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Review
, 31 (4)

Methicillin-Resistant Staphylococcus Aureus: Molecular Characterization, Evolution, and Epidemiology

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Review

Methicillin-Resistant Staphylococcus Aureus: Molecular Characterization, Evolution, and Epidemiology

Sahreena Lakhundi et al. Clin Microbiol Rev.

Abstract

Staphylococcus aureus, a major human pathogen, has a collection of virulence factors and the ability to acquire resistance to most antibiotics. This ability is further augmented by constant emergence of new clones, making S. aureus a "superbug." Clinical use of methicillin has led to the appearance of methicillin-resistant S. aureus (MRSA). The past few decades have witnessed the existence of new MRSA clones. Unlike traditional MRSA residing in hospitals, the new clones can invade community settings and infect people without predisposing risk factors. This evolution continues with the buildup of the MRSA reservoir in companion and food animals. This review focuses on imparting a better understanding of MRSA evolution and its molecular characterization and epidemiology. We first describe the origin of MRSA, with emphasis on the diverse nature of staphylococcal cassette chromosome mec (SCCmec). mecA and its new homologues (mecB, mecC, and mecD), SCCmec types (13 SCCmec types have been discovered to date), and their classification criteria are discussed. The review then describes various typing methods applied to study the molecular epidemiology and evolutionary nature of MRSA. Starting with the historical methods and continuing to the advanced whole-genome approaches, typing of collections of MRSA has shed light on the origin, spread, and evolutionary pathways of MRSA clones.

Keywords: MRSA evolution; MRSA origin; MRSA typing; SCCmec; Staphylococcus aureus; methicillin-resistant S. aureus (MRSA); molecular characterization; molecular epidemiology.

Figures

FIG 1
FIG 1
mec gene homologue classification and phylogeny. (A) Criteria. mec gene homologues are classified based on nucleotide sequence similarity to the prototype mecA gene identified in MRSA N315. Strains showing <70% similarity are classified as new mec gene types, with 4 (mecA, mecB, mecC, and mecD) currently described. The types are further subdivided into allotypes based on ≥70% to <95% sequence homology, while genes sharing ≥95% homology belong to the same allotype (144). Prototypic (asterisks) and representative strains carrying each mec gene allotype are indicated. (B) Phylogenetic tree showing the relationships between mec gene homologues identified to date. The mecA, mecB, mecC, and mecD genes cluster separately and distinct from each other, and within each group the different allotypes can be distinguished. Strain names and accession numbers for organisms in which each gene is present are indicated in parentheses. Species are indicated if they are other than S. aureus.
FIG 2
FIG 2
mec gene complex variants. Five classes (A to E) of the mec gene complex have been reported. The class A complex shows a typical IS431-mecA-mecR1-mecI structure. Class B shows the typical IS431-mecA-truncated mecR1-IS1272 structure. Class C is defined by its IS431-mecA-truncated mecR1-IS431 structure, with class C1 having the two IS431 elements coding in the same direction and C2 having them coding in opposite directions. The class D complex contains IS431-mecA-truncated mecR1 but no downstream IS element. The class E mec gene complex is composed of blaZ-mecC-mecR1-mecI. Variations within a mec gene complex type are seen as a result of differences in the lengths of the regulatory genes, as well as the presence or absence of insertion sequences (IS) and transposons. IS431 is represented by purple, mecI by black, mecA or mecC by dark blue, blaZ by pink, direct-repeat units (DRU) by orange, mecR1 by green, IS1272 by light blue, and IS1182 by yellow. Genes in the hypervariable region (HVR) are represented by white boxes. The included complexes and variants are numbered based on published data rather than being sequential representatives.
FIG 3
FIG 3
ccr gene homologue classification. (A) Novel ccr genes are defined based on DNA sequence similarity of ≤50%, and to date 3 types have been described (ccrA, ccrB, and ccrC). The types are further subdivided into allotypes based on a nucleotide sequence similarity between 50% and 85%, while sequences with >85% similarity would be classified as the same allotype (166). ccrA and ccrB each have 7 allotypes (1 to 7), with numerous reported, while ccrC has 2 allotypes reported (C1 and C2). The ccrC1 allotype is further subdivided into 10 alleles (1 to 10). Prototypic strains for each allotype, along with their corresponding accession numbers, are indicated. An asterisk indicates the prototypic strain for the ccrC1 allotype. (B) ccr gene homologues identified to date and their phylogenetic relationships to each other. Strain names and accession numbers for organisms in which each gene is present are indicated in parentheses. Species are indicated if they are other than S. aureus.
FIG 4
FIG 4
Diagrammatic illustration of the reported SCCmec types. To date, 13 SCCmec types (I to XIII) have been identified. Scale representations of the 13 SCCmec types are shown, with the mec gene complex indicated by green shading, the ccr gene complex indicated by purple shading, and the J1, J2, and J3 regions surrounding. ORFs within the SCCmec elements are represented by red boxes, while chromosomal ORFs are represented by yellow boxes. Important elements in the J regions, such as transposons and plasmids, are also indicated. Different combinations of the mec gene complex and ccr gene complexes, and occasionally different placements with respect to each other, give rise to the various SCCmec types.
FIG 5
FIG 5
Global MRSA population snapshot. (A) MRSA population structure, showing the major clones reported in each continent or region along with the commonly associated SCCmec types. While there is overlap in terms of STs between the continents, there are many STs that show marked region specificity. Red represents STs belonging to HA-MRSA, blue represents those belonging to CA-MRSA, and purple represents belonging to LA-MRSA. Alternate or traditional names for the mainly predominant epidemic strains are at the bottom. (B) Evolutionary relationships between the predominant MRSA STs listed in panel A are represented by eBURST analysis (compared with the international MLST database, updated 31 January 2018). Individual STs, as well as the clonal complexes to which they belong, are indicated. As in panel A, red (HA-MRSA), blue (CA-MRSA), and purple (LA-MRSA) are used to distinguish the types. Peach color is used to denote STs present in more than one source group.
FIG 6
FIG 6
Genetic diversity among PVL-positive Staphylococcus aureus clinical isolates (589). (A) Molecular characterization of representative PVL-positive strains, isolated over 16 years (1989 to 2004) in a large Canadian health care region, via pulsed-field gel electrophoresis (PFGE), SCCmec typing, MLST, spa typing, and accessory gene regulator (agr) typing, as well as antimicrobial susceptibility profiles of the strains. (B) Determination of clonal complexes via eBURST analysis comparing our MLST data (16 STs) with the international MLST database (updated 9 December 2008). (Adapted from reference with permission of the publisher.)
FIG 7
FIG 7
PVL-positive (PVL+) and PVL-negative (PVL−) USA400 sibling strains show no significant differences in invasion and survival abilities in human laryngeal carcinoma (HEp-2) cells (599). Sixteen-hour-old Hep-2 cells were infected with 108 CFU of S. aureus. After an hour of incubation, extracellular bacteria were removed, and intracellular bacteria were enumerated at 1 h and 4 h by serial dilution and plating. No significant differences in invasion or survival ability was noted between PVL(+) and PVL(−) isolates. (Adapted from reference with permission of the publisher.)
FIG 8
FIG 8
PVL(+) and PVL(−) USA400 sibling strains show no significant differences in cytotoxicity toward human lung epithelial (A549) cells (599). Sixteen-hour-old A549 cells were infected with 108 CFU of S. aureus, and bacteria were removed after an hour of incubation. (A) The cells were fixed and stained, using methanol and Giemsa stain, at 1, 3, and 7 h. (B) Bacterial cytotoxicity on A549 cells was determined by reading the optical density (OD) of Giemsa stain. The results are expressed as the relative percent optical density against a blank control with no bacterial infection. No significant difference in the cytotoxicity was noted between PVL(+) and PVL(−) strains. (Adapted from reference with permission of the publisher.)
FIG 9
FIG 9
PVL(+) and PVL(−) sibling strains and USA400(MW2) and USA400(MW2 PVL-knockout) control strains are both highly lethal in the C. elegans model (599). Tryptic soy agar supplemented with 7 μg/ml nalidixic acid was inoculated with bacteria 3 h prior to the addition of 30 L4-stage nematodes. Plates were incubated at 25°C, and scoring for live and dead worms was performed every 24 h. The experiment was performed thrice in triplicate, and Kaplan-Meier and log rank tests were used to analyze nematode survival data. (A) Pictorial representation of live and dead C. elegans feeding on the PVL-positive USA400 strain. (B) Kaplan-Meier survival plots of nematodes fed with PVL(+) and PVL(−) USA400 and control strains indicate that no significant difference was seen in killing activities between these two groups. (C and D) Three representative clinical isolates each from PVL(+)/(−) USA400 sibling strains demonstrate similar nematocidal activity, with no significant difference between the groups. (Adapted from reference with permission of the publisher.)
FIG 10
FIG 10
PVL(+) and PVL(−) USA400 sibling strains both have high fly-killing activities in the Drosophila melanogaster model (599). (A) For the determination of fly-killing activity, 2- to 5-day-old female Drosophila flies were pricked in the dorsal thorax with a 27.5-gauge needle dipped in bacterial suspension (8 × 108 CFU/ml of S. aureus). The flies were kept at room temperature, fed with sucrose, and monitored daily to be scored as live or dead. The experiment was performed thrice in triplicate, and Kaplan-Meier and log rank tests were used to analyze fly survival data. (B) Kaplan-Meier survival plots of flies injected with PVL(+) and PVL(−) USA400 and control strains indicate that no significant differences were seen between the two groups. (C and D) Three representative clinical isolates each from PVL(+)/(−) USA400 sibling strains show similar fly-killing activities. (Adapted from reference with permission of the publisher.)
FIG 11
FIG 11
No dermatopathological differences are seen between PVL-positive and -negative sibling strains in a murine intradermal infection model (599). No dermatopathological difference was observed on day 4 following infection with PVL(+) and PVL(−) USA400 sibling strains. Skin lesions (A to D) and histopathological sections (E to H) of mouse skin from intradermally infected BABL/c mice are shown. Similar pustules/abscesses with confined dermal abscess damage without ulceration were observed with both PVL(+) (B and F) and PVL(−) (C and G) USA400 sibling strains. In contrast, a large cutaneous ulcer with a predominantly neutrophilic inflammation was observed with the USA300 strain (D and H), and the colonizing strain M92 (A and E) caused only localized edema with a localized inflammatory reaction without ulceration. (Adapted from reference with permission of the publisher.)
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