Ribosomal Protection Research Articles

Intracellular Accumulation of Linezolid and Florfenicol in OptrA-Producing Enterococcus faecalis and Staphylococcus aureus.

The optrA gene, which confers transferable resistance to oxazolidinones and phenicols, is defined as an ATP-binding cassette (ABC) transporter but lacks transmembrane domains. The resistance mechanism of optrA and whether it involves antibiotic efflux or ribosomal protection remain unclear. In this study, we determined the MIC values of all bacterial strains by broth microdilution, and used ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry to quantitatively determine the intracellular concentrations of linezolid and florfenicol in Enterococcus faecalis and Staphylococcus aureus. Linezolid and florfenicol both accumulated in susceptible strains and optrA-carrying strains of E. faecalis and S. aureus. No significant differences were observed in the patterns of drug accumulation among E. faecalis JH2-2, E. faecalis JH2-2/pAM401, and E. faecalis JH2-2/pAM401+optrA, but also among S. aureus RN4220, S. aureus RN4220/pAM401, and S. aureus RN4220/pAM401+optrA. ANOVA scores also suggested similar accumulation conditions of the two target compounds in susceptible strains and optrA-carrying strains. Based on our findings, the mechanism of optrA-mediated resistance to oxazolidinones and phenicols obviously does not involve active efflux and the OptrA protein does not confer resistance via efflux like other ABC transporters.

Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline.

Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response.IMPORTANCEBifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.

Complete Genome Sequence and Characterization of Linezolid-Resistant Enterococcus faecalis Clinical Isolate KUB3006 Carrying a cfr(B)-Transposon on Its Chromosome and optrA-Plasmid.

Linezolid (LZD) has become one of the most important antimicrobial agents for infections caused by gram-positive bacteria, including those caused by Enterococcus species. LZD-resistant (LR) genetic features include mutations in 23S rRNA/ribosomal proteins, a plasmid-borne 23S rRNA methyltransferase gene cfr, and ribosomal protection genes (optrA and poxtA). Recently, a cfr gene variant, cfr(B), was identified in a Tn6218-like transposon (Tn) in a Clostridioides difficile isolate. Here, we isolated an LR Enterococcus faecalis clinical isolate, KUB3006, from a urine specimen of a patient with urinary tract infection during hospitalization in 2017. Comparative and whole-genome analyses were performed to characterize the genetic features and overall antimicrobial resistance genes in E. faecalis isolate KUB3006. Complete genome sequencing of KUB3006 revealed that it carried cfr(B) on a chromosomal Tn6218-like element. Surprisingly, this Tn6218-like element was almost (99%) identical to that of C. difficile Ox3196, which was isolated from a human in the UK in 2012, and to that of Enterococcus faecium 5_Efcm_HA-NL, which was isolated from a human in the Netherlands in 2012. An additional oxazolidinone and phenicol resistance gene, optrA, was also identified on a plasmid. KUB3006 is sequence type (ST) 729, suggesting that it is a minor ST that has not been reported previously and is unlikely to be a high-risk E. faecalis lineage. In summary, LR E. faecalis KUB3006 possesses a notable Tn6218-like-borne cfr(B) and a plasmid-borne optrA. This finding raises further concerns regarding the potential declining effectiveness of LZD treatment in the future.

Metagenomic Insights Into the Contribution of Phages to Antibiotic Resistance in Water Samples Related to Swine Feedlot Wastewater Treatment.

In this study, we examined the types of antibiotic resistance genes (ARGs) possessed by bacteria and bacteriophages in swine feedlot wastewater before and after treatment using a metagenomics approach. We found that the relative abundance of ARGs in bacterial DNA in all water samples was significantly higher than that in phages DNA (>10.6-fold), and wastewater treatment did not significantly change the relative abundance of bacterial- or phage-associated ARGs. We further detected the distribution and diversity of the different types of ARGs according to the class of antibiotics to which they confer resistance, the tetracycline resistance genes were the most abundant resistance genes and phages were more likely to harbor ATP-binding cassette transporter family and ribosomal protection genes. Moreover, the colistin resistance gene mcr-1 was also detected in the phage population. When assessing the contribution of phages in spreading different groups of ARGs, β-lactamase resistance genes had a relatively high spreading ability even though the abundance was low. These findings possibly indicated that phages not only could serve as important reservoir of ARG but also carry particular ARGs in swine feedlot wastewater, and this phenomenon is independent of the environment.

RiboProP: a probabilistic ribosome positioning algorithm for ribosome profiling.

Ribosome profiling has been widely used to study translation in a genome-wide fashion. It requires deep sequencing of ribosome protected mRNA fragments followed by mapping of fragments to the reference genome. For applications such as identification of ribosome pausing sites, it is not enough to map a fragment to a given gene, but the exact position of the ribosome represented by the fragment must be identified for each mRNA fragment. The assignment of the correct ribosome position is complicated by the broad length distribution of the ribosome protected fragments caused by the known sequence bias of micrococcal nuclease (MNase), the most widely used nuclease for digesting mRNAs in bacteria. Available mapping algorithms suffer from either MNase bias or low accuracy in characterizing the ribosome pausing kinetics. In this paper, we introduce a new computational method for mapping the ribosome protected fragments to ribosome locations. We first develop a mathematical model of the interplay between MNase digestion and ribosome protection of the mRNAs. We then use the model to reconstruct the ribosome occupancy profile on a per gene level. We demonstrate that our method has the capability of mitigating the sequence bias introduced by MNase and accurately locating ribosome pausing sites at codon resolution. We believe that our method can be broadly applied to ribosome profiling studies on bacteria where codon resolution is necessary. Source code implementing our approach can be downloaded under GPL3 license at http://bioserv.mps.ohio-state.edu/RiboProP. Supplementary data are available at Bioinformatics online.

Dynamics of oxytetracycline, sulfamerazine, and ciprofloxacin and related antibiotic resistance genes during swine manure composting

Understanding the dynamics of veterinary antibiotic and related antibiotic resistance genes (ARGs) during swine manure composting is crucial in assessing the environmental risk of antibiotics, which could effectively reduce their impact in natural environments. This study investigated the dissipation of oxytetracycline (OTC), sulfamerazine (SM1) and ciprofloxacin (CIP) as well as the behaviour of their corresponding ARGs during swine manure composting. These antibiotics were added at two concentration levels and two different methods of addition (single/mixture). The results indicated that the removal efficiency of antibiotics by composting were ≥85%, except for the single-SM1 treatment. The tetracycline resistance genes (TRGs) encoding ribosomal protection proteins (RPP) and efflux pump (EFP) and fluoroquinolone resistance genes (FRGs) could be effectively removed after 42 days. On the contrary, the TRGs encoding enzymatic inactivation (EI) and sulfonamide resistance genes (SRGs) were enriched up to 31-fold (sul 2 in single-low-SM1). Statistical analyses indicated that the behaviour of these class antibiotics and ARGs were controlled by microbial activity and significantly influenced by environmental factors (mainly C/N, moisture and pH) throughout the composting process.

Face mask sampling reveals antimicrobial resistance genes in exhaled aerosols from patients with chronic obstructive pulmonary disease and healthy volunteers

IntroductionThe 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...

Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ2 mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).

Quantitative Proteomics Analysis of Membrane Proteins in Enterococcus faecalis With Low-Level Linezolid-Resistance

Despite increasing reports of low-level linezolid-resistant enterococci worldwide, the mechanism of this resistance remains poorly understood. Previous transcriptome studies of low-level linezolid-resistant Enterococcus faecalis isolates have demonstrated a number of significantly up-regulated genes potentially involved in mediation of drug resistance. However, whether the transcriptome faithfully reflects the proteome remains unknown. In this study, we performed quantitative proteomics analysis of membrane proteins in an E. faecalis isolate (P10748) with low-level linezolid-resistance in comparison with two linezolid-susceptible strains 3138 and ATCC 29212, all of which have been previously investigated by whole transcriptome analysis. A total of 8,197 peptides associated with 1,170 proteins were identified in all three isolates with false discovery rate (FDR) at 1% and P < 0.05. There were 14 significantly up-regulated and 6 significantly down-regulated proteins in strain P10748 compared to strains 3138 and ATCC 29212, which were in general positively correlated with transcription levels revealed in previous transcriptome studies. Our analysis suggests that the low-level linezolid-resistance in E. faecalis is conferred primarily by the ATP-binding cassette protein OptrA through ribosomal protection and, possibly, also by the enterococcal surface protein (Esp) and other proteins through biofilm formation. The genetic transfer of optrA is potentially regulated by the surface exclusion protein Sea1, conjugal transfer protein TraB, replication protein RepA and XRE family transcription regulator protein. This report represents the first investigation of the mechanisms of linezolid-resistance in E. faecalis by a quantitative proteomics approach.

Tetracycline and Sulfonamide Antibiotic Resistance Genes in Soils From Nebraska Organic Farming Operations.

There is widespread agreement that agricultural antibiotic resistance should be reduced, however, it is unclear from the available literature what an appropriate target for reduction would be. Organic farms provide a unique opportunity to disentangle questions of agricultural antibiotic drug use from questions of antibiotic resistance in the soil. In this study, soil was collected from 12 certified organic farms in Nebraska, evaluated for the presence of tetracycline and sulfonamide resistance genes (n = 15 targets), and correlated to soil physical, chemical, and biological parameters. Tetracycline and sulfonamide antibiotic resistance genes (ARGs) were found in soils from all 12 farms, and 182 of the 196 soil samples (93%). The most frequently detected gene was tetG (55% of samples), followed by tet(Q) (49%), tet(S) (46%), tet(X) (30%), and tetA(P) (29%). Soil was collected from two depths. No differences in ARGs were observed based on soil depth. Positive correlations were noted between ARG presence and soil electrical conductivity, and concentrations of Ca, Na, and Mehlich-3 phosphorus. Data from this study point to possible relationships between selected soil properties and individual tetracycline resistance genes, including tet(O) which is a common target for environmental samples. We compared organic farm results to previously published data from prairie soils and found significant differences in detection frequency for 12 genes, eight of which were more commonly detected in prairie soils. Of interest, when tetracycline ARG results were sorted by gene mechanism, the efflux genes were generally present in higher frequency in the prairie soils, while the ribosomal protection and enzymatic genes were more frequently detected in organic farm soils, suggesting a possible ecological role for specific tetracycline resistance mechanisms. By comparing soil from organic farms with prairie soils, we can start to determine baseline effects of low-chemical input agricultural production practices on multiple measures of resistance.

Tetracycline-Inactivating Enzymes.

Tetracyclines have been foundational antibacterial agents for more than 70 years. Renewed interest in tetracycline antibiotics is being driven by advancements in tetracycline synthesis and strategic scaffold modifications designed to overcome established clinical resistance mechanisms including efflux and ribosome protection. Emerging new resistance mechanisms, including enzymatic antibiotic inactivation, threaten recent progress on bringing these next-generation tetracyclines to the clinic. Here we review the current state of knowledge on the structure, mechanism, and inhibition of tetracycline-inactivating enzymes.

High Levels of Intrinsic Tetracycline Resistance in Mycobacterium abscessus Are Conferred by a Tetracycline-Modifying Monooxygenase.

Tetracyclines have been one of the most successful classes of antibiotics. However, its extensive use has led to the emergence of widespread drug resistance, resulting in discontinuation of use against several bacterial infections. Prominent resistance mechanisms include drug efflux and the use of ribosome protection proteins. Infrequently, tetracyclines can be inactivated by the TetX class of enzymes, also referred to as tetracycline destructases. Low levels of tolerance to tetracycline in Mycobacterium smegmatis and Mycobacterium tuberculosis have been previously attributed to the WhiB7-dependent TetV/Tap efflux pump. However, Mycobacterium abscessus is ∼500-fold more resistant to tetracycline than M. smegmatis and M. tuberculosis In this report, we show that this high level of resistance to tetracycline and doxycycline in M. abscessus is conferred by a WhiB7-independent tetracycline-inactivating monooxygenase, MabTetX (MAB_1496c). The presence of sublethal doses of tetracycline and doxycycline results in a >200-fold induction of MabTetX, and an isogenic deletion strain is highly sensitive to both antibiotics. Further, purified MabTetX can rapidly monooxygenate both antibiotics. We also demonstrate that expression of MabTetX is repressed by MabTetRx, by binding to an inverted repeat sequence upstream of MabTetRx; the presence of either antibiotic relieves this repression. Moreover, anhydrotetracycline (ATc) can effectively inhibit MabTetX activity in vitro and decreases the MICs of both tetracycline and doxycycline in vivo Finally, we show that tigecycline, a glycylcycline tetracycline, not only is a poor substrate of MabTetX but also is incapable of inducing the expression of MabTetX. This is therefore the first demonstration of a tetracycline-inactivating enzyme in mycobacteria. It (i) elucidates the mechanism of tetracycline resistance in M. abscessus, (ii) demonstrates the use of an inhibitor that can potentially reclaim the use of tetracycline and doxycycline, and (iii) identifies two sequential bottlenecks-MabTetX and MabTetRx-for acquiring resistance to tigecycline, thereby reiterating its use against M. abscessus.

Ribosome protection by antibiotic resistance ATP-binding cassette protein.

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins.

Influence of zeolite and superphosphate as additives on antibiotic resistance genes and bacterial communities during factory-scale chicken manure composting

Factory-scale chicken manure composting added with zeolite (F), superphosphate (G), or zeolite and ferrous sulfate (FL) simultaneously, were evaluate for their effects on the behaviors of antibiotic resistance genes (ARGs) and bacterial communities. After composting, ARGs in manure decreased by 67.3% in the control, whereas the reductions were 86.5%, 68.6% and 72.2% in F, G and FL, respectively. ARGs encoding ribosomal protection proteins (tetO, tetB(P), and tetM) were reduced to a greater extent than tetG, tetL, sul1 and sul2. Bacteria pathogens were also effectively removed by composting. Network analysis showed that Firmicutes were the important potential host bacteria for ARGs. The bacterial communities and environmental factors, as well as the intI gene, contributed significantly to the variation of ARGs. The ARGs and integrons were reduced more when zeolite was added than when superphosphate was added; thus, it may be useful for reducing the risks of ARGs in chicken manure.

Increased occurrence of heavy metals, antibiotics and resistance genes in surface soil after long-term application of manure

The purpose of this study was to investigate the impact of long-term application of pig manure on the accumulation of heavy metals, antibiotics and ARGs in surface soil sampled from the Jiaxing long-term field experimental site with three manure treatments, N-PM (0 kg/ha/y, dw), L-PM (7720 kg/ha/y, dw), and H-PM (11,580 kg/ha/y, dw), in 2013 and 2014. The results showed that most serious metal pollution of Zn and Cu was recorded in all manured samples in both years, and their contents exceeded the soil quality standards. Among the three tetracyclines, chlortetracycline was the predominant antibiotic detected with a range of 3.04–98.03 μg·kg−1 in 2013 and 28.67–344.74 μg·kg−1 in 2014 after long-term pig manure application. Q-PCR results showed that the average accumulation of ribosomal protection protein genes (tetM, tetO, tetQ and tetW) was lower than most of the efflux pump genes (tetA and tetG). The abundance of tet and sul genes of those sites with manure application was significantly higher than that of sites without manure application in both years. Metagenomics analysis of ARGs revealed that the abundance of multidrug resistance genes was the most abundant subtype, followed by fluoroquinolone, bacitracin, sulfonamide and tetracycline. There was a positive correlation between the levels of ARGs; soil organic matter, antibiotics, Cu, As, and Zn levels in both years. These results may shed light on the mechanism underlining the effects of long-term manure application on the occurrence and dissemination of ARGs in surface soil.

Characterization of poxtA, a novel phenicol-oxazolidinone-tetracycline resistance gene from an MRSA of clinical origin.

To characterize a novel phenicol-oxazolidinone-tetracycline resistance gene, named poxtA, identified in a previously described MRSA strain that was highly resistant to linezolid and also carried the cfr gene. The poxtA gene was identified by bioinformatic analysis of the whole genome sequence of Staphylococcus aureus AOUC-0915. The poxtA gene was cloned in a shuttle plasmid vector and expressed in Escherichia coli, S. aureus and Enterococcus faecalis to investigate the protein function. Comparative sequence analyses at the protein and genetic levels were carried out using standard procedures. The poxtA gene encodes a protein that is 32% identical to OptrA and exhibits structural features typical of the F lineage of the ATP-binding cassette (ABC) protein superfamily that cause antibiotic resistance by ribosomal protection. Expression of poxtA in E. coli, S. aureus and E. faecalis was able to decrease susceptibility to phenicols, oxazolidinones and tetracyclines. A database search identified the presence of poxtA in E. faecalis, Enterococcus faecium and Pediococcus acidilactici strains, mostly of animal origin, and revealed the presence of poxtA homologues in the genomes of some Clostridiales. Analysis of the genetic context revealed that poxtA was located in a composite transposon-like structure containing two IS1216 elements. A novel resistance gene, named poxtA, encoding a protein of the antibiotic resistance (ARE) ABC-F lineage, was identified in the genome of an MRSA of clinical origin. PoxtA can confer decreased susceptibility to phenicols, oxazolidinones and tetracyclines and is associated with a putative mobile element that could contribute to its horizontal dissemination.

Diversity of antibiotic resistance genes and encoding ribosomal protection proteins gene in livestock waste polluted environment

ABSTRACTThe rapid development and increase of antibiotic resistance are global phenomena resulting from the extensive use of antibiotics in human clinics and animal feeding operations. Antibiotics can promote the occurrence of antibiotic resistance genes (ARGs), which can be transferred horizontally to humans and animals through water and the food chain. In this study, the presence and abundance of ARGs in livestock waste was monitored by quantitative PCR. A diverse set of bacteria and tetracycline resistance genes encoding ribosomal protection proteins (RPPs) from three livestock farms and a river were analyzed through denaturing gradient gel electrophoresis (DGGE). The abundance of sul(I) was 103 to 105 orders of magnitude higher than that of sul(II). Among 11 tet-ARGs, the most abundant was tet(O). The results regarding bacterial diversity indicated that the presence of antibiotics might have an evident impact on bacterial diversity at every site, particularly at the investigated swine producer. The effect of livestock waste on the bacterial diversity of soil was stronger than that of water. Furthermore, a sequencing analysis showed that tet(M) exhibited two genotypes, while the other RPPs-encoding genes exhibited at least three genotypes. This study showed that various ARGs and RPPs-encoding genes are particularly widespread among livestock.

Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest.

Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaALC and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaALC is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ2 ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ2 mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaALC inactive in antibiotic protection and the EQ2 mutant inactive in PTC inhibition.

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs from environmental hotspots such as discharge sites of pharmaceutical effluents.

Return of the tetracyclines: omadacycline, a novel aminomethylcycline antimicrobial.

Omadacycline is a novel aminomethylcycline antimicrobial agent that is available in both oral and intravenous formulations. The distinguishing structural characteristics of omadacycline from other tetracyclines allow for its continued antimicrobial activity in the presence of traditional tetracycline resistance mechanisms (efflux pumps and ribosomal protection proteins). Omadacycline has been found to have potent activity against antibiotic-resistant pathogens including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, extended spectrum beta-lactamase-producing Escherichia coli and multidrug-resistant Streptococcus pneumoniae. Currently available data indicate that omadacycline is generally well tolerated with the most common adverse effects being gastrointestinal symptoms. Omadacycline seems to be a promising new agent for the treatment of community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections. Studies for the treatment of cystitis in adult females are currently underway, and future results of these studies will further help delineate the antibacterial role of omadacycline.