Macrolide Resistance Mechanisms Research Articles

Exploring the Molecular Mechanisms of Macrolide Resistance in Laboratory Mutant Helicobacter pylori.

Resistance to clarithromycin, a macrolide antibiotic used in the first-line treatment of Helicobacter pylori infection, is the most important cause of treatment failure. Although most cases of clarithromycin resistance in H. pylori are associated with point mutations in 23S ribosomal RNA (rRNA), the relationships of other mutations with resistance remain unclear. We examined possible new macrolide resistance mechanisms in resistant strains using next-generation sequencing. Two resistant strains were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations using the agar dilution method. Sanger sequencing and whole-genome sequencing were performed to detect resistance-related mutations. Both strains carried the A2142G mutation in 23S rRNA. Candidate mutations (T1495A, T1494A, T1490A, T1476A, and G1472T) for clarithromycin resistance were detected in the Mutant-1 strain. Furthermore, a novel mutation in the gene encoding for the sulfite exporter TauE/SafE family protein was considered to be linked to clarithromycin resistance or cross-resistance, being identified as a target for further investigations. In the Mutant-2 strain, a novel mutation in the gene that encodes DUF874 family protein that can be considered as relevant with antibiotic resistance was detected. These mutations were revealed in the H. pylori genome for the first time, emphasizing their potential as targets for advanced studies.

Genetic diversity of macrolides resistant Staphylococcus aureus clinical isolates and the potential synergistic effect of vitamins, C and K3

BackgroundMacrolide antibiotics have been extensively used for the treatment of Staphylococcus aureus infections. However, the emergence of macrolide-resistant strains of S. aureus has become a major concern for public health. The molecular mechanisms underlying macrolide resistance in S. aureus are complex and diverse, involving both target site modification and efflux pump systems. In this study, we aim to overcome the molecular diversity of macrolide resistance mechanisms in S. aureus by identifying common molecular targets that could be exploited for the development of novel therapeutics.MethodsAbout 300 Staphylococcus aureus different isolates were recovered and purified from 921 clinical specimen including urine (88), blood (156), sputum (264), nasal swabs (168), pus (181) and bone (39) collected from different departments in Tanta University Hospital. Macrolide resistant isolates were detected and tested for Multi Drug Resistant (MDR). Gel electrophoresis was performed after the D test and PCR reaction for erm(A), (B), (C), msr(A), and mph(C) genes. Finally, we tried different combinations of Erythromycin or Azithromycin antibiotics with either vitamin K3 or vitamin C.ResultsMacrolide resistance S. aureus isolates exhibited 7 major resistance patterns according to number of resistance markers and each pattern included sub patterns or subgroups. The PCR amplified products of different erm genes; analysis recorded different phenotypes of the Staphylococcus aureus isolates according to their different genotypes. In addition, our new tested combinations of Erythromycin and vitamin C, Erythromycin, and vitamin K3, Azithromycin and vitamin C and Azithromycin and vitamin K3 showed significant antibacterial effect when using every antibiotic alone. Our findings provide new insights into the molecular mechanisms of macrolide resistance in S. aureus and offer potential strategies for the development of novel protocols to overcome this emerging public health threat.

Antimicrobial Susceptibility to 27 Drugs and the Molecular Mechanisms of Macrolide, Tetracycline, and Quinolone Resistance in Gemella sp.

Gemella is a catalase-negative, facultative anaerobic, Gram-positive coccus that is commensal in humans but can become opportunistic and cause severe infectious diseases, such as infective endocarditis. Few studies have tested the antimicrobial susceptibility of Gemella. We tested its antimicrobial susceptibility to 27 drugs and defined the resistant genes using PCR in 58 Gemella strains, including 52 clinical isolates and six type strains. The type strains and clinical isolates included 22 G. morbillorum, 18 G. haemolysans (GH) group (genetically indistinguishable from G. haemolysans and G. parahaemolysans), 13 G. taiwanensis, three G. sanguinis, and two G. bergeri. No strain was resistant to beta-lactams and vancomycin. In total, 6/22 (27.3%) G. morbillorum strains were erythromycin- and clindamycin-resistant ermB-positive, whereas 4/18 (22.2%) in the GH group, 7/13 (53.8%) G. taiwanensis, and 1/3 (33.3%) of the G. sanguinis strains were erythromycin-non-susceptible mefE- or mefA-positive and clindamycin-susceptible. The MIC90 of minocycline and the ratios of tetM-positive strains varied across the different species-G. morbillorum: 2 µg/mL and 27.3% (6/22); GH group: 8 µg/mL and 27.8% (5/18); G. taiwanensis: 8 µg/mL and 46.2% (6/13), respectively. Levofloxacin resistance was significantly higher in G. taiwanensis (9/13 69.2%) than in G. morbillorum (2/22 9.1%). Levofloxacin resistance was associated with a substitution at serine 83 for leucine, phenylalanine, or tyrosine in GyrA. The mechanisms of resistance to erythromycin and clindamycin differed across Gemella species. In addition, the rate of susceptibility to levofloxacin differed across Gemella sp., and the quinolone resistance mechanism was caused by mutations in GyrA alone.

Novel mechanisms of macrolide resistance revealed by in vitro selection and genome analysis in Mycoplasma pneumoniae.

Mycoplasma pneumoniae is an important pathogen causing upper and lower respiratory tract infections in children and other age groups. Macrolides are the recommended treatments of choice for M. pneumoniae infections. However, macrolide resistance in M. pneumoniae is increasing worldwide, which complicates the treatment strategies. The mechanisms of macrolide resistance have been extensively studied focusing on the mutations in 23S rRNA and ribosomal proteins. Since the secondary treatment choice for pediatric patients is very limited, we decided to look for potential new treatment strategies in macrolide drugs and investigate possible new mechanisms of resistance. We performed an in vitro selection of mutants resistant to five macrolides (erythromycin, roxithromycin, azithromycin, josamycin, and midecamycin) by inducing the parent M. pneumoniae strain M129 with increasing concentrations of the drugs. The evolving cultures in every passage were tested for their antimicrobial susceptibilities to eight drugs and mutations known to be associated with macrolide resistance by PCR and sequencing. The final selected mutants were also analyzed by whole-genome sequencing. Results showed that roxithromycin is the drug that most easily induces resistance (at 0.25 mg/L, with two passages, 23 days), while with midecamycin it is most difficult (at 5.12 mg/L, with seven passages, 87 days). Point mutations C2617A/T, A2063G, or A2064C in domain V of 23S rRNA were detected in mutants resistant to the 14- and 15-membered macrolides, while A2067G/C was selected for the 16-membered macrolides. Single amino acid changes (G72R, G72V) in ribosomal protein L4 emerged during the induction by midecamycin. Genome sequencing identified sequence variations in dnaK, rpoC, glpK, MPN449, and in one of the hsdS (MPN365) genes in the mutants. Mutants induced by the 14- or 15-membered macrolides were resistant to all macrolides, while those induced by the 16-membered macrolides (midecamycin and josamycin) remained susceptible to the 14- and 15-membered macrolides. In summary, these data demonstrated that midecamycin is less potent in inducing resistance than other macrolides, and the induced resistance is restrained to the 16-membered macrolides, suggesting a potential benefit of using midecamycin as a first treatment choice if the strain is susceptible.

Molecular Characteristics of Macrolide Resistance in Treponema pallidum from Patients with Latent Syphilis in Xinjiang, China.

Macrolide resistance in Treponema pallidum (T. pallidum) has been increasing in recent years worldwide. However, few data are available on macrolide resistance in T. pallidum from Xinjiang province, located in the western part of China, which is an area with a high incidence of syphilis. In this study, we investigated the molecular characteristics of macrolide resistance in T. pallidum from patients with latent syphilis in Xinjiang, China. In total, 204 whole blood samples were collected from patients with latent syphilis during 2016 to 2017, in the First Hospital of Xinjiang Medical University. Genomic DNA of blood samples was extracted using a QIAamp DNA Mini Kit and T. pallidum was detected by PCR with the specific polA gene of T. pallidum. The 23S rRNA gene of T. pallidum was amplified among the T. pallidum-positive samples by nested PCR, and macrolide resistance-associated mutation sites A2058G and A2059G in the 23S rRNA gene were identified using restriction enzymes MboII and BsaI. The specific polA gene of T. pallidum (T. pallidum positive) was detected in 27 blood samples (13.2%) from 204 patients with latent syphilis. The 23S rRNA gene was then amplified in all 27 T. pallidum-positive samples, among which 24/27 samples (88.9%) harbored the A2058G mutation in the 23S rRNA gene and 3/27 (11.1%) had the A2059G mutation. Our results indicated that T. pallidum macrolide resistance should not be ignored in Xinjiang, China, and that A2058G was the predominant macrolide resistance mechanism. Blood may be a suitable specimen for the detection of resistant mutations of T. pallidum in patients with latent syphilis who do not show any clinical symptoms.

Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance

ObjectivesPseudomonas aeruginosa colonizes the cystic fibrosis (CF) airways causing chronic bacterial lung infections. CF patients are routinely treated with macrolides, however, P. aeruginosa is considered insusceptible as consequence of inadequate susceptibility testing leaving resistance mechanism completely overlooked. Here, we investigated a new mechanism of macrolide resistance caused by ribosomal protein mutations. MethodsInvestigating a longitudinal collection of 529 isolates from CF patients and analysing 5758 protein sequences from different sources, mutations in P. aeruginosa's ribosomal proteins connected to macrolide resistance were identified. Using a modified susceptibility testing protocol, isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics and macrolide-induced quorum sensing modulation. Proteome and ribosome profiling were applied to assess the impact of the mutations on the bacterial physiology. ResultsFive uL4 mutations were identified in isolates from different CF patients. Most mapped to the conserved loop region of uL4 and resulted in increased macrolide tolerance (>10-fold relative to wt strains). Greater concentrations (>10-fold) of macrolide antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity of the uL4 mutants. 16 proteins involved in ribosome adaptation displayed altered expression possibly to compensate for the uL4 mutations, which changed the ribosome stoichiometry without negatively affecting bacterial physiology. ConclusionsMacrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated when patients are treated with prolonged courses of macrolides. Importantly, improved macrolide susceptibility testing is necessary for the detection of resistant bacteria.

Antimicrobial Susceptibility Profiles and Genetic Characteristics of Mycoplasma pneumoniae in Shanghai, China, from 2017 to 2019.

ObjectiveThe current study investigated the recent genetic characteristics and antimicrobial susceptibility profiles of Mycoplasma pneumoniae (M. pneumoniae) in Shanghai, becoming a clinical reference for treating M. pneumoniae infection in Shanghai.MethodsClinical strains were isolated from nasopharyngeal aspirates of the pediatric patients in Shanghai from 2017 to 2019. Nine antimicrobial agents of three antimicrobial classes macrolides, fluoroquinolones and tetracyclines, against M. pneumoniae isolates were investigated using the broth microdilution method. The mechanism of macrolide resistance was analyzed by evaluating the sequences of the 23S rRNA gene and the ribosomal protein genes L4 and L22. Molecular genotyping was undergone to classify the P1 subtypes and the multi-locus variable-number tandem-repeat analysis (MLVA) types.ResultsA total of 72 isolates were resistant to macrolides (MICs > 64 mg/L for erythromycin) based on the A2063G mutation in the 23S rRNA gene. These strains were susceptible to tetracyclines and fluoroquinolones. P1 type 1 (166/182, 91.2%) and MLVA type 4-5-7-2 (165/182, 90.7%) were the dominant subtypes. MLVA type was associated with the P1 subtypes. The distribution of the P1 subtypes and MLVA types did not change over time. The macrolide-resistant rate in P1 type 2 and MLVA type 3-5-6-2 strains were increased during the three-year study. The 5-loci MLVA typing scheme revealed the clonal expansion of MLVA type 3-4-5-7-2 strains which are macrolide-resistant in 2019.ConclusionMacrolide resistance in M. pneumoniae in Shanghai is very high and is evolving among certain subtypes. Cautions should be taken for the possible clonal spreading of macrolide-resistant genotypes within this populated region.

Clonal Distribution, Antimicrobial Resistance, and Pilus Islets in S. pneumoniae Isolates from PCV10-Vaccinated Children with Suppurative AOM in Bulgaria (2015‒2020).

Streptococcus pneumoniae is still a leading bacterial pathogen of acute otitis media (AOM), despite the availability of pneumococcal conjugate vaccines (PCVs). We conducted a study on the population structure, antibiotic nonsusceptibility, serotype distribution, and presence of pilus in middle ear fluids ‒ S. pneumoniae isolates recovered from PCV10-vaccinated children with suppurative АОМ in Bulgaria. Non-susceptibility was observed in 68.75% (n = 33) of the isolates, and multidrug resistance (MDR) was detected in 60.4% of the patients. The dual macrolide resistance mechanism was predominant. The most common serotypes were non-PCV10 serotypes 3 (27.1%, n = 13), 19A (25.0%, n = 12), and VT 19F (23.0%, n = 11). Overall, 64.6% were non-PCV10-serotypes. The presence of Pilus type I was observed mostly in the PCV10-serotypes. We found a strong association between clonal complexes (CCs), serotypes, and antimicrobial resistance. Multilocus sequence typing revealed the presence of four CCs: CC320 (39.6%), CC505 (12.5%), CC1377 8.3%), and CC230 (8.3%). The most abundant CC320 comprised MDR 19A and 19F isolates. CC230 clustered MDR isolates from serotypes 19A, 6C, and 14. CC505 and CC1377 were serotype 3 susceptible isolates. The vaccine-induced changes and trends in antimicrobial resistance and clonality must be the focus of systematic investigations.

Molecular mechanisms of macrolide and fluoroquinolone resistance among Korean isolates of Mycoplasma genitalium over a period of five years 2014-2019.

Antimicrobial resistance in Mycoplasma genitalium has become a global issue, and certain groups have a higher probability of acquiring resistant strains. Little is known about the genetic diversity and characteristics of the antimicrobial resistance-determining sites (ARDSs) of M. genitalium in the Korean population. Therefore, we examined the genetic diversity of the ARDSs of M. genitalium-positive urogenital samples obtained from Korean females (G1) and males (G2) visiting primary care clinics and DNA samples from referred males (G3) with persistent urethritis. From 2014 to 2019, 54 patients from G1, 86 patients from G2, and 68 patients from G3 were included in the study. Sanger sequencing was performed on the 2058/2059 sites in the 23S rRNA gene and quinolone resistance-determining regions (QRDRs) of M. genitalium. The rates of mutation in G1, G2, and G3 were 1.85, 5.81, and 48.53 %, respectively, for A2059G in the 23S rRNA gene (P<0.001); 1.85, 0, and 17.78 %, respectively, for M95R or I in gyrA (P<0.001); 0, 0, and 31.11 %, respectively, for D99N or G in gyrA (P<0.001); and 7.41, 16.28, and 30 %, respectively, for S83R or N or I in parC (P=0.015). A2059G significantly increased the risk of mutations at the gyrA95, gyrA99, and parC83 sites (all P<0.01). In conclusion, although the genetic diversity of the ARDSs of M. genitalium was variable among the groups, it was generally lower in isolates with macrolide resistance and higher in isolates with quinolone resistance in Korea compared with the isolates in other countries. The G3 group demonstrated increased genetic diversity at the A2059G, gyrA95, gyrA99, and parC83 sites.

Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site.

Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP-N-acetylglucosamine to yield corresponding midecamycin 2′-O-glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP-N-acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2′-O-glucopyranoside, midecamycin 2′-O-GlcNAc and midecamycin 2′-O-xylopyranoside. In contrast to midecamycin, these midecamycin 2′-O-glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.

Antimicrobial Resistance Profiles and Macrolide Resistance Mechanisms of Campylobacter coli Isolated from Pigs and Chickens.

We identified 1218 Campylobacter coli isolates from fecal and carcass samples of pigs (n = 643) and chickens (n = 575) between 2010 and 2018. About 99% of the isolates were resistant to at least one antimicrobial agent. The isolates exhibited high resistance rates (>75%) to ciprofloxacin, nalidixic acid, and tetracycline. Azithromycin and erythromycin resistance rates were the highest in isolates from pigs (39.7% and 39.2%, respectively) compared to those of chickens (15.8% and 16.3%, respectively). Additionally, a low-to-moderate proportion of the isolates were resistant to florfenicol, gentamicin, clindamycin, and telithromycin. Multidrug resistance (MDR) was found in 83.1% of the isolates, and profiles of MDR usually included ciprofloxacin, nalidixic acid, and tetracycline. We found point mutation (A2075G) in domain V of the 23S rRNA gene in the majority of erythromycin-resistant isolates. Multilocus sequence typing of 137 erythromycin-resistant C. coli isolates revealed 37 previously reported sequence types (STs) and 8 novel STs. M192I, A103VI, and G74A substitutions were frequently noted in the ribosomal proteins L4 or L22. Further, we identified a considerable proportion (>90%) of erythromycin-resistant isolates carrying virulence factor genes: flaA, cadF, ceuE, and VirB. The prudent use of antimicrobials and regular microbiological investigation in food animals will be vital in limiting the public health hazards of C. coli in Korea.

Phenotypic and Molecular Characterization of Penicillin and Macrolide-Resistant Streptococcus pneumoniae Serotypes Among Pediatric Patients in Addis Ababa, Ethiopia.

BackgroundIn several countries, introduction of the pneumococcal conjugate vaccine (PCV) has led to a decline in antimicrobial-resistant pneumococcal disease but has also resulted in a concomitant increase in antimicrobial-resistant, non-vaccine serotypes of Streptococcus pneumoniae. We sought to determine the magnitude of penicillin and macrolide resistance among pneumococcal serotypes and the mechanisms of macrolide resistance in Ethiopia, 5 years after the introduction of PCV10 in the country.MethodsSusceptibility to penicillin and erythromycin of 119 pneumococcal isolates collected from pediatric patients aged 0–15 years in Addis Ababa, Ethiopia, was tested using disc diffusion, and minimum inhibitory concentration (MIC) was also determined by Etest. Pneumococcal serotypes were determined by sequencing the cpsB gene and using Quellung reaction. Polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism analysis were used to detect and differentiate the macrolide resistance genes erm(B), mef(A), and mef(B).ResultsAmong the 119 isolates, 2.5% (3/119) were resistant to penicillin, while 58% (69/119) were intermediate. Resistance to erythromycin was observed in 33.6% (40/119) of the isolates with the highest level of resistance among isolates from middle ear discharge, i.e., 53.3% (8/15). Half (19/40) of the erythromycin resistant isolates were serotype 19A and among serotype 19A isolates, the majority i.e., 54.3% (19/35) were resistant to erythromycin. The most common macrolide resistance determinant was mef(E) with a prevalence of 50% (20/40).ConclusionFive years after introduction of PCV10 in Ethiopia, we observed that the prevalence of penicillin-resistant S. pneumoniae was low. However, there was a high level of macrolide resistance which was mostly in serotype 19A, and the resistance was mainly mediated by efflux pumps. Introduction of PCV13 (which covers serotype 19A) would significantly improve coverage of the macrolide-resistant serotypes. Continued surveillance of pneumococcal serotype distribution and their antibiotic resistance pattern in Ethiopia is warranted.

Activity of novel lactone ketolide nafithromycin against multicentric invasive and non-invasive pneumococcal isolates collected in India.

BackgroundIndia is among the nations reporting substantial healthcare burden linked to pneumococcal infections. Nafithromycin is a novel lactone ketolide antibiotic, which recently entered Phase 3 development in India for the indication of community-acquired bacterial pneumonia (CABP).ObjectivesTo assess the in vitro activity of nafithromycin against serotyped invasive and non-invasive Streptococcus pneumoniae isolates, collected from nine medical centres across India.MethodsA total of 534 isolates of S. pneumoniae were collected during 2015–20 and serotyped as per CDC protocol. A subset of erythromycin-non-susceptible S. pneumoniae (n = 200) was screened for the presence of erm(B) and mef(A/E) genes. A subset of MDR isolates (n = 54) were also subjected to MLST. The MICs of antibiotics were determined by the reference agar-dilution method (CLSI). Susceptibilities of the comparators were interpreted as per CLSI criteria.ResultsFifty-nine distinct serotypes were identified among the 534 isolates. Among erythromycin-non-susceptible isolates, erm(B) and mef(A/E) genes were found in 49% and 59% strains respectively, while MLST showed clonal diversity. Azithromycin (67.6% non-susceptible) and clindamycin (31.8% non-susceptible) showed limited activity. Penicillin (for non-meningitis) or quinolone non-susceptibility was low (<11% and <6%, respectively). Nafithromycin showed potent activity with MIC50 and MIC90 of 0.015–0.03 and 0.06 mg/L, respectively, regardless of the macrolide resistance mechanisms.ConclusionsIndian pneumococcal isolates show poor susceptibilities to macrolides, in concordance with the global trend. Nafithromycin overcomes erm as well as mef-mediated macrolide resistance mechanisms expressed individually or concurrently in S. pneumoniae. This study supports continued clinical development of nafithromycin for pneumococcal infections including CABP.

Antimicrobial susceptibility profiles of Mycobacterium abscessus complex isolates from respiratory specimens in Shanghai, China

ObjectivesThe aim of this study was to compare the antibiotic susceptibility profiles of Mycobacterium abscessus complex (MABC) isolates and to investigate the relationship between susceptibility profiles and genetic mechanisms of macrolide resistance. MethodsMore than 200 isolates collected from respiratory specimens between 2014 and 2018 were randomly analysed in this study. Minimum inhibitory concentrations (Mics) of ten potential antimicrobial agents were determined by the microplate alamarBlue assay. ResultsWe identified 43 MABC isolates, including 32 M. abscessus subsp. abscessus (M. abscessus) (6 from immunocompromised patients) and 11 M. abscessus subsp. massiliense (M. massiliense). The majority of MABC isolates were susceptible to amikacin (96.9% and 100.0% for M. abscessus and M. massiliense, respectively), linezolid (96.9% and 100.0%, respectively), cefoxitin (100.0% and 100.0%, respectively), imipenem (90.6% and 72.7%, respectively) and tobramycin (90.6% and 72.7%, respectively). The resistance rates to clarithromycin and doxycycline in isolates of M. abscessus (68.8% and 100.0%) were significantly higher than those in isolates of M. massiliense (18.2% and 63.6%) (P < 0.05), whereas the percentage of tobramycin-resistant isolates among M. abscessus (9.4%) was significantly lower than among M. massiliense (27.3%) (P = 0.007). Sequencing analyses showed significant differences between erm(41) of M. abscessus and M. massiliense. ConclusionMycobacterium abscessus is the dominant pathogen of pulmonary MABC infections in our hospital. Aminoglycosides (amikacin and tobramycin), β-lactams (cefoxitin and imipenem) and linezolid exhibited potent inhibitory activity against MABC in vitro. The erm(41) gene may be a promising marker to predict macrolide susceptibility for M. abscessus.

Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance.

Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically-relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058 located in the drug binding site, a reaction catalyzed by the Erm-type rRNA-methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4Å resolution together with the structures of unmethylated 70S ribosome functional complexes alone and in combination with macrolides. Altogether, our structural data do not support the previous models and, instead, suggest a principally new explanation of how A2058-dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome revealed a previously unknown role of desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.

Rate and Mechanism of Erythromycin Resistance in Streptococcus pyogenes in Beni-Suef University Hospital

Background: Group A streptococci (GAS) is a major cause of morbidity and mortality. Erythromycin is an effective macrolide antibiotic for treating GAS infections. However, GAS macrolide resistance has been increased due to either an efflux mechanism (M phenotype), encoded by mef A gene, or by methylation of the ribosomal target resulting in resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics. Methylase can be expressed either constitutively (cMLS phenotype) or inducibly (iMLS phenotype). Objectives: The present study aimed to find out frequency of S. pyogenes isolated from patients with upper respiratory tract infections at Beni-Suef University Hospital, determine rate and mechanism of macrolide resistance. Methodology: The present study was conducted on patients with upper respiratory tract infections attended to otorhinolaryngology clinic, Beni-Suef University Hospital, Egypt, in the period from February to December 2015. Detailed history taking was carried and clinical findings were obtained. Throat or ear swabs were taken and processed by conventional bacteriological methods. S. pyogenes isolates were further tested to determine erythromycin resistance phenotype by D- test, MIC of Erythromycin by tube broth dilution method and for mef A gene by PCR. Results: Forty two S. pyogenes isolates were identified from (100) swabs taken from either ear or throat specimens (42%), isolates resistance to erythromycin and clindamycin was 83.3% (35) and 31% (13) respectively. The pattern of macrolide resistance was 31% (13/ 42) cMLS phenotype, 52.3% (22/42) M phenotype and no isolate was iMLSB phenotype. Most strains with M phenotype expressed low-level macrolide resistance (MIC 1-4μg/ml), while cMLSB isolates showed a high level of erythromycin resistance (MIC ≥64 μg/ml) (highly significant: p-value 0.0001). The results confirmed a strong correlation between the M phenotype and the mef A gene in GAS (highly significant: p-value =0.001). Conclusion: Incidence of erythromycin resistance was evident among the isolates. To preserve the necessary efficacy, limited use of erythromycin is recommended.

Identification of the first erm(B)-positive Campylobacter jejuni and Campylobacter coli associated with novel multidrug resistance genomic islands in Australia

ObjectivesThis report describes the first identification of two Campylobacter isolates harbouring erm(B) in Australia. MethodsTwo erm(B)-positive isolates, Campylobacter coli 18V1065H1 and Campylobacter jejuni 19W1001H1, were isolated from diarrhoeal faecal samples from two travellers who had recently returned from Southeast Asia. Isolates underwent whole-genome sequencing using an Illumina NextSeq system and were analysed with the Nullarbor pipeline. Antimicrobial resistance genes were identified using AMRFinderPlus and sequence types (STs) were determined by multilocus sequence typing and the PubMLST Campylobacter jejuni/coli typing scheme. ResultsBesideserm(B), C. jejuni 19W1001H1 possessed six other resistance genes [aad9, aadE, aph(3ʹ)-Illa, blaOXA-185, catA13 and tet(O)], the gyrA T86I mutation and the RE-CmeABC multidrug efflux pump variant. Campylobacter coli 18V1065H1 also possessed six resistance genes [aad9, aadE, aph(3ʹ)-IIIa, blaOXA-61, sat4 and tet(O)] in addition to erm(B); however, this isolate lacked genetic evidence for resistance to fluoroquinolones (no gyrA mutation). The erm(B) locus differed between isolates and neither was identical to previously identified erm(B) multidrug resistance genomic island (MDRGI) types. Both erm(B)-bearing isolates belonged to novel sequence types: ST9967 (C. jejuni 19W1001H1) and ST10161 (C. coli 18V1065H1). ConclusionsThis study detected the presence oferm(B) in Campylobacter for the first time in Australia. This novel mechanism of macrolide resistance is a major concern both for human and animal health and warrants close surveillance as macrolides are often the drug of choice for treating campylobacteriosis. The erm(B) gene is associated with several MDRGIs and dissemination of this resistance mechanism will likely limit treatment options for Campylobacter infections.

Phenotypic and molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae isolated in Tunisia.

Introduction. Streptococcus pneumoniae is responsible for many community infections, with the main ones being pneumonia and meningitis. Pneumococcus has developed increased resistance to multiple classes of antibiotics. The evolution of antibiotic resistance in pneumococcus was influenced by changes in serotype distribution under vaccine selection pressure.Aim. The aim of this study was to determine the genes involved in macrolide resistance, the antimicrobial susceptibility, the serotype distribution and the spread of international antibiotic-resistant clones among clinical isolates of S. pneumoniae.Methodology. We investigated 86 erythromycin-resistant S. pneumoniae strains isolated from respiratory (n=74) or non-respiratory (n=12) samples in Tunisia. Antimicrobial susceptibility was tested using the disk diffusion method. Macrolide-resistant strains were analysed by polymerase chain reaction (PCR) for ermA, ermB, mefA and msrD. We also investigated the macrolide resistance mechanisms in eight isolates (9.3%) by sequencing the L4 and L22 riboprotein-coding genes, plus relevant segments of the three 23S rRNA genes. Capsular serotypes were detected by multiplex PCR. Sequence types (STs) were explored using multilocus sequence typing (MLST).Results. Among the 86 studied strains, 70 (81.4 %) were resistant to penicillin G. The prevalent serotypes were 19F, 14, 19A and 23F. We observed that the cMLSB phenotype (66/86, 76.7%) was the most common in these pneumococci. In addition, ermB was the most frequent resistance gene. No mutation in ribosomal protein L22 or L4 or 23S rRNA was detected. Overall, 44 STs were identified in this study, including 16 that were described for the first time. Resistance to lincomycin, tetracycline and trimethoprim/sulfamethoxazole was observed in 55 (64 %), 34 (39.5 %) and 31 (36 %) isolates, respectively. Furthermore, an increase in fluoroquinolone use in particular may lead to the emergence of levofloxacin-resistant strains. Multidrug resistance was observed in 83 isolates (96.5%). Three global antibiotic-resistant clones were identified: Denmark14 ST230, Portugal19F ST177 and Spain9V ST156.Conclusion. This study shows that macrolide resistance among S. pneumoniae isolated in Tunisia is mainly related to target site modification. Our observations demonstrate a high degree of genetic diversity and capsular types among strains resistant to macrolides.

Cloned ermTR Gene Confers Low Level Erythromycin but High Level Clindamycin Resistance in Streptococcus pyogenes NZ131.

Objectives: The most common macrolide resistance mechanisms in streptococci are the presence of methylase encoding genes ermB and ermTR or the presence of efflux encoded by mef genes. In the present study we aimed to show the effects of the ermTR gene under isogenic conditions on the activities of macrolides and lincosamides in streptococci. Materials and Methods: Total DNA was extracted from Streptococcus pyogenes C1, and the ermTR gene was amplified with or without the regulatory region using modified primer with insertion of restriction sites to clone in to pUC18. Transformants were selected after electroporation of Escherichia coli DB10. The recombinant plasmids were purified and merged to pJIM2246 to transform Gram positive bacteria. Recombinant pJIM2246 plasmids with the ermTR gene were then introduced into S. pyogenes NZ131 by electroporation. Results: After transformation with ermTR without regulatory region the minimal inhibitory concentration (MIC) for erythromycin and clindamycin increased from ≤0.06 to ≤0.06 to 8 and >128 mg/L, respectively. Induction with erythromycin affected the MICs for clindamycin of S. pyogenes transformed with ermTR with the regulatory region. Double disk testing showed that induction with erythromycin and azithromycin for the S. pyogenes transformed with ermTR, and regulatory regions decreased the clindamycin inhibition zone but not telithromycin. The ermTR gene in isogenic conditions confers low level resistance to erythromycin and high level resistance to clindamycin. Conclusion: The different induction and resistance profiles of ermTR compared to other erm genes suggest that the methylation of ErmTR may be different than well studied methylases.

665. In vitro Activity of Omadacycline Against Recent (2018) Bacterial Pathogens from the United States and Europe Obtained from Skin and Skin Structure, Respiratory, and Urinary Tract Infections

BackgroundOmadacycline (OMC) was FDA approved to treat acute bacterial skin and skin structure infection (ABSSSI) and community-acquired bacterial pneumonia (CABP) for indicated organisms in 2018. Phase 2 OMC clinical trials for uncomplicated urinary tract infection (uUTI; NCT03425396) and acute pyelonephritis (NCT03757234) are ongoing. OMC is active against bacterial isolates expressing common tetracycline, penicillin, fluoroquinolone, and macrolide resistance mechanisms.MethodsIsolates (14,000) were collected in 2018 from 31 medical centers located in the United States and 38 medical centers in Europe, including 3,458 staphylococci, 1,551 streptococci, 746 enterococci, 574 Haemophilus spp., and 5,690 Enterobacterales isolates. One isolate per patient infection episode was tested. Identifications were confirmed by MALDI-TOF MS and susceptibility testing was performed using CLSI broth microdilution methods.ResultsOMC (MIC50/90, 0.12/0.25 mg/L) was highly active against S. aureus isolates from skin and skin structure infection (SSSI; 99.3% susceptible [S]) including MRSA (97.7%S) and MSSA (99.9%S) (table). Similarly, OMC demonstrated potent activity against S. aureus isolates from respiratory tract infection (RTI; MIC50/90, 0.12/0.25 mg/L) including MSSA (98.2%S). All S. lugdunensis isolates from SSSI were S (100.0%) to OMC. All Streptococcus anginosus group (100.0%) and 97.6% of S. pyogenes isolates from SSSI were S to OMC as were 98.0% of S. pneumoniae from RTI. No streptococci were resistant (R) to OMC. OMC (MIC50/90 0.12/0.25 mg/L) had potent activity against E. faecalis isolates from SSSI (99.0%S). OMC S against E. cloacae and K. pneumoniae isolates from SSSI was 92.1% S and 89.4% S, respectively. Similarly, 86.2% of K. pneumoniae isolates from RTI were S to OMC. Susceptibility of H. influenzae isolates from RTI to OMC was 99.8%S (no isolates were R). ≥90.0% of E. coli (MIC50/90, 1/2 mg/L) and K. pneumoniae (MIC50/90, 2/4 mg/L) UTI isolates were inhibited by ≤4 mg/L of OMC.ConclusionOMC was highly active against bacterial pathogens associated with ABSSSI, CABP, and UTI including staphylococci (97.7%-100.0%S), streptococci (97.6%-100.0%S), E. faecalis (99.0%S). E. cloacae (92.1%S), K. pneumoniae (86.2%-89.4%S), and E. coli. Disclosures All authors: No reported disclosures.