Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome.

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding the cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes, but strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at internal in-frame and out-of-frame start sites, can be functionally important and contribute to the "alternative" bacterial proteome. The internal start sites may also play regulatory roles in gene expression.

Prevalence and molecular characterization of methicillin-resistant Staphylococcus aureus with mupirocin, fusidic acid and/or retapamulin resistance.

BACKGROUND: The data on the prevalence of resistance to mupirocin (MUP), fusidic acid (FA) and retapamulin (RET) in methicillin-resistant Staphylococcus aureus (MRSA) from China are still limited. This study aimed to examine these three antibiotics resistance in 1206 MRSA clinical isolates from Eastern China. Phenotypic MUP, FA and RET resistance was determined by minimum inhibitory concentrations (MICs), and genotypic by PCR and DNA sequencing of the mupA/B, fusB-D, cfr, vgaA/Av/ALC/B/C/E, lsaA-C/E and salA and mutations in ileS, fusA/E, rplC, and 23S RNA V domain. The genetic characteristics of resistance isolates were conducted by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: Overall MRSA MUP, FA and RET resistance was low (5.1, 1.0 and 0.3%, respectively). MupA was the mechanism of high-level MUP resistance. All low-level MUP resistance isolates possessed an equivocal mutation N213D in IleS; of these, 2 reported an additional V588F mutation with an impact on the Rossman fold. FusA mutations, such as L461K, H457Q, H457Y and V90I were the primary FA mechanisms among high-level resistance isolates, most of which also contained fusC; however, all low-level resistance strains carried fusB. Except lsaE gene detected in one isolate, no other resistance mechanisms tested were found among RET-resistant isolates. Additionally, sixteen PFGE types (A-P) were observed, among which type B was the most common (49/76, 64.5%), followed by types E and G (4/76, 5.3% each) and types C and M (3/76, 3.9% each). All resistant strains were divided into 15 ST types by MLST. ST764 (24/76, 31.6%), ST630 (11/76, 14.5%), ST239 (9/76, 11.8%) and ST5 (7/76, 9.2%) were the major types. PFGE type B isolates with the aforementioned STs were mainly found in mupirocin resistant isolates. CONCLUSIONS: MUP, FA and RET exhibited highly activity against the MRSA isolates. Acquired genes and chromosome-borne genes mutations were responsible for MUP and FA resistance; however, the mechanism for some RET-resistant isolates remains to be further elucidated. Also, the surveillance to MUP in MRSA should be strengthened to prevent elevated resistance due to the expansion of clones.

Unraveling the Metabolic Routes of Retapamulin: Insights into Drug Development of Pleuromutilins.

Retapamulin, a semisynthetic pleuromutilin derivative, is exclusively used for the topical short-term medication of impetigo and staphylococcal infections. In the present study, we report that retapamulin is adequately and rapidly metabolized in vitro via various metabolic pathways, such as hydroxylation, including mono-, di-, and trihydroxylation, and demethylation. Like tiamulin and valnemulin, the major metabolic routes of retapamulin were hydroxylation at the 2ß and 8α positions of the mutilin moiety. Moreover, in vivo metabolism concurred with the results of the in vitro assays. Additionally, we observed significant interspecies differences in the metabolism of retapamulin. Until now, modifying the side chain was the mainstream method for new drug discovery of the pleuromutilins. This approach, however, could not resolve the low bioavailability and short efficacy of the drugs. Considering the rapid metabolism of the pleuromutilins mediated by cytochrome P450 enzymes, we propose that blocking the active metabolic site (C-2 and C-8 motif) or administering the drug in combination with cytochrome P450 enzyme inhibitors is a promising pathway in the development of novel pleuromutilin drugs with slow metabolism and long efficacy.

Proteins à la carte: riboproteogenomic exploration of bacterial N-terminal proteoform expression.

In recent years, it has become evident that the true complexity of bacterial proteomes remains underestimated. Gene annotation tools are known to propagate biases and overlook certain classes of truly expressed proteins, particularly proteoforms-protein isoforms arising from a single gene. Recent (re-)annotation efforts heavily rely on ribosome profiling by providing a direct readout of translation to fully describe bacterial proteomes. In this study, we employ a robust riboproteogenomic pipeline to conduct a systematic census of expressed N-terminal proteoform pairs, representing two isoforms encoded by a single gene raised by annotated and alternative translation initiation, in Salmonella. Intriguingly, conditional-dependent changes in relative utilization of annotated and alternative translation initiation sites (TIS) were observed in several cases. This suggests that TIS selection is subject to regulatory control, adding yet another layer of complexity to our understanding of bacterial proteomes. IMPORTANCE: With the emerging theme of genes within genes comprising the existence of alternative open reading frames (ORFs) generated by translation initiation at in-frame start codons, mechanisms that control the relative utilization of annotated and alternative TIS need to be unraveled and our molecular understanding of resulting proteoforms broadened. Utilizing complementary ribosome profiling strategies to map ORF boundaries, we uncovered dual-encoding ORFs generated by in-frame TIS usage in Salmonella. Besides demonstrating that alternative TIS usage may generate proteoforms with different characteristics, such as differential localization and specialized function, quantitative aspects of conditional retapamulin-assisted ribosome profiling (Ribo-RET) translation initiation maps offer unprecedented insights into the relative utilization of annotated and alternative TIS, enabling the exploration of gene regulatory mechanisms that control TIS usage and, consequently, the translation of N-terminal proteoform pairs.

Erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit cytopathic effect, papain-like protease, and MPRO enzymes of SARS-CoV-2.

Background: Although tremendous success has been achieved in the development and deployment of effective COVID-19 vaccines, developing effective therapeutics for the treatment of those who do come down with the disease has been with limited success. To repurpose existing drugs for COVID-19, we previously showed, qualitatively, that erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit SARS-COV-2-induced cytopathic effect (CPE) in Vero cells. Aim: This study aimed to quantitatively explore the inhibition of SARS-CoV-2-induced CPE by erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin and to determine the effect of these drugs on SARS-CoV-2 papain-like protease and 3CL protease (MPRO) enzymes. Methods: Neutral red (3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride) cell viability assay was used to quantify CPE after infecting pre-treated Vero cells with clinical SARS-Cov-2 isolates. Furthermore, SensoLyte® 520 SARS-CoV-2 papain-like protease and SensoLyte® 520 SARS-CoV-2 MPRO activity assay kits were used to evaluate the inhibitory activity of the drugs on the respective enzymes. Results: Erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin dose-dependently inhibit SARS-CoV-2-induced CPE in Vero cells, with inhibitory concentration-50 (IC50) values of 3.27 µM, 4.23 µM, 9.29 µM, 3.19 µM, and 84.31 µM, respectively. Furthermore, erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin dose-dependently inhibited SARS-CoV-2 papain-like protease with IC50 values of 0.94 µM, 0.88 µM, 1.14 µM, 1.07 µM, and 1.51 µM, respectively, and inhibited the main protease (MPRO) with IC50 values of 1.35 µM, 1.25 µM, 7.36 µM, 1.15 µM, and 2.44 µM, respectively. Conclusion: The IC50 for all the drugs, except ivermectin, was at the clinically achievable plasma concentration in humans, which supports a possible role for the drugs in the management of COVID-19. The lack of inhibition of CPE by ivermectin at clinical concentrations could be part of the explanation for its lack of effectiveness in clinical trials.

Characterization of a vga gene variant recovered from a Staphylococcus saprophyticus causing a community-acquired urinary tract infection: report from the SENTRY Antimicrobial Surveillance Program 2017.

A patient with a history of UTI acquired an isolate of Staphylococcus saprophyticus that was resistant to clindamycin, streptogramin A, pleuromutilins (LSPs), and oxacillin. A plasmid-located vga variant was identified in this pathogen, and the encoded protein showed a 39% to 67% identity to other previously characterized vga.

A comparative review of current topical antibiotics for impetigo.

INTRODUCTION: Impetigo is a superficial bacterial skin infection largely affecting the pediatric population. The objective of this review is to provide a comparison of mechanism of action, efficacy and safety of the available topical antibiotics for impetigo. AREAS COVERED: Randomized clinical trials that evaluated the use of topical antibiotics for treatment of impetigo were included. Two thousand eighty-nine studies were initially identified, and five randomized clinical trials met the criteria for further analysis. EXPERT OPINION: Topical antibiotics had greater resolution of impetigo in comparison to vehicle in these pivotal clinical trials. Adverse events were minimal, with the most common being pruritus at the application site. Cost or insurance coverage may be a limiting factor in choosing the best therapeutic agent, with mupirocin ointment having the lowest cost. Mupirocin has shown clinical efficacy against MRSA but a bacterial culture is recommended to rule out resistance. Ozenoxacin and retapamulin are effective alternatives but may entail higher cost. Retapamulin is indicated for lesions of impetigo that are colonized by MSSA and streptococcus S. pyogenes but not MRSA based on clinical efficacy of phase III trials. Fusidic acid, available in other countries, is a non-FDA approved medication although rising resistance rates represent a growing concern.

Identification of Translation Start Sites in Bacterial Genomes.

The knowledge of translation start sites is crucial for annotation of genes in bacterial genomes. However, systematic mapping of start codons in bacterial genes has mainly relied on predictions based on protein conservation and mRNA sequence features which, although useful, are not always accurate. We recently found that the pleuromutilin antibiotic retapamulin (RET) is a specific inhibitor of translation initiation that traps ribosomes specifically at start codons, and we used it in combination with ribosome profiling to map start codons in the Escherichia coli genome. This genome-wide strategy, that was named Ribo-RET, not only verifies the position of start codons in already annotated genes but also enables identification of previously unannotated open reading frames and reveals the presence of internal start sites within genes. Here, we provide a detailed Ribo-RET protocol for E. coli. Ribo-RET can be adapted for mapping the start codons of the protein-coding sequences in a variety of bacterial species.

Translational initiation in E. coli occurs at the correct sites genome-wide in the absence of mRNA-rRNA base-pairing.

Shine-Dalgarno (SD) motifs are thought to play an important role in translational initiation in bacteria. Paradoxically, ribosome profiling studies in E. coli show no correlation between the strength of an mRNA's SD motif and how efficiently it is translated. Performing profiling on ribosomes with altered anti-Shine-Dalgarno sequences, we reveal a genome-wide correlation between SD strength and ribosome occupancy that was previously masked by other contributing factors. Using the antibiotic retapamulin to trap initiation complexes at start codons, we find that the mutant ribosomes select start sites correctly, arguing that start sites are hard-wired for initiation through the action of other mRNA features. We show that A-rich sequences upstream of start codons promote initiation. Taken together, our genome-wide study reveals that SD motifs are not necessary for ribosomes to determine where initiation occurs, though they do affect how efficiently initiation occurs.

The polymyxin derivative NAB739 is synergistic with several antibiotics against polymyxin-resistant strains of Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii.

The antibiotic crisis has reinstated polymyxins, once abandoned because of their toxicity. Now, preclinical studies have revealed better tolerated and more effective derivatives of polymyxins such as NAB739. Simultaneously, polymyxin-resistant (PMR) strains such as the mcr-1 strains have received lots of justified publicity, even though they are still very rare. Here we show that NAB739 sensitizes the PMR strains to rifampin, a classic "anti-Gram-positive" antibiotic excluded by the intact outer membrane (OM) permeability barrier, as well as to retapamulin, the surrogate of lefamulin, an antibiotic under development against Gram-positive bacteria. Polymyxin B was used as a comparator. The combination of NAB739 and rifampin was synergistic against ten out of eleven PMR strains of Escherichia coli (Fractional Synergy Indices, FICs, 0.14-0.19) and that of NAB739 and retapamulin against all the tested eleven strains (FICs 0.19-0.25). Against PMR Klebsiella pneumoniae (n = 7), the FICs were 0.13-0.27 for NAB739 + rifampin and 0.14-0.28 for NAB739+retapamulin. Against Acinetobacter baumannii (n = 2), the combination of NAB739 and rifampin had the FIC of 0.09-0.19. Furthermore, NAB739 and meropenem were synergistic (FICs 0.25-0.50) against four out of five PMR strains that were simultaneously resistant to meropenem.

Clinical and bacteriological efficacy of twice daily topical retapamulin ointment 1% in the management of impetigo and other uncomplicated superficial skin infections.

BACKGROUND: Cutaneous bacterial infections are common in children and adults and frequently are caused by Staphylococcus aureus (S. aureus). Treatment failures with topical agents are not uncommon and have been shown to be secondary to bacterial resistance. OBJECTIVE: To determine clinical and bacteriological efficacy of retapamulin ointment 1% in treatment of patients with cutaneous bacterial infections caused by methicillin-resistant S. aureus (MRSA) and other bacteria. METHODS: Prospective, nonrandomized, uncontrolled, open label, single center trial conducted between April 2008 and November 2012 that evaluated efficacy of retapamulin ointment 1% in the treatment of impetigo, folliculitis, and other minor soft tissue infections in children and adults. Fifty patients, who presented to a dermatology outpatient clinic and were clinically diagnosed with impetigo, folliculitis, or minor soft tissue infection suitable for treatment with a topical antibiotic, were screened. Thirty-eight patients were enrolled and received treatment: topical retapamulin ointment 1% twice daily for 5 days. Seven patients were MRSA positive and qualified for the primary efficacy population. One patient withdrew due to an adverse event. Clinical and microbiological exams were performed at baseline and follow-up 5 to 7 days later to assess clinical, microbiological, and therapeutic responses. Primary outcome was clinical response at follow-up in primary efficacy population with MRSA isolated as the baseline pathogen. Secondary outcomes included clinical, microbiologic, and therapeutic responses in patients who were culture positive for any species of bacteria. RESULTS: Clinical response at follow-up in the primary efficacy population (MRSA-positive patients) was not sufficiently powered to demonstrate significance; however, outcomes were excellent, with 7 of 7 patients demonstrating clinical success (5 of 7) or clinical improvement (2 of 7) at follow-up. Barring lack of significance due to small total sample size for patients who were culture positive for any species of bacteria (n = 35), overall success rates were favorable for clinical, microbiologic, and therapeutic responses with values of 66%, 97%, and 69%, respectively. Adverse events (AEs) were mild or moderate in severity. No serious AEs were reported. CONCLUSION: Safety profile appears favorable given the low number of AEs. Study design limits conclusions that can be drawn. Nevertheless, this study supports use of topical retapamulin 1% ointment in treatment of cutaneous bacterial infections, particularly those caused by S. aureus, including MRSA.

In Vitro Antimicrobial Activities of Fusidic Acid and Retapamulin against Mupirocin- and Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: The in vitro activities of retapamulin and fusidic acid against clinical isolates of mupirocin-resistant and methicillin-resistant Staphylococcus aureus (MRSA) from Korea are not well understood. OBJECTIVE: This study aimed to determine the activities of retapamulin and fusidic acid against clinical isolates of mupirocin-resistant MRSA. METHODS: Clinical isolates of mupirocin-resistant MRSA were collected from two tertiary hospitals. The minimal inhibitory concentrations of mupirocin, fusidic acid, and retapamulin were determined using agar dilution method. Polymerase chain reaction was used to confirm the identity of the species and the presence of resistance genes. Pulsed-field gel electrophoresis (PFGE) patterns of chromosomal DNA were used to determine the genetic similarity of high-level mupirocin-resistant isolates. RESULTS: Of the 497 MRSA isolates tested, 22 (4.4%) were mupirocin-resistant. Of these, 9 (1.8%) and 13 (2.6%) had high-level and low-level mupirocin resistance, respectively. Analysis of the PFGE patterns of the high-level mupirocin-resistant MRSA isolates identified five clusters. All 13 of the low-level mupirocin-resistant isolates were resistant to fusidic acid but susceptible to retapamulin. However, among the 9 high-level mupirocin-resistant isolates, 56% were resistant to fusidic acid, and all were susceptible to retapamulin. CONCLUSION: Retapamulin is highly active in vitro against Korean clinical isolates of high-level mupirocinand methicillin-resistant Staphylococcus aureus with different genetic backgrounds. Fusidic acid is more active against high-level mupirocin-resistant MRSA than low-level mupirocin-resistant MRSA.

Antibiotic resistance to Propionobacterium acnes: worldwide scenario, diagnosis and management.

Antibiotic resistance in cutaneous Propionobacterium is a global problem. As a general rule, resistance levels are high to macrolides, trimethoprim/sulfamethoxazole and clindamycin, while tetracyclines and levofloxacin have low resistance potential. Newer preparations like doxycycline MR and doxycycline 20 mg are subantimicrobial and may not lead to resistance. Sampling techniques are crucial to determine resistance. Genomic evaluation using 16S ribosomal RNA gene sequencing can be useful in diagnosing mutations and mapping phylotypes of Propionobacterium acnes. Resistance may lead to slow response and relapses. Apart from benzoyl peroxide, azelaic acid, topical dapsone, oral zinc and retinoids, novel molecules with little resistance potential include octadecenedioic acid, phytosphingosine, lauric acid, retapamulin, resveratrol, T-3912 and NB-003. The use of oral retinoids and non-antibiotics like zinc can prevent resistance and help reduce the dependence on antibiotics.

Ozenoxacin 1% cream in the treatment of impetigo: a multicenter, randomized, placebo- and retapamulin-controlled clinical trial.

AIM: We compared the efficacy and safety of ozenoxacin (a new nonfluorinated quinolone) 1% cream with placebo in the treatment of impetigo. PATIENTS & METHODS: In a randomized, double-blind, multicenter study, patients received ozenoxacin cream or placebo cream twice daily for 5 days (a third group received retapamulin 1% ointment as a control). Clinical, microbiological and laboratory evaluations were performed during follow-up (over 2 weeks). RESULTS: Ozenoxacin was superior to placebo (success rate 34.8 vs 19.2%; p = 0.003). Microbiological success was 70.8% for ozenoxacin and 38.2% for placebo after 3-4 days and 79.2% versus 56.6% after 6-7 days. Ozenoxacin produced more rapid microbiological clearance than retapamulin. All treatments were well tolerated. CONCLUSION: Ozenoxacin 1% cream was effective and safe in the treatment of impetigo.

Susceptibility of Staphylococcus aureus to topical agents in the United States: a sentinel study.

BACKGROUND: Mupirocin has been used topically for treating skin and skin structure infections and for nasal decolonization before surgical interventions. Pleuromutilin compounds, including retapamulin, provide similar treatment/interventional options. Rates of resistance of Staphylococcus aureus to mupirocin and other agents used to treat skin and skin structure infections vary between countries and medical centers, including those in the United States. These resistance rates may be associated with higher usage and/or improper epidemiologic practices. OBJECTIVE: This study aimed to determine rates of resistance to topical and other class agents against S aureus isolates collected from SSSIs. METHODS: Isolates were obtained from outpatients at 6 US dermatology centers in 5 states. Demographic data were collected from medical records, and each patient completed a study questionnaire on recent history of skin infections, antibiotic use, and hospitalization. Each isolate was tested against cephalothin, clindamycin, erythromycin, gentamicin, mupirocin, tetracycline, retapamulin, and trimethoprim/sulfamethoxazole. RESULTS: Although methicillin-resistance rates varied between centers (range, 15.8%-35.5%), macrolide resistance was ~50% at all of the sites in this study. Mupirocin-resistant isolates were observed much more frequently from 1 center (33.9%), and nearly all demonstrated high-level resistance. Only 1 retapamulin-resistant isolate (0.5%) was observed, with a minimum inhibitory concentration of 16 µg/mL. The other agents had relatively low resistance rates, which varied between centers and were dependent on susceptibility to methicillin. CONCLUSIONS: Although the rate of mupirocin-resistant S aureus isolates collected in this investigation was >10%, retapamulin resistance was infrequent. Surveillance of topical agents to determine resistance rates against targeted bacteria is necessary.