequency of TMP/SMX resistance genes among S. maltophilia clinical isolates in our region, so the adoption of prudent use of S. maltophilia antimicrobial agents and the establishment of a surveillance system are desperately needed.

Dynamics of Diversity and Abundance of Sulfonamide Resistant Bacteria in a Silt Loam Soil Fertilized by Compost.

Although composting is effective in deactivating antibiotic substances in manure, the influence of compost fertilization on the occurrence and dissemination of antibiotic resistance in arable soils remains to be controversial. Herein, the abundance and diversity of two sulfonamide resistance genes (sul1 and sul2) in soil fertilized by compost spiked with two concentrations of sulfadiazine (1 and 10 mg kg-1) were studied intensively by qPCR and high throughput sequencing based on a two-month microcosm experiment. The concentration of sulfadiazine decreased rapidly after spiking from 25% at Day 1 to less than 2.7% at Day 60. Relative abundance of both sul1 and sul2 were significantly higher in soil amended with compost than the non-amended control at Day 1 and slightly decreased with incubation time except for sul2 in the S10 treatment. Soil bacterial communities were transiently shifted by compost fertilization regardless of the presence of sulfadiazine. Relative abundance of genera in three hubs positively interlinked with sul1 and sul2 were significantly higher in compost treated soil than the control at Day 1, 7 and 21, but not at Day 60. High throughput sequencing analyses revealed that most detected (>67% in relative abundance) sul1 and sul2 genotypes sharing >99% similarity with those found in gammaproteobacterial pathogens frequently were commonly present in compost and soil. These results indicated that compost fertilization might increase the abundance rather than diversity of sulfadiazine-resistant populations in soil, which may be facilitated by the presence of sulfadiazine.

Response to sulfur in Schizosaccharomyces pombe.

Sulfur is an essential component of various biologically important molecules, including methionine, cysteine and glutathione, and it is also involved in coping with oxidative and heavy metal stress. Studies using model organisms, including budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), have contributed not only to understanding various cellular processes but also to understanding the utilization and response mechanisms of each nutrient, including sulfur. Although fission yeast can use sulfate as a sulfur source, its sulfur metabolism pathway is slightly different from that of budding yeast because it does not have a trans-sulfuration pathway. In recent years, it has been found that sulfur starvation causes various cellular responses in S. pombe, including sporulation, cell cycle arrest at G2, chronological lifespan extension, autophagy induction and reduced translation. This MiniReview identifies two sulfate transporters in S. pombe, Sul1 (encoded by SPBC3H7.02) and Sul2 (encoded by SPAC869.05c), and summarizes the metabolic pathways of sulfur assimilation and cellular response to sulfur starvation. Understanding these responses, including metabolism and adaptation, will contribute to a better understanding of the various stress and nutrient starvation responses and chronological lifespan regulation caused by sulfur starvation.

Characteristics of bacterial community and ARGs profile in engineered goldfish tanks with stresses of sulfanilamide and copper.

Abuse of antibiotics in aquaculture have been alarming and might aggravate spread of resistance genes in the environment. Holistic ARGs proliferation checks require deeper analyses of coupled absolute abundances in 16S rRNA bacteria communities at the phylum level to detect biomarkers. Sulfanilamide (sul) and copper II sulfate (CuSO4 II) were, therefore, designed and added as separate or combined treatments in 9 replicate engineered goldfish tanks comprising 3 individual sul, 3 CuSO4 II, 3 (sul + CuSO4 II) combinations, and 3 controls within 180 days. The DNA from water and fish guts was sequenced under qPCR to determine 16S rRNA bacteria biomarkers co-occurring with the correspondent ARGs. Combined chemical addition at 0.8-1.5 mg sul + 0.5-1.0 mg CuSO4 II/3 L of tank waters reduced sequenced 16S rRNA bacteria absolute abundances in fish gut and water samples while portraying the biomarkers. Absolute abundances of the entire 16S rRNA bacteria was higher in fish guts (3.4 × 1014-4.9 × 108 copies/g) than water samples (1.5 × 109-2.6 × 1015 copies/L), respectively. Much as sul 1(log) were dominant over intl 1(log) genes, and their fundamental profiles were also higher in the fish guts than water samples; the Spearman's correlation analyses revealed positive relationship (p < 0.01 and r = 0.873) among the biomarkers of both ARG pairs at the phylum level and the physicochemical parameters. In the fish gut and water samples ratios, Bacteroidetes (10-85:12-85%) > Proteobacteria (10-50:15-65%) > Planktomycetes (10-52:8-25%) featured prominently based on LEfSe use as the hot-spotted biomarkers, hence justifying its higher prospects towards innovative environmental microbiological and biotechnological studies.

Sulfate transport mutants affect hydrogen sulfide and sulfite production during alcoholic fermentation.

Hydrogen sulfide is a common wine fault, with a rotten-egg odour, which is directly related to yeast metabolism in response to nitrogen and sulfur availability. In grape juice, sulfate is the most abundant inorganic sulfur compound, which is taken up by yeast through two high-affinity sulfate transporters, Sul1p and Sul2p, and a low affinity transporter, Soa1p. Sulfate contributes to H2 S production under nitrogen limitation, by being reduced via the Sulfur Assimilation Pathway (SAP). Therefore, yeast strains with limited H2 S are highly desirable. We report on the use of toxic analogues of sulfate following ethyl methane sulfate treatment, to isolate six wine yeast mutants that produce no or reduced H2 S and SO2 during fermentation in synthetic and natural juice. Four amino acid substitutions (A99V, G380R, N588K and E856K) in Sul1p were found in all strains except D25-1 which had heterozygous alleles. Two changes were also identified in Sul2p (L268S and A470T). The Sul1p (G380R) and Sul2p (A470T) mutations were chosen for further investigation as these residues are conserved amongst SLC26 membrane proteins (including sulfate permeases). The mutations were introduced into EC1118 using Crispr cas9 technology and shown to reduce accumulation of H2 S and do not result in increased SO2 production during fermentation of model medium (chemically defined grape juice) or Riesling juice. The Sul1p (G380R) and Sul2p (A470T) mutations are newly reported as causal mutations. Our findings contribute to knowledge of the genetic basis of H2 S production as well as the potential use of these strains for winemaking and in yeast breeding programmes.