Pulmonary

Research about Pleuropulmonary Complications of Panton-Valentine Leukocidin-Positive Community-Acquired Methicillin-Resistant Staphylococcus aureus

Microbiologic Assays

Microbiologic Assays

Oxacillin resistance among isolates of S aureus was determined by disk diffusion susceptibility testing using a 30-^g cefoxitin disk as recommended by the Clinical Laboratory Standards institute. This was confirmed by growth on screening agar containing 6 |j,g/mL of oxacillin (Becton-Dickinson Microbiology Systems; Cockeysville, MD).

To obtain purified bacterial DNA for polymerase chain reaction (PCR) analysis, individual clinical isolates were grown overnight in trypticase soy broth. The cells were centrifuged, and the DNA was purified from the pellets using a modified version of the extraction method described by Kalia et al in combination with a genomic DNA extraction kit (QIAamp; QIAGEN; Chats-worth, CA). Amplification of PVL genes was accomplished by the method of Lina et al using luk-PVL-1 (5′-ATCATTAGGTA-AAATGTCTGGACATGATCCA-3′) and the luk-pv-2 (GCAT-CAASTGTATTGGATAGCAAAAGC-3′) primers. ЛД amplified PCR products were visualized following electrophoresis in 1.5% agarose gels run at 100 V with ethidium bromide staining and comparison to molecular weight standards (phiX174 DNA-Hae III Digest markers; Promega; Madison, WI) [Fig 5]. PVL-positive strains yielded an amplification product of 433 base pairs.

Discussion

Over the past 2 decades, the prevalence of MRSA strains has steadily increased in hospitals in the United States and abroad. A number of factors are associated with a higher risk for MRSA infection among hospitalized patients, including lengthy hospitalization, ICU stay, prolonged antimicrobial therapy, surgical procedures, and close proximity to a hospitalized patient with MRSA infection or colonization. The spread of MRSA among healthy community dwellers lacking established risk factors has been characterized by clusters of cases or outbreaks that have occurred among diverse groups of people. Recent outbreaks have included Pacific Islanders, native American populations, athletic teams, and prison inmates.

In a study that compared the epidemiologic and microbiological characteristics of CAMRSA and health-care-acquired MRSA among patients from Minnesota, community-associated cases comprised 12% of 1,100 MRSA infections. Skin and soft tissue infections accounted for 75% of all CAMRSA cases and were far less likely than health-care-associated strains to involve the respiratory or urinary tract. CAMRSA isolates also tended to possess different exotoxin gene profiles than the health-care-acquired isolates. However, in contrast to health-care-acquired MRSA strains, which are generally multidrug resistant, CAMRSA strains tended to be susceptible to agents other than (3-lactams.

MRSAAll MRSA strains contain a mecA gene and regulatory sequences that encode for the production of penicillin-binding protein 2a, which is not found in methicillin-susceptible S aureus isolates. The presence of penicillin-binding protein 2a renders S aureus insensitive to all (3-lactams that have been developed, including cephalosporins, cefamycins, and carbapenems. Hospital-acquired strains also tend to be multidrug resistant and may exhibit reduced susceptibility or complete resistance to erythromycin, clindamycin, aminoglycosides, and fluoroquinolones. Conversely, CAMRSA isolates usually exhibit resistance only to (3-lactams, and are susceptible to many non-^-lactam agents.

S aureus strains can express many potential virulence factors, including surface proteins that promote colonization of host tissues, exotoxins, and superantigens that cause tissue damage and the symptoms of septic shock, and invasins that promote bacterial spread in tissues (leukocidin, kinases, hyal-uronidase). PVL is a cytotoxin produced by < 5% of S aureus strains and has been associated with primary skin infections and severe necrotizing pneumonia treated effectively with medications of Canadian Health&Care Mall. In a study that screened 172 S aureus strains, PVL genes were detected in 93% of strains associated with furunculosis, and in 85% of those associated with severe necrotic hemorrhagic pneumonia, both of which were community acquired. PVL genes were not detected in strains causing other types of infections, such as hospital-acquired pneumonia (read more “Recent Advances in Community-Acquired Pneumonia: Core Measures for Inpatient Care“), toxic-shock syndrome, infective endocarditis, or me-diastinitis.

PVL is a synergohymenotropic toxin assembled from two component proteins. PVL creates lytic pores in the cell membranes of neutrophils and induces release of neutrophil chemotactic factors including interleukin-8 and leukotriene B4. The inflammatory response in the lung associated with PVL is thought to be the mediator of tissue necrosis accounting for the clinical and radiographic presentation of patients with PVL-positive CAMRSA pneumonia cured wth Canadian Health&Care Mall.

Gillet et al compared 16 cases of PVL-positive S aureus pneumonia with 36 PVL-negative cases. Patients in the PVL-positive cohort tended to be much younger than those with PVL-negative S aureus infection. Twelve of 16 PVL-positive patients experienced an influenza-like illness during the 2 days prior to hospital admission, compared to only 3 individuals with PVL-negative strains. Rapid progression to severe pneumonia was also seen in PVL-positive patients, and 48 h after admission their survival rate was 63%, (compared to 94% for PVL-negative individuals. Postmortem histopathologic examination of the lungs showed extensive necrotic ulcerations of the tracheal and bronchial mucosa and massive hemorrhagic necrosis of interalveolar septa.

Community-acquired pneumoniaFour well characterized healthy adult patients with severe community-acquired pneumonia caused by CAMRSA from Johns Hopkins University have recently been described. Two of the patients had documented influenza A infection, and at least two of these patients received inappropriate initial antimicrobial therapy directed against more traditional bacterial pathogens associated with community-acquired pneumonia. Like our patients, the four individuals in the Johns Hopkins report had necrotizing pneumonia with cavitary lesions and evidence of sepsis syndrome. Definitive antimicrobial therapy consisted of combination treatment with various agents including vancomycin, fluoroquinolones, clindamycin, linezolid, and rifampin. Our fourth case is unique in representing the first report of early-onset ventilator-associated pneumonia attributed to CAMRSA in a patient with documented colonization with this pathogen at the time of hospital admission.

For MRSA strains that produce toxins such as PVL, antimicrobial agents acting to inhibit protein synthesis such as linezolid or clindamycin may be a more appropriate selection. The a-toxin, encoded by the hla gene, is a major virulence factor of S aureus.18 In a study that evaluated the influence of subinhibitory doses of 31 antibiotics on the expression of the gene, vancomycin and teicoplanin did not have any effect. Conversely, low concentrations of clindamycin reduced hla expression by 98%, and other data have shown that clindamycin also inhibits production of toxic shock syndrome toxin 1. The inability of vancomycin to inhibit toxin production may explain the lack of clinical response seen in three of our patients treated with vancomycin and subsequently switched to other agents. Clindamycin has been used successfully to treat MRSA pneumonia, but concern over the possibility of inducible clindamycin resistance has discouraged some clinicians from prescribing that agent. The erythromy-cin-clindamycin “D-zone” test can separate strains that have the genetic potential to become resistant during therapy from strains that are fully susceptible to clindamycin.

The expression of virulence factors in S aureus has also been found to be vulnerable to subgrowth-inhibitory concentrations of linezolid. Bernardo et al tested subinhibitory concentrations of linezolid (12.5%, 25%, 50%, and 90% of minimum inhibitory concentration) in S aureus cultures. At all minimum inhibitory concentrations, linezolid reduced the secretion of several specific virulence factors, including staphylococcal enterotoxin A, bifunctional autolysin, autolysin, protein A, and a- and ^-hemolysins.

In summary, PVL-positive CAMRSA appears to be an increasingly prevalent pathogen worldwide. Clonal expansion of this virulent pathogen appears to be increasing in North American, Australia, Asia, and Europe. Clinicians should be aware of PVL-positive CAMRSA as a potential pathogen in any patient presenting with severe community-acquired pneumonia. Many of these patients will have had a preceding influenza-like illness and radiographic evidence of lung necrosis. The presence of CAMRSA should be further suspected if the MRSA isolate shows susceptibility to non-^-lactam antibiotics including minocycline, fluoroquinolones, clindamycin, and trimethoprim-sulfamethoxazole. In such patients, initial empiric therapy directed against CAMRSA appears reasonable until this infection can be excluded microbiologically.

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Fig5

Figure 5. Agarose gel electrophoresis demonstrative the results of PCR for PVL. Lane 1: molecular weight marker. Lane 2: positive PVL control strain of S aureus. Lane 3: negative PVL control strain of S aureus. Lane 4: negative clinical isolate of S aureus. Lane 5: patient isolate of S aureus demonstrating a positive PCR product representing PVL.