Lincosamides

Of the 801 combined year 2009, 2012 and 2013 IPD isolates described in this study, serotypes, antimicrobial susceptibilities, MLSTs and pilus types for 699 (87.3%) were determined by the use of our pneumococcal typing pipeline with Illumina WGS fastq files provided by the Sanger group (n = 516 from year 2009 and 2012 IPD isolates) and the CDC Biotechnology Core Facility (n = 183 from year 2013 IPD isolates). The WGS-derived data from these 699 isolates recovered during 2009, 2012 and 2013 were used to supplement the already accumulated conventional serotyping, antimicrobial susceptibility testing, MLST, pilus locus PCR and PCR/ESI-MS data (described below). WGS-based identification of serotypes, pilus loci and non-β-lactam antimicrobial resistance features employed query DNA sequences described in Table S1 (serotypes and pili) and Table S2 (non-β-lactam antimicrobial resistance). Transpeptidase domain amino acid sequences of 277–359 residues from penicillin-binding proteins (PBPs) 1a, 2b, and 2x were extracted from approximately 1600 ABCs strains characterized over the years 1998–2013. From this, databases of 69, 77 and 127 unique transpeptidase domain amino acid sequences were compiled for PBP1a, PBP2b, and PBP2x, respectively (Tables S3–S5). Each unique sequence was assigned an identifier (sequences 1a-0 to 1a-68 for PBP1a, sequences 2b-0 to 2b-78 for PBP2b, and sequences 2x-0 to 126 for PBP2x). The three-number combination from each isolate was correlated with MICs for each of the six different β-lactam antibiotics. For example, the basally β-lactam-sensitive TIGR4 strain (genbank accession AE005672) contains a composite amino acid sequence pattern of 1a-0, 2b-0, and 2x-0 (abbreviated as 0:0:0). The corresponding DNA sequences (e.g. 1a-0, 2b-0, and 2x-0) are italicized. Full-length PBP genes are referred to with standard nomenclature (pbp1a, pbp2b, and pbp2x).
Detection of non-core genome-conferred resistance loci was performed according to homology with known determinants. Core genome-encoded resistance determinants (fluoroquinolones, co-trimoxazole, ribosomal protein mutation-conferred macrolide/lincosamide/streptogramin resistance, and rifampin) were screened by identifying specific amino acid substitutions with previously described targets (Table S2). Table S2 describes the sequence coordinates used for resistance queries. The bioinformatics methods used are described in Doc. S1.

B. subtilis strains derived from CU1065 (WT) were grown on lysogeny broth (LB) medium (10 g l⁻¹ casein digest peptone, 5 g l⁻¹ yeast extract, 5 g l⁻¹ NaCl) at 37°C with vigorous shaking. The strains used in this study are listed in Table S1, available in the online version of this article. Ferrous iron (FeSO₄·7H₂O) was titrated into the media as indicated from a freshly made 100 mM iron stock solution dissolved in 1 N HCl unless otherwise indicated. SPβ phage are derivatives of SPβc2Δ2 and were constructed by integration of a promoter region–cat–lacZ operon fusion constructed in pJPM122 into strain ZB307A as described previously [38 (link)]. Ampicillin (amp; 100 μg ml⁻¹) was used to select E. coli transformants. Erythromycin (ery; 1 μg ml⁻¹) and lincomycin [linc; 25 μg ml⁻¹; for testing macrolide–lincosamide–streptogramin B (MLS) resistance], spectinomycin (spec; 100 μg ml⁻¹), kanamycin (kan; 10 μg ml⁻¹) and neomycin (neo; 10 μg ml⁻¹) were used for the selection of various B. subtilis strains.

The antimicrobial resistance profiles were provided by Phoenix BD automated system (Becton Dickinson Franklin Lakes, NJ, EUA); according to manufacturing protocols, each panel was standardized for Gram-positive and Gram-negative AST profiles comprehending the list below:
Aminoglycoside: Amikacin (AMK), Gentamicin (GEN), Synergism Gentamicin (SGEN), Synergism Streptomycin (SSTP), Tobramycin (TOB); Cephalosporins: Cefepime (FEP), Cefoxitin (FOX), Ceftaroline (CPT), Ceftazidime (CAZ), Ceftazidime + Avibactam (CZA), Ceftriaxone (CRO), Cefuroxime (CXM), Cefazolin (CZ); Quinolones: Ciprofloxacin (CIP), Norfloxacin (NX), Levofloxacin (LVX); Penicillin: Amoxicillin/Clavulanic acid (AMC), Ampicillin (AMP), Ampicillin/Sulbactam (SAM), Oxacillin (OXA), Penicillin (PEN), Piperacillin/Tazobactam (TZP); Carbapenems: Ertapenem (ETP), Imipenem (IPM), Meropenem (MEM); Glycopeptides: Teicoplanin (TEC), Vancomycin (VAN): Macrolide: Erythromycin (ERY), Rifampicin (RIP): Lincosamides: Clindamycin (CLI); Oxazolidinone: Linezolid (LZD); Tetracycline: Tetracycline (TET), Minocycline (MIN); Sulfonamides: Sulfamethoxazole/Trimethoprim (STX); Nitroimidazoles: Nitrofurantoin (NIT); Amphenicol: Chloramphenicol (C); Phosphonate: Fosfomycin (FOS); Glycylcyclines: Tigecycline (TGC); Polypeptide: Colistin (CL); Lipopeptides: Daptomycin (DAP).
The resistance profile was classified as resistant (R), and susceptible (S). Any isolate with resistance to three or more classes of antimicrobial agents was classified as multidrug-resistant (MDR) according to the definition proposed by Magiorakos et al. (2012) (link). Some of the clinical isolates were retrieved at the moment of hospitalization for epidemiological active surveillance and infection control. A total of 256 isolates were included in the study and 196 had the antimicrobial susceptibility test performed (Table 1).
Data for new COVID-19 cases for each month were obtained from the Brazilian Ministry of Health (MS) and the State Health Department of Rio de Janeiro, compiled by Cota (2020) .
The prevalence of bacteria species in pediatric, neonatal-ICU, and gynecology/obstetrics wards during the pandemic period was evaluated. In order to compare these three wards with other hospital wards, a total of 2,551 bacteria isolates were recovered from the HICC-HUAP.

The minimal inhibitory concentrations (MIC) for penicillin (β-lactams), cephalexin (β-lactams), ampicillin (β-lactams), ceftiofur (β-lactams), cefquinome (β-lactams), lincomycin (lincosamide class), oxytetracycline (Tetracycline class), marbofloxacin (Quinolone class), rifaximin (Rifamycin class), and vancomycin (Glycopeptides class) (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) were determined against 49 L. garvieae isolates using micro-broth dilution assays, following the Clinical Laboratory and Standards Institute guidelines [42 ]. All antimicrobial agents were used in concentrations ranging from 0.03 to 16μg/mL. S. aureus ATCC 29,213 was used as a quality control strain. Antimicrobial resistance was defined by combining intermediate and resistant categories into a single category. MDR was defined as resistance to ≥3 classes of antimicrobial agents [2 (link)]. Each experiment was performed in triplicate.