Publication: Virulence Factors of S. aureus

The invasiveness of methicillin resistant strains of Staphylococcus aureus (MRSA) acquired outside of the hospital setting has been linked to a gene known as the Panton Valentine leukocidin gene which codes for the production of a toxin known as Panton Valentine leukocidin (PVL). The prevalence of the PVL gene in Staphylococcus aureus in northern Utah was studied using a real-time LightCycler SYBR Green PCR assay. The study consisted of 213 S. aureus isolates, each of which was grouped into one of four categories: Hospital acquired MRSA, Community Acquired MRSA, Hospital acquired methicillin susceptible Staphylococcus aureus (MSSA), and Community acquired MSSA. There were 101 methicillin resistant S. aureus isolates tested: 52 CA-MRSA and 49 HA-MRSA. Of these isolates, 28 CA-MRSA isolates and three HA-MRSA isolates were positive for the PVL gene. The positive isolates were primarily associated with skin and/or wound infections.

In addition to the PCR testing, antimicrobial susceptibility patterns were studied to investigate if any particular pattern of susceptibility to clindamycin, levofloxacin, and trimethoprim/sulfamethoxazole was associated with these isolates. The CA-MRSA in the intermountain area was shown to be more susceptible to clindamycin than HA-MRSA.

Keywords: Staphylococcus aureus, PVL, Panton Valentine Leukocidin, CA-MRSA, clindamycin, levofloxacin

Introduction

In recent years, there has been an increase in community acquired methicillin resistant Staphylococcus aureus (MRSA). Before this increase, MRSA was predominantly seen in hospital acquired infections (infections acquired after seventy-two hours of hospitalization) and rarely in healthy individuals in the community.

CA-MRSA genotypes have been shown to be different from the hospital acquired strains. Hospital acquired strains of MRSA have undergone a genetic mutation that allows for the resistance to methicillin. When antibiotics are overused or misused, these bacteria are selected for in an individual because they will be the only strains to survive antibiotic treatment. However, CA-MRSA is said to spread clonally, which means that one strain of bacteria developed resistance to the antibiotic and it passes that resistance on when it multiplies ². The same strain is causing the infections in patients without any prior exposure to antibiotics or previous hospitalization.

These infections have shown to be very invasive and often lead to the death of the individual. Often, a patient will die within hours of showing the first signs of infection. These infections progress so quickly that, often, they will advance beyond the point where they would have been treatable before the cause of infection is even identified. Physicians will often either wait to prescribe antibiotics until a full antimicrobial susceptibility panel has been run on the patient sample, or they will prescribe an antibiotic that is not effective against the antibiotic resistant strain that is causing the infection. The invasiveness of the CA-MRSA causes the infection to progress too quickly in many cases and the patient either dies or suffers from debilitating damage to the site of infection. This invasiveness has been associated with the Panton Valentine leukocidin ⁵.

All strains of S. aureus excrete some form of toxin. Collectively, they are capable of excreting some 30 different extra-cellular products ⁴. These products include hemolysins, proteases, lipases, hyaluronidase and collagenase; some strains have the ability to produce exoprotiens that include: toxic shock syndrome toxin-1, exfoliative toxin, and Panton-Valentine leukocidin. PVL and gamma-hemolysin belong to a family of toxins known as synergohymenotropic toxins ⁴. The term synergohymenotropic means that two proteins act synergistically on cell membranes to form a pore which may lead to cellular leakage and death. In 1932, Panton and Valentine described leukocidin as a virulence factor closely linked to tissue necrosis. As a result of this, PVL has been called the most leukocytotoxic toxin produced by S. aureus ⁴. It can cause dermo-necrosis, granule secretion, secretion of leukotriene B4 , and interleukin-8 from neutrophils ⁴. The binding of PVL to neutrophils brings about chemotaxis, release of inflammatory substances, and promotes tissue necrosis and abscess formation ¹. The PVL determinant consists of two parts knows as lukS-PV which is 939 nucleotides in size, and lukF-PV which is 978 nucleotides in size ⁴.

For the past several years, LDS hospital (Salt Lake City, Utah) has been storing Staphylococcus aureus isolates from hospital patients for use in conducting research to study the prevalence of the PVL gene in the intermountain area. Analysis of these samples included 1) antimicrobial susceptibility panels to detect MRSA and Methicillin Susceptible Staphylococcus aureus (MSSA) and 2) PCR (Polymerase Chain Reaction) to detect the presence of the PVL gene. Data analysis included comparison of susceptibility patterns and the prevalence of the PVL gene in infections caused by hospital acquired MRSA (HA-MRSA), community acquired MRSA (CA-MRSA), and Methicillin Susceptible Staphylococcus aureus (MSSA) to show what role the presence of this gene plays in the severity of these infections.

Materials and Methods

The organisms investigated were comprised of S. aureus isolates identified from February to April 2005 by the microbiology department at LDS Hospital in Salt Lake City, Utah. Isolates were grown on blood agar and identified using traditional methods according to National Committee for Clinical Laboratory Standards (NCCLS). Each isolate had been previously tested by PCR for the presence of the mecA gene, and the extracted DNA was then frozen. Organisms were also inoculated into skim milk and frozen for susceptibility testing.

The isolates were separated into two categories based on the location where the culture was obtained. Isolates that were collected from acute care inpatients were considered to be hospital acquired, while isolates collected from any outpatient source, i.e. ER, InstaCare, physician office, or short stay surgery units were considered to be community acquired.

Detection of the PVL gene was accomplished using extracted genomic DNA. The extraction process was carried out using the Qiagen column, according to the manufacture’s instructions. This extraction was then used for LightCycler PCR analysis. Oligonucleotide primers (forward: 5’-GTAAAATGTCTGGACATGATCCA-3’ reverse: 5’-CAA(C/G)TGTATTGGATAGCAAAAGC-3’) were designed by Applied Biosystems (ABI) according to a previous study to amplify the last section of lukS-PV and the beginning of lukF-PV ⁴. The specificity of the primers were confirmed by sequencing at ARUP Laboratories, Salt Lake City, Utah. The PCR Mastermix (20µL) consisted of 1µL SYBR Green, 2.4µL of 4µM MgCl2, 1µL 0.73µM PVL forward primer, 1µL 0.73µM PVL reverse primer, 12.6µL molecular grade water, and 2µL template. The LightCycler program consisted of 95°C for 10 minutes, followed by 35 cycles of 95°C for 10 seconds, 52°C for 5 seconds and 72°C for 18 seconds, with fluorescence acquisition at the end of each extension step. Molecular grade water was used as a negative control. A PVL-positive S. aureus (ATCC strain 16575) isolate was used as a positive control in each PCR run. Isolates were considered positive if melting point was 80.15°C +/- 2 standard deviations of 0.34°C (range: 79.47°C to 80.83°C).

The susceptibility patterns of the MRSA and MSSA were evaluated using a Dade Behring Microscan Walkaway. The inoculated skim milk was thawed and streaked onto blood agar and incubated in a CO₂ incubator for 24-48 hours. A gram-positive pm20a MIC panel was then inoculated with colonies from these plates using Dade Behring Inoculation Prompt-D system for the anti-microbial susceptibility studies. The anti-microbials on the panel were as follows: amox/clav, amp/sulbactam, ampicillin, cefazolin, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gatifloxacin, gentamicin, imipenem, levofloxacin, linezolid, moxifloxacin, oxacillin, penicillin, pip/tazo, rifampin, synercid, tetracycline, trimeth/sulfamethoxazole, and vancomycin. Antimicrobials of interest in this study were clindamycin, levofloxacin, and trimethoprim/sulfamethoxazole.

Results

Of the 213 isolates of S. aureus that were analyzed, 101 were shown to be MRSA strains. Of the MRSA isolates, 52 were categorized as CA-MRSA and 49 were HA-MRSA. 79% of all of the isolates that were PVL positive were CA-MRSA. The methicillin resistant S. aureus (CA or HA) were equally resistant (100%) and susceptible (98%) to levofloxacin and trimethoprim/sulfamethoxazole, respectively. Seven (15%) of the hospital acquired methicillin resistant S. aureus were susceptible to clindamycin compared to 28 (64%) of the community acquired methicillin resistant S. aureus (chi-square = 15.9, p-value<<0.05). See figure 1.

Figure 1 Percent susceptible to clindamycin

Conclusion

The PVL gene is significantly more prevalent in the CA S. aureus than compared to the HA S. aureus (26% vs 3%; Chi-square value = 11.9, p-value < 0.05). See figure 2. The odds ratio of a positive PVL gene from a community acquired S. aureus is 10.4 (95% C.I - 3.1,35.5). CA-MRSA in northen Utah carries the PVL gene, a marker of increased virulence, significantly more often than HA-MRSA isolates.

Figure 2 PVL in association with S. aureus

Susceptibility studies showed that clindamycin, as an oral antibiotic for out-patient use, is significantly more active against CA-MRSA than against HA-MRSA in our region. Both CA-MRSA and HA-MRSA strains were almost completely resistant to levofloxacin and completely susceptible to trimethoprim/sulfamethoxazole. However, the variability between susceptibilities of CA-MRSA and HA-MRSA to clindamycin confirms the theory that the CA-MRSA strain is a completely different strain of S. aureus that has spread clonally.

The knowledge that the CA-MRSA in northern Utah has shown to be more susceptible to clindamycin and completely susceptible to trimethoprim/sulfamethoxazole will hopefully be used by physicians in the area to more effectively treat community acquired S. aureus infections. If the correct antibiotics could be prescribed earlier in the course of these infections, we would see a decrease in deaths and debilitation caused by CA-MRSA.

Acknowledgements

The authors wish to express their appreciation to the Infectious Disease Department and Molecular Pathology and Microbiology Department at LDS Hospital, Salt Lake City, Utah. They would also like to thank, in particular, Debbie Bennion, Addie Munson MT (ASCP), David Pombo M.D., and John Carlquist Ph.D. for all of their support and advice throughout the course of this research.

References

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2.Dufour et al, “Community-AcquiredMethicillin-ResistantStaphylococcusaureusInfectionsinFrance:EmergenceofaSingleCloneThatProducesPanton-ValentineLeukocidin,” Clinical Infectious Diseases, 3 Sept 2003, http://www.journals.uchicago.edu/CID/journal/issues/v35n7/020274/020274.html.

3.Hsu, Li-Yang, et al, “Panton-Valentine Leukocidin Positive Staphylococcus aureus, Singapore,” Emerging Infectious Diseases 10 (2004): 1509-1510.

4.Johnsson, D., et al, “Detection of Panton-Valentine Leukocidin Gene in Staphylococcus aureus by light cycler PCR: Clinical and Epidemiological Aspects,” Clinical Microbiological Infection 10(2004): 884-889.

5.Vandenesch, Francois, et al, “Community-Acquired Methicillin-Resistant Staphylococcus aureus Carrying Panton-Valentine Leukocidin Genes: Worldwide Emergence,” Emerging Infectious Disease 9 (2003): 978-984.

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Ogden, Utah 84408