Yun F. Wang

Emergence of Community-Acquired Methicillin-Resistant Staphylococcus aureus USA 300 Clone as the Predominant Cause of Skin and Soft-Tissue Infections

Context In community outbreaks of methicillin-resistant Staphylococcus aureus (MRSA), 2 clones predominate in the United States: USA 300 and USA 400. Little is known about these infections in the nonoutbreak setting. Contribution This study evaluated nonoutbreak community-acquired S. aureus skin and soft-tissue infections in patients in a large urban setting. Almost three quarters of the soft-tissue infections were caused by MRSA, and these were predominantly the USA 300 type. No MRSA USA 300 isolate was resistant to trimethoprimsulfamethoxazole or vancomycin, while most were resistant to erythromycin in addition to -lactams. Implications Antibiotic choice for serious nonoutbreak community-acquired skin and soft-tissue infection should consider high rates of MRSA in some communities. The Editors Methicillin-resistant Staphylococcus aureus (MRSA) infections have usually been associated with exposure to health care settings, but they have recently been recognized in persons without traditional risk factors. These infections have been called community-acquired or community-associated MRSA (1). Most cases have been associated with skin and soft-tissue infection and have been reported among selected populations, including correctional facility inmates, homosexual men, and sports teams (2-5). Molecular typing studies in the United States and Australia have demonstrated that most community-acquired MRSA infections are caused by one of several clones or pulsed-field types (6, 7). In the United States, 2 clones, designated as USA 300 and USA 400 by the Centers for Disease Control and Prevention (CDC), have been identified as the primary types that cause community-acquired MRSA infections (6). The outbreaks of MRSA skin and soft-tissue infections observed in correctional facilities and among athletes have been associated with the USA 300 pulsed-field type, while outbreaks associated with severe and fatal disease in children, as well as skin and soft-tissue infections in Native American populations, have been associated with the USA 400 pulsed-field type (6). The community-acquired MRSA clones have frequently been associated with the PantonValentine leukocidin virulence factor and the presence of staphylococcal chromosome cassette mec (SCCmec) type IV allele (1, 6-9). In contrast, hospital-acquired or health careassociated MRSA strains usually lack genes for PantonValentine leukocidin and are associated with other SCCmec alleles (for example, SCCmec type II) (1, 6, 7). In addition to PantonValentine leukocidin and SCCmec type IV, the community-acquired MRSA USA 300 and USA 400 genotypes have usually demonstrated resistance to -lactams and erythromycin while retaining susceptibility to clindamycin, trimethoprimsulfamethoxazole, and fluoroquinolones, whereas health careassociated genotypes are often multidrug-resistant (1, 6). Although increasingly reported as a cause of outbreaks of skin and soft-tissue infection, the proportion of S. aureus skin and soft-tissue infections caused by community-acquired MRSA in nonoutbreak settings remains poorly defined. We sought to determine the proportion of infections caused by community-acquired MRSA, the clinical characteristics associated with community-acquired MRSA, and the molecular epidemiology of community-acquired MRSA among persons with community-onset S. aureus skin and soft-tissue infection who were receiving care at a large hospital and its affiliated clinics in urban Atlanta, Georgia. Methods Laboratory Surveillance for Community-Onset S. aureus Skin and Soft-Tissue Infection We conducted active, prospective laboratory surveillance from 1 August 2003 to 15 November 2003 to identify S. aureus isolates of patients with skin and soft-tissue infections at the Grady Health System. The Grady Health System includes Grady Memorial Hospital, a 1000-bed public inner-city hospital, and its affiliated outpatient clinics. We limited surveillance to S. aureus isolates that were obtained from patients with a community-onset skin and soft-tissue infection (isolates designated either as an exudate or a body fluid culture). We defined community-onset S. aureus by positive test results from cultures that were obtained within 72 hours of admission or at an outpatient visit. An investigator classified community-onset S. aureus before we performed molecular typing studies or before we reviewed antimicrobial susceptibility results other than methicillin susceptibility. We identified S. aureus and tested antimicrobial susceptibility for all isolates in the Grady Memorial Hospital Clinical Microbiology Laboratory in accordance with the National Committee for Clinical Laboratory Standards (10). Study Sample and Data Collection The Emory University Institutional Review Board and the Grady Research Oversight Committee approved the study. Grady Memorial Hospital provides medical care for a primarily medically indigent inner-city population, approximately 80% of whom are African American, residing within inner-city Atlanta (Fulton County and DeKalb County). The hospital serves as a teaching hospital for both Emory University School of Medicine and Morehouse School of Medicine. The referral pattern for the hospital and its affiliated clinics is primarily patient self-referral on the basis of place of residence (that is, residence within DeKalb or Fulton Counties) or indigent status, with some patients referred for tertiary academic medical care. We retrospectively reviewed computerized medical and laboratory records to document patient demographic characteristics, HIV status, presence of end-stage renal disease, hospitalization within the previous 12 months, and any history of MRSA infection or colonization. The reviewer completed the abstraction process blinded to the molecular typing results of MRSA isolates. We documented the need for hospitalization for managing the S. aureus skin and soft-tissue infection. We obtained S. aureus antimicrobial susceptibility profiles from the hospitals microbiology laboratory, and we categorized infections as due to either methicillin-susceptible S. aureus (MSSA) or MRSA. We reviewed computerized pharmacy records to document the antimicrobial agent therapy that was used to treat the patients skin and soft-tissue infection. We considered antimicrobial agent therapy to be adequate if the prescribed antimicrobial agent had in vitro activity against the isolated S. aureus strain. Molecular Typing and Genetic Analyses We performed pulsed-field gel electrophoresis (PFGE) using SmaI as a restriction endonuclease on all available MRSA isolates, as described by Bannerman and colleagues (11). We digitized gels and saved them as an image for analysis with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). We performed cluster analysis by using the unweighted pair-group method based on Dice coefficients. We defined pulsed-field type clusters by using a similarity coefficient of at least 80% and the criteria of Tenover and colleagues (12). By using the nomenclature outlined by McDougal and colleagues (6), we categorized MRSA isolates into 1 of 8 lineages: USA 100 through USA 800. For some isolates, we determined SCCmec type by using polymerase chain reaction (PCR)typing of the mec gene complex as described by Okuma and colleagues (7). Similarly, we assessed the presence of PantonValentine leukocidin genes in selected MRSA isolates by using PCR as previously described by Lina and colleagues (13). Classification of Community-Onset S. aureus Skin and Soft-Tissue Infections We classified community-onset S. aureus skin and soft-tissue infections into 3 groups on the basis of either molecular typing results (pulsed-field type) of MRSA isolates or antimicrobial susceptibility pattern in cases in which the MRSA isolate was not available for PFGE. The 3 groups were 1) the community-acquired MRSA USA 300/USA 400 group, 2) the other MRSA group, and 3) the MSSA group. The community-acquired MRSA USA 300/USA 400 group was defined by MRSA skin and soft-tissue infections caused by a MRSA isolate that either demonstrated a USA 300 or USA 400 pulsed-field type (6) or had an antimicrobial susceptibility profile demonstrating resistance only to -lactams and erythromycin while retaining susceptibility to clindamycin, levofloxacin, trimethoprimsulfamethoxazole, and vancomycin. The other MRSA group was defined by MRSA skin and soft-tissue infections caused by a MRSA isolate that either demonstrated a pulsed-field type other than USA 300 or USA 400 (for example, USA 100, USA 500, or USA 800 [which have all been described as health careassociated pulsed-field types]) or had an antimicrobial susceptibility profile demonstrating resistance to -lactams, erythromycin, and at least 1 additional antibiotic. We defined the MSSA group by skin and soft-tissue infections caused by MSSA. Statistical Analysis We performed data management and statistical analyses by using Microsoft Excel 2000 software (Microsoft Corp., Redmond, Washington) and SAS software, version 8.2 (SAS Institute, Cary, North Carolina). We assessed risk factors for community-acquired MRSA skin and soft-tissue infection in 2 separate analyses. Our first analysis compared the community-acquired MRSA USA 300/USA 400 group with the MSSA group, and our second analysis compared the community-acquired MRSA USA 300/USA 400 group with the other MRSA group. In addition, we repeated each analysis using data that were limited to those skin and soft-tissue infections in which a MRSA isolate was available for PFGE to account for any potential misclassification bias introduced when skin and soft-tissue infections were classified according to antimicrobial susceptibility profile. We initially identified potential risk factors for community-acquired MRSA skin and soft-tissue infection by univariate analysis. We calculated prevalence ratios and the corresponding 95% CIs. Multivariable log-binomial regression models included variables that were

Emergence of Community-Associated Methicillin-Resistant Staphylococcus aureus USA300 Genotype as a Major Cause of Health Care—Associated Blood Stream Infections

BACKGROUND Whether community-associated methicillin-resistant Staphylococcus aureus (MRSA) genotypes (e.g., USA300) are a major cause of bloodstream infections (BSIs) and health care-associated infections has been poorly defined. METHODS Consecutive MRSA isolates recovered from patients with BSIs were prospectively collected at an urban public hospital. Molecular typing studies were performed. Prevalence and risk factors for the MRSA USA300 genotype were assessed. RESULTS One hundred thirty-two cases of MRSA BSI were documented over 7.5 months in 2004 (incidence, 6.79 per 1000 admissions); 116 isolates were available for genotyping. Characteristics of the 116 evaluable cases included: a mean age 47 years; 62% were male, 82% were African American, and 22% were HIV seropositive. The crude in-hospital mortality rate was 22%. In 107 cases (92%), there was contact with a health care facility within the year prior to infection, and a nosocomial infection (defined as positive blood culture results obtained >48 h after admission) occurred in 49 cases (42%). PFGE demonstrated that 39 (34%) of the 116 isolates were the MRSA USA300 genotype; 34 (29%) were USA100; 42 (36%) were USA500; and 1 (1%) was USA800. MRSA USA300 accounted for 28% of health care-associated BSIs and 20% of nosocomial MRSA BSIs. In multivariate analysis, isolation of the USA300 genotype was associated with injectiondrug use (OR, 3.67; 95% CI, 1.10-12.28) and skin and soft tissue infection (OR, 4.26; 95% CI, 1.08-16.84). Patients who resided in long-term care facilities (OR, 0.09; 95% CI, 0.01-0.82) and those who were treated with antimicrobials in the prior year were less likely to have MRSA USA300 genotype recovered (OR, 0.10; 95% CI, 0.02-0.49). CONCLUSIONS MRSA USA300 genotype, the predominant cause of community-associated MRSA infections in our area (Atlanta, GA), has now emerged as a significant cause of health care-associated and nosocomial BSI. MRSA USA300 as a nosocomial pathogen presents new challenges to infection control programs.

Conjugated bile acids activate the sphingosine-1-phosphate receptor 2 in primary rodent hepatocytes.

Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including extracellular regulated kinase (ERK)1/2 and protein kinase B (AKT) as well as the G‐protein–coupled receptor (GPCR) membrane‐type bile acid receptor (TGR5/M‐BAR). Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be sensitive to pertussis toxin (PTX) and dominant‐negative Gαi in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid‐activated phylogenetic family (expressed in HEK293 cells) identified sphingosine‐1‐phosphate receptor 2 (S1P2) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P‐induced activation of ERK1/2 and AKT were significantly inhibited by JTE‐013, a S1P2 antagonist, in primary rat hepatocytes. JTE‐013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knockdown of the expression of S1P2 by a recombinant lentivirus encoding S1P2 shRNA markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P2 knock out (S1P2−/−) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P2 can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily through S1P2 in primary rodent hepatocytes. (HEPATOLOGY 2012)

Rapid Identification of Bacteria and Yeasts from Positive-Blood-Culture Bottles by Using a Lysis-Filtration Method and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum Analysis with the SARAMIS Database

ABSTRACT Rapid identification of microorganisms causing bloodstream infections directly from a positive blood culture would decrease the time to directed antimicrobial therapy and greatly improve patient care. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable method for identifying microorganisms from positive culture. This study evaluates the performance of a novel filtration-based method for processing positive-blood-culture broth for immediate identification of microorganisms by MALDI-TOF with a Vitek MS research-use-only system (VMS). BacT/Alert non-charcoal-based blood culture bottles that were flagged positive by the BacT/Alert 3D system were included. An aliquot of positive-blood-culture broth was incubated with lysis buffer for 2 to 4 min at room temperature, the resulting lysate was filtered through a membrane, and harvested microorganisms were identified by VMS. Of the 259 bottles included in the study, VMS identified the organisms in 189 (73%) cultures to the species level and 51 (19.7%) gave no identification (ID), while 6 (2.3%) gave identifications that were considered incorrect. Among 131 monomicrobic isolates from positive-blood-culture bottles with one spot having a score of 99.9%, the IDs for 131 (100%) were correct to the species level. In 202 bottles where VMS was able to generate an ID, the IDs for 189 (93.6%) were correct to the species level, whereas the IDs provided for 7 isolates (3.5%) were incorrect. In conclusion, this method does not require centrifugation and produces a clean spectrum for VMS analysis in less than 15 min. This study demonstrates the effectiveness of the new lysis-filtration method for identifying microorganisms directly from positive-blood-culture bottles in a clinical setting.

Same Day Identification and Full Panel Antimicrobial Susceptibility Testing of Bacteria from Positive Blood Culture Bottles Made Possible by a Combined Lysis-Filtration Method with MALDI-TOF VITEK Mass Spectrometry and the VITEK2 System

Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001). Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship.

Outbreak of Pseudomonas aeruginosa infection associated with contamination of a flexible bronchoscope.

BACKGROUND A cluster of patients with respiratory cultures positive for Pseudomonas aeruginosa with a unique antibiogram was observed during June and July 2007 at a 1,000-bed urban teaching hospital in Atlanta, Georgia. These P. aeruginosa isolates were recovered from bronchoscopically obtained specimens. METHODS A cross-sectional study was performed to assess whether the cluster was associated with exposure to a particular bronchoscope (B1); cultures from specimens from the bronchoscopes and the environment were obtained, and the P. aeruginosa isolate type was determined using pulsed-field gel electrophoresis (PFGE). Records of patients exposed to B1 during the cluster period were reviewed. RESULTS Twelve patients with a culture positive for P. aeruginosa with the unique susceptibility pattern were identified in June-July 2007. No cases were documented from March 1 through May 31, 2007. Culture specimens obtained from B1 after high-level disinfection revealed P. aeruginosa, prompting removal of B1 from service on July 23, 2007. No cases occurred after that date. Eleven (55%) of 20 patients who were exposed to B1 during the cluster period had a culture positive for P. aeruginosa, compared with 1 (2%) of 53 patients who were exposed to other bronchoscopes (P < .001). PFGE patterns for P. aeruginosa isolates obtained from case patients and from B1 were identical. An engineering evaluation of B1 documented several internal damages. Two (10.5%) of 19 patients exposed to B1 during the cluster period may have developed P. aeruginosa infection following exposure to B1. CONCLUSIONS An outbreak or pseudo-outbreak of P. aeruginosa infection occurred in association with use of a damaged bronchoscope. Periodic engineering maintenance may be needed to prevent bronchoscope contamination that is resistant to high-level disinfection.

Sexually Transmissible Infections and Prostate Cancer Risk

Background: Sexually transmissible infections (STI) have been variably associated with increased risks of prostate cancer, largely in case-control studies. Methods: In the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, we examined risk of prostate cancer in relation to serum antibodies to Chlamydia trachomatis, human papillomavirus-16 and -18, herpes simplex virus-2, cytomegalovirus, and human herpesvirus-8 in 868 cases (765 Whites and 103 Blacks) and 1,283 controls matched by race, age, time since initial screening, and year of blood draw; all blood samples were collected at least 1 year before prostate cancer diagnosis, except for 43 Black cases. We also assessed risk associated with self-reported history of syphilis and gonorrhea. Results: Prevalences of the 7 STIs among controls were weakly correlated, and all were more frequent among Blacks than Whites, except for human herpesvirus-8. Among Whites, prostate cancer risk was not significantly associated with the individual infections or with their number (Ptrend = 0.1); however, men with one or more STI had slightly higher risk (odds ratio, 1.3; 95% confidence interval, 1.0-1.6). Among Blacks, excess risk was associated with IgA antibody to C. trachomatis (odds ratio, 2.1; 95% confidence interval, 1.2-3.6). Conclusion: This large prospective study of prostate cancer shows no consistent association with specific STIs and a borderline association with any versus none. Whether a shared response or correlated infection not directly measured underlies the weak association requires further study. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2374–81)

Conjugated bile acid–activated S1P receptor 2 is a key regulator of sphingosine kinase 2 and hepatic gene expression

Bile acids are important hormones during the feed/fast cycle, allowing the liver to coordinately regulate nutrient metabolism. How they accomplish this has not been fully elucidated. Conjugated bile acids activate both the ERK1/2 and AKT signaling pathways via sphingosine 1‐phosphate receptor 2 (S1PR2) in rodent hepatocytes and in vivo. Here, we report that feeding mice a high‐fat diet, infusion of taurocholate into the chronic bile fistula rat, or overexpression of the gene encoding S1PR2 in mouse hepatocytes significantly upregulated hepatic sphingosine kinase 2 (SphK2) but not SphK1. Key genes encoding nuclear receptors/enzymes involved in nutrient metabolism were significantly downregulated in livers of S1PR2–/– and SphK2–/– mice. In contrast, overexpression of the gene encoding S1PR2 in primary mouse hepatocytes differentially increased SphK2, but not SphK1, and mRNA levels of key genes involved in nutrient metabolism. Nuclear levels of sphingosine‐1‐phosphate, an endogenous inhibitor of histone deacetylases 1 and 2, as well as the acetylation of histones H3K9, H4K5, and H2BK12 were significantly decreased in hepatocytes prepared from S1PR2–/– and SphK2–/– mice. Conclusion: Both S1PR2–/– and SphK2–/– mice rapidly developed fatty livers on a high‐fat diet, suggesting the importance of conjugated bile acids, S1PR2, and SphK2 in regulating hepatic lipid metabolism. (Hepatology 2015;61:1216–1226)

Susceptibility of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae According to the New CLSI Breakpoints

ABSTRACT In 2010 the Clinical and Laboratory Standards Institute (CLSI) lowered the susceptibility breakpoints of some cephalosporins and aztreonam for Enterobacteriaceae and eliminated the need to perform screening for extended-spectrum β-lactamases (ESBLs) and confirmatory tests. The aim of this study was to determine how many ESBL-producing strains of three common species of Enterobacteriaceae test susceptible using the new breakpoints. As determined with the CLSI screening and confirmatory tests, 382 consecutive ESBL-producing strains were collected at Huashan Hospital between 2007 and 2008, including 158 strains of Escherichia coli, 164 of Klebsiella pneumoniae, and 60 of Proteus mirabilis. Susceptibility was determined by the CLSI agar dilution method. CTX-M-, TEM-, and SHV-specific genes were determined by PCR amplification and sequencing. bla CTX-M genes alone or in combination with bla SHV were present in 92.7% (354/382) of these ESBL-producing strains. Forty-two (25.6%) strains of K. pneumoniae harbored SHV-type ESBLs alone or in combination. No TEM ESBLs were found. Utilizing the new breakpoints, all 382 strains were resistant to cefazolin, cefotaxime, and ceftriaxone, while 85.0 to 96.7% of P. mirabilis strains tested susceptible to ceftazidime, cefepime, and aztreonam, 41.8 to 45.6% of E. coli strains appeared to be susceptible to ceftazidime and cefepime, and 20.1% of K. pneumoniae were susceptible to cefepime. In conclusion, all ESBL-producing strains of Enterobacteriaceae would be reported to be resistant to cefazolin, cefotaxime, and ceftriaxone by using the new CLSI breakpoints, but a substantial number of ESBL-containing P. mirabilis and E. coli strains would be reported to be susceptible to ceftazidime, cefepime, and aztreonam, which is likely due to the high prevalence of CTX-M type ESBLs.

Colonic inflammation and secondary bile acids in alcoholic cirrhosis

Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier and inflammation. Gut microbiota can transform primary bile acids (BA) to secondary BAs, which can adversely impact the gut barrier. The purpose of this study was to define the effect of active alcohol intake on fecal BA levels and ileal and colonic inflammation in cirrhosis. Five age-matched groups {two noncirrhotic (control and drinkers) and three cirrhotic [nondrinkers/nonalcoholics (NAlc), abstinent alcoholic for >3 mo (AbsAlc), currently drinking (CurrAlc)]} were included. Fecal and serum BA analysis, serum endotoxin, and stool microbiota using pyrosequencing were performed. A subgroup of controls, NAlc, and CurrAlc underwent ileal and sigmoid colonic biopsies on which mRNA expression of TNF-α, IL-1β, IL-6, and cyclooxygenase-2 (Cox-2) were performed. One hundred three patients (19 healthy, 6 noncirrhotic drinkers, 10 CurrAlc, 38 AbsAlc, and 30 NAlc, age 56 yr, median MELD: 10.5) were included. Five each of healthy, CurrAlc, and NAlc underwent ileal/colonic biopsies. Endotoxin, serum-conjugated DCA and stool total BAs, and secondary-to-primary BA ratios were highest in current drinkers. On biopsies, a significantly higher mRNA expression of TNF-α, IL-1β, IL-6, and Cox-2 in colon but not ileum was seen in CurrAlc compared with NAlc and controls. Active alcohol use in cirrhosis is associated with a significant increase in the secondary BA formation compared with abstinent alcoholic cirrhotics and nonalcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury.