Clonally Diverse Methicillin and Multidrug Resistant Coagulase Negative Staphylococci Are Ubiquitous and Pose Transfer Ability Between Pets and Their Owners

Abstract

Sixty-eight owners and 66 pets, from 43 unrelated pet-owning households were screened for methicillin-resistant coagulase negative staphylococci (MRCoNS), potential cases of MRCoNS interspecies transmission (IT), and persistence. MRCoNS isolates were identified by microbiological and molecular tests. MLST-based phylogenetic analysis was performed in Staphylococcus epidermidis isolates. Antimicrobial susceptibility was evaluated using phenotypic and molecular methods. SCCmec type and the presence of biofilm-related ica locus was PCR-tested. Isolates suspected for MRCoNS IT cases were subjected to SmaI-PFGE analysis and individuals from positive households were followed-up for 1 year for carriage dynamics (every 3 months, T0-T4). Nineteen MRCoNS isolates from owners (27.9%) and 12 from pets (16.7%) were detected, coming from 20 households (46.5%). S. epidermidis was predominant (90 and 67% of human and animal strains, respectively), showing high phylogenetic diversity (16 STs among 24 strains). Methicillin-resistant S. epidermidis (MRSE) strains belonged to CC5 (75%), CC11 (12.5%), singleton S556 (8.3%), and S560 (4.17%). Significant host-associated differences were observed for resistance to aminoglycosides, co-trimoxazole, chloramphenicol (higher in animal isolates) and tetracycline (higher among human strains). Multidrug resistance (MDR) was common (68.4%) and associated with human strains. Great diversity of ccr and mec complexes were detected, most strains being non-typeable, followed by SCCmecIV and V. Over one third of isolates (most from owners), carried the ica locus, all MRSE CC5. Two sporadic IT cases (T0) were identified in owners and dogs from two households (4.7%), with diverse interspecies-exchanged clones detected along the sampling year, especially in dogs. A comparative analysis of all MRCoNS, with all nasal coagulase positive staphylococci (CoPS) recovered from the same individuals at T0, revealed that CoPS alone was predominant in owners and pets, followed by co-carriage of CoPS and MRCoNS in owners but single MRCoNS in pets. Statistical analyses revealed that owners are more prone to co-carriage and that co-existence of IT cases and co-carriage are positively interrelated. MRCoNS from healthy owners and their pets are genetically heterogeneous MDR strains that are spread in the community. Therefore, pets also contribute to the dissemination of successful human clones. Owner-pet inhabitancy increases the risk for staphylococcal temporal concomitance with its subsequent risk for bacterial infection and genetic exchange.

Keywords: Staphylococcus epidermidis; carriage dynamics; co-carriage; interspecies transmission; methicillin-resistant coagulase negative staphylococci; multidrug resistance; owner; pet.

FIGURE 1

Distribution (%) of MRCoNS species recovered from the 19 positive owners (19 strains), and the 11 positive pets (12 strains). One pet (1-D1) carried one S. lentus and one S. epidermidis strain.

FIGURE 2

(A) Clustering analysis of the S. epidermidis STs detected in this study by goeBURST algorithm using Phyloviz 2 software (Nascimento et al., 2017). The most restricted level [level 1 – Single Locus Variant (SLV)] was used, requiring six of seven alleles shared to the linked ST. Cyan STs indicate probable ancestors (group founders) and green STs constitute subgroup founders. Blue STs correspond to STs that share the same background (CC). Circles in red indicate the STs detected in this study. Specific location of ST5 (CC5 ST primary founder) and ST2 (major subgroup founder of the cluster) within CC5 are indicated. (B) Distance tree of the 16 concatenate ST sequences detected among the 24 S. epidermidis isolates constructed using CLC Genomics Workbench 10.0.1 (https://www.qiagenbioinformatics.com/). Sequences were aligned using internal parameters, and the tree was built with a Neighbor Joining method using Jukes-Cantor as Nucleotide Distance measure, with a bootstrap analysis of 500 replicates. The bar length indicates the number of substitutions per site. STs in black color are those with new ST, either by the presence of a new allele or new allele combination.

FIGURE 3

Percentage of resistance to non β-lactams and antimicrobial resistance genes detected among the 31 MRCoNS isolates investigated in T0. FUS, fusidic acid; CHL, chloramphenicol; TET, tetracycline; CIP, ciprofloxacin; SXT, co-trimoxazole; GEN/TOB/KAN/STR, gentamicin/tobramycin/kanamycin/streptomycin; MUP, mupirocin; ERY/CLI, erythromycin/clindamycin. All isolates were susceptible to vancomycin and linezolid. Individual P-value (Fisher’s Exact Test for count data) to account for significant difference at 95% confidence interval is indicated at the bottom of the histogram. Asterisks (blue or red) above the bars represent those agents for which statistical differences were detected, with the asterisk color remaking the host (owner or pet, respectively) of the bacteria involved in the significance.

FIGURE 4

(A) Schematic representation of the methicillin resistant coagulase negative staphylococcal carriage dynamics of both households investigated along 1 year. IT, bacterial species responsible for interspecies transmission. T0–T4 indicate the different sampling times along the sampling year. Individuals are named H (for human) or D (dog) followed by the case number (1 or 2) and a lower-case letter to differentiate subjects per household. (B) Pulsed-field Gel Electrophoresis (PFGE) profile of genomic DNA digested with SmaI restriction enzyme of isolates recovered from individuals involved in both cases of possible direct interspecies transmission. Upper lane in PFGE per case corresponds to MidRange PFGE Marker (New England Biolabs). Antimicrobial resistance (AMR) genes detected in each strain are also indicated.

FIGURE 5

(A) Left panel, bar chart showing the percentage of owners and pets that carried Coagulase Positive Staphylococci (CoPS), i.e., S. aureus (SA) and/or S. pseudintermedius (SP); MRCoNS; or CoPS + MRCoNS in sampling T0 (Gomez-Sanz et al., 2013b). Right panel, graphical view of the distribution of CoPS and/or MRCoNS detected among the individuals positive for such bacterial species. (B) Left panel, bar chart displaying the percentage of households with individuals positive for CoPS (SA, SP), MRCoNS or CoPS + MRCoNS in sampling T0. Right panel, graphical representation of the distribution of CoPS and/or MRCoNS detected among the households with individuals positive for such bacterial species. Colored stars indicate values with significant differences between human and animal strains.