Genome sequencing defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission setting

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infection. Whole-genome sequencing of MRSA has been used to define phylogeny and transmission in well-resourced healthcare settings, yet the greatest burden of nosocomial infection occurs in resource-restricted settings where barriers to transmission are lower. Here, we study the flux and genetic diversity of MRSA on ward and individual patient levels in a hospital where transmission was common. We repeatedly screened all patients on two intensive care units for MRSA carriage over a 3-mo period. All MRSA belonged to multilocus sequence type 239 (ST 239). We defined the population structure and charted the spread of MRSA by sequencing 79 isolates from 46 patients and five members of staff, including the first MRSA-positive screen isolates and up to two repeat isolates where available. Phylogenetic analysis identified a flux of distinct ST 239 clades over time in each intensive care unit. In total, five main clades were identified, which varied in the carriage of plasmids encoding antiseptic and antimicrobial resistance determinants. Sequence data confirmed intra- and interwards transmission events and identified individual patients who were colonized by more than one clade. One patient on each unit was the source of numerous transmission events, and deep sampling of one of these cases demonstrated colonization with a "cloud" of related MRSA variants. The application of whole-genome sequencing and analysis provides novel insights into the transmission of MRSA in under-resourced healthcare settings and has relevance to wider global health.

Figure 1.

Existence of circulating clades of MRSA ST 239. Maximum likelihood phylogenetic tree based on core genome SNPs of the first and up to two repeat isolates from 46 patients and five healthcare workers. The first T number assigned to each represents the case study number. The second letter/number combination refers to the type of sample and the sample number from that particular site: (N) nasal swab; (T) throat swab; (A) axillae swab; (W) wound swab; (U) urine; (C) tracheal suction. For example, T178-N3 refers to the third nasal swab taken from case T178. Isolate labels are color-coded to signify isolation from a staff member (green), a patient on the adult ICU (red), or pediatric ICU (blue). Also shown (right-hand panel) is the presence (bold) or absence (pale) of four plasmids harboring antibiotic and antiseptic resistance genes. The colors used to denote clades 1 to 5 are cross-referenced in Figure 3.

Figure 2.

Histogram of pairwise SNP differences between 76 isolates from 46 patients and five healthcare workers. SNPs differences between isolates from the same clade are represented in red, and between isolates in different clades in blue.

Figure 3.

Dynamics of MRSA clades on the pediatric (left) and adult (right) ICUs. (Bottom) Each row provides details of a study patient (T number). Duration of admission to the hospital is shown in days, with a solid line for period(s) in the ICU ([gray] MRSA negative; [black] MRSA positive), and a dotted line for inpatient periods outside the study ICUs (MRSA status not tested). Superimposed on the lines are dots and circles ([white dot] MRSA negative; [colored circles] MRSA positive). An open colored circle signifies that the MRSA isolate was cultured from the first swab taken following ICU admission and was most likely acquired elsewhere in the hospital. A closed colored circle signifies that the MRSA isolate was cultured from a patient who had an initial negative MRSA screen in the ICU followed at a later time point by a positive one, which was interpreted as MRSA acquisition in the ICU. Colors were assigned based on the most recently sequenced isolate from the same patient and chosen to reflect sequence diversity. Each sequence was mapped to a unique color so that similar sequences have similar colors (see Methods). Representatives of the five clade colors used in Figure 1 are T35_N4 (clade 1, blue); T71_N3 (clade 2, purple); T106_N3 (clade 3, pink); T95_N1 (clade 4, green); and T126_N13 (clade 5, khaki). Isolates that were sequenced are underlined in red. (Top) The daily cumulative count of MRSA positive patients over the 3-mo study period. The colors used match the colored dots for MRSA isolates.

Figure 4.

Linear regression plot of root-to-tip distances of the phylogenetic tree for MRSA isolates from patient T126 against the day of sampling. There were 99 isolates from patient T126 and seven isolates concurrently isolated from other patients on the adult ICU that belong to the same clade. The coefficient of determination was R² = 0.22. Patient T126 was admitted to the ICU on day 29 of the study.