Comparative Prevalence of Immune Evasion Complex Genes Associated with β-Hemolysin Converting Bacteriophages in MRSA ST5 Isolates from Swine, Swine Facilities, Humans with Swine Contact, and Humans with No Swine Contact

<div><p>Livestock associated methicillin-resistant <i>Staphylococcus aureus</i> (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC) genes carried by β-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with β-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the β-hemolysin gene was intact in these strains, indicating the bacteriophage’s absence. In contrast, the prevalence of the β-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of β-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates may harbor a reduced capacity to cause severe disease in immunocompetent humans.</p></div

Agarose gel electrophoresis demonstrating inconclusive banding pattern resulting from PCR used to test an intact β-hemolysin gene in isolates obtained from humans with no swine contact.

<p>Those isolates producing only a 750bp band were found to contain an intact β-hemolysin gene, while isolates producing only a 300bp band or both bands were found to contain a disrupted β-hemolysin gene. A disrupted β-hemolysin gene is represented by the negative control ST8 Newman (lane 1), which produced a band 300bp in size. An intact β-hemolysin gene is represented by the positive control ST398 (lane 2), which produced a band 750bp in size. Of the isolates from humans with no swine contact, 9.6% (7/73) produced a 750bp band (lane 8) and 32.9% (24/73) produced a 300bp band (lane 4, 7). However, 57.5% (42/73) of the isolates produced both a 750bp and a 300bp band (lane 3, 5, 6, 9–13).</p

Primer sets with reaction components, expected product size and primer source.

<p>Primer sets with reaction components, expected product size and primer source.</p

Southern blot demonstrating the presence of an intact or disrupted β-hemolysin gene.

<p>Isolates containing an intact β-hemolysin gene produced a distinct band (20kb) as seen with the control isolate ST398 that lacked the bacteriophage (lane 1). Isolates containing a disrupted β-hemolysin gene did not produce a distinct band at 20kb and background extended in the lanes to a size greater than 40kb, as seen with the control ST8 Newman (lane 2). Swine-associated MRSA ST5 isolates are represented by lanes 3–5 and MRSA ST5 from humans with no swine contact are represented by lanes 6–17. Lanes 13 and 17 contained isolates from humans with no swine contact bearing an intact β-hemolysin gene.</p

Location of primers used for PCR used to test for the presence of the integrase gene and intact β-hemolysin gene.

<p>The integrase gene associated with the β-hemolysin converting bacteriophage (int CDS) and the 5’ end of the disrupted β-hemolysin gene (truncated beta-hemolysin CDS) are shown along with the primer specific for the the integrase gene (Int-F) and the primer specific for the β-hemolysin gene (Hlb-R3). The primers used for detection of an intact β-hemolysin gene (Hlb-TNF1 and Hlb-TNR1) spanned the disrupted portion of the gene and no product should be generated when the phage has integrated.</p

Immune-evasion complex and β-hemolysin converting bacteriophage screening results for all isolates.

<p>^a Data reported represent the results from Southern blotting.</p><p>^b Data reported as percent of isolates positive for each gene tested. Number of positive isolates is noted in parenthesis.</p><p>Immune-evasion complex and β-hemolysin converting bacteriophage screening results for all isolates.</p