An antibiotic preorganized for ribosomal binding overcomes antimicrobial resistance.

We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.

Macrolide and lincosamide resistance of Streptococcus agalactiae in pregnant women in Poland.

Knowing about the antibiotic resistance, serotypes, and virulence-associated genes of Group B Streptococcus for epidemiological and vaccine development is very important. We have determined antimicrobial susceptibility patterns, serotype, and virulence profiles. The antibiotic susceptibility was assessed for a total of 421 Streptococcus agalactiae strains, isolated from pregnant women and neonates. Then, 89 erythromycin and/or clindamycin-resistant strains (82 isolates obtained from pregnant women and seven isolates derived from neonates) were assessed in detail. PCR techniques were used to identify the studied strains, perform serotyping, and assess genes encoding selected virulence factors. Phenotypic and genotypic methods determined the mechanisms of resistance. All tested strains were sensitive to penicillin and levofloxacin. The constitutive MLSB mechanism (78.2%), inducible MLSB mechanism (14.9%), and M phenotype (6.9%) were identified in the macrolide-resistant strains. It was found that macrolide resistance is strongly associated with the presence of the ermB gene and serotype V. FbsA, fbsB, fbsC, scpB, and lmb formed the most recurring pattern of genes among the nine surface proteins whose genes were analysed. A minority (7.9%) of the GBS isolates exhibited resistance to lincosamides and macrolides, or either, including those that comprised the hypervirulent clone ST-17. The representative antibiotic resistance pattern consisted of erythromycin, clindamycin, and tetracycline resistance (71.9%). An increase in the fraction of strains resistant to macrolides and lincosamides indicates the need for monitoring both the susceptibility of these strains and the presence of the ST-17 clone.

Lincosamide Antibiotics: Structure, Activity, and Biosynthesis.

Lincosamides are naturally occurring antibiotics isolated from Streptomyces sp. Currently, lincomycin A and its semisynthetic analogue clindamycin are used as clinical drugs. Due to their unique structures and remarkable biological activities, derivatizations of lincosamides via semi-synthesis and biosynthetic studies have been reported. This review summarizes the structures and biological activities of lincosamides, and the recent studies of lincosamide biosynthetic enzymes.

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

BACKGROUND: Macrolide 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. METHODS: About 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. RESULTS: Macrolide 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.

Distribution of MecA and Erm Genes among Methicillin-resistant Staphylococcus Aureus with Inducible Resistance to Clindamycin.

BACKGROUND: The emergence of Methicillin-resistant Staphylococcus aureus and its ability to confer cross-resistance to macrolide-lincosamide-streptogramin B has complicated the treatment against it. Gene-based studies among phenotypic methicillin-resistant isolates with inducible resistance to clindamycin are less available in Nepal. This work was undertaken to detect the mecA and erm genes among such phenotypes isolated from clinical samples. METHODS: S. aureus isolated from different clinical samples was identified by standard microbiological procedures (Gram-staining, colony morphology, and different biochemical tests). Methicillin-resistant and inducible resistant to clindamycin phenotypes were detected by using cefoxitin disc (30 µg) and a double disk diffusion test according to the Clinical and Laboratory Standards Institute guidelines and mecA and erm genes were detected by polymerase chain reaction. RESULTS: Among 120 S. aureus isolates, 51.67% (n=62) were MRSA, and the prevalence of inducibly-resistant, constitutively-resistant and Macrolide-Streptogramin phenotypes were 15.83% (n=19), 28.33% (n=34) and 15.83% (n=19) respectively. While 35.84% (n=43) of isolates showed sensitivity to both antibiotics, erythromycin and clindamycin. Out of 14 inducibly-resistant phenotypes, 57.14% (n=8) were found carrying ermC and 28.57% (n=4) phenotypes contained both ermA and ermC. All phenotypes were positive for the mecA gene. CONCLUSIONS: Macrolides-Lincosamide-Streptogramin B resistance was predominant among methicillin-resistant S. aureus. While all isolates with inducible clindamycin resistance harbored mecA gene, most of them also harbored ermC gene. The higher prevalence of inducible-resistant to clindamycin indicated the need for rational use of antimicrobial agents.

Novel macrolide-lincosamide-streptogramin B resistance gene erm(56) in Trueperella pyogenes.

Whole-genome sequence analysis of a macrolide, lincosamide, streptogramin B (MLSB)-resistant Trueperella pyogenes from a dog revealed a new 23S ribosomal RNA methylase gene erm(56). Expression of the cloned erm(56) confers resistance to MLSB in T. pyogenes and Escherichia coli. The erm(56) gene was flanked by two IS6100 integrated on the chromosome next to a sul1-containing class 1 integron. GenBank query revealed additional erm(56)-containing elements in another T. pyogenes and in Rothia nasimurium from livestock. IMPORTANCE A novel 23S ribosomal RNA methylase gene erm(56) flanked by insertion sequence IS6100 was identified in a Trueperella pyogenes isolated from the abscess of a dog and was also present in another T. pyogenes and in Rothia nasimurium from livestock. It was shown to confer resistance to macrolide, lincosamide, streptogramin B antibiotics in T. pyogenes and E. coli, indicating functionality in both Gram-positive and Gram-negative bacteria. The detection of erm(56) on different elements in unrelated bacteria from different animal sources and geographical origins suggests that it has been independently acquired and likely selected by the use of antibiotics in animals.

Phenotypic and Genotypic Characterization of Antimicrobial Resistance in Streptococci Isolated from Human and Animal Clinical Specimens.

Recently, the phenomenon of infection of humans as hosts by animal pathogens has been increasing. Streptococcus is an example of a genus in which bacteria overcome the species barrier. Therefore, monitoring infections caused by new species of human pathogens is critical to their spread. Seventy-five isolates belonging to streptococcal species that have recently been reported as a cause of human infections with varying frequency, were tested. The aim of the study was to determine the drug resistance profiles of the tested strains, the occurrence of resistance genes and genes encoding the most important streptococcal virulence factors. All tested isolates retained sensitivity to ß-lactam antibiotics. Resistance to tetracyclines occurred in 56% of the tested strains. We have detected the MLSB type resistance (cross-resistance to macrolide, lincosamide, and streptogramin B) in 20% of the tested strains. 99% of the strains had tetracycline resistance genes. The erm class genes encoding MLSB resistance were present in 47% of strains. Among the strains with MLSB resistance, 92% had the streptokinase gene, 58% the streptolysin O gene and 33% the streptolysin S gene. The most extensive resistance concerned isolates that accumulated the most traits and genes, both resistance genes and virulence genes, increasing their pathogenic potential. Among the tested strains, the gene encoding streptokinase was the most common. The results of the prove that bacteria of the species S. uberis, S. dysgalactiae and S. gallolyticus are characterized by a high pathogenic potential and can pose a significant threat in case of infection of the human body.

The antimicrobial activity of cethromycin against Staphylococcus aureus and compared with erythromycin and telithromycin.

BACKGROUND: This study aims to explore the antibacterial activity of cethromycin against Staphylococcus aureus (S. aureus), and its relationship with multilocus sequence typing (MLST), erythromycin ribosomal methylase (erm) genes and macrolide-lincosamide-streptogramin B (MLSB) phenotypes of S. aureus. RESULTS: The minimum inhibitory concentrations (MICs) of cethromycin against 245 S. aureus clinical isolates ranged from 0.03125 to ≥ 8 mg/L, with the resistance of 38.8% in 121 methicillin-resistant S. aureus (MRSA). This study also found that cethromycin had strong antibacterial activity against S. aureus, with the MIC ≤ 0.5 mg/L in 55.4% of MRSA and 60.5% of methicillin-sensitive S. aureus (MSSA), respectively. The main MLSTs of 121 MRSA were ST239 and ST59, and the resistance of ST239 isolates to cethromycin was higher than that in ST59 isolates (P = 0.034). The top five MLSTs of 124 MSSA were ST7, ST59, ST398, ST88 and ST120, but there was no difference in the resistance of MSSA to cethromycin between these STs. The resistance of ermA isolates to cethromycin was higher than that of ermB or ermC isolates in MRSA (P = 0.016 and 0.041, respectively), but the resistance of ermB or ermC isolates to cethromycin was higher than that of ermA isolates in MSSA (P = 0.019 and 0.026, respectively). The resistance of constitutive MLSB (cMLSB) phenotype isolates to cethromycin was higher than that of inducible MLSB (iMLSB) phenotype isolates in MRSA (P < 0.001) or MSSA (P = 0.036). The ermA, ermB and ermC genes was mainly found in ST239, ST59 and ST1 isolates in MRSA, respectively. Among the MSSA, the ermC gene was more detected in ST7, ST88 and ST120 isolates, but more ermB genes were detected in ST59 and ST398 isolates. The cMLSB phenotype was more common in ST239 and ST59 isolates of MRSA, and was more frequently detected in ST59, ST398, and ST120 isolates of MSSA. CONCLUSION: Cethromycin had strong antibacterial activity against S. aureus. The resistance of MRSA to cethromycin may had some clonal aggregation in ST239. The resistance of S. aureus carrying various erm genes or MLSB phenotypes to cethromycin was different.

Tn5406, a staphylococcal transposon associated with macrolide-lincosamide-streptograminb resistance in clinical isolates of Staphylococcus aureus.

PURPOSE: In this study, we aimed to investigate the occurrence of MLSb resistance in clinical isolates of Staphylococcus aureus with respect to their association with transposons. METHODS: The present study was performed with clinical isolates of S. aureus. The MLSb resistant phenotypes in the obtained isolates were determined by D zone test or double disc diffusion test as per CLSI 2020 guidelines. MLSb resistance encoding genes were detected by PCR. The genes tested were ermA, ermB, ermC, msrA, mphC, vga, vgb and lnuB. The MLSb resistant Staphylococcal isolates were selected to analyze the association of the genes with mobile genetic elements Tn554, Tn5406, Tn917, Tn6133, Tn551 by PCR based method. Primer pairs were designed using sequences from transposons and the resistance genes, respectively. RESULTS: During this study, 268 isolates of S. aureus were obtained of which 233 (86.94%) isolates exhibited different MLSb resistant phenotypes. The predominant gene among the MLSb resistant isolates was msrA followed by vgaA and mphC genes. PCR assay was employed to determine whether the genes msrA, mphC and vgaA were carried by Tn554, Tn5406, Tn917, Tn6133, Tn551 transposons. PCR amplification with the designed primer pairs revealed vgaA gene being part of Tn5406. CONCLUSION: The presence of Tn5406 in all the vgaA harboring isolates highlights its potential of spread across isolates. Moreover, the co-existence of different MLSb resistance encoding genes observed in the study shows that the combination of genes involved in different mechanism mediated the nature of MLSb resistance.

Prevalence of Inducible Macrolide, Lincosamide, and Streptogramin B (inducible MLSB) Resistance in Clindamycin-Susceptible Staphylococcus aureus at Okayama University Hospital.

Inducible resistance to the macrolide, lincosamide, and streptogramin B (iMLSB) antibiotic family is a latent mechanism for antimicrobial resistance in Staphylococcus aureus. We here investigated the frequency and genotypic profiles of iMLSB resistance in clindamycin (CLDM)-susceptible S. aureus isolated in Okayama University Hospital from June 2020 to June 2021. We phenotypically screened the iMLSB resistance via D-zone test and performed PCR testing for the erythromycin ribosomal methylase (erm) genes: ermA and ermC. Among 432 CLDM-susceptible S. aureus isolates, 138 (31.9%) exhibited an iMLSB-resistance phenotype, with methicillinresistant S. aureus isolates (MRSA; 61 isolates: 58.6%) exhibiting higher positivity than methicillin-sensitive S. aureus isolates (MSSA; 77 isolates: 23.5%) (p<0.001). Male patients had a higher frequency of iMLSB resistance than females (OR [95%CI]: 1.8 [1.2-2.8]; p=0.007). Genotypically, ermA predominated in both MSSA (70.1%) and MRSA (86.9%) compared to ermC (14.3% in MSSA and 11.5% in MRSA). A single strain of MRSA possessed both ermA and ermC, while 12 (15.6%) MSSA isolates were negative for both ermA and ermC, suggesting the presence of other genetic mechanisms. Collectively, these results show that approximately 33% of CLDM-susceptible S. aureus isolates at our university hospital exhibited iMLSB resistance, predominantly caused by ermA in both MSSA and MRSA.

Emergence of lnu(C) variant conferring lincomycin resistance in Campylobacter coli of chicken origin.

Lincomycin is widely used in respiratory and gastrointestinal infection in veterinary medicine and food animal production. Campylobacter members are vital foodborne pathogens causing campylobacteriosis, and the resistance to lincosamides is seldom reported. To date, only the rRNA methyltransferase Erm(B) has been confirmed to be associated with lincosamides resistance in Campylobacter. In this study, we identified a lnu(C) variant conferring lincomycin resistance in this pathogen of chicken origin. The Lnu(C) encoded by this gene variant showed substitution at position 8 (Asn8Lys), 11 (Phe11Leu) and 112 (Leu112Phe), when compared with the firstly reported Lnu(C) from Streptococcus agalactiae. Cloning of the lnu(C) variant into lincosamide-susceptible Campylobacter jejuni NCTC 11168 confirmed its function in conferring resistance to lincomycin with the 32-fold increased MICs. Sequencing analysis showed that the lnu(C) variant was located within a MTnSag1-like transposon together with insLNU, which is inserted between panB and cj0299 genes on the chromosome. lnu(C) gene was distributed among C. coli globally, and various STs were involved in the dissemination of lnu(C). Although transposition mediated by MTnSag1-like transposon failed to occur, the horizontal transfer mediated by natural transformation and reservoir for resistance genes may facilitate their adaptation to the antimicrobial selection pressure in chickens, which should not be ignored.

Invasive Streptococcus suis isolated in Spain contain a highly promiscuous and dynamic resistome.

Introduction: Streptococcus suis is a major pathogen for swine and human. Here we aimed to know the rates of antimicrobial resistance (AMR) in invasive S. suis isolates recovered along Spain between 2016 - 2021 and elucidate their genetic origin. Methods: Antibiotic susceptibility testing was performed for 116 isolates of different genetic backgrounds and geographic origins against 18 antibiotics of 9 families. The association between AMR and genotypes and the origin of the isolates were statistically analyzed using Pearson´s chi-square test and the likelihood ratio. The antimicrobial resistant genes were identified by whole genome sequencing analysis and PCR screenings. Results: High AMR rates (>80%) were detected for tetracyclines, spectinomycin, lincosamides, and marbofloxacin, medium (20-40%) for sulphonamides/trimethoprim, tiamulin, penicillin G, and enrofloxacin, and low (< 20%) for florfenicol, and four additional ß-lactams. The occurrence of multidrug resistance was observed in 90% of isolates. For certain antibiotics (penicillin G, enrofloxacin, marbofloxacin, tilmicosin, and erythromycin), AMR was significantly associated with particular sequence types (STs), geographic regions, age of pigs, and time course. Whole genome sequencing comparisons and PCR screenings identified 23 AMR genes, of which 19 were previously reported in S. suis (aph(3')-IIIa, sat4, aadE, spw, aac(6')-Ie-aph(2'')-Ia, fexA, optrA, erm(B), mef(A/E), mrs(D), mph(C), lnu(B), lsa(E), vga(F), tet(M), tet(O), tet(O/W/32/O), tet(W)), and 4 were novel (aph(2'')-IIIa, apmA, erm(47), tet(T)). These AMR genes explained the AMR to spectinomycin, macrolides, lincosamides, tiamulin, and tetracyclines. Several genes were located on mobile genetic elements which showed a variable organization and composition. As AMR gene homologs were identified in many human and animal pathogens, the resistome of S. suis has a different phylogenetic origin. Moreover, AMR to penicillin G, fluoroquinolones, and trimethoprim related to mutations in genes coding for target enzymes (pbp1a, pbp2b, pbp2x, mraY, gyrA, parC, and dhfr). Bioinformatic analysis estimated traits of recombination on target genes, also indicative of gene transfer events. Conclusions: Our work evidences that S. suis is a major contributor to AMR dissemination across veterinary and human pathogens. Therefore, control of AMR in S. suis should be considered from a One Health approach in regions with high pig production to properly tackle the issue of antimicrobial drug resistance.

Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes.

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

[Historical Antibiotic Stress Changed the Effects of Sulfamethoxazole and Trimethoprim on Activated Sludge: ARGs and Potential Hosts].

The co-exposure of antibiotics has important effects on antibiotic resistance genes (ARGs) and microbial community aggregation in wastewater treatment plants (WWTPs). However, it is unclear whether differences in historical antibiotic exposure stress can determine responses of microbes and ARGs to combined antibiotics. By selecting a high concentration (30 mg·L-1) of sulfamethoxazole (SMX) and trimethoprim (TMP) as historical exposure stress conditions, the effects of SMX and TMP-combined pollution on ARGs, bacterial communities, and their interactions were explored in short-term experiments. Based on high-throughput quantitative PCR, a total of 13 ARGs were detected, and the absolute abundance was 2.21-5.42 copies·µL-1 (logarithm, DNA, the same below). Among them, sul2, ermB, mefA, and tetM-01 were the main subtypes in the samples, and the absolute abundance was between 2.95 and 5.40 copies·µL-1. The combined contamination of SMX and TMP could cause the enrichment of ARGs and mobile genetic elements (MGEs); however, their effects on each subtype were different, and the historical legacy effect of SMX was higher than that of TMP. Under the different exposure histories, the co-occurrence and co-exclusion patterns existed between ARGs. Moreover, MGEs (especially intI-1) were significantly correlated with sulfonamides (sul1 and sul2), tetracyclines[tet(32)], and macrolide-lincosamide-streptogramin (MLSB) resistance genes (ermB). Based on the full-scale classification of microorganisms, it was found that the microbial community structure of various groups responded differently to combined pollution, and the conditionally abundant taxa (CAT) were obviously enriched. Thauera, Pseudoxanthomonas, and Paracoccus were the dominant resistant bacterial genera. Furthermore, a total of 31 potential hosts of ARGs were identified with network analysis, which were dominated with conditionally rare taxa (CRT). Particularly, Candidatus_Alysiosphaera and Fusibacter were positively correlated with most of the ARGs, being the common protentional hosts. Importantly, some rare genera (RT, Variibacter, Aeromonas, Cloacibacterium, etc.) were potential hosts of transposon IS613, which played an important role in the proliferation and spread of ARGs. In conclusion, this study revealed the legacy effects of historical antibiotic stress on ARGs and their hosts, which could provide new ideas and theoretical basis for reducing ARGs pollution in WWTPs.

Human enterococcal isolates as reservoirs for macrolide-lincosamide-streptogramin and other resistance genes.

According to recent studies, the importance of MLS (macrolide-lincosamide-streptogramin) resistance phenotypes and genes in enterococci are reflected in the fact that they represent reservoirs of MLS resistance genes. The aim of this study was to investigate distribution of MLS resistance genes and phenotypes in community- and hospital-acquired enterococcal isolates and to determine their prevalence. The MLS resistance phenotypes (cMLSb, iMLSb, M/MSb, and L/LSa) were determined in 245 enterococcal isolates were characterized using the double-disc diffusion method. Specific primers were chosen from database sequences for detection of the MLS resistance genes (ermA, ermB, ermC, msrA/B, lnuA, lnuB, and lsaA) in 60 isolates of enterococci by end-point PCR. There was no linezolid-resistant enterococcal isolate. Only one vancomycin-resistant (0.6%) isolate was found and it occurred in a community-acquired enterococcal isolate. The most frequent MLS resistance phenotype among enterococcal isolates was cMLSb (79.7% community- and 67.9% hospital-acquired). The most common identified MLS resistance genes among enterococcal isolates were lsaA (52.9% community- and 33.3% hospital-acquired) and ermB (17.6% community- and 33.3% hospital-acquired). The most prevalent MLS gene combination was lnuA + lsaA (five enterococcal isolates). The ermB gene encoded cMLSb phenotype, and it was identified in only one isolate that displayed iMLSb resistance phenotype. Based on the results obtained, we can conclude that the most frequent MLS resistance phenotype among enterococcal isolates was cMLSb. Surprisingly, a vancomycin-resistant enterococcal isolate was identified in a community-acquired enterococcal isolate. This study shows that enterococci may represent a major reservoir of ermB, lsaA, and lnuA genes.

erm(T)-Mediated Macrolide-Lincosamide Resistance in Streptococcus suis.

To investigate the presence and location of erm(T) in clinical Streptococcus suis isolates and explore the transmission ability and fitness cost of erm(T)-carrying mobile genetic elements among S. suis isolates, MICs were determined by broth microdilution. The presence of erm(T) in S. suis was detected by PCR. The genetic environment of erm(T) in S. suis was explored by whole-genome sequencing (WGS) analysis. Intraspecies and interspecies transmission were examined by electrotransformation. The fitness cost associated with the carriage of an erm(T)-harboring plasmid or an integrative and conjugative element (ICE) was examined by competition experiments. Of 237 nonduplicate strains, erm(T) was detected in 2 S. suis strains (SC262-ST954 and SC117-ST1314), with its location on a 5,125-bp plasmid in S. suis SC262 and on a 64,013-bp ICESsuSC117 in S. suis SC117, respectively. Both the erm(T)-carrying plasmid pSC262 and the ICESsuSC117 were transmissible by transformation. Plasmid pSC262 can replicate and express macrolide-lincosamide resistance in heterologous hosts, including S. aureus and S. pneumoniae. Both the erm(T)-carrying plasmid and the ICE posed a fitness cost to the host S. suis isolate. To the best of our knowledge, this is the first report of the macrolide-lincosamide-streptogramin B resistance gene erm(T) in S. suis. Its location on a plasmid or an ICE will aid in its transmission. The low detection rate of erm(T) gene among the S. suis population might be due to the fitness cost of the erm(T)-carrying plasmid and ICE. IMPORTANCE Macrolide and lincosamide resistance due to the presence of erm(T) have posed a challenge for the treatment of Gram-positive pathogens. Although the low detection rate of erm(T) gene among the S. suis population due to the fitness cost of the erm(T)-carrying plasmid and ICE, the presence of erm(T) in S. suis and its potential transmission to other Gram-positive pathogens will be of important significance.

Clarithromycin Inhibits Pneumolysin Production via Downregulation of ply Gene Transcription despite Autolysis Activation.

Streptococcus pneumoniae, the most common cause of community-acquired pneumonia, causes severe invasive infections, including meningitis and bacteremia. The widespread use of macrolides has been reported to increase the prevalence of macrolide-resistant S. pneumoniae (MRSP), thereby leading to treatment failure in patients with pneumococcal pneumonia. However, previous studies have demonstrated that several macrolides and lincosamides have beneficial effects on MRSP infection since they inhibit the production and release of pneumolysin, a pneumococcal pore-forming toxin released during autolysis. In this regard, we previously demonstrated that the mechanisms underlying the inhibition of pneumolysin release by erythromycin involved both the transcriptional downregulation of the gene encoding pneumolysin and the impairment of autolysis in MRSP. Here, using a cell supernatant of the culture, we have shown that clarithromycin inhibits pneumolysin release in MRSP. However, contrary to previous observations in erythromycin-treated MRSP, clarithromycin upregulated the transcription of the pneumococcal autolysis-related lytA gene and enhanced autolysis, leading to the leakage of pneumococcal DNA. On the other hand, compared to erythromycin, clarithromycin significantly downregulated the gene encoding pneumolysin. In a mouse model of MRSP pneumonia, the administration of both clarithromycin and erythromycin significantly decreased the pneumolysin protein level in bronchoalveolar lavage fluid and improved lung injury and arterial oxygen saturation without affecting bacterial load. Collectively, these in vitro and in vivo data reinforce the benefits of macrolides on the clinical outcomes of patients with pneumococcal pneumonia. IMPORTANCE Pneumolysin is a potent intracellular toxin possessing multiple functions that augment pneumococcal virulence. For over 10 years, sub-MICs of macrolides, including clarithromycin, have been recognized to decrease pneumolysin production and release from pneumococcal cells. However, this study indicates that macrolides significantly slowed pneumococcal growth, which may be related to decreased pneumolysin release recorded by previous studies. In this study, we demonstrated that clarithromycin decreases pneumolysin production through downregulation of ply gene transcription, regardless of its inhibitory activity against bacterial growth. Additionally, administration of clarithromycin resulted in the amelioration of lung injury in a mouse model of pneumonia induced by macrolide-resistant pneumococci. Therefore, therapeutic targeting of pneumolysin offers a good strategy to treat pneumococcal pneumonia.

Ribosome Protection as a Mechanism of Lincosamide Resistance in Mycobacterium abscessus.

Mycobacterium abscessus has emerged as a successful pathogen owing to its intrinsic drug resistance. Macrolide and lincosamide antibiotics share overlapping binding sites within the ribosome and common resistance pathways. Nevertheless, while M. abscessus is initially susceptible to macrolides, they are completely resistant to the lincosamide antibiotics. Here, we have used RNA sequencing to determine the changes in gene expression in M. abscessus upon exposure to the lincosamide, clindamycin (CLY). We show that Mab_1846, encoding a putative ARE-ABCF protein, was upregulated upon exposure to macrolides and lincosamides but conferred resistance to CLY alone. A Mycobacterium smegmatis homologue of Mab_1846, Ms_5102, was similarly found to be required for CLY resistance in M. smegmatis. We demonstrate that Ms5102 mediates CLY resistance by directly interacting with the ribosomes and protecting it from CLY inhibition. Additional biochemical characterization showed that ribosome binding is not nucleotide dependent, but ATP hydrolysis is required for dissociation of Ms5102 from the ribosome as well as for its ability to confer CLY resistance. Finally, we show that in comparison to the macrolides, CLY is a potent inducer of Mab_1846 and the whiB7 regulon, such that exposure of M. abscessus to very low antibiotic concentrations induces a heightened expression of erm41, hflX, and Mab_1846, which likely function together to result in a particularly antibiotic-resistant state.