Kunyan Zhang

Novel Multiplex PCR Assay for Characterization and Concomitant Subtyping of Staphylococcal Cassette Chromosome mec Types I to V in Methicillin-Resistant Staphylococcus aureus

ABSTRACT Staphylococcal cassette chromosome mec (SCCmec) typing is essential for understanding the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA). SCCmec elements are currently classified into types I to V based on the nature of the mec and ccr gene complexes, and are further classified into subtypes according to their junkyard region DNA segments. Previously described traditional SCCmec PCR typing schemes require multiple primer sets and PCR experiments, while a previously published multiplex PCR assay is limited in its ability to detect recently discovered types and subtypes such as SCCmec type V and subtypes IVa, b, c, and d. We designed new sets of SCCmec type- and subtype-unique and specific primers and developed a novel multiplex PCR assay allowing for concomitant detection of the methicillin resistance (mecA gene) (also serving as an internal control) to facilitate detection and classification of all currently described SCCmec types and subtypes I, II, III, IVa, b, c, d, and V. Our assay demonstrated 100% sensitivity and specificity in accurately characterizing 54 MRSA strains belonging to the various known SCCmec types and subtypes, when compared with previously described typing methods. Further application of our assay in 453 randomly selected local clinical isolates confirmed its feasibility and practicality. This novel assay offers a rapid, simple, and feasible method for SCCmec typing of MRSA, and may serve as a useful tool for clinicians and epidemiologists in their efforts to prevent and control infections caused by this organism.

A Novel Mechanism of Rapid Nuclear Neutrophil Extracellular Trap Formation in Response to Staphylococcus aureus

Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3–4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5–60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton–Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton–Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.

Classification of staphylococcal cassette chromosome mec (SCCmec) : guidelines for reporting novel SCCmec elements.

Classification of staphylococcal cassette chromosome mec (SCCmec) : guidelines for reporting novel SCCmec elements.

Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.Neutrophil extracellular traps (NETs) are released, as neutrophils die in vitro, in a process requiring hours, leaving a temporal gap for invasive microbes to exploit. Functional neutrophils undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live PMN in vivo rapidly releasing NETs, which prevented bacterial dissemination. NETosis occurred during crawling thereby casting large areas of NETs. NET-releasing PMN developed diffuse decondensed nuclei ultimately becoming devoid of DNA. Cells with abnormal nuclei displayed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A combined requirement of Tlr2 and complement mediated opsonization tightly regulated NET release. Additionally live human PMN developed decondensed nuclei and formed NETS in vivo and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection, non-cell death NETosis occurs in vivo during Gram-positive infection in mice and humans.

New Quadriplex PCR Assay for Detection of Methicillin and Mupirocin Resistance and Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci

ABSTRACT Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.

Novel Multiplex PCR Assay for Detection of the Staphylococcal Virulence Marker Panton-Valentine Leukocidin Genes and Simultaneous Discrimination of Methicillin-Susceptible from -Resistant Staphylococci

ABSTRACT We developed a new multiplex PCR assay for detection of Panton-Valentine leukocidin virulence genes and simultaneous discrimination of methicillin-susceptible from -resistant staphylococci. This assay is simple, rapid, and accurate and offers the potential for prompt detection of newly emerging community-associated methicillin-resistant Staphylococcus aureus.

Outbreak in Alberta of community-acquired (USA300) methicillin-resistant Staphylococcus aureus in people with a history of drug use, homelessness or incarceration

Background: The USA300 strain of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) can cause severe infection and is increasingly recognized as a cause of community outbreaks. In 2004, an outbreak was identified in the Calgary Health Region (CHR). Methods: MRSA isolates were identified with standard methods at a central regional laboratory and typed via pulsed-field gel electrophoresis (PFGE). Isolates were tested by PCR for mecA, Panton–Valentine leukocidin (PVL), SCCmec, and spa genes. Cases were defined as such if a clinical isolate of the USA300 strain was noted between January 1 and September 30, 2004, and the patient had lived or traveled in CHR within 2 years before symptom onset. Demographic, clinical and risk data on all such cases were collected from several sources for statistical analysis. A case was defined as high-risk if the patient had a history of drug use, homelessness or incarceration. Results: Of 40 isolates with the USA300 PFGE pattern, all tested positive for PVL, SCCmec type IVa and spa type 008. Almost all infections (39/40, 98%) involved skin and soft tissues, except for 1 death from necrotizing hemorrhagic pneumonia; a notable proportion (38%) required hospital admission or intravenous antimicrobial therapy. The outbreak centred on the high-risk population in CHR (70%; risk ratio 169.4, 95% confidence interval 86.1–333.0). Interpretation: People with histories of illicit drug use, homelessness or recent incarceration were at highest risk for infection with CA-MRSA. The emergence and spread of this virulent strain has important implications for treatment and public health in Canada.

Novel Staphylococcal Cassette Chromosome mec Type, Tentatively Designated Type VIII, Harboring Class A mec and Type 4 ccr Gene Complexes in a Canadian Epidemic Strain of Methicillin-Resistant Staphylococcus aureus

ABSTRACT Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element characterized by flanking terminal direct and, in most cases, inverted repeat sequences, the mec and ccr gene complexes, and their surrounding DNA regions. Unique combinations of the mec and ccr gene complexes generate various SCCmec types. Six SCCmec types have been reported to date. We describe here a novel SCCmec type identified in a Canadian methicillin-resistant Staphylococcus aureus (MRSA) epidemic strain. MRSA clinical isolates were screened for known SCCmec types by multiplex and conventional PCR methods. Three phenotypically and genotypically identical MRSA clinical isolates with a pulsotype identical to CMRSA9 were identified locally and found to be nontypeable by available SCCmec typing schemes. Complete sequencing of the SCCmec element revealed a nucleotide fragment of 32,168 bp integrated at an identical chromosomal integration site (attBscc) at the 3′ end of the orfX gene. The nucleotide sequences at the chromosome-SCCmec junction regions were typical of other SCCmec types, but the element harbored a unique combination of class A mec and type 4 ccr gene complexes. Sequence recombination analysis suggested that this unique SCCmec type may be derived from homologous recombination between the previously described SCCRP62A of S. epidermidis strain RP62A and SCC composite island of S. epidermidis ATCC 12228, respectively, or via recombination of other staphylococcal strains that carry the same or similar mobile cassettes. We identified a previously undescribed type of SCCmec from isolate C10682, tentatively designated type VIII, and we provide compelling evidence supporting the ability of SCC elements to transfer horizontally or undergo recombination to generate new SCCmec types.

Novel Multiplex PCR Assay for Simultaneous Identification of Community-Associated Methicillin-Resistant Staphylococcus aureus Strains USA300 and USA400 and Detection of mecA and Panton-Valentine Leukocidin Genes, with Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci

ABSTRACT We developed a novel multiplex PCR assay for rapid identification and discrimination of the USA300 and USA400 strains and concomitant detection of Panton-Valentine leukocidin genes, with simultaneous discrimination of methicillin-resistant Staphylococcus aureus strains from methicillin-susceptible S. aureus strains, S. aureus strains from coagulase-negative staphylococci, and staphylococci from other bacteria.

Coexistence of Panton-Valentine Leukocidin—Positive and —Negative Community-Associated Methicillin-Resistant Staphylococcus aureus USA400 Sibling Strains in a Large Canadian Health-Care Region

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains often carry the Panton-Valentine leukocidin (PVL) genes. However, the specific role that PVL plays in the epidemiological features and pathogenesis of CA-MRSA infections has remained undefined and controversial. Conducting a retrospective study on a natural population of MRSA clinical isolates recovered from community and hospital patients in a large Canadian health-care region during a 6-year period, we identified the coexistence of 2 USA400 (a major clonal group of CA-MRSA) sibling strains with and without PVL genes. Polymerase chain reaction and sequence analysis indicated that the PVL-carrying prophage phiSa2mw was present in PVL(+) but absent in PVL(-) USA400 isolates. These strains shared identical genotypic and phenotypic properties and similar clinical characteristics. This study provides direct evidence that PVL genes are not necessarily the key determinants associated with the increasing dissemination of CA-MRSA strains, suggesting that the genomic milieu may play a greater role in this regard.