Three critical regions of the erythromycin resistance methyltransferase, ErmE, are required for function supporting a model for the interaction of Erm family enzymes with substrate rRNA

6-methyladenosine modification of DNA and RNA is widespread throughout the three domains of life and often accomplished by a Rossmann-fold methyltransferase domain which contains conserved sequence elements directing S-adenosylmethionine cofactor binding and placement of the target adenosine residue into the active site. Elaborations to the conserved Rossman-fold and appended domains direct methylation to diverse DNA and RNA sequences and structures. Recently the first atomic resolution structure of a Ribosomal RNA Adenine Dimethylase (RRAD) family member bound to rRNA was solved, TFB1M bound to helix 45 of 12S rRNA. Since erythromycin resistance methyltransferases are also members of the RRAD family and understanding how these enzymes recognize rRNA could be used to combat their role in antibiotic resistance, we constructed a model of ErmE bound to a 23S rRNA fragment based on the TFB1M-rRNA structure. We designed site-directed mutants of ErmE based on this model and assayed the mutants by in vivo phenotypic assays and in vitro assays with purified protein. Our results and additional bioinformatic analyses suggest our structural model captures key ErmE-rRNA interactions and suggest three regions of Erm proteins play a critical role in methylation: the target adenosine binding pocket, the basic ridge and the α4-cleft.

184. Inducible Clindamycin Resistance Testing on Pediatric Streptococcus pneumoniae Isolates

Background In 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. Methods We 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. Results We 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). Conclusion Erythromycin-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)

Incidence of Tetracycline and Erythromycin Resistance in Meat-Associated Bacteria: Impact of Different Livestock Management Strategies

The extensive use of antibiotics as growth promoters, or their continued abusive misuse to cure or prevent the onset of bacterial infections as occurs in the intensive farming, may have played a pivotal role in the spread of reservoirs of antibiotic resistance (AR) among food-associated bacteria including pathogens representing risks to human health. The present study compares the incidence of tetracycline and erythromycin resistances in lactic acid bacteria (LAB) and coagulase negative staphylococci (CNS) from fermented products manufacturing using meat from intensive animal husbandry (industrialized manufacturing Italian salami) and from extensive farms (artisanal sausages facilities pork and llama Argentinean sausages). A higher incidence of tetracycline resistance (TET-R) compared to erythromycin resistance (ERY-R) was observed among the 205 isolates. Unlike CNS strains, the LAB showed a significant correlation between the TET-R and the ERY-R phenotypes. Genotypic assessment shows a high correlation with tetK and tetM for the TET-R strains and with ermB and ermC for the ERY-R strains. Multiple correspondence analyses have highlighted the association between AR phenotypes and CNS species isolated from Italian salami, while the susceptible phenotypes were associated with the LAB species from Argentinean sausages. Since antibiotic resistance in meat-associated bacteria is a very complex phenomenon, the assessment of bacterial resistance in different environmental contexts with diverse farming practices and food production technologies will help in monitoring the factors influencing AR emergence and spread in animal production.

Emergence of erythromycin resistance methyltransferases in Campylobacter coli strains in France

Antimicrobial resistance in Campylobacters is described worldwide. The emergence of multiresistant isolates, particularly among C. coli , is concerning. New resistance mechanisms appear frequently, and DNA-sequence-based methods such as whole genome sequencing (WGS) have become useful tools to monitor their emergence. The genomes of 51 multiresistant French Campylobacter sp. clinical strains from 2018 to 2019 were analyzed to identify associated resistance mechanisms. Analyses of erythromycin-resistant strains revealed 23S ribosomal RNA mutations among most of them and two different methyltransferases in 4 strains: Erm(B) and a novel methyltransferase, here named Erm(N). The erm(B) gene was found in multidrug-resistant genomic islands, whereas erm(N) was inserted within CRISPR arrays of the CRISPR- cas9 operon. Moreover, using PCR screening in erythromycin-resistant strains from our collection, we showed that erm(N) was already present in 3 French clinical strains 2 years before its first report in 2018 in Quebec. Bacterial transformations confirmed that insertion of erm(N) into a CRISPR- cas9 operon can confer macrolide resistance. Campylobacter species are easily able to adapt to their environment and acquire new resistance mechanisms, and the emergence of methyltransferases in Campylobacters in France is a matter of concern in the coming years.

Changing characteristics of S. aureus bacteremia caused by PVL-negative, MRSA strain over 11 years

Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) has emerged as an important cause of infection. We conducted a longitudinal study to evaluate changes in clinical and microbiological characteristics as well as outcomes of sequence type (ST) 72 MRSA bacteremia. We reviewed adult patients enrolled in a prospective cohort with ST72 MRSA bacteremia from August 2008 to December 2018 at Asan Medical Center, Seoul, South Korea. Changes in clinical characteristics, outcomes, and microbiological characteristics of patients over time were evaluated. Generalized linear and linear regression models were used to evaluate changes. Of the 1,760 isolates, 915 (62%) were MRSA bacteremia and 292 (31.9%) were ST72 MRSA. During the study period, the relative risk (RR) of MRSA bacteremia decreased annually by 3.7%; however, among MRSA bacteremia, RR of ST72 MRSA increased annually by 8.5%. Vancomycin minimum inhibitory concentration (MIC) decreased over the study period. Metastatic infection, persistent bacteremia, and recurrence of bacteremia within 12 weeks decreased significantly. There were no significant changes in 30-d and 12-week mortality. Antibiotic susceptibility of ST72 MRSA was evaluated, and the resistance rate to erythromycin decreased significantly. ST72 MRSA incidence increased annually; its vancomycin MIC and erythromycin resistance rate decreased over the 11 years.

Directed Recovery and Molecular Characterization of Antibiotic Resistance Plasmids from Cheese Bacteria

Resistance to antimicrobials is a growing problem of worldwide concern. Plasmids are thought to be major drivers of antibiotic resistance spread. The present work reports a simple way to recover replicative plasmids conferring antibiotic resistance from the bacteria in cheese. Purified plasmid DNA from colonies grown in the presence of tetracycline and erythromycin was introduced into plasmid-free strains of Lactococcus lactis, Lactiplantibacillus plantarum and Lacticaseibacillus casei. Following antibiotic selection, the plasmids from resistant transformants were isolated, analyzed by restriction enzyme digestion, and sequenced. Seven patterns were obtained for the tetracycline-resistant colonies, five from L. lactis, and one each from the lactobacilli strains, as well as a single digestion profile for the erythromycin-resistant transformants obtained in L. lactis. Sequence analysis respectively identified tet(S) and ermB in the tetracycline- and erythromycin-resistance plasmids from L. lactis. No dedicated resistance genes were detected in plasmids conferring tetracycline resistance to L. casei and L. plantarum. The present results highlight the usefulness of the proposed methodology for isolating functional plasmids that confer antibiotic resistance to LAB species, widen our knowledge of antibiotic resistance in the bacteria that inhabit cheese, and emphasize the leading role of plasmids in the spread of resistance genes via the food chain.

Phenotypic Detection of Methicillin and Inducible Clindamycin Resistant Staphylococcus Aureus: An Effort to Combat Antimicrobial Resistance and Improve Patient Safety in Gombe, Nigeria

Introduction: Antimicrobial resistance (AMR) is a major threat to patient safety. Methicillin and inducible clindamycin resistant Staphylococcus aureus are important multidrug resistant organisms (MDROs). Timely reporting of MDROs is necessary for rational antibiotic prescription and in combating AMR. We present the prevalence and distribution of Methicillin and inducible clindamycin resistant (iCR) isolates of Staphylococcus aureus from Gombe Nigeria. Materials and Methods: This descriptive cross-sectional study included 260 isolates of Staphylococcus aureus from clinical specimens in Federal Teaching Hospital Gombe (FTHG). Isolates identification was done using conventional biochemical methods. Methicillin resistance was detected by cefoxitin disc diffusion method while iCR isolates by erythromycin and clindamycin disc approximation test (D-test). Data analysis was done using SPSS version 23.0. Results: Methicillin resistant Staphylococcus aureus (MRSA) was detected in 178 (68.5%) isolates while 214 (82.3%) were iCR (D-test positive). Among the MRSA, 87.1% were also iCR while 72.4% of the iCR isolates were MRSA. There was significant association between MRSA and iCR (p = 0.03), MRSA and clindamycin resistance (p = 0.05) and MRSA and erythromycin resistance (p < 0.01). Conclusion: Prevalence of MDR Staphylococcus aureus is high in Gombe Nigeria. Antimicrobial stewardship programme (ASP) and good Infection Prevention and Control (IPC) are necessary in combating AMR and improving patient safety.