Sameer Elsayed

Burn Wound Infections

SUMMARY Burns are one of the most common and devastating forms of trauma. Patients with serious thermal injury require immediate specialized care in order to minimize morbidity and mortality. Significant thermal injuries induce a state of immunosuppression that predisposes burn patients to infectious complications. A current summary of the classifications of burn wound infections, including their diagnosis, treatment, and prevention, is given. Early excision of the eschar has substantially decreased the incidence of invasive burn wound infection and secondary sepsis, but most deaths in severely burn-injured patients are still due to burn wound sepsis or complications due to inhalation injury. Burn patients are also at risk for developing sepsis secondary to pneumonia, catheter-related infections, and suppurative thrombophlebitis. The introduction of silver-impregnated devices (e.g., central lines and Foley urinary catheters) may reduce the incidence of nosocomial infections due to prolonged placement of these devices. Improved outcomes for severely burned patients have been attributed to medical advances in fluid resuscitation, nutritional support, pulmonary and burn wound care, and infection control practices.

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.

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.

Community-Wide Outbreaks of Clonally Related CTX-M-14 β-Lactamase-Producing Escherichia coli Strains in the Calgary Health Region

ABSTRACT Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs) typically cause nosocomial infections. Previous surveillance in the Calgary Health Region showed that Escherichia coli strains producing ESBLs were common among community patients. During the period (2000 to 2002): 23 of 157 (15%) of the strains were positive for blaCTX-M genes from the CTX-M-I group (CTX-M-1-like) and 87 of 157 (55%) of the strains were positive for blaCTX-M genes from the CTX-M-III group (CTX-M-14-like). The objective of this study was to investigate the molecular epidemiology of these strains. The β-lactamases were characterized, and the genetic relatedness of the isolates was analyzed by digesting genomic DNA with the restriction endonuclease XbaI and by performing pulse-field gel electrophoresis (PFGE). PFGE revealed two closely related restriction patterns (clusters CTXM14A and CTXM14AR) among 67 (77%) CTX-M-14 producers. These strains from CTXM14A had nearly identical susceptibility patterns and were isolated most often from urine samples obtained at community sites during 2000 and 2001. Strains from the CTX-M-1-like and CTX-M-negative groups were unrelated to clusters CTXM14, CTXM14AR, and CTXM14NR. We conclude that clonally related strains of E. coli producing CTX-M-14 β-lactamases were responsible for a predominantly community-wide outbreak. Further studies are warranted to investigate whether community-onset diseases caused by ESBL-producing E. coli are related to a point source or transmission within the community.

Population‐Based Epidemiological Study of Infections Caused by Carbapenem‐Resistant Pseudomonas aeruginosa in the Calgary Health Region: Importance of Metallo‐β‐Lactamase (MBL)–Producing Strains

BACKGROUND A study was conducted in the Calgary Health Region between May 2002 and April 2004 to define the population-based epidemiological characteristics of infections caused by imipenem-resistant Pseudomonas aeruginosa and to explore the clinical outcomes due to metallo- beta -lactamase (MBL)-producing and non-MBL-producing strains. METHODS Detailed clinical information was obtained by chart review, and phenotypic and molecular characterizations were performed using the MBL E-test, polymerase chain reaction with sequencing, and pulsed-field gel electrophoresis. RESULTS A total of 228 patients with infections caused by imipenem-resistant P. aeruginosa were identified (annual incidence, 10.5 cases/100,000 population), with the highest incidence rate in those >or=75 years old. MBL-producing strains (98/228) were associated with higher rates of multidrug resistance and bacteremia. Ninety MBL-producing strains also produced VIM-2, 4 produced IMP-7, and 4 were unclassified. A cluster of VIM-2-producing strains was responsible for a nosocomial outbreak during 2003. The case-fatality rate was significantly higher for infections caused by MBL-producing strains than for those caused by non-MBL-producing strains (25% vs. 13%; relative risk, 1.98 [95% confidence interval, 1.00-3.90]; P=.05). CONCLUSION MBL-producing P. aeruginosa strains were associated with a higher case-fatality rate and invasive disease. Our study highlights the potential importance of molecular laboratory techniques in infection control and patient care.

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.

Association between Handling of Pet Treats and Infection with Salmonella enterica Serotype Newport Expressing the AmpC β-Lactamase, CMY-2

ABSTRACT Resistance to the extended-spectrum cephalosporins can occur in Salmonella species via the production of extended-spectrum and AmpC β-lactamases. We describe human infections with Salmonella enterica serotype Newport phage type 14 strains resistant to ceftazidime (CAZ) and cefoxitin (FOX) related to the handling of pet treats containing dried beef. These strains were isolated from five patients in Calgary, Alberta, Canada, during 2002 and were compared to a strain cultured from a commercial pet treat present at the property of one of the patients. The strains were resistant to FOX, CAZ, cefpodoxime, ampicillin, and chloramphenicol; intermediate resistant to ceftriaxone and cefotaxime; and sensitive to the aminoglycosides, ciprofloxacin, cefepime, and imipenem. Isoelectric focusing, multiplex PCR, and sequencing of the amplicons showed that all strains produced the plasmid-encoded AmpC β-lactamase, CMY-2. Restriction analysis of plasmid DNA following transformation demonstrated that blaCMY-2 was encoded on an approximately 140-kb plasmid. Pulsed-field gel electrophoresis showed the human and pet treat Salmonella strains to be highly related. This study is the first to implicate the transfer of multidrug-resistant Salmonella species through the handling of commercial pet treats containing animal products. In addition to documenting the first cases of human infection caused by CMY-2-producing S. enterica serotype Newport strains in Canada, this study illustrates the necessity of rapid and accurate laboratory-based surveillance in the identification of novel types of antimicrobial resistance.