George Killgore

Pulsed-Field Gel Electrophoresis Typing of Oxacillin-Resistant Staphylococcus aureus Isolates from the United States: Establishing a National Database

ABSTRACT Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care-associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of U.S. strains, the database contains patterns of representative epidemic-type strains from the United Kingdom, Canada, and Australia; previously described ORSA clonal-type isolates; 13 vancomycin-intermediate S. aureus (VISA) isolates, and two high-level vancomycin-resistant, vanA-positive strains (VRSA). Among the isolates from the United States, we identified eight lineages, designated as pulsed-field types (PFTs) USA100 through USA800, seven of which included both ORSA and oxacillin-susceptible S. aureus isolates. With the exception of the PFT pairs USA100 and USA800, and USA300 and USA500, each of the PFTs had a unique multilocus sequence type and spa type motif. The USA100 PFT, previously designated as the New York/Tokyo clone, was the most common PFT in the database, representing 44% of the ORSA isolates. USA100 isolates were typically multiresistant and included all but one of the U.S. VISA strains and both VRSA isolates. Multiresistant ORSA isolates from the USA200, -500, and -600 PFTs have PFGE patterns similar to those of previously described epidemic strains from Europe and Australia. The USA300 and -400 PFTs contained community isolates resistant only to β-lactam drugs and erythromycin. Noticeably absent from the U.S. database were isolates with the previously described Brazilian and EMRSA15 PFGE patterns. These data suggest that there are a limited number of ORSA genotypes present in the United States.

Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe

BACKGROUND Toxins A and B are the primary virulence factors of Clostridium difficile. Since 2002, an epidemic of C difficile-associated disease with increased morbidity and mortality has been present in Quebec province, Canada. We characterised the dominant strain of this epidemic to determine whether it produces higher amounts of toxins A and B than those produced by non-epidemic strains. METHODS We obtained isolates from 124 patients from Centre Hospitalier Universitaire de Sherbrooke in Quebec. Additional isolates from the USA, Canada, and the UK were included to increase the genetic diversity of the toxinotypes tested. Isolate characterisation included toxinotyping, pulsed-field gel electrophoresis (PFGE), PCR ribotyping, detection of a binary toxin gene, and detection of deletions in a putative negative regulator for toxins A and B (tcdC). By use of an enzyme-linked immunoassay, we measured the in-vitro production of toxins A and B by epidemic strain and non-dominant strain isolates. FINDINGS The epidemic strain was characterised as toxinotype III, North American PFGE type 1, and PCR-ribotype 027 (NAP1/027). This strain carried the binary toxin gene cdtB and an 18-bp deletion in tcdC. We isolated this strain from 72 patients with C difficile-associated disease (58 [67%] of 86 with health-care-associated disease; 14 [37%] of 38 with community-acquired disease). Peak median (IQR) toxin A and toxin B concentrations produced in vitro by NAP1/027 were 16 and 23 times higher, respectively, than those measured in isolates representing 12 different PFGE types, known as toxinotype 0 (toxin A, median 848 microg/L [IQR 504-1022] vs 54 microg/L [23-203]; toxin B, 180 microg/L [137-210] vs 8 microg/L [5-25]; p<0.0001 for both toxins). INTERPRETATION The severity of C difficile-associated disease caused by NAP1/027 could result from hyperproduction of toxins A and B. Dissemination of this strain in North America and Europe could lead to important changes in the epidemiology of C difficile-associated disease.

Changes in the Prevalence of Nasal Colonization with Staphylococcus aureus in the United States, 2001–2004

BACKGROUND Staphylococcus aureus is a common cause of infection, particularly in persons colonized by this organism. Virulent strains of methicillin-resistant S. aureus (MRSA) have emerged in the general community. METHODS A nationally representative survey of nasal colonization with S. aureus was conducted from 2001 through 2004 as part of the National Health and Nutrition Examination Survey. MRSA isolates were identified by the oxacillin disk-diffusion method. The pulsed-field gel electrophoresis (PFGE) type was determined for all MRSA isolates. A t statistic was used to compare the prevalence of colonization across biennia and across population subgroups. Cofactors independently associated with colonization were determined with backward stepwise logistic modeling. RESULTS The prevalence of colonization with S. aureus decreased from 32.4% in 2001-2002 to 28.6% in 2003-2004 (P < .01), whereas the prevalence of colonization with MRSA increased from 0.8% to 1.5% (P < .05). Colonization with MRSA was independently associated with healthcare exposure in males and with having been born in the United States, age > or =60 years, diabetes, and poverty in females. In 2003-2004, a total of 19.7% (95% confidence interval, 12.4%-28.8%) of MRSA-colonized persons carried a PFGE type associated with community transmission. CONCLUSIONS Nasal colonization with MRSA has increased in the United States, despite an overall decrease in nasal colonization with S. aureus. PFGE types associated with community transmission only partially account for the increase in MRSA colonization.

Prevalence of Staphylococcus aureus Nasal Colonization in the United States, 2001–2002

BACKGROUND Staphylococcus aureus is a common cause of disease, particularly in colonized persons. Although methicillin-resistant S. aureus (MRSA) infection has become increasingly reported, population-based S. aureus and MRSA colonization estimates are lacking. METHODS Nasal samples for S. aureus culture and sociodemographic data were obtained from 9622 persons > or = 1 year old as part of the National Health and Nutrition Examination Survey, 2001-2002. After screening for oxacillin susceptibility, MRSA and selected methicillin-susceptible S. aureus isolates were tested for antimicrobial susceptibility, pulsed-field gel electrophoresis clonal type, toxin genes (e.g., for Panton-Valentine leukocidin [PVL]), and staphylococcal cassette chromosome mec (SCCmec) type I-IV genes. RESULTS For 2001-2002, national S. aureus and MRSA colonization prevalence estimates were 32.4% (95% confidence interval [CI], 30.7%-34.1%) and 0.8% (95% CI, 0.4%-1.4%), respectively, and population estimates were 89.4 million persons (95% CI, 84.8-94.1 million persons) and 2.3 million persons (95% CI, 1.2-3.8 million persons), respectively. S. aureus colonization prevalence was highest in participants 6-11 years old. MRSA colonization was associated with age > or = 60 years and being female but not with recent health-care exposure. In unweighted analyses, the SCCmec type IV gene was more frequent in isolates from participants of younger age and of non-Hispanic black race/ethnicity; the PVL gene was present in 9 (2.4%) of 372 of isolates tested. CONCLUSIONS Many persons in the United States are colonized with S. aureus; prevalence rates differ demographically. MRSA colonization prevalence, although low nationally in 2001-2002, may vary with demographic and organism characteristics.

Characterization of a Strain of Community-Associated Methicillin-Resistant Staphylococcus aureus Widely Disseminated in the United States

ABSTRACT A highly stable strain of Staphylococcus aureus with a pulsed-field gel electrophoresis type of USA300 and multilocus sequence type 8 has been isolated from patients residing in diverse geographic regions of the United States. This strain, designated USA300-0114, is a major cause of skin and soft tissue infections among persons in community settings, including day care centers and correctional facilities, and among sports teams, Native Americans, men who have sex with men, and military recruits. The organism is typically resistant to penicillin, oxacillin, and erythromycin (the latter mediated by msrA) and carries SCCmec type IVa. This strain is variably resistant to tetracycline [mediated by tet(K)]; several recent isolates have decreased susceptibility to fluoroquinolones. S. aureus USA300-0114 harbors the genes encoding the Panton-Valentine leucocidin toxin. DNA sequence analysis of the direct repeat units within the mec determinant of 30 USA300-0114 isolates revealed differences in only a single isolate. Plasmid analysis identified a common 30-kb plasmid that hybridized with blaZ and msrA probes and a 3.1-kb cryptic plasmid. A 4.3-kb plasmid encoding tet(K) and a 2.6-kb plasmid encoding ermC were observed in a few isolates. DNA microarray analysis was used to determine the genetic loci for a series of virulence factors and genes associated with antimicrobial resistance. Comparative genomics between USA300-0114 and three other S. aureus lineages (USA100, USA400, and USA500) defined a set of USA300-0114-specific genes, which may facilitate the strains pathogenesis within diverse environments.

Vancomycin-Resistant Staphylococcus aureus Isolate from a Patient in Pennsylvania

ABSTRACT A vancomycin-resistant Staphylococcus aureus (VRSA) isolate was obtained from a patient in Pennsylvania in September 2002. Species identification was confirmed by standard biochemical tests and analysis of 16S ribosomal DNA, gyrA, and gyrB sequences; all of the results were consistent with the S. aureus identification. The MICs of a variety of antimicrobial agents were determined by broth microdilution and macrodilution methods following National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The isolate was resistant to vancomycin (MIC = 32 μg/ml), aminoglycosides, β-lactams, fluoroquinolones, macrolides, and tetracycline, but it was susceptible to linezolid, minocycline, quinupristin-dalfopristin, rifampin, teicoplanin, and trimethoprim-sulfamethoxazole. The isolate, which was originally detected by using disk diffusion and a vancomycin agar screen plate, was vancomycin susceptible by automated susceptibility testing methods. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA indicated that the isolate belonged to the USA100 lineage (also known as the New York/Japan clone), the most common staphylococcal PFGE type found in hospitals in the United States. The VRSA isolate contained two plasmids of 120 and 4 kb and was positive for mecA and vanA by PCR amplification. The vanA sequence was identical to the vanA sequence present in Tn1546. A DNA probe for vanA hybridized to the 120-kb plasmid. This is the second VRSA isolate reported in the United States.

Epidemics of Diarrhea Caused by a Clindamycin-Resistant Strain of Clostridium difficile in Four Hospitals

BACKGROUND Large outbreaks of diarrhea caused by a newly recognized strain of Clostridium difficile occurred in four hospitals located in different parts of the United States between 1989 and 1992. Since frequent use of clindamycin was associated with the outbreak in one of the hospitals, we examined the resistance genes of the epidemic-strain isolates and studied the role of clindamycin use in these outbreaks. METHODS Case-control studies were performed at three of the four hospitals to assess the relation of the use of clindamycin to C. difficile-associated diarrhea. All isolates of the epidemic strain and representative isolates of other strains identified during each outbreak were tested for susceptibility to clindamycin. Chromosomal DNA from these representative isolates was also analyzed by dot blot hybridization and amplification with the polymerase chain reaction (PCR) with the use of probes and primers from a previously described determinant of erythromycin resistance - the erythromycin ribosomal methylase B (ermB) gene - found in C. perfringens and C. difficile. RESULTS In a stratified analysis of the case-control studies with pooling of the results according to the Mantel-Haenszel method, we found that the use of clindamycin was significantly increased among patients with diarrhea due to the epidemic strain of C. difficile, as compared with patients whose diarrhea was due to nonepidemic strains (pooled odds ratio, 4.35; 95 percent confidence interval, 2.02 to 9.38; P<0.001). Exposure to other types of antibiotics or hospitalization in a surgical ward was not significantly associated with the risk of C. difficile-associated diarrhea due to the epidemic strain. All epidemic-strain isolates were highly resistant to clindamycin (minimal inhibitory concentration, >256 microg per milliliter). DNA hybridization and PCR analysis showed that all these isolates had an ermB gene, which encodes a 23S ribosomal RNA methylase that mediates resistance to macrolide, lincosamide, and streptogramin antibiotics. Only 15 percent of the nonepidemic strains were resistant to clindamycin. CONCLUSIONS A strain of C. difficile that is highly resistant to clindamycin was responsible for large outbreaks of diarrhea in four hospitals in different states. The use of clindamycin is a specific risk factor for diarrhea due to this strain. Resistance to clindamycin further increases the risk of C. difficile-associated diarrhea, an established complication of antimicrobial use.

Comparison of Seven Techniques for Typing International Epidemic Strains of Clostridium difficile: Restriction Endonuclease Analysis, Pulsed-Field Gel Electrophoresis, PCR-Ribotyping, Multilocus Sequence Typing, Multilocus Variable-Number Tandem-Repeat Analysis, Amplified Fragment Length Polymorphism, and Surface Layer Protein A Gene Sequence Typing

ABSTRACT Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

Severe community-acquired pneumonia due to Staphylococcus aureus, 2003-04 influenza season

S. aureus community-acquired pneumonia has been reported from 9 states.

Outbreak of Clostridium difficile Infection in a Long-Term Care Facility: Association with Gatifloxacin Use

To determine the cause of an increase in the rate of Clostridium difficile-associated diarrhea (CDAD) in a long-term care facility (LTCF), we analyzed CDAD cases among LTCF patients from October 2001 through June 2002. CDAD cases were identified from review of all enzyme immunoassays positive for C. difficile toxin A. The increase coincided with a formulary change from levofloxacin to gatifloxacin. We performed a case-control study in which we randomly selected control subjects from 612 LTCF admissions during this period. Although we examined a variety of risk factors, logistic regression analysis only demonstrated associations between CDAD and use of clindamycin (P=.005) and gatifloxacin, the latter being associated with an increasing risk of CDAD with increasing duration of gatifloxacin therapy (P<.0001). We concluded that an outbreak of CDAD in an LTCF was associated with a formulary change from levofloxacin to gatifloxacin. The rate of CDAD in the LTCF decreased after a change back to levofloxacin.