C-672b First report of Group B Streptococcus Isolates with Cross-resistance to Lincosamides, Streptogramin A, and Pleuromutilins in the United States P. A. Hawkins1,2, C. S. Law2, D. M. Jackson2, B. J. Metcalf2, L. F. Westblade3, R. C. Jerris4, B. W. Beall2, L. McGee2 1. Rollins School of Public Health, Emory University, Atlanta, GA. 2. Centers for Disease Control and Prevention, Atlanta, GA. 3. Emory University School of Medicine, Atlanta, GA. 4. Children’s Healthcare of Atlanta, Atlanta, GA. Revised Abstract Background: Group B Streptococcus (GBS) is the leading cause of sepsis and meningitis in neonates, and less commonly of pneumonia and soft-tissue infections in older adults with comorbidities. While resistance to penicillin remains low among GBS isolates, the proportion of isolates resistant to erythromycin and clindamycin has increased in the last few years. Resistance to clindamycin usually occurs in association with erythromycin resistance as a result of the modification of ribosomal targets, and is most commonly mediated by an erm methylase. Resistance to clindamycin in the absence of erythromycin resistance is rare, but has been previously reported in clinical GBS isolates from Spain, New Zealand, Argentina, Canada, and Korea, and is due to the presence of either one of two genes: lsaC or lnuB. Methods: In this study, we screened the minimum inhibitory concentration (MIC) results for 49,310 GBS isolates in the Active Bacterial Core Surveillance (ABCs) system (collected between 1998-2014) at CDC, to identify isolates resistant to clindamycin, but susceptible to erythromycin. We found forty-four isolates (0.09%) that expressed this phenotype. The whole genome sequences for these isolates were obtained and mapped against known macrolide, lincosamide, and streptogramin (MLS) resistance genes: ermA, ermB, cfr, lnuA, lnuB, lnuC, lnuD, lnuE, vgaALC, and lsaC using SRST2. Since cross-resistance to streptogramin A and pleuromutilins has been previously reported in association with lincosamide resistance, we also determined the MIC of these isolates to Virginiamycin M1, a streptogramin A, and Tiamulin, a pleuromutilin. Results and conclusions: Of the 44 clindamycin-resistant isolates, 39 were non-susceptible to both virginiamycin M1 and tiamulin (LSAP phenotype), one was susceptible to virginiamycin M1, but non-susceptible to tiamulin (LP phenotype) and 3 were susceptible to both antibiotics (L phenotype). Thirty-two isolates encoded the lsaC gene and seven carried the lnuB gene; the MIC to tiamulin was much higher among isolates harboring the lnuB gene. The five isolates expressing the L and LP phenotypes did not carry any of the genes we screened for and thus the mechanism involved remains undetermined. To the best of our knowledge, this is the first report of this phenotype and of GBS isolates carrying these genes in the United States. Methods We screened MIC results from 49,310 GBS isolates in the Active Bacterial Core Surveillance (ABCs) system (collected between 1998-2014) at CDC, in order to identify isolates resistant to clindamycin, but susceptible to erythromycin. We found forty-four isolates (0.09%) that expressed this phenotype. The whole genome sequences for these isolates were obtained and mapped against known MLS resistance genes: ermA, ermB, cfr, lnuA, lnuB, lnuC, lnuD, lnuE, vgaALC, and lsaC, using SRST2, a read mapping-based tool for fast and accurate detection of genes from whole genome sequencing data7. Since cross-resistance to streptogramin A and pleuromutilins has been previously reported in association with lincosamide resistance, we also determined the MIC of these isolates to virginiamycin M1, a streptogramin A, and tiamulin, a pleuromutilin. Wild-type cutoff values were determined by constructing MIC distributions of 45 control GBS isolates plus the 44 test isolates as described by Turnidge and Paterson8, since breakpoints for virginiamycin M1 and tiamulin have not been established. The 45 control isolates are non-clonal and either susceptible to both erythromycin and clindamycin (15 isolates), resistant to erythromycin but susceptible to clindamycin (15 isolates), or resistant to both erythromycin and clindamycin (15 isolates). Table 1. Year and State of isolate collection. OR: Oregon, GA: Georgia, MN: Minnesota, MD: Maryland, CA: California. Table 2. Phenotypes observed and corresponding MIC values. L: lincosamide resistant, LP: lincosamide and pleuromutilin resistant, LSAP: lincosamide, streptogramin A, and pleuromutilin resistant. CLD: clindamycin, WM1: virginiamycin M1, TIA: tiamulin, Year n OR GA MN MD CA 1998-2009 15 2 4 1 2010 3 2011 6 2012 2013 2014 14 7 Total 44 11 13 Results and Discussion Table 1 shows the number of isolates separated out by year and state. Fourteen of the isolates were collected in 2014, more than in any other year. There does not seem to be a pattern in terms of the origin of the isolates. Table 2 shows the phenotypes observed and corresponding MIC values. Figure 1 shows the MIC distribution of virginiamycin M1 against the control and test isolates. According to these results, we classified isolates with an MIC value ≥16 µg/mL as non-susceptible to virginiamycin M1 and therefore streptogramin A. Figure 2 shows the MIC distribution of tiamulin against the control and test isolates. Using these data, we classified isolates with an MIC value ≥1 µg/mL as non-susceptible to tiamulin and thus pleuromutilins. These results match what has been previously reported for these antibiotics against GBS2. Based on these cutoff values, of the 44 clindamycin-resistant isolates identified and tested, 39 were non-susceptible to both virginiamycin M1 and tiamulin (LSAP phenotype), 4 were susceptible to both antibiotics (L phenotype), and 1 was non-susceptible to tiamulin, yet susceptible to virginiamycin M1 (LP phenotype). Of the 39 LSAP isolates, 32 harbored the lsaC gene and 7 carried the lnuB gene. It is of interest to note that the lnuB gene confers much higher resistance to tiamulin than the lsaC gene (3 to 6 fold higher MIC value). The 5 L and LP isolates did not harbor any of the genes we screened for and thus the mechanism of resistance remains undetermined. MIC range (mg/mL) Phenotype n CLD VM1 TIA L 4 1 - 32 1 - 4 0.25 - 0.5 LP 1 0.5 LSAP lsaC 32 16 1 -4 lnuB 7 16 - 32 64+ Conclusions To the best of our knowledge, this is the first report of the L, LP, and LSAP phenotypes in GBS isolates from the United States. The LSAP phenotype was explained by the presence of the lsaC and lnuB genes among our isolates, while the other two phenotypes (L and LP) remain unexplained and are to be the subject of ongoing studies. The Active Bacterial Core surveillance (ABCs) system offers unique opportunities to identify atypical resistance patterns and, with the advent of more accessible whole genome sequencing technologies, the elucidation of the mechanisms responsible behind these phenotypes. It is important to identify and monitor emerging antimicrobial resistance phenotypes, because these may determine clinical decisions, such as the choice of antibiotics used in patients allergic to penicillin. References 1. Malbruny et al. 2004. J. Antimicrob. Chemother. 54:1040. 2. Malbruny et al. 2011. Antimicrob. Agents Chemother. 55:1470 3. Arana et al. 2014. Rev Esp Quimioter. 27: 106. 4. Faccone et al. 2010. J Infect Dev Ctries. 4:580. 5. Azavedo et al. 2001. Antimicrob. Agents Chemother. 45:3504. 6. Seo et al. 2010. J Korean Med Sci. 25: 817. 7. Inouye et al. 2014. Genome Med. 6:90 8. Turnidge and Paterson. 2007. Clin. Microbiol. Rev. 20:391 Contact Information Lars F. Westblade Emory University School of Medicine lars.westblade@emory.edu Paulina Hawkins Rollins School of Public Health, Emory University pahues@emory.edu Lesley McGee Centers for Disease Control and Prevention lmcgee@cdc.gov Background Streptococcus agalactiae, group B streptococcus (GBS), is part of the commensal flora of the genital and digestive tracts. This bacterial species is also a common cause of severe infections in neonates and can cause various infections in adults, including bacteremia and endocarditis1. While resistance to penicillin remains low among GBS isolates, the proportion of isolates resistant to erythromycin and clindamycin has increased in the last few years. Resistance to clindamycin usually occurs in association with erythromycin resistance as a result of the modification of ribosomal targets, and is most commonly mediated by an erm methylase. Resistance to clindamycin in the absence of erythromycin resistance is rare, but has been previously reported in clinical GBS isolates from New Zealand1,2, Spain3, Argentina4, Canada5, and Korea6, and is due to the presence of either one of two genes: lsaC or lnuB. The lsaC gene was first identified in New Zealand and is responsible for the LSAP phenotype in GBS2. Inactivation of lincosamides mediated by the lnu genes was firstly described in Enterococcus faecium HM1025 strain6. Figure 1. Virginiamycin M1 MIC (mg/mL) values against 45 controls (magenta bars) and 44 test isolates (purple bars). Isolates with MIC values of 16 and above were classified as non-susceptible. Figure 2. Tiamulin MIC (mg/mL) values against 45 controls (magenta bars) and 44 test isolates (purple bars). Isolates with MIC values of 1 and above were classified as non-susceptible. Division of Bacterial Diseases, Respiratory Diseases Branch