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476100.9976Antimicrobial resistance and biofilm formation of penile prosthesis isolates: insights from in-vitro analysis. BACKGROUND: Inflatable penile prostheses (IPPs) have been shown to harbor biofilms in the presence and absence of infection despite exposure to various antimicrobials. Microbes persisting on IPPs following antibiotic exposure have not been adequately studied to assess biofilm formation capacity and antibiotic resistance. AIM: In this study, we aimed to assess these properties of microbes obtained from explanted infected and non-infected IPPS using an in vitro model. METHODS: 35 bacterial isolates were grown and tested against various single-agent or multiple agent antibiotic regimens including: bacitracin, cefaclor, cefazolin, gentamicin, levofloxacin, trimethoprim-sulfamethoxazole, tobramycin, vancomycin, piperacillin/tazobactam, gentamicin + piperacillin/tazobactam, gentamicin + cefazolin, and gentamicin + vancomycin. Zones of inhibition were averaged for each sample site and species. Statistics were analyzed with Holm's corrected, one-sample t-tests against a null hypothesis of 0. Isolates were also allowed to form biofilms in a 96-well polyvinyl plate and absorbance was tested at 570 nm using a microplate reader. OUTCOMES: Resistance was determined via clinical guidelines or previously established literature, and the mean and standard deviation of biofilm absorbance values were calculated and normalized to the optical density600 of the bacterial inoculum. RESULTS: Every species tested was able to form robust biofilms with the exception of Staphylococcus warneri. As expected, most bacteria were resistant to common perioperative antimicrobial prophylaxis. Gentamicin dual therapy demonstrated somewhat greater efficacy. STRENGTHS AND LIMITATIONS: This study examines a broad range of antimicrobials against clinically obtained bacterial isolates. However, not all species and antibiotics tested had standardized breakpoints, requiring the use of surrogate values from the literature. The microbes included in this study and their resistance genes are expectedly biased towards those that survived antibiotic exposure, and thus reflect the types of microbes which might "survive" in vivo exposure following revisional surgery. CLINICAL TRANSLATION: Despite exposure to antimicrobials, bacteria isolated during penile prosthesis revision for both infected and non-infected cases exhibit biofilm forming capacity and extensive antibiotic resistance patterns in vitro. These microbes merit further investigation to understand when simple colonization vs re-infection might occur. CONCLUSIONS: Although increasing evidence supports the concept that all IPPs harbor biofilms, even in the absence of infection, a deeper understanding of the characteristics of bacteria that survive revisional surgery is warranted. This study demonstrated extensive biofilm forming capabilities, and resistance patterns among bacteria isolated from both non-infected and infected IPP revision surgeries. Further investigation is warranted to determine why some devices become infected while others remain colonized but non-infected.202540062463
580310.9974Face mask sampling reveals antimicrobial resistance genes in exhaled aerosols from patients with chronic obstructive pulmonary disease and healthy volunteers. INTRODUCTION: The degree to which bacteria in the human respiratory tract are aerosolised by individuals is not established. Building on our experience sampling bacteria exhaled by individuals with pulmonary tuberculosis using face masks, we hypothesised that patients with conditions frequently treated with antimicrobials, such as chronic obstructive pulmonary disease (COPD), might exhale significant numbers of bacteria carrying antimicrobial resistance (AMR) genes and that this may constitute a previously undefined risk for the transmission of AMR. METHODS: Fifteen-minute mask samples were taken from 13 patients with COPD (five paired with contemporaneous sputum samples) and 10 healthy controls. DNA was extracted from cell pellets derived from gelatine filters mounted within the mask. Quantitative PCR analyses directed to the AMR encoding genes: blaTEM (β-lactamase), ErmB (target methylation), mefA (macrolide efflux pump) and tetM (tetracycline ribosomal protection protein) and six additional targets were investigated. Positive signals above control samples were obtained for all the listed genes; however, background signals from the gelatine precluded analysis of the additional targets. RESULTS: 9 patients with COPD (69%), aerosolised cells containing, in order of prevalence, mefA, tetM, ErmB and blaTEM, while three healthy controls (30%) gave weak positive signals including all targets except blaTEM. Maximum estimated copy numbers of AMR genes aerosolised per minute were mefA: 3010, tetM: 486, ErmB: 92 and blaTEM: 24. The profile of positive signals found in sputum was not concordant with that in aerosol in multiple instances. DISCUSSION: We identified aerosolised AMR genes in patients repeatedly exposed to antimicrobials and in healthy volunteers at lower frequencies and levels. The discrepancies between paired samples add weight to the view that sputum content does not define aerosol content. Mask sampling is a simple approach yielding samples from all subjects and information distinct from sputum analysis. Our results raise the possibility that patient-generated aerosols may be a significant means of AMR dissemination that should be assessed further and that consideration be given to related control measures.201830271606
364720.9973Biofilm Forming Antibiotic Resistant Gram-Positive Pathogens Isolated From Surfaces on the International Space Station. The International Space Station (ISS) is a closed habitat in a uniquely extreme and hostile environment. Due to these special conditions, the human microflora can undergo unusual changes and may represent health risks for the crew. To address this problem, we investigated the antimicrobial activity of AGXX®, a novel surface coating consisting of micro-galvanic elements of silver and ruthenium along with examining the activity of a conventional silver coating. The antimicrobial materials were exposed on the ISS for 6, 12, and 19 months each at a place frequently visited by the crew. Bacteria that survived on the antimicrobial coatings [AGXX® and silver (Ag)] or the uncoated stainless steel carrier (V2A, control material) were recovered, phylogenetically affiliated and characterized in terms of antibiotic resistance (phenotype and genotype), plasmid content, biofilm formation capacity and antibiotic resistance transferability. On all three materials, surviving bacteria were dominated by Gram-positive bacteria and among those by Staphylococcus, Bacillus and Enterococcus spp. The novel antimicrobial surface coating proved to be highly effective. The conventional Ag coating showed only little antimicrobial activity. Microbial diversity increased with increasing exposure time on all three materials. The number of recovered bacteria decreased significantly from V2A to V2A-Ag to AGXX®. After 6 months exposure on the ISS no bacteria were recovered from AGXX®, after 12 months nine and after 19 months three isolates were obtained. Most Gram-positive pathogenic isolates were multidrug resistant (resistant to more than three antibiotics). Sulfamethoxazole, erythromycin and ampicillin resistance were most prevalent. An Enterococcus faecalis strain recovered from V2A steel after 12 months exposure exhibited the highest number of resistances (n = 9). The most prevalent resistance genes were ermC (erythromycin resistance) and tetK (tetracycline resistance). Average transfer frequency of erythromycin, tetracycline and gentamicin resistance from selected ISS isolates was 10(-5) transconjugants/recipient. Most importantly, no serious human pathogens such as methicillin resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococci (VRE) were found on any surface. Thus, the infection risk for the crew is low, especially when antimicrobial surfaces such as AGXX® are applied to surfaces prone to microbial contamination.201930941112
568730.9973The effect of short-course antibiotics on the resistance profile of colonizing gut bacteria in the ICU: a prospective cohort study. BACKGROUND: The need for early antibiotics in the intensive care unit (ICU) is often balanced against the goal of antibiotic stewardship. Long-course antibiotics increase the burden of antimicrobial resistance within colonizing gut bacteria, but the dynamics of this process are not fully understood. We sought to determine how short-course antibiotics affect the antimicrobial resistance phenotype and genotype of colonizing gut bacteria in the ICU by performing a prospective cohort study with assessments of resistance at ICU admission and exactly 72 h later. METHODS: Deep rectal swabs were performed on 48 adults at the time of ICU admission and exactly 72 h later, including patients who did and did not receive antibiotics. To determine resistance phenotype, rectal swabs were cultured for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). In addition, Gram-negative bacterial isolates were cultured against relevant antibiotics. To determine resistance genotype, quantitative PCR (qPCR) was performed from rectal swabs for 87 established resistance genes. Within-individual changes in antimicrobial resistance were calculated based on culture and qPCR results and correlated with exposure to relevant antibiotics (e.g., did β-lactam antibiotic exposure associate with a detectable change in β-lactam resistance over this 72-h period?). RESULTS: Of 48 ICU patients, 41 (85%) received antibiotics. Overall, there was no increase in the antimicrobial resistance profile of colonizing gut bacteria during the 72-h study period. There was also no increase in antimicrobial resistance after stratification by receipt of antibiotics (i.e., no detectable increase in β-lactam, vancomycin, or macrolide resistance regardless of whether patients received those same antibiotics). This was true for both culture and PCR. Antimicrobial resistance pattern at ICU admission strongly predicted resistance pattern after 72 h. CONCLUSIONS: Short-course ICU antibiotics made little detectable difference in the antimicrobial resistance pattern of colonizing gut bacteria over 72 h in the ICU. This provides an improved understanding of the dynamics of antimicrobial resistance in the ICU and some reassurance that short-course antibiotics may not adversely impact the stewardship goal of reducing antimicrobial resistance.202032646458
528440.9972Long-term impact of oral surgery with or without amoxicillin on the oral microbiome-A prospective cohort study. Routine postoperative antibiotic prophylaxis is not recommended for third molar extractions. However, amoxicillin still continues to be used customarily in several clinical practices worldwide to prevent infections. A prospective cohort study was conducted in cohorts who underwent third molar extractions with (group EA, n = 20) or without (group E, n = 20) amoxicillin (250 mg three times daily for 5 days). Further, a control group without amoxicillin and extractions (group C, n = 17) was included. Salivary samples were collected at baseline, 1-, 2-, 3-, 4-weeks and 3 months to assess the bacterial shift and antibiotic resistance gene changes employing 16S rRNA gene sequencing (Illumina-Miseq) and quantitative polymerase chain reaction. A further 6-month follow-up was performed for groups E and EA. Seven operational taxonomic units reported a significant change from baseline to 3 months for group EA (adjusted p < 0.05). No significant change in relative abundance of bacteria and β-lactamase resistance genes (TEM-1) was observed over 6 months for any group (adjusted p > 0.05). In conclusion, the salivary microbiome is resilient to an antibiotic challenge by a low-dose regimen of amoxicillin. Further studies evaluating the effect of routinely used higher dose regimens of amoxicillin on gram-negative bacteria and antibiotic resistance genes are warranted.201931822712
352950.9972High dietary zinc supplementation increases the occurrence of tetracycline and sulfonamide resistance genes in the intestine of weaned pigs. BACKGROUND: Dietary zinc oxide is used in pig nutrition to combat post weaning diarrhoea. Recent data suggests that high doses (2.5 g/kg feed) increase the bacterial antibiotic resistance development in weaned pigs. Therefore, the aim of this study was to investigate the development of enterobacterial antibiotic resistance genes in the intestinal tract of weaned pigs. FINDINGS: Weaned pigs were fed diets for 4 weeks containing 57 (low), 164 (intermediate) or 2425 (high) mg kg(-1) analytical grade ZnO. DNA extracts from stomach, mid-jejunum, terminal ileum and colon ascendens were amplified by qPCR assays to quantify copy numbers for the tetracycline (tetA) and sulfonamide (sul1) resistance genes in Gram-negative bacteria. Overall, the combined data (n = 336) showed that copy numbers for tetracycline and sulfonamide resistance genes were significantly increased in the high zinc treatment compared to the low (tetA: p value < 10(-6); sul1: p value = 1 × 10(-5)) or intermediate (tetA: P < 1.6 × 10(-4); sul1: P = 3.2 × 10(-4)) zinc treatment. Regarding the time dependent development, no treatment effects were seen 1 week after weaning, but significant differences between high and low/intermediate zinc treatments evolved 2 weeks after weaning. The increased number of tetA and sul1 copies was not confined to the hind gut, but was already present in stomach contents. CONCLUSIONS: The results of this study suggest that the use of high doses of dietary zinc beyond 2 weeks after weaning should be avoided in pigs due to the possible increase of antibiotic resistance in Gram-negative bacteria.201526322131
765560.9971Impact of manure fertilization on the abundance of antibiotic-resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest. Consumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.201323851089
255070.9971Comparative gut microbiota and resistome profiling of intensive care patients receiving selective digestive tract decontamination and healthy subjects. BACKGROUND: The gut microbiota is a reservoir of opportunistic pathogens that can cause life-threatening infections in critically ill patients during their stay in an intensive care unit (ICU). To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed "selective decontamination of the digestive tract" (SDD), is used in some countries where it improves clinical outcome in ICU patients. Yet, the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes ("the resistome") of ICU patients during SDD and of healthy subjects. RESULTS: From ten patients that were acutely admitted to the ICU, 30 fecal samples were collected during ICU stay. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. Feces from ten healthy subjects were collected twice, with a 1-year interval. Gut microbiota and resistome composition were determined using 16S rRNA gene phylogenetic profiling and nanolitre-scale quantitative PCRs. The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by lower microbial diversity, decreased levels of Escherichia coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes (aac(6')-Ii, ermC, qacA, tetQ), providing resistance to aminoglycosides, macrolides, disinfectants, and tetracyclines, respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene (catA) and a tetracycline resistance gene (tetW) were more abundant in healthy subjects. CONCLUSIONS: The gut microbiota of SDD-treated ICU patients deviated strongly from the gut microbiota of healthy subjects. The negative effects on the resistome were limited to selection for four resistance genes. While it was not possible to disentangle the effects of SDD from confounding variables in the patient cohort, our data suggest that the risks associated with ICU hospitalization and SDD on selection for antibiotic resistance are limited. However, we found evidence indicating that recolonization of the gut by antibiotic-resistant bacteria may occur upon ICU discharge and cessation of SDD.201728803549
352680.9971The impact of antibiotic residues on resistance patterns in leek at harvest. When crops are cultivated on fields fertilized with animal manure, the risk exists that plants may take up antibiotic residues and may be exposed to antibiotic resistance genes and antibiotic resistant bacteria. During cultivation in a greenhouse pot experiment, leek (Allium porrum) was fertilized with either pig slurry or mineral fertilizer and exposed to either no antibiotics, doxycycline (10,000 μg/kg manure), sulfadiazine (1000 μg/kg manure), or lincomycin (1000 μg/kg manure). At harvest, 4.5 months later, lincomycin, sulfadiazine or doxycycline were not detected in any of the leek samples nor in their corresponding soil samples. Further, antimicrobial susceptibility testing was performed on 181 Bacillus cereus group isolates and 52 Pseudomonas aeruginosa isolates from the grown leek. For the B. cereus group isolates, only a small shift in MIC50 for lincomycin was observed among isolates from the lincomycin and control treatment. For P. aeruginosa, only in the setup with doxycycline treatment a higher MIC50 for doxycycline was observed compared to the control, specifically the isolates selected from growth media supplemented with 8 mg/L doxycycline. Nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) were investigated at harvest in the leek and soil samples. In the leek samples, none of the antibiotic resistance genes were detected. In the soil samples fertilized with pig slurry, the genes erm(B), erm(F), tet(M), sul2, tet(W) and tet(O) were detected in significantly higher copy numbers in the lincomycin treatment as compared to the other antibiotic treatments. This could be due to a shift in soil microbiota induced by the addition of lincomycin. The results of this study indicate that consumption of leek carries a low risk of exposure to antibiotic residues or antibiotic resistance to doxycycline, sulfadiazine or lincomycin.202337215782
314690.9971Resistomes from oxytetracycline-treated pigs are readily transferred to untreated pen mates. Pork is currently a major part of Danish food export and is also a key dietary source of protein across the world. Industrial pork production, however, comes with high antibiotic usage in many countries, including Denmark. This has created consumer demand for meat Raised Without Antibiotics (RWA). Previous work has demonstrated that levels of antibiotic resistance genes (ARGs) are indeed increased in antibiotically treated animals, but also suggest that these ARGs are transferred to untreated pen-mates. In a Danish commercial farm, we studied four groups of physically separated pigs: one group of only antibiotic treated pigs (n = 20), one group of only untreated pigs (n = 30 total, n = 15 analysed), and one group combining treated (n = 15) and untreated pigs (n = 15). These groups were followed for 16 weeks during which all pigs were profiled for both their faecal microbiome (through 16 S rRNA gene sequencing) and resistome (by use of a high-throughput qPCR platform targeting 82 ARGs and their variants). We found that the resistome of treated pigs was substantially enriched in resistance genes compared to untreated pigs but, importantly, observed that untreated pigs co-reared with treated pigs had levels of resistance genes approaching their treated pen mates, suggesting that the treated enterotype is readily transferred to the untreated animal. From this, we conclude that mixing of treated and untreated pigs causes spill-over of antibiotic resistant bacteria and/or resistance genes from treated pigs when these are co-reared. To optimize RWA production, treated and untreated pigs should be physically separated to limit the proliferation of ARGs.202439578929
3144100.9971Impact of florfenicol dosing regimen on the phenotypic and genotypic resistance of enteric bacteria in steers. The food animal sector's use of antimicrobials is heavily critiqued for its role in allowing resistance to develop against critically important antimicrobials in human health. The WHO recommends using lower tier antimicrobials such as florfenicol for disease treatment. The primary objective of this study was to assess the differences in resistance profiles of enteric microbes following administration of florfenicol to steers using both FDA-approved dosing regimens and two different detection methods. Our hypothesis was that we would identify an increased prevalence of resistance in the steers administered the repeated, lower dose of florfenicol; additionally, we hypothesized resistance profiles would be similar between both detection methods. Twelve steers were administered either two intramuscular (20 mg/kg q 48 h; n = 6) or a single subcutaneous dose (40 mg/kg, n = 6). Fecal samples were collected for 38 days, and E. coli and Enterococcus were isolated and tested for resistance. Fecal samples were submitted for metagenomic sequencing analysis. Metagenomics revealed genes conferring resistance to aminoglycosides as the most abundant drug class. Most multidrug resistance genes contained phenicols. The genotypic and phenotypic patterns of resistance were not similar between drug classes. Observed increases in resistant isolates and relative abundance of resistance genes peaked after drug administration and returned to baseline by the end of the sampling period. The use of a "lower tier" antimicrobial, such as florfenicol, may cause an increased amount of resistance to critically important antimicrobials for a brief period, but these changes largely resolve by the end of the drug withdrawal period.202438418677
5283110.9971Persistent effects of intramammary ceftiofur treatment on the gut microbiome and antibiotic resistance in dairy cattle. BACKGROUND: Intramammary (IMM) ceftiofur treatment is commonly used in dairy farms to prevent mastitis, though its impact on the cattle gut microbiome and selection of antibiotic-resistant bacteria has not been elucidated. Herein, we enrolled 40 dairy (Holstein) cows at the end of the lactation phase for dry-cow therapy: 20 were treated with IMM ceftiofur (Spectramast®DC) and a non-antibiotic internal teat sealant (bismuth subnitrate) and 20 (controls) received only bismuth subnitrate. Fecal grab samples were collected before and after treatment (weeks 1, 2, 3, 5, 7, and 9) for bacterial quantification and metagenomic next-generation sequencing. RESULTS: Overall, 90% and 24% of the 278 samples had Gram-negative bacteria with resistance to ampicillin and ceftiofur, respectively. Most of the cows treated with ceftiofur did not have an increase in the number of resistant bacteria; however, a subset (25%) shed higher levels of ceftiofur-resistant bacteria for up to 2 weeks post-treatment. At week 5, the antibiotic-treated cows had lower microbiota abundance and richness, whereas a greater abundance of genes encoding extended-spectrum β-lactamases (ESBLs), CfxA, ACI-1, and CMY, was observed at weeks 1, 5 and 9. Moreover, the contig and network analyses detected associations between β-lactam resistance genes and phages, mobile genetic elements, and specific genera. Commensal bacterial populations belonging to Bacteroidetes most commonly possessed ESBL genes followed by members of Enterobacteriaceae. CONCLUSION: This study highlights variable, persistent effects of IMM ceftiofur treatment on the gut microbiome and resistome in dairy cattle. Antibiotic-treated cattle had an increased abundance of specific taxa and genes encoding ESBL production that persisted for 9 weeks. Fecal shedding of ESBL-producing Enterobacteriaceae, which was classified as a serious public health threat, varied across animals. Together, these findings highlight the need for additional studies aimed at identifying factors associated with shedding levels and the dissemination and persistence of antibiotic resistance determinants on dairy farms across geographic locations.202337946266
3323120.9971Minimal Impact on the Resistome of Children in Botswana After Azithromycin Treatment for Acute Severe Diarrheal Disease. BACKGROUND: Macrolide antibiotics, including azithromycin, can reduce under 5 years of age mortality rates and treat various infections in children in sub-Saharan Africa. These exposures, however, can select for antibiotic-resistant bacteria in the gut microbiota. METHODS: Our previous randomized controlled trial (RCT) of a rapid-test-and-treat strategy for severe acute diarrheal disease in children in Botswana included an intervention (3-day azithromycin dose) group and a control group that received supportive treatment. In this prospective matched cohort study using stools collected at baseline and 60 days after treatment from RCT participants, the collection of antibiotic resistance genes or resistome was compared between groups. RESULTS: Certain macrolide resistance genes increased in prevalence by 13%-55% at 60 days, without differences in gene presence between the intervention and control groups. These genes were linked to tetracycline resistance genes and mobile genetic elements. CONCLUSIONS: Azithromycin treatment for bacterial diarrhea for young children in Botswana resulted in similar effects on the gut resistome as the supportive treatment and did not provide additional selective pressure for macrolide resistance gene maintenance. The gut microbiota of these children contains diverse macrolide resistance genes that may be transferred within the gut upon repeated exposures to azithromycin or coselected by other antibiotics. CLINICAL TRIALS REGISTRATION: NCT02803827.202439052715
4734130.9971Antibiotic resistance gene-free probiont administration to tilapia for growth performance and Streptococcus agalactiae resistance. BACKGROUND AND AIM: The rapid development of aquaculture as a major food sector is accompanied by challenges, including diseases that affect tilapia farming worldwide. One such infectious disease caused by Streptococcus agalactiae poses a serious threat to tilapia populations. Probiotics have emerged as a potentially safe preventive measure against S. agalactiae infection. However, antimicrobial resistance from antibiotic-resistant bacteria remains a concern because it can lead to the spread of resistant bacteria and serve as a reservoir of antibiotic-resistant genes in fishes and the surrounding environment. This study aimed to identify candidate probiotic bacteria capable of promoting tilapia growth, providing resistance to S. agalactiae infection, devoid of potential pathogenicity, and free from antibiotic resistance genes. Subsequently, the performance of these probiotic candidates in tilapia was evaluated. MATERIALS AND METHODS: Lactococcus garvieae, Priestia megaterium, Bacterium spp., Bacillus megaterium, Bacillus subtilis, and Bacillus pumilus were examined to assess their antibacterial properties, hemolytic patterns, and antibiotic resistance genes. We used the specific primers tetA, tetB, tetD, tetE, tetO, tetQ, ermB, and qnrS that were used for antibiotic resistance gene detection. In vivo probiotic efficacy was evaluated by administering probiotic candidates in tilapia feed at a concentration of 1 × 10(6) colonies/mL/50 g of feed over a 60-day maintenance period. Resistance to S. agalactiae infection was observed for 14 days after the challenge test. RESULTS: Lactococcus garvieae, P. megaterium, and Bacterium spp. were identified as promising probiotic candidates among the bacterial isolates. On the other hand, B. megaterium, B. subtilis, and B. pumilus carried resistance genes and exhibited a β hemolytic pattern, rendering them unsuitable as probiotic candidates. The selected probiotic candidates (L. garvieae, P. megaterium, and Bacterium spp.) demonstrated the potential to enhance tilapia growth, exhibited no pathogenic tendencies, and were free from antibiotic resistance genes. Supplementation with L. garvieae and Bacterium spp. enhanced tilapia resistance to S. agalactiae infection, whereas P. megaterium supplementation showed an insignificant survival rate compared with controls after the challenge test period. CONCLUSION: Probiotics, particularly L. garvieae, P. megaterium, and Bacterium spp., enhance growth and resistance against S. agalactiae infection, without harboring antibiotic resistance genes. Selecting probiotic candidates based on antibiotic resistance genes is essential to ensure the safety of fish, the environment, and human health.202338328352
3150140.9971Macrolide-susceptible probiotic Enterococcus faecium ST296 exhibits faecal-environmental-oral microbial community cycling among beef cattle in feedlots. Enterococci are included in the United States National Antimicrobial Resistance Monitoring System to track antibiotic resistance among commensal Gram-positive enteric bacteria, largely due to their high abundance in food animals and in retail meat. In the U.S. cattle industry, macrolides are used to prevent and control liver abscesses, which cause significant economic losses. Previous studies have suggested that feeding tylosin and the intensity of the pen environment, both expand and sustain respectively the prevalence of multidrug resistance among enterococci in feedlot cattle. This has led to research into alternative feed supplements and improved stewardship practices. In a randomized controlled trial, we measured the impact of a probiotic and an altered pen environment on antimicrobial resistance among faecal Enterococcus spp. in cattle fed tylosin. Supplementing cattle with an Enterococcus faecium and Saccharomyces cerevisiae-based probiotic yielded the isolation of E. faecium of the probiotic sequence type (ST296) from faecal and environmental samples in treatment groups, as well as from cattle and the manure pack in nearby pens. Of importance, the probiotic strain also was found in a desiccated and milled manure pack sample taken 120 days after the initial trial ended. Phylogenetic and SNP analyses revealed clonal survival and spread compatible with faecal-environmental-oral recycling of the probiotic strain within and among cattle and pens. The increase in prevalence of the ST296 strain occurred concomitant with a decrease in ST240, the dominant sequence type associated with ermB and tet(M) resistance genes in this trial. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrate that a macrolide-susceptible probiotic Enterococcus faecium ST296 strain fed to beef cattle becomes fully embedded in the microbial community cycling of bacteria via faecal-environmental-oral transmission within and among feedlot pens. An initial investment in feeding the probiotic is thereby leveraged into expanding numbers of susceptible bacteria in cattle and their environment, even among those cattle fed tylosin.202031883125
4584150.9971High-Pressure Processing Influences Antibiotic Resistance Gene Transfer in Listeria monocytogenes Isolated from Food and Processing Environments. The study aimed to assess the high-pressure processing (HPP) impact on antibiotic resistance gene transfer in L. monocytogenes from food and food processing environments, both in vitro (in microbiological medium) and in situ (in carrot juice), using the membrane filter method. Survival, recovery, and frequency of antibiotic resistance gene transfer analyses were performed by treating samples with HPP at different pressures (200 MPa and 400 MPa). The results showed that the higher pressure (400 MPa) had a significant effect on increasing the transfer frequency of genes such as fosX, encoding fosfomycin resistance, and tet_A1, tet_A3, tetC, responsible for tetracycline resistance, both in vitro and in situ. In contrast, the Lde gene (the gene encoding ciprofloxacin resistance) was not transferred under any conditions. In the food matrix (carrot juice), greater variability in results was observed, suggesting that food matrices may have a protective effect on bacteria and modify HPP efficacy. In general, an increase in MIC values for antibiotics was noted in transconjugants compared to donors. Genotypic analysis of transconjugants showed differences in genetic structure, especially after exposure to 400 MPa pressure, indicating genotypic changes induced by pressure stress. The study confirms the possibility of antibiotic resistance genes transfer in the food environment, even from strains showing initial susceptibility to antibiotics carrying so-called silent antibiotic resistance genes, highlighting the public health risk of the potential spread of antibiotic-resistant strains through the food chain. The findings suggest that high-pressure processing can increase and decrease the frequency of resistance gene transfer depending on the strain, antibiotic combination, and processing conditions.202439684674
7126160.9971Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics. BACKGROUND: Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA. RESULTS: The concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments. CONCLUSION: The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.201121261985
4765170.9971Enhancing the Antibacterial Impact of Lipopeptide Extracted from Bacillus licheniformis as a Probiotic against MDR Acinetobacter baumannii. BACKGROUND: The antibiotic resistance of microorganisms is escalating rapidly. Infections caused by opportunistic pathogens in immunocompromised individuals have prompted researchers to seek for potent and safe antibacterial agents. The purpose of this investigation was to explore the suppression of virulence gene expression, specifically the pga operon genes responsible in biofilm formation in Acinetobacter baumannii, through the utilization of metabolites obtained from probiotic bacteria. METHODS: To assess the antimicrobial properties, standard strains of five probiotic bacteria were tested against a standard strain of multidrug-resistant (MDR) A. baumannii employing the agar gel diffusion technique. Following the identification of the most potent probiotic strain (Bacillus licheniformis), the existence of its LanA and LanM genes was confirmed using the polymerase chain reaction (PCR) test. High-performance liquid chromatography (HPLC) and fourier-transform infrared spectroscopy (FTIR) techniques were employed to identify the intended metabolite, which was found to be a lipopeptide nature. The minimum inhibitory concentration (MIC) values and anti-biofilm activity of the targeted metabolite were determined using a dilution method in 96-well microplates and field emission scanning electron microscopy (FE-SEM). Real-time PCR (qPCR) was utilized for comparing the expression of pga operon genes, including pgaABCD, in A. baumannii pre- and post-exposure to the derived lipopeptide. RESULTS: The MIC results indicated that the probiotic product inhibited the growth of A. baumannii at concentrations lower than those needed for conventional antibiotics. Furthermore, it was observed that the desired genes' expression decreased due to the effect of this substance. CONCLUSIONS: This research concludes that the B. licheniformis probiotic product could be a viable alternative for combating drug resistance in A. baumannii.202438812307
3430180.9970Benzalkonium chloride alters phenotypic and genotypic antibiotic resistance profiles in a source water used for drinking water treatment. Antibiotic resistance is a major public health concern. Triclosan is an antimicrobial compound with direct links to antibiotic resistance that was widely used in soaps in the U.S. until its ban by the U.S. Food and Drug Administration. Benzalkonium chloride (BAC), a quaternary ammonium compound, has widely replaced triclosan in soaps marketed as an antibacterial. BAC has been detected in surface waters and its presence will likely increase following increased use in soap products. The objective of this study was to determine the effect of BAC on relative abundance of antibiotic resistance in a bacterial community from a surface water used as a source for drinking water treatment. Bench-scale microcosm experiments were conducted with microbial communities amended with BAC at concentrations ranging from 0.1 μg L(-1) to 500 μg L(-1). Phenotypic antibiotic resistance was quantified by culturing bacteria in the presence of different antibiotics, and genotypic resistance was determined using qPCR to quantify antibiotic resistance genes (ARGs). BAC at concentrations ranging from 0.1 μg L(-1) to 500 μg L(-1) was found to positively select for bacteria resistant to ciprofloxacin and sulfamethoxazole, and negatively select against bacteria with resistance to six other antibiotics. Exposure to BAC for 14 days increased the relative abundance of sul1 and bla(TEM). This study re-highlights the importance of employing both culture and non-culture-based techniques to identify selection for antibiotic resistance. The widespread use of BAC will likely impact antibiotic resistance profiles of bacteria in the environment, including in source waters used for drinking water, wastewater treatment plants, and natural waterways.202031767233
7654190.9970Impact of fertilizing with raw or anaerobically digested sewage sludge on the abundance of antibiotic-resistant coliforms, antibiotic resistance genes, and pathogenic bacteria in soil and on vegetables at harvest. The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.201425172864