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757800.9103Dissemination of sulfonamide resistance genes in digester microbiome during anaerobic digestion of food waste leachate. The preeminence of sulfonamide drug resistance genes in food waste (FW) and the increased utilization of high-strength organic FW in anaerobic digestion (AD) to enhance methane production have raised severe public health concerns in wastewater treatment plants worldwide. In this regard, the dissemination patterns of different sulfonamide resistance genes (sul1 and sul2) and their impact on the digester core microbiota during AD of FW leachate (FWL) were evaluated. The presence of various sulfonamide antibiotics (SAs) in FWL digesters improved the final methane yield by 37 % during AD compared with FWL digesters without SAs. Microbial population shifts towards hydrolytic, acidogenic, and acetogenic bacteria in the phyla Actinobacteriota, Bacteroidota, Chloroflexi, Firmicutes, Proteobacteria, and Synergistota occurred due to SA induced substrate digestion and absorption through active transport; butanoate, propanoate, and pyruvate metabolism; glycolysis; gluconeogenesis; the citrate cycle; and pentose phosphate pathway. The initial dominance of Methanosaeta (89-96 %) declined to 47-53 % as AD progressed and shifted towards Methanosarcina (40 %) in digesters with the highest SA concentrations at the end of AD. Dissemination of sul1 depended on class 1 integron gene (intl1)-based horizontal gene transfer to pathogenic members of Chloroflexi, Firmicutes, and Patescibacteria, whereas sul2 was transmitted to Synergistota independent of intl1. Low susceptibility and ability to utilize SAs during methanogenesis shielded methanogenic archaea against selection pressure, thus preventing them from interacting with sul or intl1 genes, thereby minimizing the risk of antibiotic resistance development. The observed emergence of cationic antimicrobial peptide, vancomycin, and β-lactam resistance in the core microbiota during AD of FWL in the presence of SAs suggests that multidrug resistance caused by bacterial transformation could lead to an increase in the environmental resistome through wastewater sludge treatment.202336958158
523410.9061A Multidrug-Resistant Escherichia coli Caused the Death of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis). The rapid increase in drug resistance in recent years has become a significant global public health concern. Escherichia coli are ubiquitous bacteria, widely distributed in various environments. This study isolated a bacterial strain (HD-593) from diseased Chinese soft-shelled turtles (Pelodiscus sinensis). The bacterium was identified based on morphology, biochemical tests, and 16S rRNA sequencing, confirming it as E. coli. Drug susceptibility tests revealed that the HD-593 strain was highly resistant to ceftriaxone, enrofloxacin, doxycycline, sulfadiazine, gentamicin, neomycin, florfenicol, carbenicillin, cefradine, erythromycin, penicillin, ampicillin, midecamycin, and streptomycin. Resistance gene analysis confirmed the presence of quinolone resistance genes (oqxA and oqxB), aminoglycoside resistance genes (aac(3)-II and aphA1), a β-lactam resistance gene (blaTEM), and an acylaminol resistance gene (floR) in HD-593. The median lethal dose (LD50) of HD-593 for P. sinensis was 6.53 × 10(5) CFU/g. Biochemical analysis of serum revealed that HD-593 infection caused a significant reduction in total protein, albumin, and globulin levels, while markedly increasing the levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Histopathological analysis revealed severe intestinal damage characterized by villi detachment and muscle cell necrosis. Additionally, extensive splenocyte necrosis with nuclear marginalization, glomerular swelling, and pronounced hepatic steatosis accompanied by distended sinusoids were observed. This study identified a multidrug-resistant E. coli strain from deceased P. sinensis, suggesting that drug resistance genes may circulate in aquaculture ecosystems, posing potential risks to aquaculture.202540431566
121620.9057Coexistence of multidrug resistance and ESBL encoding genes - bla(TEM), bla(SHV), and bla(CTX-M); its amplification and dispersion in the environment via municipal wastewater treatment plant. Municipal wastewater treatment plants (MWWTPs) are a global source of antibiotic resistance genes (ARGs), collecting wastewater from a variety of sources, including hospital wastewater, domestic wastewater, runoff from agricultural and livestock farms, etc. These sources are contaminated with organic and inorganic pollutants, ARGs and antibiotic-resistant bacteria (ARB). Such pollutants aided eutrophication and encouraged bacterial growth. During bacterial growth horizontal gene transfer (HGT) and vertical gene transfer (VGT) of ARGs and extended-spectrum β-lactamase (ESBL) encoding genes may facilitate, resulting in the spread of antibiotic resistance exponentially. The current study investigated the prevalence of multidrug resistance (MDR) and ESBL encoding genes in various treatment units of MWWTP and their spread in the environment. A total of three sampling sites (BUT, BRO, and BFB) were chosen, and 33 morphologically distinct bacterial colonies were isolated. 14 of the 33 isolates tested positive for antibiotic resistance and were further tested for the coexistence of MDR and ESBL production. The selected 14 isolates showed the highest resistance to trimethoprim (85.71%), followed by ciprofloxacin, azithromycin, and ampicillin (71.42%), tetracycline (57.14%), and vancomycin, gentamicin, and colistin sulphate (50%). A total of 9 isolates (64.28%) were phenotypically positive for ESBL production (BUT2, BUT3, BUT5, BRO1, BRO2, BRO3, BRO4, BRO5 and BFB1). The molecular detection of ESBL encoding genes, i.e. bla(TEM), bla(SHV), and bla(CTX-M) was carried out. The most prevalent gene was bla(TEM) (69.23%), followed by bla(SHV) (46.15%), and bla(CTX-M) (23.07%). In this study, 9 isolates (64.28%) out of 14 showed the coexistence of MDR and ESBL encoding genes, namely BUT3, BUT4, BUT5, BUT6, BUT7, BRO1, BRO2, BRO4, and BFB1. The coexistence of ESBL encoding genes and resistance to other antibiotic classes exacerbates human health and the environment.202438992444
134630.9053High prevalence of multidrug resistant Escherichia coli isolated from fresh vegetables sold by selected formal and informal traders in the most densely populated Province of South Africa. Contaminated fresh produce has increasingly been implicated in foodborne disease outbreaks. As microbiological safety surveillance in South Africa is limited, a total of 545 vegetable samples (spinach, tomato, lettuce, cucumber, and green beans) were purchased from retailers, street traders, trolley vendors and farmers' markets. Escherichia coli, coliforms and Enterobacteriaceae were enumerated and the prevalence of Escherichia coli, Salmonella spp. and Listeria monocytogenes determined. E. coli isolates were characterized phenotypically (antibiotic resistance) and genotypically (diarrheagenic virulence genes). Coliforms, E. coli and Enterobacteriaceae counts were mostly not significantly different between formal and informal markets, with exceptions noted on occasion. When compared to international standards, 90% to 98% tomatoes, 70% to 94% spinach, 82% cucumbers, 93% lettuce, and 80% green bean samples, had satisfactory (≤ 100 CFU/g) E. coli counts. Of the 545 vegetable samples analyzed, 14.86% (n = 81) harbored E. coli, predominantly from leafy green vegetables. Virulence genes (lt, st, bfpA, eagg, eaeA, stx1, stx2, and ipaH) were not detected in the E. coli isolates (n = 67) characterized, however 40.30% were multidrug-resistant. Resistance to aminoglycosides (neomycin, 73.13%; gentamycin, < 10%), penicillins (ampicillin, 38.81%; amoxicillin, 41.79%; augmentin, < 10%), sulfonamides (cotrimoxazole, 22.39%), tetracycline (19.4%), chloramphenicol (11.94%), cephalosporins (cefepime, 34.33%), and carbapenemases (imipenem, < 10%) were observed. This study highlights the need for continued surveillance of multidrug resistant foodborne pathogens in fresh produce retailed formally and informally for potential consumer health risks. PRACTICAL APPLICATION: The results indicate that the microbiological quality of different vegetables were similar per product type, regardless of being purchased from formal retailers or informal street traders, trolley vendors or farmers' markets. Although no pathogenic bacteria (diarrheagenic E. coli, Salmonella spp. or L. monocytogenes) were isolated, high levels of multidrug-resistance was observed in the generic E. coli isolates. These findings highlight the importance of microbiological quality surveillance of fresh produce in formal and informal markets, as these products can be a reservoir of multidrug resistant bacteria harboring antibiotic resistance and virulence genes, potentially impacting human health.202133294974
133840.9052Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan. Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.202438551906
537850.9046Genome-Wide Analysis of Staphylococcus aureus Sequence Type 72 Isolates Provides Insights Into Resistance Against Antimicrobial Agents and Virulence Potential. Staphylococcus aureus sequence type 72 (ST72) is a major community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) that has rapidly entered the hospital setting in Korea, causing mild superficial skin wounds to severe bloodstream infections. In this study, we sequenced and analyzed the genomes of one methicillin-resistant human isolate and one methicillin-sensitive human isolate of ST72 from Korea, K07-204 and K07-561, respectively. We used a subtractive genomics approach to compare these two isolates to other 27 ST72 isolates to investigate antimicrobial resistance (AMR) and virulence potential. Furthermore, we validated genotypic differences by phenotypic characteristics analysis. Comparative and subtractive genomics analysis revealed that K07-204 contains methicillin (mecA), ampicillin (blaZ), erythromycin (ermC), aminoglycoside (aadD), and tetracycline (tet38, tetracycline efflux pump) resistance genes while K07-561 has ampicillin (blaZ) and tetracycline (tet38) resistance genes. In addition to antibiotics, K07-204 was reported to show resistance to lysostaphin treatment. K07-204 also has additional virulence genes (adsA, aur, hysA, icaABCDR, lip, lukD, sdrC, and sdrE) compared to K07-561, which may explain the differential virulence potential of these human isolates of ST72. Unexpectedly, the virulence potential of K07-561 was higher in an in vivo wax-worm infection model than that of K07-204, putatively due to the presence of a 20-fold higher staphyloxanthin concentration than K07-204. Comprehensive genomic analysis of these two human isolates, with 27 ST72 isolates, and S. aureus USA300 (ST8) suggested that acquisition of both virulence and antibiotics resistance genes by ST72 isolates might have facilitated their adaptation from a community to a hospital setting where the selective pressure imposed by antibiotics selects for more resistant and virulent isolates. Taken together, the results of the current study provide insight into the genotypic and phenotypic features of various ST72 clones across the globe, delivering more options for developing therapeutics and rapid molecular diagnostic tools to detect resistant bacteria.202033552024
149460.9043Characterization of a Novel Chromosomal Class C β-Lactamase, YOC-1, and Comparative Genomics Analysis of a Multidrug Resistance Plasmid in Yokenella regensburgei W13. Yokenella regensburgei, a member of the family Enterobacteriaceae, is usually isolated from environmental samples and generally resistant to early generations of cephalosporins. To characterize the resistance mechanism of Y. regensburgei strain W13 isolated from the sewage of an animal farm, whole genome sequencing, comparative genomics analysis and molecular cloning were performed. The results showed that a novel chromosomally encoded class C β-lactamase gene with the ability to confer resistance to β-lactam antibiotics, designated bla (YOC) (-) (1), was identified in the genome of Y. regensburgei W13. Kinetic analysis revealed that the β-lactamase YOC-1 has a broad spectrum of substrates, including penicillins, cefazolin, cefoxitin and cefotaxime. The two functionally characterized β-lactamases with the highest amino acid identities to YOC-1 were CDA-1 (71.69%) and CMY-2 (70.65%). The genetic context of the bla (YOC) (-) (1) -ampR-encoding region was unique compared with the sequences in the NCBI nucleotide database. The plasmid pRYW13-125 of Y. regensburgei W13 harbored 11 resistance genes (bla (OXA) (-) (10), bla (LAP) (-) (2), dfrA14, tetA, tetR, cmlA5, floR, sul2, ant(3″)-IIa, arr-2 and qnrS1) within an ∼34 kb multidrug resistance region; these genes were all related to mobile genetic elements. The multidrug resistance region of pYRW13-125 shared the highest identities with those of two plasmids from clinical Klebsiella pneumoniae isolates, indicating the possibility of horizontal transfer of these resistance genes between bacteria of various origins.202032973731
135470.9034The prevalence, antibiotic resistance and multilocus sequence typing of colistin-resistant bacteria isolated from Penaeus vannamei farms in earthen ponds and HDPE film-lined ponds in China. The aquaculture environment, especially the culture ponds and aquaculture products, is considered to be an important reservoir of colistin resistance genes. However, systematic investigations of colistin resistance in Penaeus vannamei farming in different culture modes are scarce. In this study, a total of 93 non-duplicated samples were collected from P. vannamei farms in five cities in China from 2019 to 2021. The prevalence, antibiotic resistance and multilocus sequence typing (MLST) of colistin-resistant bacteria were measured and analysed. The results showed that among the 1601 isolates in P. vannamei and its environmental samples, the pollution of colistin-resistant bacteria was serious (the overall prevalence was 37.3% and 28.8%, respectively), regardless of the earthen pond or high-density polyethylene (HDPE) film-lined pond. Among 533 isolates, the prevalence of mobile colistin resistance (mcr) genes, mcr-1, was the highest (60%, 320/533), followed by mcr-4 (1.5%, 8/533), mcr-8 (0.9%, 5/533), mcr-10 (0.6%, 3/533) and mcr-7 (0.4%, 2/533). The prevalence of mcr-1 in earthen ponds was significantly higher than that in HDPE film-lined ponds (67.5% vs. 49.1%, p < .001). The dominant strain carrying mcr-1 was Bacillus spp. (54.1%, 173/320), followed by Enterobacter spp. (8.1%, 26/320), Staphylococcus spp. (6.3%, 20/320) and Aeromonas spp. (5.3%, 17/320). The antibiotic resistance profiles of 173 Bacillus spp. varied among different sampling locations and culture types. These isolates were highly resistant to cefepime, ceftriaxone, trimethoprim-sulfamethoxazole and ceftiofur (>45%), and multidrug-resistant isolates were common (62.4%, 108/173). Sequence type (ST) 26 (37/66, 56%) was found to be the most prevalent ST in mcr-1-positive Bacillus cereus isolated from the aquaculture environment. In summary, our study pointed out that it is necessary to continuously monitor antibiotic usage and its residues regardless of the pond types, especially with regard to critical drugs such as colistin.202235841601
209780.9033Effective Photodynamic Therapy with Ir(III) for Virulent Clinical Isolates of Extended-Spectrum Beta-Lactamase Klebsiella pneumoniae. BACKGROUND: The extended-spectrum beta-lactamase (ESBL) Klebsiella pneumoniae is one of the leading causes of health-associated infections (HAIs), whose antibiotic treatments have been severely reduced. Moreover, HAI bacteria may harbor pathogenic factors such as siderophores, enzymes, or capsules, which increase the virulence of these strains. Thus, new therapies, such as antimicrobial photodynamic inactivation (aPDI), are needed. METHOD: A collection of 118 clinical isolates of K. pneumoniae was characterized by susceptibility and virulence through the determination of the minimum inhibitory concentration (MIC) of amikacin (Amk), cefotaxime (Cfx), ceftazidime (Cfz), imipenem (Imp), meropenem (Mer), and piperacillin-tazobactam (Pip-Taz); and, by PCR, the frequency of the virulence genes K2, magA, rmpA, entB, ybtS, and allS. Susceptibility to innate immunity, such as human serum, macrophages, and polymorphonuclear cells, was tested. All the strains were tested for sensitivity to the photosensitizer PSIR-3 (4 µg/mL) in a 17 µW/cm(2) for 30 min aPDI. RESULTS: A significantly higher frequency of virulence genes in ESBL than non-ESBL bacteria was observed. The isolates of the genotype K2+, ybtS+, and allS+ display enhanced virulence, since they showed higher resistance to human serum, as well as to phagocytosis. All strains are susceptible to the aPDI with PSIR-3 decreasing viability in 3log10. The combined treatment with Cfx improved the aPDI to 6log10 for the ESBL strains. The combined treatment is synergistic, as it showed a fractional inhibitory concentration (FIC) index value of 0.15. CONCLUSIONS: The aPDI effectively inhibits clinical isolates of K. pneumoniae, including the riskier strains of ESBL-producing bacteria and the K2+, ybtS+, and allS+ genotype. The aPDI with PSIR-3 is synergistic with Cfx.202133922077
134790.9033Microbiological quality and antimicrobial resistance characterization of Salmonella spp. in fresh milk value chains in Ghana. Consumer perception of poor hygiene of fresh milk products is a major barrier to promotion of milk consumption as an intervention to alleviate the burden of malnutrition in Ghana. Fresh milk is retailed raw, boiled, or processed into unfermented cheese and spontaneously fermented products in unlicensed outlets. In this study, we have determined microbiological quality of informally retailed fresh milk products and characterized the genomic diversity and antimicrobial resistance (AMR) patterns of non-typhoidal Salmonella (NTS) in implicated products. A total of 159 common dairy products were purchased from five traditional milk markets in Accra. Samples were analysed for concentrations of aerobic bacteria, total and fecal coliforms, Escherichia coli, staphylococci, lactic acid bacteria and yeast and moulds. The presence of Salmonella, E. coli O157:H7, Listeria monocytogenes and Staphylococcus aureus were determined. AMR of Salmonella against 18 antibiotics was experimentally determined. Genome sequencing of 19 Salmonella isolates allowed determination of serovars, antigenic profiles, prediction of AMR genes in silico and inference of phylogenetic relatedness between strains. Raw and heat-treated milk did not differ significantly in overall bacterial quality (P = 0.851). E. coli O157:H7 and Staphylococcus aureus were present in 34.3% and 12.9% of dairy products respectively. Multidrug resistant (MDR) Salmonella enterica serovars Muenster and Legon were identified in 11.8% and 5.9% of unfermented cheese samples respectively. Pan genome analysis revealed a total of 3712 core genes. All Salmonella strains were resistant to Trimethoprim/Sulfamethoxazole, Cefoxitin, Cefuroxime Axetil and Cefuroxime. Resistance to Chloramphenicol (18%) and Ciprofloxacin (100%), which are first line antibiotics used in treatment of NTS bacteremia in Ghana, was evident. AMR was attributed to presence and/or mutations in the following genes: golS, sdiA for cephalosporins, aac(6')-Iy, ant(9) for aminoglycosides, mdtK, gyrA, gyrB, parC, parE for quinolones and cat1, cat4 for phenicols. Phylogenetic analysis based on accessory genes clustered S. Legon strains separately from the S. Muenster strains. These strains were from different markets suggesting local circulation of related strains. Our study justifies consumer resistance to consumption of unripened soft cheese without further lethal heat treatment, and provides evidence that supports the Ghana Health Service recommendation for use of 3rd generation cephalosporins for the treatment of MDR NTS infections.201829680695
7989100.9032Feasibility of sulfate-calcined eggshells for removing pathogenic bacteria and antibiotic resistance genes from landfill leachates. High abundance of human pathogen and antibiotic resistance genes (ARGs) in landfill leachate has become an emerging threat against human health. Therefore, sulfate- and calcination-modified eggshells as green agricultural bioresource were applied to test the feasibility of removing pathogenic bacteria and ARGs from leachate. The highest removal of Escherichia coli (E. coil) and gentamycin resistant gene (gmrA) from artificial contaminated landfill leachate was achieved by the application of eggshell with combined treatment of sulfate and calcination. The 16S and gmrA gene copies of E. coil declined significantly from 1.78E8±8.7E6 and 4.12E8±5.9E6 copies mL(-1) to 1.32E7±2.6E6 and 2.69E7±7.2E6 copies mL(-1), respectively, within 24h dynamic adsorption equilibrium process (p<0.05). Moreover, according to the Langmuir kinetic model, the greatest adsorption amount (1.56×10(9) CFU E. coil per gram of modified eggshells) could be obtained at neutral pH of 7.5. The optimal adsorption eggshells were then screened to the further application in three typical landfill leachates in Nanjing, eastern China. Significant decrease in species and abundance of pathogenic bacteria and ARGs (tet, sul, erm, qnr, and ampC) indicated its great efficiency to purify landfill leachates. This study demonstrated that sulfate-calcined eggshells can be an environmentally-friendly and highly efficient bioadsorbent to the management of reducing dissemination risk of pathogen and ARGs in landfill leachate.201728343745
1994110.9031Antibiotic resistance genes, mobile elements, virulence genes, and phages in cultivated ESBL-producing Escherichia coli of poultry origin in Kwara State, North Central Nigeria. The paucity of information on the genomic diversity of drug-resistant bacteria in most food-producing animals, including poultry in Nigeria, has led to poor hazard characterization and the lack of critical control points to safeguard public health. Hence, this study used whole genome sequencing (WGS) to assess the presence and the diversity of antibiotic resistance genes, mobile genetic elements, virulence genes, and phages in Extended Spectrum Beta Lactamase producing Escherichia coli (ESBL - E. coli) isolates obtained from poultry via the EURL guideline of 2017 in Ilorin, Nigeria. The prevalence of ESBL - E. coli in poultry was 10.5 % (n = 37/354). The phenotypic antibiotic susceptibility testing showed that all the ESBL- E. coli isolates were multi-drug resistant (MDR). The in-silico analysis of the WGS raw-read data from 11 purposively selected isolates showed that the isolates had a wide array of ARGs that conferred resistance to beta-lactam antibiotics, and 8 other classes of antibiotics (fluoroquinolones, foliate pathway antagonists, aminoglycoside, phenicol, tetracycline, epoxide, macrolides, and rifamycin). All the ARGs were in the bacterial chromosome except in two isolates where plasmid-mediated quinolone resistance (PMQR) was detected. Two isolates carried the gyrAp.S83L mutation which confers resistance to certain fluoroquinolones. The mobilome consisted of several Col-plasmids and the predominant IncF plasmids belonged to the IncF64:A-:B27 sequence type. The virulome consisted of genes that function as adhesins, iron acquisition genes, toxins, and protectins. Intact phages were found in 8 of the 11 isolates and the phageome consisted of representatives of four families of viruses: Myoviridae (62.5 %, n = 5/8), Siphoviridae (37.5 %, n = 3/8), Inoviridae (12.5 %, n = 1), and Podoviridae (12.5 %, n = 1/8). ESBL - E. coli isolates harboured 1-5 intact phages and no ARGs were identified on any of the phages. Although five of the isolates belonged to phylogroup A, the isolates were diverse as they belonged to different serotype and sequence types. Our findings demonstrate the high genomic diversity of ESBL - E. coli of poultry origin in Ilorin, Nigeria. These diverse isolates harbor clinically relevant ARGs, mobile elements, virulence genes, and phages that may have detrimental zoonotic potentials on human health.202336738714
7756120.9031Mitigation of antibiotic resistance: the efficiency of a hybrid subsurface flow constructed wetland in the removal of resistant bacteria in wastewater. This research investigates the effectiveness of a lab-scale hybrid subsurface flow constructed wetland (HSSFCW) for removing wastewater contaminants, including antibiotic-resistant bacteria (ARB), genes (ARGs) and antibiotics. The results suggested that HSSFCW demonstrated a high removal efficiency for COD (89%) and BOD (88.9%), while lower efficiencies were observed for salts, TDS, EC, and TKN. Further, various bacteria such as Enterobacter cloacae, Serratia liquefaciens and Serratia odorifera were detected in the plant rhizosphere, while Acinetobacter baumanii and Staphylococcus spp. were identified as biofilm formers on the wetland media. The mean removal efficiency of 70.44, 65.99, 70.66 and 51.49% was observed for total heterotrophic bacteria; Cefixime (Cef)-, Ciprofloxacin (Cip)-, and Linezolid (Lzd)-resistant bacteria. Upon chlorination of effluent samples, Cef-, Cip- and Lzd-resistant bacteria were effectively inactivated at 30, 15 and 7.5 mg Cl(2) min/L, respectively. The wetland achieved a removal efficiency of 83.85% for Cip and 100% for Lzd at week 12 with p = 0.040 and p < 0.001, respectively. Further, a log reduction of 0.66 for 16S, 0.82 for blaTEM, 0.61 for blaCTX, and 0.48 for blaOXA was observed. Thus, HSSFCW was observed to be efficient in removing organic contaminants, ARBs, ARGs and antibiotics from domestic wastewater and can be upgraded under natural environments.202540536145
1996130.9030Conjugation of plasmid harboring bla (NDM-1) in a clinical Providencia rettgeri strain through the formation of a fusion plasmid. Providencia rettgeri has recently gained increased importance owing to the New Delhi metallo-β-lactamase (NDM) and other β-lactamases produced by its clinical isolates. These enzymes reduce the efficiency of antimicrobial therapy. Herein, we reported the findings of whole-genome sequence analysis and a comprehensive pan-genome analysis performed on a multidrug-resistant P. rettgeri 18004577 clinical strain recovered from the urine of a hospitalized patient in Shandong, China, in 2018. Providencia rettgeri 18004577 was found to have a genome assembly size of 4.6 Mb with a G + C content of 41%; a circular plasmid p18004577_NDM of 273.3 Kb, harboring an accessory multidrug-resistant region; and a circular, stable IncT plasmid p18004577_Rts of 146.2 Kb. Additionally, various resistance genes were identified in its genome, including bla (NDM-1), bla (OXA-10), bla (PER-4), aph(3')-VI, ant(2'')-Ia, ant(3')-Ia, sul1, catB8, catA1, mph(E), and tet. Conjugation experiments and whole-genome sequencing revealed that the bla (NDM-1) gene could be transferred to the transconjugant via the formation of pJ18004577_NDM, a novel hybrid plasmid. Based on the genetic comparison, the main possible formation process for pJ18004577_NDM was the insertion of the [ΔISKox2-IS26-ΔISKox2]-aph(3')-VI-bla (NDM-1) translocatable unit module from p18004577_NDM into plasmid p18004577_Rts in the Russian doll insertion structure (ΔISKox2-IS26-ΔISKox2), which played a role similar to that of IS26 using the "copy-in" route in the mobilization of [aph(3')-VI]-bla (NDM-1). The array, multiplicity, and diversity of the resistance and virulence genes in this strain necessitate stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of the resistance genes. Roary analysis based on 30 P. rettgeri strains pan genome identified 415 core, 756 soft core, 5,744 shell, and 12,967 cloud genes, highlighting the "close" nature of P. rettgeri pan-genome. After a comprehensive pan-genome analysis, representative biological information was revealed that included phylogenetic distances, presence or absence of genes across the P. rettgeri bacteria clade, and functional distribution of proteins. Moreover, pan-genome analysis has been shown to be an effective approach to better understand P. rettgeri bacteria because it helps develop various tailored therapeutic strategies based on their biological similarities and differences.202236687647
6046140.9028Safety Evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI. Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.201829747442
1218150.9028Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania. We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.202031999747
6150160.9026Redox biotransformation of arsenic along with plant growth promotion by multi-metal resistance Pseudomonas sp. MX6. Remediation of toxic metal-polluted sites by microorganisms is an environment-friendly remediation technique. Multi-metal-resistant bacteria were isolated from a wastewater treatment plant showing resistance against As(III), As(V), Cr, Co, Cu, Cd, Hg, Ni, Pb, Se and Zn. Maximum resistance against all metals was shown by the bacterial isolate MX-6 (As 20mM, Cd 30mM, Cr 5.0mM, Co 25mM, Cu 25mM, Ni 20mM, Zn 30mM, Pb 15mM, Se 20mM and Hg 2.5mM), which was identified as Pseudomonas sp. through 16S rDNA sequencing. Pseudomonas sp. MX-6 reduced 506μM As(V) and also oxidized 160μM As(III). The genes for As, Cd, Se and Zn resistance in Pseudomonas sp. MX-6 were found to be plasmid borne, as indicated by transformation. Pseudomonas sp. MX-6 produced 49.37μg·mL(-1) IAA and was also positive for HCN production and phosphate solubilisation. The bacterial isolate also supported Vigna radiata growth, both in the absence and presence of the aforementioned metals. Such bacteria can be used as biofertilizers to reclaim the polluted lands and to enhance crop production in metal-contaminated soils.201728684222
813170.9026Fighting against evolution of antibiotic resistance by utilizing evolvable antimicrobial drugs. Antibiotic resistance is a worldwide public health problem (Bush et al. in Nat Rev Microbiol 9:894-896, 2011). The lack of effective therapies against resistant bacteria globally leads to prolonged treatments, increased mortality, and inflating health care costs (Oz et al. in Mol Biol Evol 31:2387-2401, 2014; Martinez in Science 321:365-367, 2008; Lipsitch et al. in Proc Natl Acad Sci USA 97:1938-1943, 2000; Taubes in Science 321:356-361, 2008; Laxminarayan et al. in Lancet, 2016; Laxminarayan et al. in Lancet Infect Dis 13:1057-1098, 2013). Current efforts towards a solution of this problem can be boiled down to two main strategies: (1) developing of new antimicrobial agents and (2) searching for smart strategies that can restore or preserve the efficacy of existing antimicrobial agents. In this short review article, we discuss the need for evolvable antimicrobial agents, focusing on a new antimicrobial technology that utilizes peptide-conjugated phosphorodiamidate morpholino oligomers to inhibit the growth of pathogenic bacteria by targeting bacterial genes.201728497241
2373180.9026Antimicrobial Resistance Profiles, Virulence Determinants, and Biofilm Formation in Enterococci Isolated from Rhesus Macaques (Macaca mulatta): A Potential Threat for Wildlife in Bangladesh? Enterococci are commensal bacteria that inhabit the digestive tracts of animals and humans. The transmission of antibiotic-resistant genes through human-animal contact poses a potential public health risk worldwide, as zoonoses from wildlife reservoirs can occur on every continent. The purpose of this study was to detect Enterococcus spp. in rhesus macaques (Macaca mulatta) and to investigate their resistance patterns, virulence profiles, and biofilm-forming ability. Conventional screening of rectal swabs (n = 67) from macaques was followed by polymerase chain reaction (PCR). The biofilm-forming enterococci were determined using the Congo red agar plate assay. Using the disk diffusion test (DDT), antibiogram profiles were determined, followed by resistance and virulence genes identification by PCR. PCR for bacterial species confirmation revealed that 65.7% (44/67) and 22.4% (15/67) of the samples tested positive for E. faecalis and E. faecium, respectively. All the isolated enterococci were biofilm formers. In the DDT, enterococcal isolates exhibited high to moderate resistance to penicillin, rifampin, ampicillin, erythromycin, vancomycin, and linezolid. In the PCR assays, the resistance gene bla(TEM) was detected in 61.4% (27/44) of E. faecalis and 60% (9/15) of E. faecium isolates. Interestingly, 88.63 % (39/44) of E. faecalis and 100% (15/15) of E. faecium isolates were phenotypically multidrug-resistant. Virulence genes (agg, fsrA, fsrB, fsrC, gelE, sprE, pil, and ace) were more frequent in E. faecalis compared to E. faecium; however, isolates of both Enterococcus spp. were found negative for the cyl gene. As far as we know, the present study has detected, for the first time in Bangladesh, the presence of virulence genes in MDR biofilm-forming enterococci isolated from rhesus macaques. The findings of this study suggest employing epidemiological surveillance along with the one-health approach to monitor these pathogens in wild animals in Bangladesh, which will aid in preventing their potential transmission to humans.202337508046
2100190.9025Prevalence of Bacteria and Antimicrobial Resistance Genes in Hospital Water and Surfaces. Purpose Antimicrobial resistance (AMR) has become a worldwide environmental and public health problem, causing more than 250,000 deaths per year. Unregulated usage, unsafe hospital practices, and misuse in veterinary contribute to the development of multidrug resistance in various bacteria. Hospital water was hypothesized to be a hotspot for AMR transmission because of (1) increased exposure to antibiotic load, (2) poor drainage and sanitation system, (3) interaction between environmental and clinical microbes. The purpose of the research was to assess the biodiversity and AMR in hospital tap waters. Methodology In this study, the microflora of the hospital tap water and hospital surfaces was observed by obtaining water samples from the intensive care unit (ICU), surgical wards, and washrooms. These were processed through membrane filtration and spread on seven different media (Aeromonas Medium, Azide Dextrose Agar, MacConkey Agar, Mannitol Salt Agar, Pseudomonas Cetrimide Agar, Salmonella Shigella Agar, and Thiosulfate Citrate Bile Salts Sucrose Agar). Surface samples were collected from the faucet, basin, and drain and directly spread on the media plates. Isolates were identified using standard bacteriological and biochemical tests. Kirby-Bauer disk diffusion method was performed using 21 antibiotic disks from 10 different antibiotic classes. They included ampicillin (AMP), amoxicillin (AML), piperacillin-tazobactam (TZP), cefipime (FEP), cefoxitin (FOX), ceftazidime (CAZ), ceftriaxone (CRO), imipenem (IMP), meropenem (MEM), ciprofloxacin (CIP), moxifloxacin (MXF), levofloxacin (LEV), amikacin (AK), gentamicin (CN), tigecycline (TGC), aztreonam (ATM), erythromycin (E), clindamycin (DA), rifampicin (RD), colistin (CT), and chloramphenicol (C). The results were interpreted according to EUCAST guidelines for the antibiogram of the isolates; 38 isolates were selected out of 162 based on different parameters for genotyping and detection of six beta-lactamase genes (blaSHV, blaTEM, blaCTX-M, blaOXA, blaKPC, blaNDM). Results Among these 162 isolates, 82 were obtained from water sources and 80 were collected from surfaces (faucet, basin, drain). The isolates included a variety of bacteria including Aeromonas spp. (20%), Klebsiella spp. (13%), Staphylococcus aureus (13%), Pseudomonas spp.(10%), Escherichia coli (9%), Vibrio spp. (8%), Enterococcus spp. (6%), Shigella spp. (6%), Salmonella spp. (4%), Acinetobacter spp. (3%), Staphylococcus epidermitis (3%), Streptococci spp. (2%), Proteus spp. (1%), Citrobacter spp. (1%), and Serratia spp. (1%). A diverse range of microbes were identified including clinically relevant bacteria, which shows that the urban water cycle is already contaminated with multidrug-resistant microflora of the hospital settings. Macrolide and lincosamide showed the highest resistance followed by penicillin, monobactam, and cephalosporins. blaSHV and blaTEM were prevalent in samples. blaNDM was also found which manifests as a real threat since it causes resistance against carbapenems and colistin, antibiotics reserved as a last resort against infections. Conclusions This study presented the ground reality of antibiotic resistance in Pakistan and how its subsequent spread poses a great threat to the strides made in the field of medicine and public health. Strict regulations regarding antibiotic usage, hospital effluent, and urban water sanitation must be imposed to curb the devastating effects of this increasing phenomenon.202134790487