Antibiotic Levels Research Articles

Screening and detection of antibiotic residues on broiler meat based on trade system variations, seasonal differences, and the impact on final consumer safety in Romania

IntroductionOne key factor contributing to microbial resistance is the deliberate and inappropriate use of antibiotics in human and animal health management. Recent studies point out various ways to tackle this controversy to mitigate the unnatural rapid evolution of pathogenic bacteria. Chicken meat remains at the top of Romanian consumers' preferences, being the most consumed type of meat, desired for its nutritional and dietetic attributes.MethodsThis research was conducted in 2022, aiming to evaluate the antibiotic residues (quinolones, aminoglycosides, tetracycline, and sulfonamides) in broiler chicken meat with various trade strategies [retail market (RM), n = 40, traditional market (TM), n = 185, and door-to-door vendors (DTD), n = 121] during two seasonal periods, spring (March–April) and summer (July–September). An efficient and precise protocol was employed for determining the meat organoleptic attributes, qualitative screening, and quantitative assay antibiotic of six antibiotics (enrofloxacin, marbofloxacin, streptomycin, oxytetracycline, doxycycline, and sulfamethoxazole), consumers' antibiotic exposure (estimated daily intake), and potential risk assessment (hazard quotient).ResultsThe antibiotic quality assessment revealed an overall antibiotic residue presence in groups TM (75%) and DTD (82%), while no antibiotic residues were detected in the RM group. Our results show that 32% (n = 110) of the total chicken meat samples were free of antibiotic residues, 4.5% (n = 16) contained antibiotic residues belonging to one class of antibiotics, 40% (n = 139) had two antibiotics groups, 22% (n = 77) had three antibiotics groups, and 1.5% of the chicken meat samples presented four groups of antibiotics.DiscussionDuring the spring season, the enrofloxacin antibiotic residue present in the meat samples was higher, showing an 84% presence in TM meat samples group when compared with the DTD meat sampled group (75%). The analysis data processing showed a strong correlation between the antibiotic residue's meat samples origin (trade market and door-to-door traded meat antibiotic residue variations) and seasonal variations. As a result of the hazard quotient assay, the meat antibiotic residue levels had subunit values, indicating the meat quality was proper for consumption. It is mandatory to strengthen the level of knowledge by continuously monitoring and providing updated information to each group of farmers to increase their understanding of and adherence to the proper handling of antibiotics when growing chickens. Regarding the use of prohibited growth-promoting antibiotics in chicken-rearing systems, local authorities should increase the guard level, at antibiotic supplier and end user levels.

Prioritized regional management for antibiotics and heavy metals in animal manure across China

High levels of antibiotics and heavy metals in animal manure pose a potential threat to both the ecological environment and public health. A regional knowledge of their distribution and risk assessment across China remains unclear. A dataset containing 4082 records covering a total of forty-two antibiotics and eight heavy metals was established for animal manure across China. The results showed that the residual concentration of antibiotics was in the order of tetracyclines > aminoglycosides > fluoroquinolones > macrolides > sulfonamides > β-lactams, and that of heavy metals is Zn > Cu > Cr > Pb > Ni > As > Cd > Hg. The mean concentration of antibiotics and heavy metals was higher in pig manure compared to chicken and cow manure (Kruskal-Wallis test). The lowest level of antibiotics was observed in Northwest China based on geographic distribution characteristics. It was related to the high ratio of cow and sheep farming that less antibiotics were administered to. The pollution status of heavy metals was more severe in East China. Furthermore, high correlations were observed between antibiotics (tetracyclines) and heavy metals (Cu, Zn, and As). Especially, tetracycline in North China and Cd in Northeast China exhibited a high risk in manure; thus, they were priority regions for antibiotics/heavy metals pollution control. This study identified risk assessment of typical antibiotics and heavy metals in animal manure and emphasized the necessity of regional management across China.

Occurrence and Health Risk Assessment of Sulfonamide Antibiotics in Different Freshwater Fish in Northeast China.

This study aimed to investigate the levels of 12 sulfonamide antibiotics in freshwater fish species obtained from three cities in northeastern China (Harbin, Changchun, and Shenyang). The analysis was conducted using HPLC-MS/MS to accurately quantify the antibiotic concentrations in the fish samples. The results showed that the average levels of sulfonamide antibiotics in fish samples from Harbin, Changchun, and Shenyang were 1.83 ng/g ww, 0.98 ng/g ww, and 1.60 ng/g ww, respectively. Sulfamethoxazole displayed the highest levels and detection rates in all three cities, whereas sulphapyridine exhibited the lowest concentrations in all the fish samples. The levels of sulfonamide antibiotic residues in the different fish species varied widely among the cities, and the highest level of antibiotic residues was found in the muscle of carnivorous fish. The results from a health risk evaluation on the consumption of these fish indicated that the risk from long-term antibiotic exposure to local residents from the intake of the sampled fish was small and not sufficient to pose a significant health risk to consumers.

Short notes on molecular mechanisms behind antimicrobial drug resistance

Infectious disease mortality have occurred often, raising public awareness of their hazards and advancing global antibiotic research. On the other hand, higher antibiotic levels in natural ecosystems are a result of increased antibiotic demand and use. Gene mutations causing antibiotic resistance in bacterial populations were brought on by natural selection and adaption. The emergence of antibiotic-resistant bacteria was caused by the spread of antibiotic resistance genes in ecosystems, which resulted in a number of antibiotic-resistant diseases on a global scale. An overview of the history of antibiotic discovery, antibiotic use, and antibiotic resistance mechanisms is given in this review study. Bioinformatics is also used to combat antibiotic resistance. To control bacteria that no longer respond to the therapy, the creation of new medications appears to be the primary requirement today.

Development and Validation of UPLC-MS/MS Method for Simultaneous Determination of Multi-Class Antibiotics, Antivirals and Antifungals in Water

According to a WHO report, antimicrobial resistance is one of the top 10 health threats. Environmental sources are extremely contaminated with high levels of antibiotics, antivirals and antifungals due to their excessive and unsafe usage. Hence, there is a need to detect and quantitate the antimicrobial agents in water. Consequently, a simultaneous analytical method is developed and validated, employing a solid-phase extraction methodology with an HLB cartridge. This method was utilized to quantify a minimum of twenty different chemicals derived from antibiotics, antivirals and antifungal drugs. Antibiotics include quinolones, isonicotinic acid, macrolides, oxazolidinones, penicillin, sulphonamides, antivirals that contain nucleotide class compounds and antifungals that comprising of triazoles and imidazoles. Ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC- S/MS) was used to quantify the analytes. Detection was achieved using multiple reaction monitoring with positive electron spray ionization. All the compounds were well separated with the column, Xterra MS C18 4.6 × 50 mm 2.5 μm, using water and acetonitrile with 0.1% formic acid as a mobile phase. The calibration curve range was set at 50 to 2000 ng mL-1. Validation parameters like precision and accuracy, matrix effect, sensitivity, autosampler stability, the limit of detection and recovery were performed.

Evaluation of the impact before and after the application of an antimicrobial stewardship program at Dong Thap General Hospital, Vietnam, from 2017 to 2021

Vietnam has one of the highest rates of antibiotic resistance in Asia. In 2020, the Vietnam Minister of Health introduced new legislation for the implementation of an antimicrobial stewardship program (ASP). The evidence for the effectiveness of ASP in small hospitals and hospitals located in provinces was limited compared with larger-scale and central city hospitals. Evaluation of the impact before and after the introduction of an antimicrobial stewardship program at Dong Thap General Hospital, from 2017 to 2021. Retrospective data was collected from June 2017 to June 2021. The impact of the ASP on changes in antibiotic use and the clinical outcome associated with the implementation of the ASP was evaluated using autoregressive integrated moving average modelling of controlled interrupted time-series analysis. There was a significant and sustained decrease in antibiotic consumption level (step change) in 2 indicators, DOT/1000PD (129.55; P<0.01) and LOT/1000PD (99.95, P<0.01), immediately after the ASP intervention. There were no statistically significant changes identified in terms of consumption with DDD/1000PD, or in the clinical outcomes. The results showed no statistically significant change in consumption trend (ramps) in all evaluated indicators. No statistically significant changes in consumption levels and trends were observed in the control group. The ASP implemented in Dong Thap General Hospital from 2017 to 2021 showed a considerable influence on antibiotic consumption as indicated by the DOT/1000 PD and LOT/1000 PD during the initial stages. Moreover, controlling antibiotic consumption did not negatively impact patient outcomes.

First Report of Antibiotic Resistance Markers cfiA and nim Among Bacteroides fragilis Group Strains in Ecuadorian Patients.

In recent years, increasing resistance of Bacteroides fragilis to several antibiotics has been reported in different countries. The aim of this study was to evaluate the antibiotic resistance profiles of Bacteroides spp. isolated from clinical samples by phenotypic and molecular methods. A total of 40 nonrepetitive isolates of the B. fragilis group were studied from 2018 to 2019. The species was identified by API 20A system. The minimum inhibitory concentrations (MICs) were determined by Sensititre anaerobe MIC plate. The presence of the nim and cfiA genes was checked by conventional PCR. The association between genes and insertion sequence (IS) was performed by whole genome sequencing. Eleven isolates were categorized as metronidazole-resistant and only 2 isolates harbored the nim gene. Five isolates were imipenem-resistant, but cfiA gene was detected in two isolates. cfiA gene was closely related to the cfiA-4 allele and associated with IS614B. The nim gene was not related to any nim gene type and was considered a new variant named nimL. IS612 was found upstream of nimL gene. In view of the scarcity of data on B. fragilis, there is a need to surveil antibiotic resistance levels and molecular mechanisms to implement better antimicrobial therapies against this important group of bacteria.

Microbial community and gene dynamics response to high concentrations of gadolinium and sulfamethoxazole in biological nitrogen removal system

The impact of high antibiotic and heavy metal pollution levels on biological nitrogen removal in wastewater treatment plants (WWTPs) remains poorly understood, posing a global concern regarding the issue spread of antibiotic resistance induced by these contaminants. Herein, we investigated the effects of gadolinium (Gd) and sulfamethoxazole (SMX), commonly found in medical wastewater, on biological nitrogen removal systems and microbial characteristics, and the fate of antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and mobile genetic elements (MGEs). Our findings indicated that high SMX and Gd(III) concentrations adversely affected nitrification and denitrification, with Gd(III) exerting a strong inhibitory effect on microbial activity. Metagenomic analysis revealed that high SMX and Gd(III) concentrations could reduce microbial diversity, with Thauera and Pseudomonas emerging as dominant genera across all samples. While the relative abundance of most ARGs decreased under single Gd(III) stress, MRGs increased, and nitrification functional genes were inhibited. Conversely, combined SMX and Gd(III) pollution increased the relative abundance of intl1. Correlation analysis revealed that most genera could host ARGs and MRGs, indicating co-selection and competition between these resistance genes. However, most denitrifying functional genes exhibited a positive correlation with MRGs. Overall, our study provides novel insights into the impact of high concentrations of antibiotics and heavy metal pollution in WWTPs, and laying the groundwork for the spread and proliferation of resistance genes under combined SMX and Gd pollution.

The Abnormal Accumulation of Lipopolysaccharide Secreted by Enriched Gram-Negative Bacteria Increases the Risk of Rotavirus Colonization in Young Adults.

Human rotavirus (HRV) is an enteric virus that causes infantile diarrhea. However, the risk factors contributing to HRV colonization in young adults have not been thoroughly investigated. Here, we compared the differences in dietary habits and composition of gut microbiota between asymptomatic HRV-infected young adults and their healthy counterparts and investigated potential risk factors contributing to HRV colonization. Our results indicated that asymptomatic HRV-infected adults had an excessive intake of milk and dairy and high levels of veterinary antibiotics (VAs) and preferred veterinary antibiotic (PVAs) residues in urine samples. Their gut microbiota is characterized by abundant Gram-negative (G-) bacteria and high concentrations of lipopolysaccharide (LPS). Several opportunistic pathogens provide discriminatory power to asymptomatic, HRV-infected adults. Finally, we observed an association between HRV colonization and disrupted gut microbiota caused by the exposure to VAs and PVAs. Our study reveals the traits of disrupted gut microbiota in asymptomatic HRV-infected adults and provides a potential avenue for gut microbiota-based prevention strategies for HRV colonization.

Source Apportionment and Source-specific Risk of Typical Antibiotics in Baiyangdian Lake

The current situation of antibiotic pollution in lakes is critical. At present, most of the previous studies on antibiotics in lakes have focused on the spatiotemporal distribution and risk assessment, while less attention has been paid to the source apportionment. Ultra-high performance liquid chromatography-mass spectrometry was used to determine the concentration of tetracyclines (TCs), sulfonamides (SAs), and quinolones (QNs) in the samples. The source apportionment and source-specific risk of typical antibiotics in the study area were analyzed using the combination of a PMF model and risk quotients (RQ). The results showed that ① the total concentrations of target antibiotics (Σ antibiotics) ranged from ND to 2635 ng·L-1 for surface water and from ND to 259.8 ng·g-1 for sediments. ② The spatial distribution of QNs in surface water decreased from west to east, SAs decreased from middle to north and south, and TCs increased from middle to north and south. In the sediment, QNs decreased from middle to east and west, whereas SAs and TCs increased from east to west. ③ Aquaculture was the major antibiotic source, accounting for the highest proportion (33.2%), followed by sewage treatment plants (29.2%), livestock activities (18.9%), and domestic sewage (18.7%). ④ The ecological risk assessment results showed that enrofloxacin and flumequine were at a medium-high risk level. ⑤ For the spatial distribution of source-specific risk, the results showed that the aquaculture at S1 was at a high risk level, whereas the source-specific risks for other sites were at a medium-low risk level. In terms of source types, aquaculture was at a medium-high risk level, whereas the other sources were at a medium-low risk level. Therefore, considering the major sources and source-specific risk level of antibiotics, more precise and scientific antibiotic risk control should be adopted in Baiyangdian Lake.

Critically Resistant Pseudomonas aeruginosa: a Short Review on the Mechanisms of Resistance and Therapeutic Strategies

Pseudomonas aeruginosa is a critically resistant pathogen that is responsible for the morbidity of immunodeficient patients throughout the world. The bacterium possesses unique resistance to several antibiotics. Numerous resistance mechanisms like reliable efflux pumps, reduced permeability of the cell membrane, inactivation of antimicrobial drugs by enzymatic mechanisms and modification of antibiotic target sites enable the survival of the bacteria in adverse conditions. The causes and extent of antibiotic resistance in this bacterial species have been elucidated clearly. The ability of the pathogen to express several mutations in crucial genes makes it lethal. The everlasting threat of intrinsic as well as acquired antimicrobial resistance in common bacterial species is studied extensively to devise newer and effective therapeutics for the general public. Several studies have worked on the methods to deal with P. aeruginosa infections. The highest level of the newest antibiotics along with combinatorial therapy is administered to severely affected patients. Modern therapeutic strategies of bacteriophage therapy have been tested preclinically. The problem statement of curing P. aeruginosa infections thoroughly is yet to be resolved constructively.

Determination of the antibacterial effect of bee venom against rainbow trout pathogens and antibiotic resistance gene expression

Abstract Bee venom (BV) is a rich source of secondary metabolites from honeybees (Apis mellifera L.). It contains a variety of bioactive ingredients including peptides, proteins, enzymes, and volatile metabolites. This study investigated the antibacterial effects of the bee venom obtained from honey bees (Apis mellifera L.) against bacterial fish pathogens, such as Lactococcus garvieae (Lg1, Lg2, Lg3), Vibrio anguillarum (Va1, Va2, Va3), Yersinia ruckeri (Yr1, Yr2, Yr3), and Aeromonas hydrophila (Ah1, Ah2, Ah3) and the expression levels on the antibiotic resistance genes hly and fbp (hemolysin and fibronectin-binding prot) of them. It was determined that bee venom had an antibacterial effect against L. garvieae, L. anguillarum, and Y. ruckeri strains, while it had no effect only against Ah3 and Ah2 bacterial strains. As staded by the gene expression of hly (hemolysin) and fbp (fibronectin-binding protein), among the antibiotic resistance genes the effect levels of bee venom on bacterial species varied, although it affected antibiotic resistance and gene expression level in all bacteria. It was revealed that the expression level was the highest for V. anguillarum strains, whereas it was below the control group for L. garvieae. i.e the effect of bee venom on the resistance mechanism for L. garvieae was much less compared to V. anguillarum. Based on the results in the current study it could be concluded that applying bee venom to pathogenic bacteria that cause mortality in the aquaculture sector could induce the defense-related gene and change the broad-spectrum biocontrol activity at the molecular level.

Young people exposure to antibiotics: Implication for health risk and the impact from eating habits of takeaway food

Exposure to antibiotics, mainly from animal food ingestion, may have adverse effects on human health. Takeaway food is the preferred choice for the dietary of most Chinese young people nowadays, but the relationship between takeaway eating and antibiotic exposure is not yet adequately understood. In the present study, 297 young people were recruited to collect urine samples and questionnaires with an emphasis on their takeaway eating habits. The internal exposure to 16 antibiotics and three metabolites was measured in urine samples by high-performance liquid chromatography-tandem mass spectrometry, as well as a DNA oxidative damage marker, 8-hydroxydeoxyguanosine (8-OHdG). At least one kind of antibiotic was found in over 90 % of urine samples, with total concentrations from 0.667 to 3.02 × 104 ng/mL. High exposure levels of antibiotics were more likely to be found in individuals with a larger body mass index. The concentrations of six antibiotics were significantly different among people with different overall weekly eating frequencies, usually an upward trend. The estimated daily intakes of antibiotics were on the levels of 0.001–1.0 μg/kg/day, mainly contributed by clarithromycin, ciprofloxacin and oxytetracycline, indicating a potential health risk based on the microbiological effect. A significantly positive correlation was found between DNA oxidative damage and exposure for four categories of antibiotics, conformed by both Spearman correlation and multiple linear regression analysis. The levels of 8-OHdG were 355 %, 239 %, 234 %, and 334 % higher with elevated levels of phenicols, macrolides, tetracyclines and sulfonamides from quartiles 2 to 4. Our results suggest that high-frequency consumption of takeaways may exacerbate oxidative stress trends through human exposure to antibiotics.

Experimental facility had a greater effect on growth performance, gut microbiome, and metabolome in weaned pigs than feeding diets containing subtherapeutic levels of antibiotics: A case study.

The objective of this study was to define changes in the intestinal metabolome and microbiome associated with growth performance of weaned pigs fed subtherapeutic concentrations of antibiotics. Three experiments with the same antibiotic treatments were conducted on the same research farm but in two different facilities (nursery and wean-finish) using pigs weaned at 20-days of age from the same source herd and genotype, and fed the same diets formulated without antibiotics (NC) or with 0.01% chlortetracycline and 0.01% sulfamethazine (AB). Pigs were weighed and feed disappearance was determined on days (d) 10, 21, and 42 post-weaning to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain:feed (G:F). On d 42, one pig/pen was selected for blood and ileal and cecal content collection. Targeted and untargeted metabolomic profiles were determined in serum and cecal contents using liquid chromatography-mass spectrometry, and composition of bacterial communities in intestinal content samples was determined by sequencing the V4 region of the 16s rRNA gene. Metabolomics and microbiome data were analyzed using diverse multivariate and machine learning methods. Pigs fed AB had significantly greater (P < 0.05) overall ADG and ADFI compared with those fed NC, and pig body weight, ADG, and G:F were also significantly different (P < 0.05) between experiments. Differences (P < 0.05) in serum metabolome along with ileal and cecal microbiome beta diversity were observed between experiments, but there were no differences in microbiome alpha diversity between experiments or treatments. Bacteria from the families Clostridiaceae, Streptomycetaceae, Peptostreptomycetaceae, and Leuconostocaceae were significant biomarkers for the AB treatment. In addition, pigs fed AB had increased serum arginine, histidine, lysine, and phenylalanine concentrations compared with NC. Percentage error from a random forest analysis indicated that most of the variation (8% error) in the microbiome was explained by the facility where the experiments were conducted. These results indicate that facility had a greater effect on growth performance, metabolome, and microbiome responses than feeding diets containing subtherapeutic levels of antibiotics.

Removal Rule, Ecological Risk, and Full-Scale Assessment of Antibiotics in Three Organic Fertilizer Production Methods

The occurrence of antibiotic residues present in organic fertilizers produced by diverse techniques is largely unknown. Large-scale evaluation and standardization are imperative to identify the concentration and risks of residual antibiotics during different treatment techniques of livestock manure (i.e., compost, carbonization, and anaerobic treatment). In this study, we assessed the concentration and ecological risks of 30 antibiotics in three types of commercial organic fertilizers by the risk quotient (RQ) method. The highest levels of residual antibiotics in the organic fertilizers produced by composting, carbonization, and anaerobic treatment were 142.58 μg/kg (dry weight) oxytetracycline, 2.53 μg/kg (dry weight) oxytetracycline, and 341.86 mg/L chlortetracycline. We observed that the compost and carbon-based organic fertilizers had an RQ of less than 0.1, indicating a low risk. In contrast, liquid organic fertilizer had an RQ of 0.93, indicating a medium risk. After a comprehensive comparison and evaluation, the “carbonization + anaerobic treatment” mode was found to be more appropriate for most cases. Moreover, the preparation method of organic fertilizers needs further optimization to minimize the concentration of residual antibiotics within the range of low ecotoxicity.

Risk assessment and dissemination mechanism of antibiotic resistance genes in compost

In recent years, the excessive of antibiotics in livestock and poultry husbandry, stemming from extensive industry experience, has resulted in the accumulation of residual antibiotics and antibiotic resistance genes (ARGs) in livestock manure. Composting, as a crucial approach for the utilization of manure resources, has the potential to reduce the levels of antibiotics and ARGs in manure, although complete elimination is challenging. Previous studies have primarily focused on the diversity and abundance of ARGs in compost or have solely examined the correlation between ARGs and their carriers, potentially leading to a misjudgment of the actual risk associated with ARGs in compost. To address this gap, this study investigated the transfer potential of ARGs in compost and their co-occurrence with opportunistic pathogenic bacteria by extensively analyzing metagenomic sequencing data of compost worldwide. The results demonstrated that the potential risk of ARGs in compost was significantly lower than in manure, suggesting that composting effectively reduces the risk of ARGs. Further analysis showed that the microbes shifted their life history strategy in manure and compost due to antibiotic pressure and formed metabolic interactions dominated by antibiotic-resistant microbes, increasing ARG dissemination frequency. Therefore, husbandry practice without antibiotic addition was recommended to control ARG evolution, dissemination, and abatement both at the source and throughout processing.

Review on application of probiotics in poultry farm

Poultry farming, raising of birds domestically or commercially, primarily for meat and eggs but also for feathers. In poultry industry, probiotics applications have widely been shown to improve the barrier function of intestine and reduce pathogenic problems in gastrointestinal tract thus leading to the enhancement of immune response and replacement of sub-therapeutic antibiotics. Probiotics, the novel feed or food supplements are naturally occurring beneficial live microbes including bacteria, fungi and yeasts. Probiotics are proven to improve the health and wellbeing of birds. As a group of growth promoters, the addition of probiotics to the diet of poultry has been found to improve growth performance, feed conversion efficiency, immune responses and help in combating enteric pathogens. The addition of probiotics to poultry diet also improves egg production in layers. Three basic mechanisms of probiotics include competitive exclusion of pathogenic microbes, production of antibacterial substances and immune modulation of host. Generally, live a pathogenic microbial strain, singly or as multistrain probiotics, belonging to genus Lactobacillus, Streptococcus, Bacillus, Enterococcus, Pediococcus, Aspergillus and Saccharomyces are used in poultry. The injudicious and prolific use of sub-therapeutic levels of antibiotics in animal feed to suppress or eliminate harmful organisms, and to improve growth and feed efficiency, have paved way for their presence as residues in animal by-products and have also given rise to drug resistant microorganisms. Because of public health concerns, nowadays, the use of probiotics as substitute for antibiotics in poultry production has become an area of great interest. The present article reviews the beneficial effects and potential applications of probiotics or direct fed microbial (DFM) on health and production performances in poultry.

Beta-Lactam Antibiotics Can Be Measured in the Exhaled Breath Condensate in Mechanically Ventilated Patients: A Pilot Study.

Different techniques have been proposed to measure antibiotic levels within the lung parenchyma; however, their use is limited because they are invasive and associated with adverse effects. We explore whether beta-lactam antibiotics could be measured in exhaled breath condensate collected from heat and moisture exchange filters (HMEFs) and correlated with the concentration of antibiotics measured from bronchoalveolar lavage (BAL). We designed an observational study in patients undergoing mechanical ventilation, which required a BAL to confirm or discard the diagnosis of pneumonia. We measured and correlated the concentration of beta-lactam antibiotics in plasma, epithelial lining fluid (ELF), and exhaled breath condensate collected from HMEFs. We studied 12 patients, and we detected the presence of antibiotics in plasma, ELF, and HMEFs from every patient studied. The concentrations of antibiotics were very heterogeneous over the population studied. The mean antibiotic concentration was 293.5 (715) ng/mL in plasma, 12.3 (31) ng/mL in ELF, and 0.5 (0.9) ng/mL in HMEF. We found no significant correlation between the concentration of antibiotics in plasma and ELF (R2 = 0.02, p = 0.64), between plasma and HMEF (R2 = 0.02, p = 0.63), or between ELF and HMEF (R2 = 0.02, p = 0.66). We conclude that beta-lactam antibiotics can be detected and measured from the exhaled breath condensate accumulated in the HMEF from mechanically ventilated patients. However, no correlations were observed between the antibiotic concentrations in HMEF with either plasma or ELF.

The Establishment of a Tobramycin-Responsive Whole-Cell Micro-Biosensor Based on an Artificial Ribozyme Switch.

In this study, a tobramycin concentration-dependent whole-cell micro-biosensor (tob-HHAz) was constructed by fusing a tobramycin aptamer with a hammerhead ribozyme (HHR) from Schistosoma mansoni. The biosensor was obtained by integrating all the modules into one complete RNA sequence, which was easily introduced into E. coli without suffering from harsh external environments. Three independent tobramycin-sensitive RNA structures were identified via high-throughput screening in vivo and were further verified in vitro to undergo the desired self-cleavage reaction. The computation prediction of the RNA structure was performed to help analyze the mechanisms of various conformations by performing a qualitative and rapid detection of tobramycin in practical samples; two sensors exhibited high responsiveness to spiked milk, with a detection limit of around 40 nM, which is below the EU's antibiotic maximum residual level. One of the structures provides a linear range from 30 to 650 nM with a minimum detection limit of 30 nM and showed relatively good selectivity in spiked urine. This study is the first in which in vivo screening was combined with computation analysis to optimize the pivotal structure of sensors. This strategy enables researchers to use artificial ribozyme-based biosensors not only for antibiotic detection but also as a generally applicable method for the further detection of substances in living cells.

Genetic Determinants of Acinetobacter baumannii Serum-Associated Adaptive Efflux-Mediated Antibiotic Resistance.

Acinetobacter baumannii is a nosocomial pathogen of serious healthcare concern that is becoming increasingly difficult to treat due to antibiotic treatment failure. Recent studies have revealed that clinically defined antibiotic-susceptible strains upregulate the expression of a repertoire of putative drug efflux pumps during their growth under biologically relevant conditions, e.g., in human serum, resulting in efflux-associated resistance to physiologically achievable antibiotic levels within a patient. This phenomenon, termed Adaptive Efflux Mediated Resistance (AEMR), has been hypothesized to account for one mechanism by which antibiotic-susceptible A. baumannii fails to respond to antibiotic treatment. In the current study, we sought to identify genetic determinants that contribute to A. baumannii serum-associated AEMR by screening a transposon mutant library for members that display a loss of the AEMR phenotype. Results revealed that mutation of a putative pirin-like protein, YhaK, results in a loss of AEMR, a phenotype that could be complemented by a wild-type copy of the yhaK gene and was verified in a second strain background. Ethidium bromide efflux assays confirmed that the loss of AEMR phenotype due to pirin-like protein mutation correlated with reduced overarching efflux capacity. Further, flow cytometry and confocal microscopy measures of a fluorophore 7-(dimethylamino)-coumarin-4-acetic acid (DMACA)-tagged levofloxacin isomer, ofloxacin, further verified that YhaK mutation reduces AEMR-mediated antibiotic efflux. RNA-sequencing studies revealed that YhaK may be required for the expression of multiple efflux-associated systems, including MATE and ABC families of efflux pumps. Collectively, the data indicate that the A. baumannii YhaK pirin-like protein plays a role in modulating the organism's adaptive efflux-mediated resistance phenotype.