Patient-level analysis of incident vancomycin-resistant enterococci colonization and antibiotic days of therapy.

Vancomycin-resistant enterococci (VRE) infections are a public health threat associated with increased patient mortality and healthcare costs. Antibiotic usage, particularly cephalosporins, has been associated with VRE colonization and VRE bloodstream infections (VRE BSI). We examined the relationship between antimicrobial usage and incident VRE colonization at the individual patient level. Prospective, weekly surveillance was undertaken for incident VRE colonization defined by negative admission but positive surveillance swab in a medical intensive care unit over a 17-month period. Antimicrobial exposure was quantified as days of therapy (DOT)/1000 patient-days. Multiple logistic regression was used to analyse incident VRE colonization and antibiotic DOT, controlling for demographic and clinical covariates. Ninety-six percent (1398/1454) of admissions were swabbed within 24 h of intensive care unit (ICU) arrival and of the 380 patients in the ICU long enough for weekly surveillance, 83 (22%) developed incident VRE colonization. Incident colonization was associated in bivariate analysis with male gender, more previous hospital admissions, longer previous hospital stay, and use of cefepime/ceftazidime, fluconazole, azithromycin, and metronidazole (P < 0·05). After controlling for demographic and clinical covariates, metronidazole was the only antibiotic independently associated with incident VRE colonization (odds ratio 2·0, 95% confidence interval 1·2-3·3, P < 0·009). Our findings suggest that risk of incident VRE colonization differs between individual antibiotic agents and support the possibility that antimicrobial stewardship may impact VRE colonization and infection.

Comparative genotyping of Streptococcus mutans by repetitive extragenic palindromic polymerase chain reaction and multilocus sequence typing.

The genetic diversity of Streptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) is a genotyping approach used for screening large numbers of bacterial isolates. This two-part study used multilocus sequence typing (MLST) analysis to evaluate genotypes previously identified as unique using rep-PCR. In part one, an isolate was selected from each of the 22 S. mutans rep-PCR genotype groups representing 8000 clinical isolates. For part two, four additional isolates were selected from the six most commonly occurring genotype groups (GG) for further analysis. Real-time PCR was performed using eight housekeeping S. mutans gene loci and the amplicons were sequenced. Sequence data analysis was performed using CLC DNA Workbench and alleles were compared with the PubMLST database for Oral Streptococcus using the Nakano scheme. Concatenated sequences were evaluated with MEGA using a minimum evolution method with bootstrap. All 22 rep-PCR genotypes were unique by MLST analysis. Within rep-PCR GGs, MLST matched rep-PCR in three groups demonstrating clonality; three groups exhibited more diversity with MLST. The discovery of three clonal groups is unique to this study and suggests that S. mutans genotypes are shared between unrelated subjects. Furthermore, MLST defined 19 new alleles and 26 new sequence types that have been confirmed and registered with PubMLST. Methods for processing were streamlined and a process for using MLST with rep-PCR is suggested. In conclusion, MLST verified that rep-PCR is a reliable and cost-effective method for screening large numbers of S. mutans strains for epidemiological study.

Pneumocystis pneumonia in hospitalized patients: a detailed examination of symptoms, management, and outcomes in human immunodeficiency virus (HIV)-infected and HIV-uninfected persons.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) is a life-threatening infection for immunocompromised individuals. Robust data and clear guidelines are available for prophylaxis and treatment of human immunodeficiency virus (HIV)-related PCP (HIV-PCP), yet few data and no guidelines are available for non-HIV-related PCP (NH-PCP). We postulated that prevention and inpatient management of HIV-PCP differed from NH-PCP. METHODS: We performed a retrospective case review of all pathologically confirmed cases of PCP seen at the University of Alabama Medical Center from 1996 to 2008. Data on clinical presentation, hospital course, and outcome were collected using a standardized data collection instrument. Bivariate analysis compared prophylaxis, adjunctive corticosteroids, and clinical outcomes between patients with HIV-PCP and NH-PCP. RESULTS: Our analysis of the cohort included 97 cases of PCP; 65 HIV and 32 non-HIV cases. Non-HIV cases rarely received primary prophylaxis (4% vs. 38%, P = 0.01) and received appropriate antibiotics later in the course of hospitalization (5.2 days vs. 1.1 days, P < 0.005). Among transplant patients, NH-PCP was diagnosed a mean of 1066 days after transplantation and most patients were on low-dose corticosteroids (87%) at the time of disease onset. No significant differences in adjunctive corticosteroid use (69% vs. 77%, P = 0.39) and 90-day mortality (41% vs. 28%, P = 0.20) were detected. CONCLUSIONS: Patients who have undergone organ or stem cell transplant remain at risk for PCP for many years after transplantation. In our cohort, patients who developed NH-PCP were rarely given prophylaxis, and initiation of appropriate antibiotics was significantly delayed compared to cases of HIV-PCP. Medical providers should be aware of the ongoing risk for NH-PCP, even late after transplantation, and consider more aggressive approaches to both prophylaxis and earlier empirical therapy for PCP.

Genetic diversity of plaque mutans streptococci with rep-PCR.

Mutans streptococci (MS) are key organisms associated with the etiology of dental caries. Using probabilities that were tested by oversampling, we designed this study to determine the minimal number of MS isolates from an individual required to evaluate diversity of genotypes. MS isolates were genotyped by repetitive extragenic palindromic-polymerase chain-reaction (rep-PCR). Analysis of 20 isolates from individuals resulted in a mean of 1.6 and 2.4 genotypes in children (N = 12) and adults (N = 10), respectively. In a follow-up study, reducing the number of isolates to 7-10 resulted in a theoretical probability of up to 78% for detecting up to 4 genotypes. A mean of 1.5 genotypes was found in 35 children and 10 adults. These findings provide evidence for the design of studies of MS genotyping that can serve as a model for the analysis of genotypes within individuals.

Observational study of the epidemiology and outcomes of vancomycin-resistant Enterococcus bacteraemia treated with newer antimicrobial agents.

Vancomycin-resistant Enterococcus bloodstream infections (VRE-BSI) are a growing problem with few clinical trials to guide therapy. We conducted a retrospective study of management and predictors of mortality for VRE-BSI at a tertiary-care centre from January 2005 to August 2008. Univariate and multivariable analyses examined the relationship of patient characteristics and antibiotic therapy with 30-day all-cause mortality. Rates of VRE-BSI increased from 0·06 to 0·17 infections/1000 patient-days (P=0·03). For 235 patients, 30-day mortality was 34·9%. Patients were primarily treated with linezolid (44·2%) or daptomycin (36·5%). Factors associated with mortality were haemodialysis [odds ratio (OR) 3·2, 95% confidence interval (CI) 1·6-6·3, P=0·007], mechanical ventilation (OR 3·7, 95% CI 1·3-10·4, P=0·01), and malnutrition (OR 2·0, 95% CI 1·0-4·0, P=0·046). Use of linezolid, but not daptomycin (P=0·052) showed a trend towards an association with survival. In conclusion, VRE-BSI is a growing problem, associated with significant 30-day mortality. Multiple factors were associated with poor outcomes at our hospital.

Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR.

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

Repetitive extragenic palindromic PCR for study of Streptococcus mutans diversity and transmission in human populations.

Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for molecular epidemiological study. Repetitive extragenic palindromic PCR (rep-PCR) is less time-consuming and more suitable for analyzing large numbers of bacterial strains in human populations. PFGE and rep-PCR provide comparable genotyping results for investigating Streptococcus mutans diversity and transmission.

Multiple-locus variable-number tandem-repeat analysis of meticillin-resistant Staphylococcus aureus discriminates within USA pulsed-field gel electrophoresis types.

Many isolates of meticillin-resistant Staphylococcus aureus (MRSA) are indistinguishable when compared using the standard pulsed-field gel electrophoresis (PFGE) typing method. This may present a problem when investigating local outbreaks of MRSA transmission in a healthcare setting. It also impedes investigation of the widely disseminated community-acquired MRSA (USA 300-0114) in the inpatient setting, which is displacing other traditional hospital-acquired PFGE types. Combination of methods, including multiple-locus sequence typing (MLST), spa typing and staphylococcal cassette chromosome mec (SCCmec) typing, have been used with, or in place of, PFGE to characterise MRSA for epidemiological purposes. These methods are technically challenging, time-consuming and expensive and are rarely feasible except in large laboratories in tertiary care medical centres. Another method, which is simpler and with faster turnaround time, is multiple-locus variable-number tandem-repeat analysis (MLVA). We investigated the utility of MLVA to distinguish common PFGE types. The results suggest that MLVA can be used to identify unrelated strains with identical PFGE patterns or confirm close genetic composition of linked isolates. MLVA could potentially be used in conjunction with PFGE to validate relationships, but further prospective evaluation of these relationships will be required in order to define the proper role, if any, for use of this method in hospital epidemiology.

Emergence of USA300 MRSA in a tertiary medical centre: implications for epidemiological studies.

Community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) has become a major pathogen, particularly in outbreaks of skin and soft-tissue infection (SSTI). A preliminary study conducted at our institution in 2004 revealed that up to 45% of inpatient and 70% of outpatient MRSA isolates tested were the USA300 genotype. In this report, we used pulsed-field gel electrophoresis (PFGE) in a retrospective analysis to determine the time when CA-MRSA USA300 moved from the community to the inpatient population. During the five-year period 2000 to 2004, unique MRSA isolates (N=253) were selected from inpatients in surgical and medical intensive care units, the general hospital population and outpatients. The most common PFGE types found in all populations from 2000 to 2003 were USA100, USA200 and USA600. USA300 was absent from all inpatients from 2000 to 2003 and only sporadic numbers found in the outpatient group. However, in 2004 the USA300 strain emerged in both outpatient and hospitalised patients. There was no difference in the distribution of USA300 between ICUs and the general inpatient population. The emergence of CA-MRSA has resulted in a shift of the MRSA strains that are implicated in healthcare-associated infections in our institution. This has been a recent development that has implications as to the use of PFGE to determine transmission of MRSA in the inpatient setting. Further evaluation of these data in the context of the epidemiology of these infections is needed to determine if more discriminatory approaches to typing will be required for monitoring the spread of the more virulent CA-MRSA phenotype within the inpatient population.

Genotypic characterization of Ureaplasma species by pulsed field gel electrophoresis.

We describe the first use of pulsed field gel electrophoresis to genotype human Ureaplasma species. This technique can distinguish between U. urealyticum and U. parvum, differentiate most of the 14 serovars from one another, and identify differences among clinical isolates of the same serovar.

Association between antecedent intravenous antimicrobial exposure and isolation of vancomycin-resistant enterococci.

Vancomycin-resistant enterococci (VRE) have become important causes of nosocomial infections. This study evaluated the association between a variety of intravenous antimicrobial exposures and the isolation of VRE using two control groups: (1) a vancomycin-susceptible enterococci (VSE) group, to assess factors associated with development of VRE, and (2) a nonenterococci control group, to assess factors associated with positive cultures for enterococci without regard to vancomycin resistance. After adjusting for the effect of other antimicrobials, time at risk, and patient morbidity, compared to vancomycin-susceptible enterococci controls, exposures to imipenem (OR = 4.9, 95% CI = 1.6-14.1) and ceftazidime (OR = 2.6, 95% CI = 1.1-6.1) were significant predictors of VRE. When compared to nonenterococci controls, exposures to ampicillin (OR = 20.1, 95% CI = 1.5-263.1) and imipenem (OR = 5.1, 95% CI = 1.5-17.1) were significantly associated with VRE. Neither piperacillin nor vancomycin was associated with VRE compared to either control group. This study offers further evidence that the replacement of broad-spectrum cephalosporins by extended-spectrum penicillins, specifically piperacillin, may be effective in reducing VRE.

Vancomycin-resistant enterococci: 15 years and counting.

We review the history of vancomycin-resistant enterococci (VRE) and propose a causal model illustrating the roles of exposure to VRE reservoirs, patient characteristics, antimicrobial exposure, and prevalence of VRE in the progression from potential VRE reservoirs to active disease in hospitalized patients. Differences in VRE colonization and VRE infection are discussed with respect to hospital surveillance methodology and implications for interventions. We further document clonal transmission of VRE in a large, urban, teaching hospital and demonstrate VRE susceptibility to a wide array of antimicrobial agents. This model can guide the identification of mutable factors that are focal points for intervention.

Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.

Lactobacillus johnsonii strain 100-100 expresses two antigenically distinct conjugated bile salt hydrolases (BSH), alpha and beta, that combine to form native homo- and heterotrimers. This paper reports characterization of loci within the genome that encode this capacity. A locus that encodes BSH beta (cbsH beta), a partial (cbsT1) and a complete conjugated bile salt transporter (cbsT2) was identified previously. DNA sequence analysis at this locus was extended and revealed a complete ORF for cbsT1 and no other ORFs in tandem. The three genes, cbsT1, cbsT2 and cbsH beta, probably constitute an operon; a putative promoter was identified upstream of cbsT1. A second locus that expresses BSH activity in strain 100-100 was identified. Sequence analysis of the clone predicted a 978 nt ORF that did not share tandem organization with other ORFs, was similar in sequence to other BSH genes, and matched, in predicted protein sequence, the first 25 amino acids of BSH alpha. A phenotypic screen for BSH activity and a genetic screen for the cbsH beta locus were performed on 50 Lactobacillus isolates from humans or dairy products. Nearly all of the isolates that were positive for cbsH beta were from human sources. Variability in the BSH phenotype and cbsH beta genotype was identified in isolates of the same species. DNA sequence was obtained and analysed from the cbsH beta locus of one human isolate, L. acidophilus strain KS-13. This organism has cbsT1, cbsT2 and cbs beta genes that are 84, 87 and 85% identical in DNA sequence to those of strain 100-100. DNA sequence identity to strain 100-100 ends in regions flanking this locus. The findings of this study suggest that BSH genes have been acquired horizontally and that BSH activity is important at some level for lactobacilli to colonize the lower gastrointestinal tract.

Bacteremia after spinal cord injury in initial versus subsequent hospitalizations.

BACKGROUND: Individuals with spinal cord injury (SCI) have a high lifelong risk for systemic infection. For optimal therapy, it is important to characterize the organisms involved in bacteremic episodes and the sites of primary infection. The increase in drug-resistant bacteria in recent years underscores the importance of gathering accurate microbiological information. METHODS: We performed a retrospective study of hospitalized people with SCI using a computerized Microbiology Laboratory Database. We compared the microbiology of bacteremic episodes during initial versus unplanned subsequent hospitalizations. Data were collected on 55 bacteremic episodes in 30 people during initial hospitalization for SCI and 50 episodes in 29 people who were rehospitalized. RESULTS: Among cases in which a site of origin could be identified, the respiratory tract was the origin of the majority of bacteremias during initial hospitalizations, and the urinary tract was the primary origin during rehospitalizations. Polymicrobial bacteremia occurred in 14 of 55 (25%) initial versus 14 of 50 (28%) subsequent hospitalization episodes. The most common pathogens were coagulase-negative staphylococci, followed by Staphylococcus aureus and Enterobacteriaceae. Bacteremia was more common in people with tetraplegia and complete neurologic lesions than in those with paraplegia and incomplete lesions. One person in the rehospitalization group died from complications of bacteremia. All others were successfully treated. CONCLUSIONS: This study describes the frequency and characteristics of bacteremia during initial and subsequent hospitalizations following SCI and examines differences in original sites of infection. This information should be considered when planning infection control measures and empiric antibiotic regimens for patients with SCI.

Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli.

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.

Trends in frequency and susceptibilities of Candida glabrata bloodstream isolates at a university hospital.

The frequency of isolation and antifungal susceptibility patterns to fluconazole and itraconazole were determined for 166 Candida glabrata isolates causing bloodstream infection at a single institution from 1995-2000. Findings demonstrated a trend of increasing resistance to itraconazole among the isolates, but no trend in resistance to fluconazole. The frequency of C. glabrata isolates among all blood culture isolates of Candida spp. causing bloodstream infection remained stable during the study period and ranged from 18-31%.

Reduction of vancomycin-resistant enterococcal infections by limitation of broad-spectrum cephalosporin use in a trauma and burn intensive care unit.

Both vancomycin and third-generation cephalosporin use are believed to contribute to a rise in vancomycin-resistant enterococci (VRE) infections. In 1998, the largest number of VRE infections in our hospital occurred in the trauma/burn intensive care unit (TBICU), accounting for nearly 20% of hospital infections. In an attempt to control the VRE infection rate, antibiotic protocols for prophylaxis, empiric, and definitive therapy were initiated during the final quarter of 1998 to minimize cephalosporin use by the introduction of piperacillin/tazobactam. Therefore, we undertook a study of the VRE infection rate for the TBICU in relation to vancomycin, piperacillin/tazobactam, piperacillin, third-generation cephalosporin, and total cephalosporin use before and after efforts to limit cephalosporins. These data were compared to those in the medical and surgical intensive care units. During 1998, seven VRE infections occurred in the TBICU. Following initiation of antibiotic protocols, one case of VRE infection occurred in the subsequent month and no cases in the 17 months since. The decrease in the VRE infection rate corresponded with a significant increase in the use of piperacillin/tazobactam and a reduction in third-generation and total cephalosporin use. In contrast, cephalosporin use in the medical and surgical intensive care units remains significantly higher than in the TBICU, and neither unit has had a reduction in their VRE infection rates.