Identification of Hemolytic Activity and Hemolytic Genes of Methicillin-Resistant Staphylococcus aureus Isolated from Chinese Children

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Identification of Hemolytic Activity and Hemolytic Genes of Methicillin-Resistant Staphylococcus aureus Isolated from Chinese Children

Methicillin-resistant Staphylococcus aureus (MRSA) remains a significant pathogen causing diverse infections in children, ranging from mild skin conditions to life-threatening invasive diseases. Understanding the molecular epidemiology and virulence characteristics of circulating MRSA clones is critical for infection control and clinical management. This study investigated the temporal shifts in MRSA clones isolated from Chinese children over three periods (2005–2011, 2012–2013, and 2016), with a focus on their hemolytic activities and associated genetic determinants. The findings highlight the emergence of a dominant ST59 clone with distinct hemolytic properties and genetic features, providing insights into its potential adaptation and spread in both community and hospital settings.

Molecular Epidemiology of MRSA Clones

A total of 259 MRSA isolates were collected from Beijing Children’s Hospital during the study periods: 70 isolates from 2005–2011, 90 from 2012–2013, and 99 from 2016. Multilocus sequence typing (MLST) revealed dynamic changes in the prevalence of MRSA clones. ST59 emerged as the predominant sequence type, increasing significantly from 48.6% (34/70) in 2005–2011 to 79.8% (79/99) in 2016 (P < 0.001). Conversely, the previously prevalent ST239 clone disappeared entirely by 2016.

The molecular characteristics of these isolates were further defined by SCCmec, spa, and agr typing. SCCmec type IV, associated with community-acquired MRSA (CA-MRSA), became increasingly dominant, rising from 61.4% in 2005–2011 to 84.8% in 2016 (P < 0.001). SCCmec type III, historically linked to hospital-associated MRSA (HA-MRSA), was undetected in 2016. Similarly, the spa-t437 lineage surged from 31.4% to 63.6% over the study period (P < 0.001). The agr type I predominated across all periods, escalating from 72.9% in 2005–2011 to 92% in 2016 (P < 0.01). The composite genotype ST59-SCCmecIV-spa-t437-agrI represented 59.6% (59/99) of isolates in 2016, up from 24.3% in 2005–2011.

Hemolytic Phenotypes and Genetic Correlates

Hemolytic activity, a key virulence trait, was assessed using the CAMP test on sheep blood agar (SBA). Four distinct hemolytic patterns were observed:

  1. α- and δ-hemolysis: Characterized by a wide zone of complete hemolysis (α) and a narrow zone of incomplete hemolysis (δ).
  2. β- and δ-hemolysis: Featuring incomplete hemolysis (β) with sharp edges and δ-hemolysis.
  3. β-hemolysis alone: Incomplete hemolysis without δ activity.
  4. δ-hemolysis alone: Narrow zones of incomplete hemolysis.

Notably, β-hemolysis partially inhibited α-hemolysis, producing turbid zones, while δ-hemolysis synergized with β-hemolysis to broaden the turbid area (Figure 1). Among the 259 isolates, 153 exhibited β- and δ-hemolysis, with 93.5% (143/153) belonging to the ST59 lineage. The α-hemolytic phenotype was rare and primarily associated with the declining ST239 clone.

Genetic analysis revealed that all isolates harbored the hla (α-hemolysin) and hld (δ-hemolysin) genes. The intact hlb gene, responsible for β-hemolysin production, was present in 97.4% (149/153) of β-hemolytic isolates. The absence of the bacteriophage φ13, which typically disrupts hlb, likely explains the functional β-hemolysin in these strains. Among α-hemolytic isolates, 94.1% (80/85) carried the int gene, a marker for bacteriophage integration that may modulate α-hemolysin expression.

Clinical Correlations and Epidemiological Implications

Of the 259 cases, 40 (15.4%) were classified as invasive infections, including bacteremia, pneumonia, and soft tissue infections. Patients originated from multiple regions across China, including Anhui, Hubei, Hebei, Shandong, Shanxi, and Beijing. No significant association was found between hemolytic phenotypes and clinical variables such as age, sex, pediatric intensive care unit (PICU) admission, or specific diagnoses (P > 0.05).

The displacement of ST239 by ST59 suggests a shift in MRSA epidemiology in North China. ST59-SCCmecIV-spa-t437-agrI, now the dominant clone, exhibits β- and δ-hemolytic activity but lacks α-hemolysis. This contrasts with ST239, which produced α-hemolysis and was historically associated with severe hospital-acquired infections. The decline of α-hemolysis in prevalent clones raises questions about the role of this toxin in MRSA adaptation. While α-hemolysin is a key virulence factor in many S. aureus infections, its absence in ST59 may reflect selective pressures favoring immune evasion or enhanced transmission.

The predominance of intact hlb and functional β-hemolysin contrasts with global trends, where hlb is often disrupted by φ13 in human isolates. This finding underscores the genetic diversity of Chinese MRSA clones and their potential divergence from international strains.

Conclusions

This study documents the rapid clonal replacement of ST239 by ST59 in Chinese pediatric MRSA isolates, accompanied by distinct shifts in hemolytic activity and genetic profiles. The ST59 clone’s β- and δ-hemolytic phenotype, coupled with the absence of α-hemolysis, highlights its unique virulence strategy. The persistence of intact hlb and the role of bacteriophage dynamics in modulating hemolysin expression warrant further investigation. These findings emphasize the need for ongoing surveillance to track MRSA evolution and inform targeted interventions.

doi.org/10.1097/CM9.0000000000000571

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