Bacitracin Resistance Research Articles

Spatiotemporal profiles and underlying mechanisms of the antibiotic resistome in two water-diversion lakes

Human-induced interventions have altered the local characteristics of the lake ecosystems through changes in hydraulic exchange, which in turn impacts the ecological processes of antibiotic resistance genes (ARGs) in the lakes. However, the current understanding of the spatiotemporal patterns and driving factors of ARGs in water-diversion lakes is still seriously insufficient. In the present study, we investigated antibiotic resistome in the main regulation and storage hubs, namely Nansi Lake and Dongping Lake, of the eastern part of the South-to-North Water Diversion project in Shandong Province (China) using a metagenomic-based approach. A total of 653 ARG subtypes belonging to 25 ARG types were detected with a total abundance of 0.125–0.390 copies/cell, with the dominance of bacitracin, multidrug, and macrolide-lincosamide streptogramin resistance genes. The ARG compositions were sensitive to seasonal variation and also interfered by artificial regulation structures along the way. Human pathogenic bacteria such as Acinetobacter calcoaceticus, Acinetobacter lwoffii, Klebsiella pneumoniae, along with the multidrug resistance genes they carried, were the focus of risk control in the two studied lakes, especially in summer. Plasmids were the key mobile genetic elements (MGEs) driving the horizontal gene transfer of ARGs, especially multidrug and sulfonamide resistance genes. The null model revealed that stochastic process was the main driver of ecological drift for ARGs in the lakes. The partial least squares structural equation model further determined that seasonal changes of pH and temperature drove a shift in the bacterial community, which in turn shaped the profile of ARGs by altering the composition of MGEs, antibacterial biocide- and metal-resistance genes (BMGs), and virulence factor genes (VFGs). Our results highlighted the importance of seasonal factors in determining the water transfer period. These findings can aid in a deeper understanding of the spatiotemporal variations of ARGs in lakes and their driving factors, offering a scientific basis for antibiotic resistance management.

Genome-centric metagenomes unveiling the hidden resistome in an anchialine cave

BackgroundAntibiotic resistance is a critical global concern, posing significant challenges to human health and medical treatments. Studying antibiotic resistance genes (ARGs) is essential not only in clinical settings but also in diverse environmental contexts. However, ARGs in unique environments such as anchialine caves, which connect both fresh and marine water, remain largely unexplored despite their intriguing ecological characteristics.ResultsWe present the first study that comprehensively explores the occurrence and distribution of ARGs and mobile genetic elements (MGEs) within an anchialine cave. Utilizing metagenomic sequencing we uncovered a wide array of ARGs with the bacitracin resistance gene, bacA and multidrug resistance genes, being the most dominant. The cave’s microbial community and associated resistome were significantly influenced by the salinity gradient. The discovery of novel β-lactamase variants revealed the cave’s potential as a reservoir for previously undetected resistance genes. ARGs in the cave demonstrated horizontal transfer potential via plasmids, unveiling ecological implications.ConclusionsThese findings highlight the need for further exploration of the resistome in unique environments like anchialine caves. The interconnected dynamics of ARGs and MGEs within anchialine caves offer valuable insights into potential reservoirs and mechanisms of antibiotic resistance in natural ecosystems. This study not only advances our fundamental understanding but also highlights the need for a comprehensive approach to address antibiotic resistance in diverse ecological settings.

Evolution and distribution of antibiotic resistance genes in submerged macrophytes and biofilm systems: From seasonal monitoring to mesocosm experiments

The aquatic ecosystem has been extensively investigated as a hotspot for the spread of antibiotic resistance genes (ARGs); yet, the evolution and distribution of ARGs profiles in submerged macrophytes biofilms and surrounding water remained unclear. In this study, the dynamic distribution and seasonal variations of microbial communities and ARGs profiles were investigated, alongside their assembly processes and mutual interactions. Bacitracin and multidrug resistance genes were predominant, constituting more than 60% of the total ARGs abundance. The deterministic processes (<65%), influenced by the physicochemical properties of the river environment, governed the assembly and composition of ARGs profiles, exhibiting significant seasonal variation. The peak diversity (21 types) and abundance (0.316 copy ratios) of ARGs were detected during the summer. Proteobacteria and Actinobacteria were the dominant bacterial phyla, accounting for 38.41–85.50% and 4.03–27.09% of the microbial community, respectively. Furthermore, Proteobacteria, especially genera such as Acinetobacter, Burkholderia, and Pseudomonas, with various resistance sequences, were the primary carriers of multiple ARGs. Notably, the genetic exchanges between biofilms and surrounding water facilitated the further propagation of high-risk ARGs, posing greater ecological risks. Redundancy analysis indicated that the total nitrogen and temperature in water determined the fate of pathogenic-resistant species. These findings provided theoretical support for the mitigation of ARGs contamination in aquatic environments.

Metagenomic approach revealed the mobility and co-occurrence of antibiotic resistomes between non-intensive aquaculture environment and human

BackgroundAquaculture is an important food source worldwide. The extensive use of antibiotics in intensive large-scale farms has resulted in resistance development. Non-intensive aquaculture is another aquatic feeding model that is conducive to ecological protection and closely related to the natural environment. However, the transmission of resistomes in non-intensive aquaculture has not been well characterized. Moreover, the influence of aquaculture resistomes on human health needs to be further understood. Here, metagenomic approach was employed to identify the mobility of aquaculture resistomes and estimate the potential risks to human health.ResultsThe results demonstrated that antibiotic resistance genes (ARGs) were widely present in non-intensive aquaculture systems and the multidrug type was most abundant accounting for 34%. ARGs of non-intensive aquaculture environments were mainly shaped by microbial communities accounting for 51%. Seventy-seven genera and 36 mobile genetic elements (MGEs) were significantly associated with 23 ARG types (p < 0.05) according to network analysis. Six ARGs were defined as core ARGs (top 3% most abundant with occurrence frequency > 80%) which occupied 40% of ARG abundance in fish gut samples. Seventy-one ARG-carrying contigs were identified and 75% of them carried MGEs simultaneously. The qacEdelta1 and sul1 formed a stable combination and were detected simultaneously in aquaculture environments and humans. Additionally, 475 high-quality metagenomic-assembled genomes (MAGs) were recovered and 81 MAGs carried ARGs. The multidrug and bacitracin resistance genes were the most abundant ARG types carried by MAGs. Strikingly, Fusobacterium_A (opportunistic human pathogen) carrying ARGs and MGEs were identified in both the aquaculture system and human guts, which indicated the potential risks of ARG transfer.ConclusionsThe mobility and pathogenicity of aquaculture resistomes were explored by a metagenomic approach. Given the observed co-occurrence of resistomes between the aquaculture environment and human, more stringent regulation of resistomes in non-intensive aquaculture systems may be required.4-S8N7kaEyTj6WBvQ6hX9pVideo

Bacitracin-resistant Staphylococcus aureus induced in chicken gut and in vitro under bacitracin exposure

It is common knowledge that prolonged and excessive use of antibiotics can lead to antimicrobial resistance. However, the characteristics and mechanism of resistant-bacteria induced by clinically recommended and prophylactic dose drugs remain largely unclear. This study aimed to observe the trends of drug resistance of the bacitracin-susceptible Staphylococcus aureus strain FS127 under exposure to bacitracin (BAC), which were induced in vitro and in chicken gut. Antimicrobial susceptibility testing was used to detect the susceptibility of S. aureus induced in vitro and in the chicken gut to gentamicin, chloramphenicol, tetracycline, doxycycline, penicillin and chloramphenicol. The research results showed that bacitracin could induce drug resistance in S. aureus both in vitro and in vivo. The bacitracin-resistance rate of S. aureus isolated from chicken gut was positively correlated with the dose and time of bacitracin administration. The findings revealed that bacitracin-resistant S. aureus induced in vivo had enhanced susceptibility to chloramphenicol but no such change in vitro. Meanwhile, RT-qPCR assay was used to detect the expression levels of vraD, braD, braR and bacA in typical strains with different bacitracin-resistance levels. It was found that BacA may play a key role in the bacitracin resistance of S. aureus. In conclusion, this work reveals the characteristics and mechanism of bacitracin-resistant S. aureus induced by bacitracin in vivo and in vitro respectively.

Regulatory interactions between daptomycin- and bacitracin-responsive pathways coordinate the cell envelope antibiotic resistance response of Enterococcus faecalis.

Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope-acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two-component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP-resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS-target genes under dual control, where expression is directly controlled by SapRS, which itself is up-regulated through LiaFSR. The network structure described here shows how Enterococcus faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components.

Assessing the Risk of Antimicrobial Resistant Enterococcal Infections in Humans Due to Bacitracin Usage in Poultry

Bacitracin is an antimicrobial used in the feed or water of poultry in the U.S. for the prevention, treatment, and control of clostridial diseases such as necrotic enteritis. Concern has been raised that bacitracin can select for antimicrobial-resistant bacteria that can be transmitted to humans and subsequently cause disease that is more difficult to treat because of the resistance. The objective of the present study was to perform a quantitative risk assessment (QRA) to estimate the potential risk in the U.S. of human infection with antimicrobial-resistant Enterococcus faecalis and E. faecium derived from chicken and turkey products as a result of bacitracin usage in U.S. poultry. The modeling approach estimated the annual number of healthcare-associated enterococcal infections in the U.S. that would be resistant to antimicrobial therapy and that would be derived from poultry sources because of bacitracin use in poultry. Parameter estimates were developed to be “maximum risk” to overestimate the risk to humans. While approximately 60% of E. faecalis and E. faecium derived from poultry were predicted to possess bacitracin resistance based on the presence of the bcrABDR gene locus, very few human-derived isolates possessed this trait. Furthermore, no vancomycin or linezolid-resistant strains of E. faecalis or E. faecium were detected in poultry sources between the years 2002 and 2019. The model estimated the number of antimicrobial-resistant E. faecalis and E. faecium cases per year that might resist therapy due to bacitracin use in poultry as 0.86 and 0.14, respectively, which translates to an annual risk estimate for E. faecalis of less than 1 in 350 million and for E. faecium of less than 1 in 2 billion for members of the U.S. population. Even with the use of risk-maximizing assumptions, the results indicate that there is a high probability that the use of bacitracin according to label instructions in U.S. poultry presents a negligible risk to human health.

The Bacillus subtilis cell envelope stress-inducible ytpAB operon modulates membrane properties and contributes to bacitracin resistance.

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σM responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σM-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σM-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.IMPORTANCEPeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the σM regulon, which is critical for intrinsic antibiotic resistance. The σM-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.

High Incidence of Multiple-Drug-Resistant Pheromone-Responsive Plasmids and Transmissions of VanA-Type Vancomycin-Resistant Enterococcus faecalis between Livestock and Humans in Taiwan.

A total of seventy VanA-type vancomycin-resistant enterococci (VRE) isolates obtained in Taiwan in the early 2000s were retrospectively characterized. Forty isolates were obtained from human patients and thirty from livestock. Of these VRE isolates, twenty-three (57.5%) of the human VRE and thirty (100%) of the livestock VRE were Enterococcus faecalis, and the remaining seventeen (42.5%) of the human VRE were E. faecium. Of the 53 E. faecalis isolates, twenty-two (96%) of the human VRE and thirty (100%) of the livestock VRE exhibited a high level of resistance to vancomycin and sensitivity to teicoplanin. They also had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. The vancomycin resistance of all of the 22 human isolates, and 20 of the 30 livestock isolates, transferred to E. faecalis FA2-2 at a frequency of 10-5 to 10-3 per donor cell in broth. Each of the transconjugants responded to E. faecalis pheromone (i.e., E. faecalis FA2-2 culture filtrate), indicating that the conjugative plasmids were pheromone-responsive plasmids. Three of the conjugative plasmids originated from human isolates, and five plasmids from livestock isolates were corresponded and classified as type A plasmid. Two plasmids originated from human isolates and six plasmids from livestock isolates were corresponded and classified as type B plasmid. E. faecalis FA2-2 containing either the type A or type B plasmid responded to the synthetic pheromone cAD1. The type A and type B plasmids transferred between E. faecalis FA2-2 and JH2SS at a frequency of about 10-2 per donor cell and conferred vancomycin, bacitracin, and erythromycin resistances. The complete DNA sequence of the representative type A plasmid pTW9 (85,068 bp) showed that the plasmid carried a Tn1546-like element encoding vanA-type resistance, erythromycin resistance (ermB), and bacitracin resistance (bcrABDR). The plasmid contained the regulatory region found in the pheromone-responsive plasmid and encoded the genes traA, traD and iad1, which are the key negative regulatory elements, and traE1, a key positive regulator of plasmid pAD1, indicating that plasmid pTW9 was pAD1-type pheromone-responsive plasmid. PFGE analysis of SmaI-digested chromosomal DNAs showed that several E. faecalis strains harboring an identical type A pheromone-responsive plasmid were indistinguishable, and that these were identified both in human and livestock isolates, indicating the transmissions of the VRE strains between livestock and humans. These data showed that the multiple-drug-resistant pheromone-responsive conjugative plasmids have been widely spread in both human and livestock VRE, and there was high potential for transfers of VRE from food animals to humans in Taiwan in the early 2000s.

Rare resistome rather than core resistome exhibited higher diversity and risk along the Yangtze River

As important freshwater ecosystems, the occurrence and distribution of antibiotic resistance genes (ARGs) in rivers are relevant to public health. However, studies investigating ARGs of different environmental media in river ecosystems are limited. In this study, we analyzed the ARGs of microbes in free-living setting, particle-associated setting, sediment and bank soil of the Yangtze River using metagenomics. Twenty-six ARGs were found in all samples regardless of media (core resistome) with a diversity of 8.6 %-34.7 %, accounting for 22.7 %-89.2 % of the relative abundance of the overall ARGs. The core resistome of the Yangtze River was dominated by multidrug resistance genes consisting mainly of efflux pumps and bacitracin resistance genes. The rare resistome was dominated by multidrug, sulfonamide, and aminoglycoside resistance genes. The core resistome was more prevalent in chromosomes, implying that these ARGs with low diversity and high relative abundance may be intrinsic to microbes in the Yangtze River. The rare resistome was more prevalent in plasmids, suggesting these ARGs with high diversity and low relative abundance were acquired under environmental stresses and had transfer potential. Additionally, we found that core and rare resistome were mainly carried by specific bacteria. Noteworthily, twenty-two ARGs of high clinical concern were identified in rare resistome, especially aac(6′)-I, sul1, and tetM, which were plasmid-borne and hosted by clinically relevant pathogens. Both core and rare resistome hosts showed the highest niche breadths in particle-associated setting compared to other media, and particle-associated setting could provide more stable and ideal conditions for resistome hosts to survive. This study elucidated the genetic locations of ARGs and the community assembly mechanisms of ARG hosts in freshwater environments.

Occurrence and fate of antibiotic-resistance genes and their potential hosts in high-moisture alfalfa silage treated with or without formic acid bactericide

Silage as the main forage for ruminants could be a reservoir for antibiotic resistance genes (ARGs) through which these genes got access into the animals' system causing a latent health risk. This study employed metagenomics and investigated the ARGs’ fate and transmission mechanism in high-moisture alfalfa silage treated with formic acid bactericide. The results showed that there were 22 ARGs types, in which multidrug, macrolide-lincosamide-streptogramine, bacitracin, beta-lactam, fosmidomycin, kasugamycin, and polymycin resistance genes were the most prevalent ARGs types in the ensiled alfalfa. The natural ensiling process increased ARGs enrichment. Intriguingly, after 5 days of ensiling, formic acid-treated silage reduced ARGs abundances by inhibiting host bacterial and plasmids. Although formic acid bactericide enhanced the fermentation characteristics of the high-moisture alfalfa by lowering silage pH, butyric acid concentration, dry matter losses and proteolysis, it increased ARGs abundances in alfalfa silage owing to increases in abundances of ARGs carriers and transposase after 90 days of ensiling. Notably, several pathogens like Staphylococcus, Clostridium, and Pseudomonas were inferred as potential ARGs hosts in high-moisture alfalfa silage, and high-moisture alfalfa silage may harbor a portion of the clinical ARGs. Fundamentally, microbes were distinguished as the foremost driving factor of ARGs propagation in ensiling microecosystem. In conclusion, although formic acid bactericide improved the fermentation characteristics of high-moisture alfalfa during ensiling and reduced ARGs enrichment at the initial ensiling stage, it increased ARGs enrichment at the end of ensiling.

Expanding our grasp of two-component signaling in Clostridioides difficile.

The intestinal pathogen Clostridioides difficile encodes roughly 50 TCS, but very few have been characterized in terms of their activating signals or their regulatory roles. A. G. Pannullo, B. R. Zbylicki, and C. D. Ellermeier (J Bacteriol 205:e00164-23, 2023, https://doi.org/10.1128/jb.00164-23) have identified both for the novel C. difficile TCD DraRS. DraRS responds to antibiotics that target lipid-II molecules in the bacterial cell envelope, and regulates the production of a novel glycolipid necessary for bacitracin and daptomycin resistance in C. difficile.

Molecular Characterization and Antimicrobial Susceptibilities of Corynebacterium pseudotuberculosis Isolated from Caseous Lymphadenitis of Smallholder Sheep and Goats.

Caseous lymphadenitis (CLA) is a bacterial infection caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) that affects sheep and goats, leading to abscess formation in their lymph nodes. The present study aimed to isolate and identify C. pseudotuberculosis from CLA in smallholder sheep and goats, and determine the resistance patterns, virulence, and resistance genes of the isolates. Additionally, genotypic and phylogenetic analysis of the isolates was conducted using ERIC-PCR and DNA sequencing techniques. A cross-sectional study examined 220 animals (130 sheep and 90 goats) from 39 smallholder flocks for clinical signs of CLA. Fifty-four (24.54%) animals showed CLA-compatible lesions, confirmed by C. pseudotuberculosis isolation and PCR identification. Sheep had a lower infection rate of CLA (18.46%) compared with goats (33.3%). Antimicrobial susceptibility testing of 54 C. pseudotuberculosis isolates to 24 antimicrobial drugs revealed that they were 100% resistant to bacitracin and florfenicol, while none of the isolates were resistant to norfloxacin. A high resistance rate was observed for penicillin and erythromycin (92.6% each). Interestingly, 16.7% of C. pseudotuberculosis isolates recovered from sheep showed vancomycin resistance. Molecular characterization of C. pseudotuberculosis isolates revealed that PLD, PIP, and FagA virulence genes were present in all examined isolates. However, the FagB, FagC, and FagD genes were detected in 24 (100%), 20 (83%), and 18 (75%) of the sheep isolates, and 26 (87%), 26 (87%), and 18 (60%) of the goat isolates, respectively. The β-lactam resistance gene was present in all isolates. Furthermore, 83% of the sheep isolates carried the aminoglycoside (aph(3″)-lb), chloramphenicol (cat1), and bacitracin (bcrA) resistance genes. Among the isolates recovered from goats, 73% were found to contain macrolides (ermX), sulfonamide (sul1), and bacitracin (bcrA) resistance genes. It is worrisome that the glycopeptide (vanA) resistance gene was detected in 8% of the sheep isolates as a first report. ERIC-PCR genotyping of 10 multi-drug-resistant C. pseudotuberculosis isolates showed a high similarity index of 83.6% between isolates from sheep and goats. Nucleotide sequence analysis of partial 16S rRNA sequences of C. pseudotuberculosis revealed 98.83% similarity with biovar Ovis of globally available reference sequences on the Genbank database. Overall, our findings might indicate that C. pseudotuberculosis infection in smallholders in Egypt might be underestimated despite the significant financial impact on animal husbandry and potential health hazards it poses. Moreover, this study highlights the importance of implementing a sustainable control strategy and increasing knowledge and awareness among smallholder breeders to mitigate the economic impact of CLA.

Identification of DraRS in Clostridioides difficile, a Two-Component Regulatory System That Responds to Lipid II-Interacting Antibiotics.

Clostridioides difficile is a Gram-positive opportunistic pathogen that results in 220,000 infections, 12,000 deaths, and upwards of $1 billion in medical costs in the United States each year. C. difficile is highly resistant to a variety of antibiotics, but we have a poor understanding of how C. difficile senses and responds to antibiotic stress and how such sensory systems affect clinical outcomes. We have identified a spontaneous C. difficile mutant that displays increased daptomycin resistance. We performed whole-genome sequencing and found a nonsense mutation, S605*, in draS, which encodes a putative sensor histidine kinase of a two-component system (TCS). The draSS605* mutant has an ~4- to 8-fold increase in the daptomycin MIC compared to the wild type (WT). We found that the expression of constitutively active DraRD54E in the WT increases daptomycin resistance 8- to 16-fold and increases bacitracin resistance ~4-fold. We found that a selection of lipid II-inhibiting compounds leads to the increased activity of the luciferase-based reporter PdraR-slucopt, including vancomycin, bacitracin, ramoplanin, and daptomycin. Using RNA sequencing (RNA-seq), we identified the DraRS regulon. Interestingly, we found that DraRS can induce the expression of the previously identified hex locus required for the synthesis of a novel glycolipid produced in C. difficile. Our data suggest that the induction of the hex locus by DraR explains some, but not all, of the DraR-induced daptomycin and bacitracin resistance. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. C. difficile encodes ~50 annotated two-component systems (TCSs); however, only a few have been studied. The function of these unstudied TCSs is not known. Here, we show that the TCS DraRS plays a role in responding to a subset of lipid II-inhibiting antibiotics and mediates resistance to daptomycin and bacitracin in part by inducing the expression of the recently identified hex locus, which encodes enzymes required for the production of a novel glycolipid in C. difficile.

Microbial community and antibiotic resistance gene distribution in food waste, anaerobic digestate, and paddy soil

The study assessed the occurrence and distribution of microbial community and antibiotic resistance genes (ARGs) in food waste, anaerobic digestate, and paddy soil samples, and revealed the potential hosts of ARGs and factors influencing their distribution. A total of 24 bacterial phyla were identified, of which 16 were shared by all samples, with Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria accounting for 65.9–92.3 % of the total bacterial community. Firmicutes was the most abundant bacteria in food waste and digestate samples, accounting for 33–83 % of the total microbial community. However, in paddy soil samples with digestate, Proteobacteria had the highest relative abundance of 38–60 %. Further, 22 ARGs were detected in food waste and digestate samples, with multidrug, macrolide-lincosamide-streptogramin (MLS), bacitracin, aminoglycoside, tetracycline, vancomycin, sulfonamide, and rifamycin resistance genes being the most abundant and shared by all samples. The highest total relative abundance of ARGs in food waste, digestate, and soil without and with digestate was detected in samples from January 2020, May 2020, October 2019, and May 2020, respectively. The MLS, vancomycin, tetracycline, aminoglycoside, and sulfonamide resistance genes had higher relative abundance in food waste and anaerobic digestate samples, whereas multidrug, bacteriocin, quinolone, and rifampin resistance genes were more abundant in paddy soil samples. Redundancy analysis demonstrated that aminoglycoside, tetracycline, sulfonamide, and rifamycin resistance genes were positively correlated with total ammonia nitrogen and pH of food waste and digestate samples. Vancomycin, multidrug, bacitracin, and fosmidomycin resistance genes had positive correlations with potassium, moisture, and organic matter in soil samples. The co-occurrence of ARG subtypes with bacterial genera was investigated using network analysis. Actinobacteria, Proteobacteria, Bacteroidetes, and Acidobacteria were identified as potential hosts of multidrug resistance genes.

Reforestation substantially changed the soil antibiotic resistome and its relationships with metal resistance genes, mobile genetic elements, and pathogens

Revealing the effects of reforestation on soil antibiotic resistome is essential for assessing ecosystem health, yet related studies remain scarce. Here, to determine the responses of the soil antibiotic resistome to reforestation, 30 pairs of cropland and forest soil samples were collected from southwestern China, a region with high environmental heterogeneity. All the forests had been derived from croplands more than one decade ago. The diversity and abundance of soil antibiotic resistance genes (ARGs), metal resistance genes (MRGs), mobile genetic elements (MGEs), and pathogens were determined by metagenomic sequencing and real-time PCR. The results showed that reforestation significantly increased soil microbial abundance and the contents of Cu, total carbon, total nitrogen, total organic carbon, and ammonium nitrogen. Nevertheless, it decreased the contents of soil Zn, Ba, nitrate nitrogen, and available phosphorus. The main soil ARGs identified in this region were vancomycin, multidrug, and bacitracin resistance genes. Reforestation significantly increased the soil ARG abundance by 62.58%, while it decreased the ARG richness by 16.50%. Reforestation exerted no significant effects on the abundance of heavy metal resistance genes and pathogens, but it doubled the abundance of MGEs. Additionally, reforestation substantially decreased the co-occurrence frequencies of ARGs with MRGs and pathogens. In contrast, the correlation between ARGs and MGEs was greatly enhanced by reforestation. Similarly, the correlations between soil ARG abundance and environmental factors were also strengthened by reforestation. These findings suggest that reforestation can substantially affect the soil antibiotic resistome and exerts overall positive effects on soil health by decreasing ARG richness, providing critical information for assessing the effects of “grain for green” project on soil health.

In silico identification of auxiliary factors genes required for β-lactam resistance

Staphylococcus aureus is a type of bacteria commonly found on the skin and in the nasal passages of healthy individuals. However, it can also cause a range of infections in clinical settings. One of the most concerning aspects of S. aureus is its ability to develop antibiotic resistance. Methicillin-resistant S. aureus (MRSA) is a strain of the bacteria that is resistant to many antibiotics and can be difficult to treat. The primary mechanism of methicillin resistance in MRSA is the presence of the mecA gene, which encodes for a modified penicillin-binding protein known as PBP2a. This protein has a lower affinity for β-lactam antibiotics. Another gene, blaZ, is also present in MRSA and encodes for a β-lactamase enzyme that can hydrolyse and inactivate β-lactam antibiotics such as penicillin. In addition, there are several auxiliary factors that can contribute to β-lactam resistance. They can include efflux pumps, enzymes that modify or degrade antibiotics, and bacterial cell wall modifications that reduce the affinity of antibiotics for their targets. In this study, with the aid of the in silico identification method, we identify the novel auxiliary factors aux1, aux2, aux4, aux11, aux14, aux16 and aux19. Next, we show that aux2, aux4, aux11, aux14 are not directly involved in β-lactam resistance, but may contribute through other mechanisms that decrease the efficacy of these antibiotics, whereas aux16 and aux19 are directly associated with β-lactam and bacitracin resistance, respectively. Understanding the various auxiliary factors that contribute to beta-lactam resistance can help guide the development of new antibiotics and other therapeutic strategies.

HexSDF Is Required for Synthesis of a Novel Glycolipid That Mediates Daptomycin and Bacitracin Resistance in C. difficile.

Clostridioides difficile is a Gram-positive opportunistic pathogen responsible for 250,000 hospital-associated infections, 12,000 hospital-associated deaths, and $1 billion in medical costs in the United States each year. There has been recent interest in using a daptomycin analog, surotomycin, to treat C. difficile infections. Daptomycin interacts with phosphatidylglycerol and lipid II to disrupt the membrane and halt peptidoglycan synthesis. C. difficile has an unusual lipid membrane composition, as it has no phosphatidylserine or phosphatidylethanolamine, and ~50% of its membrane is composed of glycolipids, including the unique C. difficile lipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS), HexRK, that is required for C. difficile resistance to daptomycin. Using transcriptome sequencing (RNA-seq), we found that HexRK regulates expression of hexSDF, a three-gene operon of unknown function. Based on bioinformatic predictions, hexS encodes a monogalactosyldiacylglycerol synthase, hexD encodes a polysaccharide deacetylase, and hexF encodes an MprF-like flippase. Deletion of hexRK leads to a 4-fold decrease in daptomycin MIC, and that deletion of hexSDF leads to an 8- to 16-fold decrease in daptomycin MIC. The ΔhexSDF mutant is also 4-fold less resistant to bacitracin but no other cell wall-active antibiotics. Our data indicate that in the absence of HexSDF, the phospholipid membrane composition is altered. In wild-type (WT) C. difficile, the unique glycolipid HNHDRG makes up ~17% of the lipids in the membrane. However, in a ΔhexSDF mutant, HNHDRG is completely absent. While it is unclear how HNHDRG contributes to daptomycin resistance, the requirement for bacitracin resistance suggests it has a general role in cell membrane biogenesis. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. Little is understood about C. difficile membrane lipids, but a unique glycolipid, HNHDRG, has been previously identified in C. difficile and, currently, has not been identified in other organisms. Here, we show that HexSDF and HexRK are required for synthesis of HNHDRG and that production of HNHDRG impacts resistance to daptomycin and bacitracin.

Metagenomics insights into the effects of lactic acid bacteria inoculation on the biological reduction of antibiotic resistance genes in alfalfa silage

Antibiotic resistance genes (ARGs) are a new type of pollutant and pose major threats to public health. However, the distribution and transmission risk of ARGs in alfalfa silage as the main forage for ruminants have not been studied. This study first deciphered the effects of Lactobacillus plantarum MTD/1 or Lactobacillus buchneri 40788 inoculations on distribution and transmission mechanism of ARGs in alfalfa silage by metagenomics. Results showed that multidrug and bacitracin resistance genes were the dominant ARGs in ensiled alfalfa. The natural ensiling process increased the abundances of bacitracin, beta_lactam, and aminoglycoside in alfalfa silage with 30% DM, and vancomycin in alfalfa silage with 40% DM. Meanwhile, prolonged wilting increased ARG enrichment in fresh alfalfa. Interestingly, alfalfa silage inoculated with L. plantarum MTD/1 or L. buchneri 40788 reduced the abundances of total ARG, and multidrug, MLS, vancomycin, aminoglycoside, tetracycline, and fosmidomycin resistance genes by reductions of the host bacteria and the enrichment of ARGs located in the plasmid. The hosts of ARG in alfalfa silage were mainly derived from harmful bacteria or pathogens, and some of the clinical ARGs were observed in alfalfa silage. Basically, the combined effect of microbes, MGEs, and fermentation quality was the major driver of ARG transfer and dissemination in microecosystem of ensiling, where the microbes appeared to be the crucial factor. In summary, inoculation with the present lactic acid bacteria could reduce ARG abundance in ensiled alfalfa, and a better effect was observed in L. plantarum-treated silage than in L. buchneri treated silage.

Monsoon affects the distribution of antibiotic resistome in Tibetan glaciers

Antibiotic-resistance gene (ARG) is a biological pollutant and is globally distributed due to increased anthropogenic activities. ARGs in the cryosphere have received increased attention due to global warming, and ARGs in glaciers are predicted to be released into downstream ecosystems during glacier melting. In this study, ARG distribution and influential factors were investigated in 85 samples from 21 Tibetan glaciers, covering snow, ice, and cryoconite habitats. The results revealed ARGs against 29 antibiotics in Tibetan glaciers, dominated by tetracycline, bacitracin, macrolide, and fluoroquinolone resistance. ARGs in snow exhibited biogeographic patterns influenced by atmospheric circulation. Specifically, monsoon-dominated glaciers exhibited a significantly higher abundance of ARGs than the westerly-dominated glaciers, which could be associated with higher antibiotic usage in the Indian subcontinent. Of the 3241 metagenome-assembled genomes obtained, 36.8% of which were identified as ARG hosts and 33.8% were multidrug-resistant. In addition, 90 ARGs were linked to mobile genetic elements (MGEs). 90.9% and 9.1% of MGEs were identified as plasmid and phage in 45 MAGs carrying both ARGs and MGEs. Our study suggests a greater risk of ARGs being released from the monsoon-dominated glaciers, which were the glaciers that melt at high rates and thus need to be carefully monitored.