Macrolide-resistant Isolates Research Articles

Ultrastructural, metabolic and genetic characteristics of determinants facilitating the acquisition of macrolide resistance by Streptococcus pneumoniae

AimsTo investigate the molecular events associated with acquiring macrolide resistance genes [mefE/mel (Mega) or ermB] in Streptococcus pneumoniae (Spn) during nasopharyngeal colonization. Methods and resultsGenomic analysis of 128 macrolide-resistant Spn isolates revealed recombination events in genes of the conjugation apparatus, or the competence system, in strains carrying Tn916-related elements. Studies using confocal and electron microscopy demonstrated that during the transfer of Tn916-related elements in nasopharyngeal cell biofilms, pneumococcal strains formed clusters facilitating their acquisition of resistance determinants at a high recombination frequency (rF). Remarkably, these aggregates comprise both encapsulated and nonencapsulated pneumococci that span extracellular and intracellular compartments. rF assessments showed similar rates regardless Mega was associated with large integrative and conjugative elements (ICEs) (>23 kb) or not (∼5.4 kb). The rF for Mega Class IV(c) insertion region (∼53 kb) was three orders of magnitude higher than the transformation of the capsule locus. Metabolomics studies of the microenvironment created by colonization of human nasopharyngeal cells revealed a link between the acquisition of ICEs and the pathways involving nicotinic acid and sucrose. ConclusionsPneumococcal clusters, both extracellular and intracellular, facilitate macrolide resistance acquisition, and ICEs were acquired at a higher frequency than the capsule locus. Metabolic changes could serve as intervention targets.

Resurgence of Bordetella pertussis, including one macrolide-resistant isolate, France, 2024.

As other European countries, France is experiencing a resurgence of pertussis in 2024. Between 1 January and 31 May 2024, 5,616 (24.9%) positive Bordetella pertussis qPCR tests were identified, following a 3-year period of almost null incidence. Of 67 cultured and whole genome sequenced B. pertussis isolates, 66 produced pertactin and 56 produced FIM2, in contrast to pre-COVID-19 years. One isolate of genotype Bp-AgST4 was resistant to macrolides. Pertussis resurgence may favour isolates that produce FIM2 and pertactin.

Whole-genome comparison of two same-genotype macrolide-resistant Bordetella pertussis isolates collected in Japan.

The emergence of macrolide-resistant Bordetella pertussis (MRBP) is a significant problem because it reduces treatment options for pertussis and exacerbates the severity and spread of the disease. MRBP has been widely prevalent in mainland China since the 2010s and has been sporadically detected in other Asian countries. In Japan, two MRBP clinical strains were first isolated in Tokyo and Osaka between June and July 2018. The isolates BP616 in Osaka and BP625 in Tokyo harbored the same virulence-associated allelic genes (including ptxP1, ptxA1, prn1, fim3A, and fhaB3) and MT195 genotype and exhibited similar antimicrobial susceptibility profiles. However, despite their simultaneous occurrence, a distinguishable epidemiological link between these isolates could not be established. To gain further insight into the genetic relationship between these isolates in this study, we performed whole-genome analyses. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms revealed that the isolates belonged to one of the three clades of Chinese MRBP isolates, but there were 11 single-nucleotide polymorphism differences between BP616 and BP625. Genome structure analysis revealed two large inversions (202 and 523 kbp) and one small transposition (3.8 kbp) between the genomes. These findings indicate that the two Japanese MRBP isolates are closely related to Chinese MRBP isolates but are genomically distinct, suggesting that they were introduced into Japan from mainland China through different transmission routes.

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.

Molecular typing and antimicrobial resistance of group B Streptococcus clinical isolates in Saudi Arabia

Group B Streptococcus (GBS) has emerged as an important cause of severe infections in adults. However, limited data are available regarding the epidemiology of GBS in Saudi Arabia. Isolates were collected over a period of eight months from colonized (n=104) and infected adults (n=95). Serotypes and virulence determinants were detected by polymerase chain reactions (PCRs). Genetic relatedness was assessed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Antimicrobial susceptibilities were determined by disk diffusion. Serotypes III and V (25% each) were the most prevalent, followed by serotypes II (16.18%), Ia (13.24%), VI (9.31%), and Ib (8.82%), while five isolates remained non-typeable (2.45%). Hypervirulent serotype III/CC17 clone (n=21) accounted for 41.18% of the serotype III isolates. Most isolates (53.92%) harboured pilus island (PI) 1 and 2a types, while PI-2b was predominantly detected in the hypervirulent clone. Isolates were variably resistant to tetracycline (76.47%), erythromycin (36.76%), clindamycin (25.49%), and levofloxacin (6.37%), but remained susceptible to penicillin. Macrolide resistant isolates exhibited constitutive (55.42%) and inducible macrolide-lincosamide-streptogramin B resistance phenotypes (33.74%), while a few had L (9.64%) or M (1.2%) phenotypes. MLVA patterns of dominant serotypes III and V revealed 40 different types divided into 12 clusters and 28 singletons. Interestingly, macrolide resistance was significantly associated with two major MLVA types. GBS isolates belonged predominantly to serotypes III and V, but there were no clear associations between serotypes and patient groups. The studied isolates exhibited high levels of resistance to erythromycin and clindamycin that need further surveillance.

Plasmid-mediated drug resistance in mycobacteria: the tip of the iceberg?

Macrolides, such as clarithromycin, are crucial in the treatment of nontuberculous mycobacteria (NTM). NTM are notoriously innately drug resistant, which has made the dependence on macrolides for their treatment even more important. Not surprisingly, resistance to macrolides has been documented in some NTM, including Mycobacterium avium and Mycobacterium abscessus, which are the two NTM species most often identified in clinical isolates. Resistance is mediated by point mutations in the 23S ribosomal RNA or by methylation of the rRNA by a methylase (encoded by an erm gene). Chromosomally encoded erm genes have been identified in many of the macrolide-resistant isolates, but not in Mycobacterium chelonae. Now, Brown-Elliott et al. (J Clin Microbiol 61:e00428-23, 2023, https://doi.org/10.1128/JCM.00428-23) describe the identification of a new erm variant, erm(55), which was found either on the chromosome or on a plasmid in highly macrolide-resistant clinical isolates of M. chelonae. The chromosomal erm(55) gene appears to be associated with mobile elements; one gene is within a putative transposon and the second is in a large (37 kb) insertion/deletion. The plasmid carrying erm(55) also encodes type IV and type VII secretion systems, which are often linked on large mycobacterial plasmids and are hypothesized to mediate plasmid transfer. While the conjugative transfer of the erm(55)-containing plasmid between NTM has yet to be demonstrated, the inferences are clear, as evidenced by the dissemination of plasmid-mediated drug resistance in other medically important bacteria. Here, we discuss the findings of Brown-Elliott et al., and the potential ramifications on treatment of NTM infections.

Repurposing Selamectin as an Antimicrobial Drug against Hospital-Acquired Staphylococcus aureus Infections.

The emergence of multidrug-resistant strains requires the urgent discovery of new antibacterial drugs. In this context, an antibacterial screening of a subset of anthelmintic avermectins against gram-positive and gram-negative strains was performed. Selamectin completely inhibited bacterial growth at 6.3 μg/mL concentrations against reference gram-positive strains, while no antibacterial activity was found against gram-negative strains up to the highest concentration tested of 50 μg/mL. Given its relevance as a community and hospital pathogen, further studies have been performed on selamectin activity against Staphylococcus aureus (S. aureus), using clinical isolates with different antibiotic resistance profiles and a reference biofilm-producing strain. Antibacterial studies have been extensive on clinical S. aureus isolates with different antibiotic resistance profiles. Mean MIC90 values of 6.2 μg/mL were reported for all tested S. aureus strains, except for the macrolide-resistant isolate with constitutive macrolide-lincosamide-streptogramin B resistance phenotype (MIC90 9.9 μg/mL). Scanning Electron Microscopy (SEM) showed that selamectin exposure caused relevant cell surface alterations. A synergistic effect was observed between ampicillin and selamectin, dictated by an FIC value of 0.5 against methicillin-resistant strain. Drug administration at MIC concentration reduced the intracellular bacterial load by 81.3%. The effect on preformed biofilm was investigated via crystal violet and confocal laser scanning microscopy. Selamectin reduced the biofilm biomass in a dose-dependent manner with minimal biofilm eradication concentrations inducing a 50% eradication (MBEC50) at 5.89 μg/mL. The cytotoxic tests indicated that selamectin exhibited no relevant hemolytic and cytotoxic activity at active concentrations. These data suggest that selamectin may represent a timely and promising macrocyclic lactone for the treatment of S. aureus infections.

In vitro and intracellular activities of novel thiopeptide derivatives against macrolide-susceptible and macrolide-resistant Mycobacterium avium complex.

Unsatisfactory outcomes following long-term multidrug treatment in patients with Mycobacterium avium complex (MAC) pulmonary disease have urged us to develop novel antibiotics. Thiopeptides, a class of peptide antibiotics derived from natural products, have potential as drug candidates that target bacterial ribosomes, but drug development has been hampered due to their extremely poor solubility. Here, we evaluated three new compounds (AJ-037, AJ-039, and AJ-206) derived from the thiopeptide micrococcin P2 with enhanced aqueous solubility; the derivatives were generated based on structure-activity relationship analysis. We conducted in vitro drug susceptibility and intracellular antimycobacterial activity testing of the three thiopeptide derivatives against various MAC strains, including macrolide-resistant MAC clinical isolates. These compounds showed low MICs against MAC, similar to that of clarithromycin (CLR). In particular, two compounds, AJ-037 and AJ-206, had intracellular antimycobacterial activities, along with synergistic effects with CLR, and inhibited the growth of MAC inside macrophages. Moreover, these two compounds showed in vitro and intracellular anti-MAC activities against macrolide-resistant MAC strains without showing cross-resistance with CLR. Taken together, the results of the current study suggest that AJ-037 and AJ-206 can be promising anti-MAC agents for the treatment of MAC infection, including for macrolide-resistant MAC strains. IMPORTANCE Novel antibiotics for the treatment of MAC infection are urgently needed because the treatment outcomes using the standard regimen for Mycobacterium avium complex (MAC) pulmonary disease remain unsatisfactory. Here, we evaluated three novel thiopeptide derivatives (AJ-037, AJ-039, and AJ-206) derived from the thiopeptide micrococcin P2, and they were confirmed to be effective against macrolide-susceptible and macrolide-resistant MAC. Our thiopeptide derivatives have enhanced aqueous solubility through structural modifications of poorly soluble thiopeptides. The thiopeptide derivatives showed minimal inhibitory concentrations against MAC that were comparable to clarithromycin (CLR). Notably, two compounds, AJ-037 and AJ-206, exhibited intracellular antimycobacterial activities and acted synergistically with CLR to hinder the growth of MAC within macrophages. Additionally, these compounds demonstrated in vitro and intracellular anti-MAC activities against macrolide-resistant MAC strains without showing any cross-resistance with CLR. We believe that AJ-037 and AJ-206 can be promising anti-MAC agents for the treatment of MAC infections, including macrolide-resistant MAC strains.

Monotherapy: Key cause of macrolide-resistant Mycobacterium avium complex disease

BackgroundMacrolide-resistant Mycobacterium avium complex (MAC) disease is very difficult to cure. Macrolide-resistance emerges in patients and is largely preventable by appropriate screening and treatment practices. MethodsPatients with macrolide-resistant MAC isolates between March 2019 and March 2022 were retrieved from the mycobacteriology reference laboratory database at Radboudumc, Nijmegen, the Netherlands. Clinical consultation reports were extracted from the database to assess the cause of macrolide resistance. ResultsSixteen patients with macrolide-resistant MAC disease were included, from a total of 815 patients with MAC isolates (2%); Macrolide monotherapy in bronchiectasis or CF was the most frequent cause of development of macrolide-resistance MAC disease (n = 8; 50%). Short (n = 3; mean duration 9 months, range 6–12) or guideline non-compliant (n = 2) treatment regimens and patient non-adherence (n = 2) were other key causes of macrolide-resistance. ConclusionsMacrolide monotherapy after inappropriate screening is the most frequent cause of macrolide-resistant Mycobacterium avium complex disease in the Netherlands. Educational efforts are needed to prevent this.

Antibiotic dispensing during first year of COVID-19 pandemic in Brazilian public hospitals.

Aim: To evaluate the impact of the first year of the COVID-19 pandemic on antibiotic dispensation in three Brazilian hospitals. Methods: Stock movement was accessed frompharmacy databases and microbiological reports from 2018 to 2021. Results: Reduced antibiotic dispensation occurred during 2020 in all hospitals. The most significant reduction was in April at Hospital Doutor Jayme dos Santos Neves (∼58%), andin May at Hospital Doutor Roberto Arnizaut Silvares (∼66%)and Hospital Doutor Dório Silva (∼29%). However, azithromycin dispensation increased in all hospitals in 2020. Macrolide-resistant bacterial isolates rose from 66.6% in 2019 to 77.1% in 2020 and 88.3% in 2021. Conclusion: Despitereduced antibiotic dispensation, the increase in azithromycin consumption in 2020 highlights the urgency to monitor macrolide resistance after the pandemic and improve stewardship activities.

Emergence of Erythromycin-Resistant Invasive Group A Streptococcus, West Virginia, USA, 2020-2021.

Clindamycin and β-lactam antibiotics have been mainstays for treating invasive group A Streptococcus (iGAS) infection, yet such regimens might be limited for strains displaying MLSB phenotypes. We investigated 76 iGAS isolates from 66 patients in West Virginia, USA, during 2020-2021. We performed emm typing using Centers for Disease Control and Prevention guidelines and assessed resistance both genotypically and phenotypically. Median patient age was 42 (range 23-86) years. We found 76% of isolates were simultaneously resistant to erythromycin and clindamycin, including all emm92 and emm11 isolates. Macrolide resistance was conferred by the plasmid-borne ermT gene in all emm92 isolates and by chromosomally encoded ermA, ermB, and a single mefA in other emm types. Macrolide-resistant iGAS isolates were typically resistant to tetracycline and aminoglycosides. Vulnerability to infection was associated with socioeconomic status. Our results show a predominance of macrolide-resistant isolates and a shift in emm type distribution compared with historical reports.

Correlation of Moraxella catarrhalis macrolide susceptibility with the ability to adhere and invade human respiratory epithelial cells

ABSTRACT Recently, the prevalence of macrolide-resistant Moraxella catarrhalis has been reported, especially among Chinese children. The fitness cost of resistance is reported to render the resistant bacteria less virulent. To investigate the correlation between macrolide susceptibility of M. catarrhalis and pathogenicity, the whole genome of 70 M. catarrhalis isolates belonging to four clonal complexes with different macrolide susceptibilities was sequenced. The gene products were annotated with the Gene Ontology terms. Based on 46 extracted essential virulence genes, 19 representative isolates were selected to infect type II alveolar cells (A549 cells). The ability of these isolates to adhere and invade human epithelial cells and to produce cytokines was comparatively analysed. Furthermore, mice were infected with a pair of M. catarrhalis isolates with different pathogenic behaviours and macrolide susceptibilities to examine pulmonary clearance, histological findings, and the production of cytokines. The percentages of annotations for binding, metabolic process, cellular process, and cell were non-significantly different between the macrolide-resistant and macrolide-susceptible groups. The presence of uspA2, uspA2H, pilO, lbpB, lex1, modM, mboIA, and mboIB significantly differed among the four clonal complexes and macrolide susceptibility groups. Furthermore, compared with those in macrolide-susceptible isolates, the adhesion ability was stronger (P = 0.0019) and the invasion ability was weaker (P < 0.0001) in the macrolide-resistant isolates. Mouse experiments revealed that pulmonary macrophages elicit immune responses against M. catarrhalis infection by significantly upregulating the Csf2, Il4, Il13, Il1b, Il6, Tnf, and Il18. Therefore, M. catarrhalis populations exhibited diverse pathogenicity in vitro and in vivo.

Induction of the macrolide-resistance efflux pump Mega inhibits intoxication of Staphylococcus aureus strains by Streptococcus pneumoniae

Streptococcus pneumoniae (Spn) kills Staphylococcus aureus (Sau) through a contact-dependent mechanism that is catalyzed by cations, including iron, to convert hydrogen peroxide (H2O2) to highly toxic hydroxyl radicals (•OH). There are two well-characterized ABC transporters that contribute to the pool of iron in Spn, named Pia and Piu. Some Spn strains have acquired genes mef(E)/mel encoding another ABC trasporter (Mega) that produces an inducible efflux pump for resistance to macrolides. In macrolide-resistant Spn clinical isolates the insertion of Mega class 1. IV and 2. IVc deleted the locus piaABCD and these strains were attenuated for intoxicating Sau. The goal of this study was to investigate if the disruption of iron acquisition, or the antimicrobial-resistance activity of Mega, contributed to inhibiting the killing mechanism. Neither depletion of iron with 2,2'-dipyridyl-d8 (DP) nor incubating with a double knockout mutant SpnΔpiaAΔpiuA, inhibited killing of Sau. Clinical Spn strains carrying Mega1. IV or Mega2. IVc showed a significant delay for killing Sau. An ex vivo recombination system was used to transfer Mega1. IV or Mega2. IVc to reference Spn strains, which was confirmed by whole genome sequencing, and recombinants TIGR4Mega2. IVc, D39Mega2. IVc, and D39Mega1. IV were delayed for killing Sau. We then compared Sau killing of selected Mega-carrying Spn strains when incubated with sub-inhibitory erythromycin (Mega-induced) or sub-inhibitory cefuroxime. Remarkably, killing of Sau was completely inhibited under the Mega-induced condition whereas incubation with cefuroxime did not interfere with killing. Both mef(E) and mel were upregulated > 400-fold, and spxB (encoding an enzyme responsible for production of most H2O2) was upregulated 14.2-fold, whereas transcription of the autolysin (lytA) gene was downregulated when incubated with erythromycin. We demonstrated that erythromycin induction of Mega inhibits the •OH-mediated intoxication of Sau and that the inhibition occurred at the post-translational level suggesting that an imbalance of ions in the membrane inhibits these reactions.

Serotype distribution and antimicrobial resistance of invasive Streptococcus pneumoniae isolates among Croatian adults during a fifteen-year period (2005-2019)

AimTo assess serotype distribution, antibiotic resistance, and vaccine coverage against Streptococcus pneumoniae causing invasive infections in Croatian adults from 2005 to 2019.MethodsIn this retrospective study, invasive pneumococcal strains were collected through a microbiological laboratory network with country coverage >95%. Capsular typing was performed with the Quellung reaction. In vitro susceptibility testing was carried out according to the European Committee on Antimicrobial Susceptibility Testing guidelines. In macrolide-resistant isolates, the presence of ermB and mefA genes was evaluated.ResultsDuring the fifteen-year study period, 1123 invasive pneumococcal isolates were obtained. The most prevalent serotypes were 3, 14, 19A, 9V, 7F, and 23F, comprising 60% of all invasive pneumococcal isolates. Serotype 3 was the dominant serotype, with the highest prevalence in patients ≥65 years of age. Penicillin susceptibility, increased exposure was 18.6%, mostly associated with serotypes 14 and 19A. Resistance to penicillin was low (<1%). Macrolide resistance was 23%, mostly associated with serotypes 14, 19A, and 19F. The coverage with 13-valent conjugate vaccine (PCV13) and 23-valent polysaccharide vaccine (PPV23) was 80.2% and 93.6%, respectively.ConclusionsThe incidence of invasive pneumococcal disease in adults is highest in patients ≥65 years of age. Penicillin susceptibility, increased exposure and macrolide resistance were mostly associated with serotypes 14 and 19A. PCV13 and PPV23 provide very high serotype coverage. Future studies should evaluate the effects of the 10-valent vaccine, introduced in the Croatian National Immunization Program in June 2019, on serotype distribution and antibiotic resistance rates.

Population structure of ocular Streptococcus pneumoniae is highly diverse and formed by lineages that escape current vaccines.

Streptococcus pneumoniae is a leading cause of ocular infections including serious and sight-threatening conditions. The use of pneumococcal conjugate vaccines (PCV) has substantially reduced the incidence of pneumonia and invasive pneumococcal diseases, but has had limited impact on ocular infections. Additionally, widespread vaccine use has resulted in ongoing selective pressure and serotype replacement in carriage and disease. To gain insight into the population structure of pneumococcal isolates causing ocular infections in a post-PCV-13 time period, we investigated the genomic epidemiology of ocular S. pneumoniae isolates (n=45) collected at Massachusetts Eye and Ear between 2014 and 2017. By performing a series of molecular typing methods from draft genomes, we found that the population structure of ocular S. pneumoniae is highly diverse with 27 sequence types (grouped into 18 clonal complexes) and 17 serotypes being identified. Distribution of these lineages diverged according to the site of isolation, with conjunctivitis being commonly caused by isolates grouped in the Epidemic Conjunctivitis Cluster-ECC (60 %), and ST448 (53.3 %) being most frequently identified. Conversely, S. pneumoniae keratitis cases were caused by a highly diverse population of isolates grouping within 15 different clonal complexes. Serotyping inference demonstrated that 95.5 % of the isolates were non-PCV-13 vaccine types. Most of the conjunctivitis isolates (80 %) were unencapsulated, with the remaining belonging to serotypes 15B, 3 and 23B. On the other hand, S. pneumoniae causing keratitis were predominantly encapsulated (95.2 %) with 13 different serotypes identified, mostly being non-vaccine types. Carriage of macrolide resistance genes was common in our ocular S. pneumoniae population (42.2 %), and usually associated with the mefA +msrD genotype (n=15). These genes were located in the Macrolide Efflux Genetic Assembly cassette and were associated with low-level in vitro resistance to 14- and 15-membered macrolides. Less frequently, macrolide-resistant isolates carried an ermB gene (n=4), which was co-located with the tetM gene in a Tn-916-like transposon. Our study demonstrates that the population structure of ocular S. pneumoniae is highly diverse, mainly composed by isolates that escape the PCV-13 vaccine, with patterns of tissue/niche segregation, adaptation and specialization. These findings suggest that the population structure of ocular pneumococcus may be shaped by multiple factors including PCV-13 selective pressure, microbial-related and niche-specific host-associated features.

Impact of intra-partum azithromycin on carriage of group A streptococcus in the Gambia: a posthoc analysis of a double-blind randomized placebo-controlled trial

BackgroundGroup A Streptococcus (GAS) is a major human pathogen and an important cause of maternal and neonatal sepsis. Asymptomatic bacterial colonization is considered a necessary step towards sepsis. Intra-partum azithromycin may reduce GAS carriage.MethodsA posthoc analysis of a double-blind, placebo-controlled randomized-trial was performed to determine the impact of 2 g oral dose of intra-partum azithromycin on maternal and neonatal GAS carriage and antibiotic resistance. Following screening, 829 mothers were randomized who delivered 843 babies. GAS was determined by obtaining samples from the maternal and newborn nasopharynx, maternal vaginal tract and breastmilk. Whole Genome Sequencing (WGS) of GAS isolates was performed using the Illumina Miseq platform.ResultsGAS carriage was lower in the nasopharynx of both mothers and babies and breast milk among participants in the azithromycin arm. No differences in GAS carriage were found between groups in the vaginal tract. The occurrence of azithromycin-resistant GAS was similar in both arms, except for a higher prevalence in the vaginal tract among women in the azithromycin arm. WGS revealed all macrolide-resistant vaginal tract isolates from the azithromycin arm were Streptococcus dysgalactiae subspecies equisimilis expressing Lancefield group A carbohydrate (SDSE(A)) harbouring macrolide resistant genes msr(D) and mef(A). Ten of the 45 GAS isolates (22.2%) were SDSE(A).ConclusionsOral intra-partum azithromycin reduced GAS carriage among Gambian mothers and neonates however carriage in the maternal vaginal tract was not affected by the intervention due to azithromycin resistant SDSE(A). SDSE(A) resistance must be closely monitored to fully assess the public health impact of intrapartum azithromycin on GAS.Trial registration ClinicalTrials.gov Identifier NCT01800942

184. Inducible Clindamycin Resistance Testing on Pediatric Streptococcus pneumoniae Isolates

BackgroundIn 2013, the Clinical and Laboratory Standards Institute recommended inducible clindamycin resistance (ICR) testing on macrolide-resistant Streptococcus pneumoniae isolates, which arises due to the ermB gene. Ribosomal methylation by ermB confers resistance to macrolides (high-level resistance), lincosamides and streptogramin B. The goal of our study is to characterize the prevalence of ICR among pediatric pneumococcal isolates.MethodsWe identified erythromycin-resistant(R) (minimum inhibitory concentration [MIC] ≥ 1 µg/mL) and clindamycin-susceptible(S) (MIC ≤ 0.25 µg/mL) pneumococcal isolates from pediatric patients seen at Children’s Mercy Hospital from 2007 to 2017. Determination of ICR was achieved via disk approximation (D-zone test) with standard erythromycin (15 µg) and clindamycin (2 µg) disks. Isolates with high-level erythromycin resistance (MIC ≥ 32µg/mL) were also tested for ermB gene by PCR. Positive and negative controls were used for D-zone test and ermB PCR.ResultsWe identified 289 erythromycin-R pneumococcal isolates; of those 194 (67.1%) were clindamycin-S (Figure 1). One-hundred and sixty-nine isolates were available for ICR testing, 166 (98%) isolates represented non-invasive disease samples. Median age of patients with erythromycin-R and clindamycin-S isolates was 19 (range 0.1 – 180) months. None of the isolates expressed ICR based on the D-zone test. Thirteen of those isolates (7.7%) expressed high-level erythromycin-R (MIC range 32-128 µg/mL); all were negative for ermB. The most common serotypes/serogroups among erythromycin-R and clindamycin-S isolates were: 15 (n=22), 35B (n=19), 11 (n=16), 6 (n=16), 19A (n=14) and 33 (n=12). ConclusionErythromycin-R and clindamycin-S pneumococcal isolates did not express ICR and isolates with high-level erythromycin-R did not carry ermB. Multicenter studies are needed to determine if ICR testing is required for macrolide-resistant pneumococcal isolates in the PCV13 era.Disclosures Liset Olarte, MD, MSc, GSK (Research Grant or Support)Merck (Research Grant or Support)Pfizer (Research Grant or Support)Sanofi (Research Grant or Support) Douglas S. Swanson, MD, Merck (Research Grant or Support)Pfizer (Research Grant or Support)Sanofi (Research Grant or Support)

Phenotypic Antimicrobial Susceptibility and Genotypic Characterization of Clinical Ureaplasma Isolates Circulating in Shanghai, China.

There is a growing global concern regarding the rise of antimicrobial resistance among Ureaplasma spp. isolates. However, studies on the antimicrobial susceptibility profiles, resistance mechanisms, and clonality of Ureaplasma spp. clinical isolates are still limited and cover only some geographic regions. Firstly, Ureaplasma species from the urogenital tracts of patients in Shanghai, China, were isolated by using the culture medium (A8 and 10B broth), and identified the genotype by polymerase chain reaction (PCR). Secondly, the antimicrobial susceptibility tests were determined by using broth microdilution assay. Then, the resistance genetic determinants to fluoroquinolones (FQs), macrolides, and tetracyclines were investigated through PCR/DNA sequencing. Finally, the molecular epidemiology of Ureaplasma species was studied by multilocus sequence typing (MLST). Among 258 isolates, Ureaplasma parvum (UPA) and Ureaplasma urealyticum (UUR) were found in 226 (87.60%) and 32 (12.40%) isolates, respectively. The minimum inhibitory concentrations (MICs) of 258 Ureaplasma spp. strains ranged from 0.015 to 64μg/ml for all 11 kinds of antimicrobials. Regardless of species, the isolates were most sensitive to AZI (1.94%), JOS (3.49%), and CLA (4.23%). Among them, there were 39 (15.12%) multidrug-resistant (MDR) strains, including 32 UPA isolates. The resistance rates of UPA to CIP (91.59%), and ROX (36.28%) were significantly higher than those of UUR. Twenty six FQ-resistant isolates had amino acid substitutions in gyrA and in parC (Ser83Leu). Mutations were detected in genes encoding ribosomal proteins L4 (Thr84Ile) and L22 (Ser81Pro) in macrolide-resistant isolates. Tet(M) was found in four UPA isolates. These mutations were mainly found in UPA isolates. Sequence type 1 (ST1) was the predominant ST, which contained 18 isolates. In conclusion, this study showed a higher resistance rate (especially to ROX and CIP), higher substitution rate, and higher MDR rate among UPA strains. The most active antimicrobial agents were AZI, JOS, and CLA. Identifying UPA or UUR in clinical isolates could help clinicians to choose appropriate drugs for treatment. The main resistance mechanisms may involve gene substitution of Ser83Leu in parC and Ser81Pro in L22. ST1 was the predominant ST of Ureaplasma isolates with MDR to FQs and macrolides in Shanghai, China.

First Emergence of Resistance to Macrolides and Tetracycline Identified in Mannheimia haemolytica and Pasteurella multocida Isolates from Beef Feedlots in Australia.

Bovine respiratory disease (BRD) causes high morbidity and mortality in beef cattle worldwide. Antimicrobial resistance (AMR) monitoring of BRD pathogens is critical to promote appropriate antimicrobial stewardship in veterinary medicine for optimal treatment and control. Here, the susceptibility of Mannheimia haemolytica and Pasteurella multicoda isolates obtained from BRD clinical cases (deep lung swabs at post-mortem) among feedlots in four Australian states (2014–2019) was determined for 19 antimicrobial agents. The M. haemolytica isolates were pan-susceptible to all tested agents apart from a single macrolide-resistant isolate (1/88; 1.1%) from New South Wales (NSW). Much higher frequencies of P. multocida isolates were resistant to tetracycline (18/140; 12.9%), tilmicosin (19/140; 13.6%), tulathromycin/gamithromycin (17/140; 12.1%), and ampicillin/penicillin (6/140; 4.6%). Five P. multocida isolates (3.6%), all obtained from NSW in 2019, exhibited dual resistance to macrolides and tetracycline, and a further two Queensland isolates from 2019 (1.4%) exhibited a multidrug-resistant phenotype to ampicillin/penicillin, tetracycline, and tilmicosin. Random-amplified polymorphic DNA (RAPD) typing identified a high degree of genetic homogeneity among the M. haemolytica isolates, whereas P. multocida isolates were more heterogeneous. Illumina whole genome sequencing identified the genes msr(E) and mph(E)encoding macrolide resistance, tet(R)-tet(H) or tet(Y) encoding tetracycline resistance, and blaROB-1 encoding ampicillin/penicillin resistance in all isolates exhibiting a corresponding resistant phenotype. The exception was the tilmicosin-resistant, tulathromycin/gamithromycin-susceptible phenotype identified in two Queensland isolates, the genetic basis of which could not be determined. These results confirm the first emergence of AMR in M. haemolytica and P. multocida from BRD cases in Australia, which should be closely monitored.

Deciphering mobile genetic elements disseminating macrolide resistance in Streptococcus pyogenes over a 21 year period in Barcelona, Spain.

To phenotypically and genetically characterize the antibiotic resistance determinants and associated mobile genetic elements (MGEs) among macrolide-resistant (MR) Streptococcus pyogenes [Group A streptococci (GAS)] clinical isolates collected in Barcelona, Spain. Antibiotic susceptibility testing was performed by microdilution. Isolates were emm and MLST typed and 55 were whole-genome sequenced to determine the nature of the macrolide resistance (MR) determinants and their larger MGE and chromosomal context. Between 1998 and 2018, 142 of 1028 GAS (13.8%) were MR. Among 108 isolates available for molecular characterization, 41.7% had cMLSB, 30.5% iMLSB and 27.8% M phenotype. Eight erm(B)-containing strains were notable in having an MDR phenotype conferred by an MGE encoding several antibiotic resistance genes. MR isolates were comprised of several distinct genetic lineages as defined by the combination of emm and ST. Although most lineages were only transiently present, the emm11/ST403 clone persisted throughout the period. Two lineages, emm9/ST75 with erm(B) and emm77/ST63 with erm(TR), emerged in 2016-18. The erm(B) was predominantly encoded on the Tn916 family of transposons (21/31) with different genetic contexts, and in other MGEs (Tn6263, ICESpHKU372 and one harbouring an MDR cluster called ICESp1070HUB). The erm(TR) was found in ICESp2905 (8/17), ICESp1108-like (4/17), ICESpHKU165 (3/17) and two structures described in this study (IMESp316HUB and ICESp3729HUB). The M phenotype [mef(A)-msr(D)] was linked to phage φ1207.3. Eight integrative conjugative element/integrative mobilizable element (ICE/IME) cluster groups were classified on the basis of gene content within conjugation modules. These groups were found among MGEs, which corresponded with the MR-containing element or the site of integration. We detected several different MGEs harbouring erm(B) or erm(TR). This is the first known description of Tn6263 in GAS and three MGEs [IMESp316HUB, ICESp3729HUB and ICESp1070HUB] associated with MR. Periods of high MR rates in our area were mainly associated with the expansion of certain predominant lineages, while in low MR periods different sporadic and low prevalence lineages were more frequent.