Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak

Abstract

Background: Isolates of methicillin-resistant Staphylococcus aureus (MRSA) belonging to a single lineage are often indistinguishable by means of current typing techniques. Whole-genome sequencing may provide improved resolution to define transmission pathways and characterize outbreaks.

Methods: We investigated a putative MRSA outbreak in a neonatal intensive care unit. By using rapid high-throughput sequencing technology with a clinically relevant turnaround time, we retrospectively sequenced the DNA from seven isolates associated with the outbreak and another seven MRSA isolates associated with carriage of MRSA or bacteremia in the same hospital.

Results: We constructed a phylogenetic tree by comparing single-nucleotide polymorphisms (SNPs) in the core genome to a reference genome (an epidemic MRSA clone, EMRSA-15 [sequence type 22]). This revealed a distinct cluster of outbreak isolates and clear separation between these and the nonoutbreak isolates. A previously missed transmission event was detected between two patients with bacteremia who were not part of the outbreak. We created an artificial "resistome" of antibiotic-resistance genes and demonstrated concordance between it and the results of phenotypic susceptibility testing; we also created a "toxome" consisting of toxin genes. One outbreak isolate had a hypermutator phenotype with a higher number of SNPs than the other outbreak isolates, highlighting the difficulty of imposing a simple threshold for the number of SNPs between isolates to decide whether they are part of a recent transmission chain.

Conclusions: Whole-genome sequencing can provide clinically relevant data within a time frame that can influence patient care. The need for automated data interpretation and the provision of clinically meaningful reports represent hurdles to clinical implementation. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.).

Figure 1. Timeline of the Neonatal Intensive Care Unit (NICU) Outbreak and Methicillin-Resistant Staphylococcus aureus (MRSA) Bacteremia Cases on Other Wards

The index patient (Patient 1) and 11 other infants (Patients 2 through 12) were involved in a MRSA outbreak that was characterized by a distinct antibiogram (resistance to cefoxitin, erythromycin, ciprofloxacin, gentamicin, and mupirocin). Patient 13 was colonized with an isolate with a different antibiogram (resistance to only cefoxitin, erythromycin, and ciprofloxacin), and Patients 14 and 15 had been in the NICU before the outbreak; none of these three were considered to be part of the outbreak. Five other patients (Patients 16 through 20) with bacteremia that developed during the time of the outbreak are shown, in addition to key events throughout this period. Cases of MRSA bacteremia (B) and MRSA carriage (C) are indicated along with the number of the affected patient (e.g., Patient 14’s MRSA carriage is indicated as “14C”).

Figure 2. Phenotypic Antibiograms, Resistome, and Toxome

Panel A shows the antibiogram, or antimicrobial susceptibility pattern, for each of the 14 isolates sequenced, and Panel B shows the resistome and toxome. The samples are identified with the patient number followed by a B for bacteremia or a C for carriage of MRSA, along with the sequence type (ST). For each antimicrobial drug, resistance is indicated with an R; its absence indicates susceptibility. The staphylococcal cassette chromosome (SCC) mec types are also shown. In Panel B, at the top are genes responsible for drug resistance (the resistome) and toxin genes (the toxome). The toxin genes considered were limited to those that are tested for in the United Kingdom (staphylococcal enterotoxins [sea and sej], exfoliative toxins [eta and etd], toxic shock syndrome toxin [tst], and Panton–Valentine leukocidin [PVL] [lukS-PV and lukF-PV]). Only those genes present in at least 1 isolate are shown. Underneath these is a “heat map” showing the presence (red) or absence (blue) of the relevant genes for each of the samples (sequences used in the resistome and toxome are listed in Table S2 in the Supplementary Appendix). Antimicrobial resistance in MRSA can arise by way of either gene acquisition or chromosomal mutations. Gene acquisition accounted for resistance to 10 antimicrobial drugs in the 14 isolates studied. Consistent with a prior study in our hospital of sequence type 1 community-associated MRSA, both transmission isolates 16B and 17B were PVL-negative and carried sea and seh. Not shown are the chromosomal mutations detected that accounted for resistance to rifampin (RIF) or ciprofloxacin (CIP): mutations in rpoB (Leu466Ser and His481Asn) accounted for RIF resistance in isolate 14C and two mutations (Ser80Phe mutation in grlA and Ser84Leu in gyrA) were responsible for CIP resistance in all ST 22 and ST 36 isolates, and no mutations responsible for linezolid (LIN) resistance were found. CLIN denotes clindamycin, CXT cefoxitin, ERY erythromycin, FUS fusidic acid, GEN gentamicin, KAN kanamycin, MUP mupirocin, TET tetracycline, TMP trimethoprim, and TOB tobramycin.

Figure 3. Results of Phylogenetic Analysis of the 10 MRSA Isolates of Sequence Type 22

An unrooted maximum likelihood tree of the 10 sequence type 22 MRSA isolates identified, together with a reference sequence type 22 isolate (HO 5096 0412), is shown. The isolates are identified with the patient number followed by a B for bacteremia or a C for carriage of MRSA. Bootstrap values are shown in red. Outbreak isolates are circled in blue. Six of the seven outbreak isolates clustered closely together. The seventh isolate (6C) had an extended branch length that could be explained by the fact that it had a hypermutator phenotype. The remaining sequence type 22 isolates associated with carriage by an infant in the NICU (15C) or bacteremia in patients on other wards of the same hospital (19B, 20B) were distantly related to the outbreak isolates and to each other. SNP denotes single-nucleotide polymorphism.