Methicillin-resistant Staphylococcus aureus: comparison of susceptibility testing methods and analysis of mecA-positive susceptible strains.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the mecA gene used as the "gold standard" assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecA was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that mecA- and PBP 2a-positive S. aureus isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA.

Activities of taurolidine in vitro and in experimental enterococcal endocarditis.

In vitro, the antimicrobial agent taurolidine inhibited virtually all of the bacteria tested, including vancomycin-resistant enterococci, oxacillin-resistant staphylococci, and Stenotrophomonas maltophilia, at concentrations between 250 and 2,000 microg/ml. Taurolidine was not effective in experimental endocarditis. While it appears unlikely that this antimicrobial would be useful for systemic therapy, its bactericidal activity and the resistance rates found (<10(-9)) are favorable indicators for its possible development for topical use.

Molecular typing of coagulase-negative staphylococci from blood cultures does not correlate with clinical criteria for true bacteremia.

PURPOSE: Determining whether a blood culture that contains coagulase-negative staphylococci represents bacteremia or contamination is a clinical dilemma. We compared molecular-typing results of coagulase-negative staphylococcal blood culture isolates with clinical criteria for true bacteremia. SUBJECTS AND METHODS: Pulsed-field gel electrophoresis and arbitrary primed polymerase chain reaction (PCR) were used to determine whether patients with two or more blood cultures with coagulase-negative staphylococcal isolates had the same strain of organism in each culture (same strain bacteremia). We evaluated three different clinical criteria for bacteremia: whether the patient received more than 4 days of antibiotics, whether there was an explicit note in the medical chart in which the physician diagnosed a true bacteremia, and the Centers for Disease Control surveillance criteria for primary bloodstream infection. Agreement between same-strain bacteremia and each definition was examined, based on the assumption that most true infections should be the result of a single strain. RESULTS: The study sample consisted of 42 patients and 106 isolates. Nineteen of the 42 bacteremias (45%) were the same strain. Classification of bacteremias as same-strain correlated poorly with all three clinical assessments (range of percent agreement, 50% to 57%; range of kappa statistic, 0.01 to 0.15). There were both false-positive and false-negative errors. Patients with three or more positive blood cultures were more likely to have same-strain bacteremia than those with only two positive cultures [11 of 15 (73%) vs 8 of 27 (30%), P = 0.006]. Pulsed-field gel electrophoresis was more discriminating than arbitrary primed PCR (percent agreement, 83%; kappa, 0.67). CONCLUSION: Molecular typing correlated poorly with clinical criteria for true bacteremia, suggesting either that true bacteremias are frequently the result of multiple strains or that the commonly used clinical criteria are not accurate for distinguishing contamination from true bacteremia. Vancomycin treatment of clinically defined coagulase-negative staphylococcal bacteremia may frequently be unnecessary.

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.

Epidemiology and clinical outcomes of patients with multiresistant Pseudomonas aeruginosa.

We conducted a case-series study of multiresistant Pseudomonas aeruginosa in patients who did not have cystic fibrosis. Patient characteristics, antibiotic exposures, time course of emergence of resistance, and clinical outcomes were examined. Twenty-two patients were identified from whom P. aeruginosa resistant to ciprofloxacin, imipenem, ceftazidime, and piperacillin was isolated. Nineteen (86%) had clinical infection. Patients received prolonged courses of antipseudomonal antibiotics before isolation of multiresistant P. aeruginosa. Nine of 11 patients with soft-tissue infection exhibited resolution of clinical infection but usually required surgical removal of infected tissue with or without revascularization. Overall, three patients died. In two instances in which multiple isolates with different susceptibility profiles from the same patient were available, pulsed-field gel electrophoresis profiles of serial isolates were indistinguishable or closely related. This study illustrates that multiresistant P. aeruginosa emerges in a stepwise manner after exposure to antipseudomonal antibiotics and results in adverse outcomes.

Molecular epidemiology of ceftazidime-resistant gram-negative bacilli on inanimate surfaces and their role in cross-transmission during nonoutbreak periods.

We described the molecular epidemiology of expanded-spectrum cephalosporin-resistant gram-negative bacilli (RGN) recovered from inanimate surfaces. RGN were isolated from 9% of environmental cultures. Numerous species, each with multiple unique strains, were recovered. Epidemiological links between environmental, personnel, and patient strains suggested the exogenous acquisition of RGN from the hospital environment.

Colonization with broad-spectrum cephalosporin-resistant gram-negative bacilli in intensive care units during a nonoutbreak period: prevalence, risk factors, and rate of infection.

OBJECTIVE: To define the epidemiology of broad-spectrum cephalosporin-resistant gram-negative bacilli in intensive care units (ICUs) during a nonoutbreak period, including the prevalence, the risk factors for colonization, the frequency of acquisition, and the rate of infection. DESIGN: Prospective cohort study. SETTING: Tertiary care hospital. PATIENTS: Consecutive patients admitted to two surgical ICUs. MAIN OUTCOME MEASUREMENTS: Serial patient surveillance cultures screened for ceftazidime (CAZ) resistance, antibiotic and hospital exposure, and infections. RESULTS: Of the 333 patients enrolled, 60 (18%) were colonized with CAZ-resistant gram-negative bacilli (CAZ-RGN) at admission. Clinical cultures detected CAZ-RGN in only 5% (3/60) of these patients. By using logistic regression, CAZ-RGN colonization was associated with duration of exposure to cefazolin (odds ratio, 10.3; p < or = .006) and to broad-spectrum cephalosporins/penicillins (odds ratio, 2; p < or = .03), Acute Physiology and Chronic Health Evaluation III score (odds ratio, 1.2; p < or = .008), and previous hospitalization (odds ratio, 3.1; p < or = .006). Of the 100 patients who remained in the surgical ICU for > or = 3 days, 26% acquired a CAZ-RGN. Of the 14 infections caused by CAZ-RGN, 11 (79%) were attributable to the same species present in surveillance cultures at admission to the surgical ICU. CONCLUSIONS: Colonization with CAZ-RGN was common and was usually not recognized by clinical cultures. Most patients colonized or infected with CAZ-RGN had positive surveillance cultures at the time of admission to the surgical ICU, suggesting that acquisition frequently occurred in other wards and institutions. Patients exposed to first-generation cephalosporins, as well as broad-spectrum cephalosporins/penicillins, were at high risk of colonization with CAZ-RGN. Empirical treatment of nosocomial gram-negative infections with broad-spectrum cephalosporins, especially in the critically ill patient, should be reconsidered.

Vancomycin-resistant enterococci in intensive care units: high frequency of stool carriage during a non-outbreak period.

BACKGROUND: We aimed to define the epidemiological associations of vancomycin-resistant enterococci (VRE) in intensive care units (ICUs) during a non-outbreak period by examining prevalence, risk factors for colonization, frequency of acquisition, and molecular strain types. DESIGN: A prospective cohort design was followed. Consecutive patient admissions to 2 surgical ICUs at a tertiary care hospital were enrolled. The main outcome measures were results of serial surveillance cultures screened for VRE. RESULTS: Of 290 patients enrolled, 35 (12%) had colonization with VRE on admission. The VRE colonization or infection had been previously detected by clinical cultures in only 4 of these patients. Using logistic regression, VRE colonization at the time of ICU admission was associated with second- and third-generation cephalosporins (odds ratio [OR] = 6.0, P<.0001), length of stay prior to surgical ICU admission (OR = 1.06, P = .001) greater than 1 prior ICU stay (OR = 9.6, P = .002), and a history of solid-organ transplantation (OR = 3.8, P = .021). Eleven (12.8%) of 78 patients with follow-up cultures acquired VRE. By pulsed-field gel electrophoresis, 2 strains predominated, one of which was associated with an overt outbreak on a non-ICU ward near the end of the study period. CONCLUSIONS: Colonization was common and usually not recognized by clinical culture. Most patients who had colonization with VRE and were on the surgical ICU acquired VRE prior to surgical ICU entry. Exposure to second- and third-generation cephalosporins, but not vancomycin, was an independent risk factor for colonization. Prospective surveillance of hospitalized patients may yield useful insights about the dissemination of nosocomial VRE beyond what is appreciated by clinical cultures alone.

Epidemiological analysis of imipenem-resistant Serratia marcescens in hospitalized patients.

Our objective was to examine epidemiological characteristics of hospitalized patients with imipenem-resistant Serratia marcescens. We performed a case-control study using data collected from computerized databases and chart review. Molecular typing by pulsed field gel electrophoresis of available isolates was performed. One hundred and ten patients had Serratia spp isolated during the 23-month study period. Twelve were infected or colonized with S. marcescens resistant or of intermediate susceptibility to imipenem. Eleven of the 12 patients were detected during a seven-month period between August 1994 and February 1995, suggesting the possible occurrence of an outbreak. However, the patients were admitted to different wards and services and, in eight patients, imipenem-resistant S. marcescens were isolated within 48 h or admission. None of the patients had epidemiological links within other institutions. The 12 cases were not more likely to have been exposed to beta-lactam antibiotics, including imipenem, than patients with imipenem-susceptible isolates. Six isolates were available for typing by PFGE; three were indistinguishable or closely related whereas each of the other three isolates were unique. In conclusion both the prevalence of imipenem-resistant S. marcescens and its unusual epidemiologic characteristics warrant further study.

The molecular and clinical epidemiology of enterobacteriaceae-producing extended-spectrum beta-lactamase in a tertiary care hospital.

To describe the epidemiology of Enterobacteriaceae-producing extended-spectrum beta-lactamase (EP-ESBL) in a non-outbreak setting, and to define the risk factors associated with colonization, a 5-month surveillance study was initiated. Ten of 333 patients were colonized with EP-ESBL, as defined by isoelectric focusing. Klebsiella sp. and Escherichia coli were the species most commonly harbouring these plasmid-mediated enzymes. Of the 16 SHV-producing isolates, 10 were SHV-3-like (pI 7.0) and six were SHV-5-like (pI 8.2). All isolates were resistant to ceftriaxone. Ceftazidime resistance was detected in 50% and 100% of SHV-3-like and SHV-5-like producing isolates, respectively. One patient was colonized with four different SHV-5-like producing Enterobacteriaceae. These isolates carried plasmids that were indistinguishable by restriction endonuclease analysis, indicating broad plasmid transfer within the patient. By logistic regression, haemodialysis was a strong risk factor for colonization with EP-ESBL, suggesting that, in our hospital, horizontal transmission is an important mechanism of dissemination of these resistant pathogens.

Carriage of methicillin-resistant Staphylococcus aureus at hospital admission.

OBJECTIVES: To measure the prevalence of, and to establish predictors for, the nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) at hospital admission. To evaluate mannitol-salt agar with oxacillin for the simultaneous detection and identification of MRSA from nasal swabs. DESIGN: Three-month prospective case-control survey, with data collected from interviews and computerized databases. The criterion standard for MRSA detection was culture on Mueller-Hinton agar with oxacillin 6 microg/mL (National Committee for Clinical Laboratory Standards method). SETTING: 320-bed tertiary-care hospital. PATIENTS: 387 patients screened within 24 hours after admission, including 10 MRSA carriers (cases), 291 patients with no S aureus, and 86 patients with methicillin-susceptible S aureus. RESULTS: The prevalence of MRSA nasal carriage was 2.6%, whereas the prevalence of carriage was 3.1% when both nasal and wound cultures were performed. The significant predictors of carriage were a prior detection of MRSA, open wounds, diabetes mellitus, treatments by injection, prior nursing home stays, visits at home by a nurse, and prior antibiotic treatments. Cases had stayed for longer periods in hospitals and had received longer antibiotic treatments within a year. Eighty patients (including the 10 cases) had diabetes, had been exposed to healthcare facilities within a year, and had antibiotics within 6 months. The sensitivity and negative predictive value of nasal swabs on mannitol-salt agar with oxacillin were 60% and 71%, respectively. CONCLUSION: MRSA carriage on admission to the hospital may be an increasing and underestimated problem. Further studies are needed to develop and validate a sensitive and specific prediction rule.

Stool colonization of healthcare workers with selected resistant bacteria.

We examined the carriage of selected resistant bacteria in the stools of healthcare workers who provided direct patient care. Neither vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, nor Clostridium difficile was recovered from the 55 stool specimens collected. A ceftazidime-resistant Citrobacter freundii was isolated from one specimen. We conclude that the stool of healthcare workers is colonized infrequently with these resistant organisms.

Failure of quality control measures to prevent reporting of false resistance to imipenem, resulting in a pseudo-outbreak of imipenem-resistant Pseudomonas aeruginosa.

False results showing an outbreak of Pseudomonas aeruginosa with resistance to imipenem were traced to a defective lot of microdilution MIC testing panels. These panels contained two- to threefold lower concentrations of imipenem than expected and resulted in artifactual two- to fourfold increases in MICs of imipenem. The quality-control MIC results for Pseudomonas aeruginosa ATCC 27853 were 4 microg/ml, the highest value within the range recommended by the National Committee for Clinical Laboratory Standards. We recommend that this value be considered out of the quality-control range.

Multicenter typing comparison of sporadic and outbreak Clostridium difficile isolates from geographically diverse hospitals.

In a collaborative study by three laboratories, arbitrarily primed polymerase chain reaction (AP-PCR), HindIII restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE) using SmaI were compared for typing of Clostridium difficile. The study included 30 isolates from nosocomial outbreaks in six geographically disparate hospitals and 15 isolates from sporadic cases of C. difficile diarrhea. REA distinguished a total of 23 types representing 10 groups; AP-PCR performed at Deaconess Hospital resolved 19 types; AP-PCR performed at the Centers for Disease Control resolved 15 types. Thirty isolates exhibited degradation of larger sized fragments during processing and therefore were nontypeable by PFGE; among the remaining 15 isolates, PFGE resolved 11 types. Outbreak isolates in five different hospitals represented REA group J and constituted a single AP-PCR strain. In summary, nosocomial outbreaks of C. difficile diarrhea in five hospitals were associated with a single genetic lineage as resolved by multiple strain typing systems.

Molecular epidemiology of acquisition of ceftazidime-resistant gram-negative bacilli in a nonoutbreak setting.

We prospectively studied the acquisition of ceftazidime-resistant gram-negative bacilli (CAZ-RGN) in two surgical intensive care units (SICU) during a nonoutbreak period. Surveillance cultures were obtained from patients at the time of admission and serially thereafter. CAZ-RGN isolates were typed by pulsed-field gel electrophoresis (PFGE). Three hundred and forty-three patients were enrolled from whom 1,621 baseline and follow-up cultures were obtained. The most common species isolated from patients were Pseudomonas aeruginosa (22), Enterobacter cloacae (21), Acinetobacter spp. (13), Enterobacter aerogenes (11), Citrobacter spp. (10), Pseudomonas spp. (non P. aeruginosa) (9), and Stenotrophomonas spp. (7). For each species, PFGE strain types were highly diverse; no single type was recovered from more than four patients. Twenty-eight patients acquired a CAZ-RGN during the SICU stay; in six (21%), emergence of resistance from a previously susceptible strain was documented on the basis of matching serial strain types. Transmission of CAZ-RGN between patients occurred but was infrequent, as judged by analyzing strain types of epidemiologically linked patients. In conclusion, colonization with CAZ-RGN in SICU was associated with diverse species and strains, as determined by molecular typing. Emergence of resistance from previously susceptible strains appeared to be more important than horizontal transmission in acquisition of CAZ-RGN in a nonoutbreak period.

Parasitic sinusitis and otitis in patients infected with human immunodeficiency virus: report of five cases and review.

We describe five cases of parasitic sinusitis and otitis in patients infected with human immunodeficiency virus (HIV) and review 14 reported cases. The pathogens identified in our group of patients included agents such as Microsporidium, Cryptosporidium, and Acanthamoeba species. The clinical features common to these patients included a long history of HIV seropositivity associated with advanced immunosuppression and multiple opportunistic infections as well as long-standing local symptoms refractory to multiple courses of antibacterial agents. Symptoms often included fever and chills in addition to local tenderness and discharge. Invasive diagnostic procedures were necessary to obtain the final diagnosis and to initiate appropriate therapy. Although most patients responded at least partially to specific therapy, relapses and recurrences were frequent in patients who did not receive long-term suppressive therapy. The general outcome for HIV-infected patients with parasitic sinusitis and otitis was poor; however, deaths were generally associated with other complications of the underlying HIV infection.

A medium chain triglyceride-based diet in patients with HIV and chronic diarrhea reduces diarrhea and malabsorption: a prospective, controlled trial.

Our objective was to determine whether a medium-chained triglyceride (MCT)-based diet, compared to a long-chain triglyceride (LCT)-based diet, conveys a beneficial effect on diarrhea and fat malabsorption in human immunodeficiency virus (HIV)-infected individuals with chronic diarrhea and weight loss. A secondary objective was to evaluate the pathogens associated with the diarrhea and to evaluate whether the etiologic agent was a determinant of response to the nutritional intervention. Prospective, randomized double-blind comparative trial was conducted in 24 adult patients with HIV, diarrhea of greater than 4-wk duration, fat malabsorption, and loss of 10-20% of ideal body weight, these patients were recruited from our outpatient infectious disease clinic. Evaluations of diarrheal pathogens were made by complete stool examination, upper and lower endoscopy with quantitative culture, and biopsy. Body composition determinations, and measurements of fat, carbohydrate, and vitamin absorption pre- and postintervention. Patients were randomly assigned to one of two complete nutritional products with either medium- or long-chain triglyceride fat exclusively for 12 d followed by treatment of infectious pathogens. Ten patients were found to have Microsporidium and 9 patients had no identifiable pathogen. All patients responded to intervention with both nutritional products overall with 45% fewer stools, decreased stool fat and weight, and a significant increase in urine nitrogen. The group that received the MCT product demonstrated significantly decreased stool number (mean 4 to 2.5), stool fat (mean 14 to 5.4 g), and stool weight (mean 428 to 262 g) compared with baseline (P < 0.01 for all). Patients with both species of microsporidia and with pathogen negative diarrhea had good response. We found that HIV patients with diarrhea, regardless of etiology, and documented fat malabsorption may benefit symptomatically from a diet composed of an MCT-based liquid supplement.

Enterocytozoon bieneusi infection and diarrheal disease in patients who were not infected with human immunodeficiency virus: case report and review.

We describe the identification of the protozoan parasite Enterocytozoon bieneusi in the stool of a patient who was not infected with HIV but who presented with persistent diarrheal disease and severe abdominal complaints. The patient was not infected with HIV but had been noted to have a decreased CD4 cell count since at least 1992 and had had a prior episode of cryptococcal meningitis. The organisms were detected in stool smears with a modified trichrome stain and were identified to the species level by transmission electron microscopy of the stool. The patient responded readily and dramatically to treatment with albendazole, with resolution of symptoms and clearance of the organisms from the stool. Eight or possibly nine other cases of E. bieneusi infection associated with diarrheal disease in individuals who were not infected with HIV were identified in the English-language literature. In two individuals with intact immune function, symptoms were self-limited and diarrheal disease resolved within 2 weeks. The cases summarized herein suggest that E. bieneusi may be more commonly associated with sporadic diarrheal disease than was previously suspected and that the immune system may play a role in the control of this organism within the intestine.

Comparison of PCR-based approaches to molecular epidemiologic analysis of Clostridium difficile.

Representative isolates of the 10 serogroups of Clostridium difficile and 39 clinical isolates (30 toxigenic and 9 nontoxigenic), including 5 isolates from a confirmed nosocomial outbreak, were analyzed by using two previously described arbitrary-primer PCR (AP-PCR) molecular typing methodologies (AP-PG05 and AP-ARB11) and PCR ribotyping. The two AP-PCR methods investigated gave comparable results; AP-PG05 and AP-ARB11 identified 8 and 7 groups among the serogroup isolates and classified the clinical isolates into 21 and 20 distinct groups, respectively. PCR ribotyping also identified 8 unique groups among the serogroup isolates but classified the clinical isolates into 23 groups. In addition, when results obtained by the PCR methods were compared with typing data generated by pulsed-field gel electrophoresis (PFGE), PCR ribotyping and PFGE were found to be in agreement for 83% (29 of 35) of isolates typeable by both techniques while AP-PG05 was in agreement with PFGE for 60% (20 of 33) and AP-ARB11 was in agreement with PFGE for only 44% (17 of 36). These results indicate that PCR ribotyping is a more discriminatory approach than AP-PCR for typing C. difficile and, furthermore, that this technique generates results that are in higher concordance with those obtained by using an established method for differentiating isolates of this organism on a molecular level than are results generated by using AP-PCR.