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ABSTRACTOver a 2-year period (2003 to 2005) patients with community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA) and community-acquired methicillin-susceptible Staphylococcus aureus(CA-MSSA) infections were prospectively identified. Patients infected with CA-MRSA (n= 102 patients) and CA-MSSA (n= 102 patients) had median ages of 46 and 53 years, respectively; the most common sites of infection in the two groups were skin/soft tissue (80 and 93%, respectively), respiratory tract (13 and 6%, respectively), and blood (4 and 1%, respectively). Fourteen percent of patients with CA-MRSA infections and 3% of patients with CA-MSSA infections had household contacts with similar infections (P< 0.01). Among the CA-MRSA isolates, the pulsed-field gel electrophoresis (PFGE) groups detected were USA300 (49%) and USA100 (13%), with 27 PFGE groups overall; 71% of the isolates were staphylococcal chromosome cassette mec(SCCmec) type IV, 29% were SCCmectype II, and 54% had the Panton-Valentine leucocidin (PVL) gene. Among the CA-MSSA isolates there were 33 PFGE groups, with isolates of the USA200 group comprising 11%, isolates of the USA600 group comprising 11%, isolates of the USA100 group comprising 10%, and isolates of the PVL type comprising 10%. Forty-six and 18% of the patients infected with CA-MRSA and CA-MSSA, respectively, were hospitalized (P< 0.001). Fifty percent of the patients received antibiotic therapy alone, 5% received surgery alone, 30% received antibiotics and surgery, 3% received other therapy, and 12% received no treatment. The median durations of antibiotic therapy were 12 and 10 days in the CA-MRSA- and CA-MSSA-infected patients, respectively; 48 and 56% of the patients in the two groups received adequate antimicrobial therapy, respectively (P< 0.001). The clinical success rates of the initial therapy in the two groups were 61 and 84%, respectively (P< 0.001); recurrences were more common in the CA-MRSA group (recurrences were detected in 18 and 6% of the patients in the two groups, respectively [P< 0.001]). CA-MRSA was an independent predictor of clinical failure in multivariate analysis (odds ratio, 3.4; 95% confidence interval, 1.7 to 6.9). In the community setting, the molecular characteristics of the S. aureusstrains were heterogeneous. CA-MRSA infections were associated with a more adverse impact on outcome than CA-MSSA infections.

ABSTRACTThree hundred sixty-one quinupristin-dalfopristin (Q-D)-resistant Enterococcus faecium(QDREF) isolates were isolated from humans, turkeys, chickens, swine, dairy and beef cattle from farms, chicken carcasses, and ground pork from grocery stores in the United States from 1995 to 2003. These isolates were evaluated by pulsed-field gel electrophoresis (PFGE) to determine possible commonality between QDREF isolates from human and animal sources. PCR was performed to detect the streptogramin resistance genes vatD, vatE, and vgbAand the macrolide resistance gene ermBto determine the genetic mechanism of resistance in these isolates. QDREF from humans did not have PFGE patterns similar to those from animal sources. vatEwas found in 35%, 26%, and 2% of QDREF isolates from turkeys, chickens, and humans, respectively, and was not found in QDREF isolates from other sources. ermBwas commonly found in QDREF isolates from all sources. Known streptogramin resistance genes were absent in the majority of isolates, suggesting the presence of other, as-yet-undetermined, mechanisms of Q-D resistance.

ABSTRACTWe evaluated the molecular mechanism for resistance of 360 enterococci for which the gentamicin MICs were =128 µg/ml. The aac(6')-Ie-aph(2")-Ia, aph(2")-Ic, and aph(2")-Idgenes were identified by PCR in isolates from animals, food, and humans. The aph(2")-Ibgene was not identified in any of the isolates. Two Enterococcus faecalisisolates (MICs > 1,024 µg/ml) from animals failed to generate a PCR product for any of the genes tested and likely contain a new unidentified aminoglycoside resistance gene. Pulsed-field gel electrophoresis (PFGE) analysis showed a diversity of strains. However, 1 human and 18 pork E. faecalisisolates from Michigan with the aac(6')-Ie-aph(2")-Iagene had related PFGE patterns and 2 E. faecalisisolates from Oregon (1 human and 1 grocery store chicken isolate) had indistinguishable PFGE patterns. We found that when a gentamicin-resistant gene was present in resistant enterococci from animals, that gene was also present in enterococci isolated from food products of the same animal species. Although these data indicate much diversity among gentamicin-resistant enterococci, the data also suggest similarities in gentamicin resistance among enterococci isolated from humans, retail food, and farm animals from geographically diverse areas and provide evidence of the spread of gentamicin-resistant enterococci from animals to humans through the food supply.

ABSTRACTThirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a 2-year period (1996 to 1998). Isolated species included Enterococcus faecium(n= 13), Enterococcus faecalis(n= 7), Enterococcus gallinarum(n= 11), and Enterococcus casseliflavus(n= 4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to three or more antibiotics. One E. faeciumisolate, CVM1869, displayed high-level resistance to vancomycin (MIC > 32 µg/ml) and gentamicin (MIC > 2,048 µg/ml). Molecular analysis of this isolate revealed the presence of Tn1546(vanA), responsible for high-level vancomycin resistance, and Tn5281carrying aac6'-aph2", conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine E. faeciumclone that had acquired Tn1546, perhaps from a human vancomycin-resistant E. faecium. Transposons Tn5281and Tn1546were located on two different conjugative plasmids. Sequence analysis revealed that in Tn1546, ORF1 had an 889-bp deletion and an IS1216Vinsertion at the 5' end and an IS1251insertion between vanSand vanH. To date, this particular form of Tn1546has only been described in human clinical vancomycin-resistant enterococcus isolates unique to the United States. Additionally, this is the first report of a vancomycin-resistant E. faeciumisolated from a companion animal in the United States.

Thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a 2-year period (1996 to 1998). Isolated species included Enterococcus faecium (n = 13), Enterococcus faecalis (n = 7), Enterococcus gallinarum (n = 11), and Enterococcus casseliflavus (n = 4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to three or more antibiotics. One E. faecium isolate, CVM1869, displayed high-level resistance to vancomycin (MIC > 32 micro g/ml) and gentamicin (MIC > 2,048 micro g/ml). Molecular analysis of this isolate revealed the presence of Tn1546 (vanA), responsible for high-level vancomycin resistance, and Tn5281 carrying aac6'-aph2", conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine E. faecium clone that had acquired Tn1546, perhaps from a human vancomycin-resistant E. faecium. Transposons Tn5281 and Tn1546 were located on two different conjugative plasmids. Sequence analysis revealed that in Tn1546, ORF1 had an 889-bp deletion and an IS1216V insertion at the 5' end and an IS1251 insertion between vanS and vanH. To date, this particular form of Tn1546 has only been described in human clinical vancomycin-resistant enterococcus isolates unique to the United States. Additionally, this is the first report of a vancomycin-resistant E. faecium isolated from a companion animal in the United States.

Between 1986 and 1988, the incidence of ampicillin-resistant enterococci increased sevenfold at a university-affiliated hospital. Forty-three patients acquired nosocomial infections with ampicillin-resistant enterococci, most of which were also resistant to mezlocillin, piperacillin, and imipenem. An analysis of plasmid and chromosomal DNAs of isolates revealed that the increase was due to an epidemic of 19 nosocomial infections that yielded closely related strains of Enterococcus faecium and to a significant increase in the incidence of nonepidemic, largely unrelated strains of ampicillin-resistant enterococci. The nonepidemic strains were identified as E. faecium, E. raffinosus, E. durans, and E. gallinarum. A logistic regression analysis revealed that patients with nonepidemic resistant strains were 16 times more likely than controls to have received preceding therapy with imipenem. In our institution, the increase in the incidence of ampicillin-resistant enterococci appears to be due to the selection of various strains of resistant enterococci by the use of imipenem and to the nosocomial transmission of E. faecium and E. raffinosus.

Plasmids from the first six reported beta-lactamase-producing (Bla+) enterococci were compared for genetic relatedness. Bla+ enterococcal plasmids from strains isolated in Houston, Tex.; Philadelphia, Pa.; Connecticut; and Pittsburgh, Pa., had heterogeneous HaeIII and MspI-ClaI restriction endonuclease digestion patterns. A staphylococcal beta-lactamase probe hybridized to all six Bla+ enterococcal plasmids, but hybridization was detected on different HaeIII and MspI-ClaI fragments of the six plasmids. An enterococcal gentamicin resistance (Gmr) probe hybridized to a common 3.9-kilobase HaeIII fragment from the five Gmr plasmids. The Houston plasmid was cross-hybridized to the other five strains, and moderate to extensive homology was demonstrated. Bla+ enterococcal plasmids from a broad geographic range are heterogeneous with respect to size and restriction endonuclease digestion patterns but contain homologous genetic material, including Bla+ and Gmr determinants.

Candida species are important nosocomial pathogens; however, little is known about the epidemiology of Candida lusitaniae, an organism frequently resistant to amphotericin B. We evaluated 98 patients admitted to the bone marrow transplant and medical intensive care units of a tertiary-care hospital. Each patient with C. lusitaniae was matched with control patients. Restriction fragment analysis of DNA was used to determine strain relatedness. Seven patients (7.1%) with C. lusitaniae were identified; five acquired C. lusitaniae after admission to the study unit. All isolates were susceptible to amphotericin B. There were no differences between patients and controls with regard to duration of stay in the study unit, antibiotic administration, antifungal therapy, immunosuppressive therapy, catheter use, or underlying disease. Temporal and geographic clustering of five patients with identical strains occurred. No common source was identified. Restriction fragment analysis revealed a total of eight strains, and five patients shared one strain type. These results demonstrate exogenous acquisition of C. lusitaniae. The mechanism of acquisition is probably indirect contact transmission between patients.

Candida species are an important cause of infection in immunocompromised hosts and the leading cause of nosocomial fungal infections. Study of the epidemiology of Candida infection has been difficult because of lack of a reliable typing system. We describe a typing system utilizing contour-clamped homogeneous electric fields (CHEF), which is a modified version of pulsed-field gradient gel electrophoresis, and compared it with restriction enzyme analysis (REA) of genomic DNA. The study was done with 35 Candida albicans clinical isolates from separate patients. CHEF and REA were performed on each isolate, and the patterns were compared. The REA procedure revealed 17 strain types while the CHEF procedure was able to distinguish 23 strain types of C. albicans. The CHEF technique yields unique patterns of chromosomal bands that can be used to distinguish clinical isolates and demonstrates greater sensitivity than REA.

Enterococci are important causes of community-acquired and nosocomial infection. They cause endocarditis, bacteremia, urinary tract infections and neonatal sepsis. As causes of intra-abdominal and pelvic infection, enterococci are more commonly associated with abscess, biliary tract infection, spontaneous bacterial peritonitis, post-operative infection, post-partum endomyometritis and chronic or recurrent infection. As causes of soft tissue infection, enterococci are more commonly identified in burns, decubitus or diabetic foot ulcers, and wounds associated with intestinal surgery. Enterococci are often cultured in association with other pathogens when identified in intra-abdominal, pelvic or skin and soft tissue infection. Enterococcal superinfection after therapy with cephalosporins has been well described, and occurs as a result of the low in vitro activity of cephalosporins against enterococci. The epidemiology of enterococcal infection is complex and includes both endogenous and exogenous acquisition of the organism. Antibiotic resistance is an ever-increasing problem complicating therapy in patients with enterococcal infection.

Candida glabrata has emerged as an important nosocomial pathogen, yet little is known about its epidemiology. We prospectively followed 98 patients admitted to a medical intensive care unit and the bone marrow transplant unit of a university hospital. Samples from environmental surfaces and the hands of hospital personnel were also cultured. Patients with newly acquired C. glabrata strains were compared to controls who were culture negative for C. glabrata. C. glabrata was recovered from multiple sites from 24 patients and three environmental surfaces. Sixteen patients (17%) acquired C. glabrata after admission to the study units. Significant risk factors for the nosocomial acquisition of C. glabrata were prolonged duration of hospitalization in the unit and prior antimicrobial use. Strain delineation by restriction enzyme analysis revealed 28 different strains of C. glabrata; three strain types were common to nine patients. The environmental isolates were of the same strain type and common to five patients (four patients with newly acquired strains). These results suggest the possibility of exogenous nosocomial acquisition of C. glabrata, including the possible acquisition from the hospital environment. Transmission may be by indirect contact since identical strains of C. glabrata were recovered from patients who were geographically and temporally associated.

Direct comparisons between contour-clamped homogeneous electric field (CHEF) electrophoresis and field inversion gel electrophoresis (FIGE) to determine the epidemiology of antibiotic-resistant enterococci have not been previously published. Fifty non-beta-lactamase-producing, ampicillin-resistant Enterococcus faecium isolates and 10 vancomycin-resistant E. faecium strains collected from multiple centers were analyzed in a blinded fashion by CHEF electrophoresis and FIGE after digestion with SmaI. Isolates were considered clonally related if there was a difference of three of fewer bands between electrophoretic patterns. Agreement between CHEF electrophoresis and FIGE was seen for 12 of 13 identified groups of ampicillin-resistant E. faecium and 7 of 7 groups of vancomycin-resistant E. faecium. The lone discordance was accounted for by a fourth band difference between two strains recognized near 350 kb by CHEF electrophoresis but not by FIGE, placing them into different clonal groups. Better band separation was noted in the 50- to 200-kb range for FIGE, while CHEF electrophoresis revealed better resolution over 250 kb more reliably, including detection of some bands not seen on FIGE. Molecular epidemiologic investigations of E. faecium by either technique should provide comparable results.

Enterococcus faecium strains resistant to ampicillin, high levels of gentamicin, and vancomycin but susceptible to teicoplanin (vanB class vancomycin resistance) were recovered from 37 patients during an outbreak involving a 250-bed university-affiliated hospital. Three isolates with vancomycin MICs ranging from 8 to 256 micrograms/ml all hybridized with a vanB probe. Restriction endonuclease analysis of chromosomal and plasmid DNA suggested that all isolates tested were derived from a single clone. Vancomycin resistance was shown to be transferable. Risk factors for acquiring the epidemic strain included proximity to another case patient (P, 0.0005) and exposure to a nurse who cared for another case patient (P, 0.007). Contamination of the environment by the epidemic strain occurred significantly more often when case patients had diarrhea (P, 0.001). Placing patients in private rooms and requiring the use of gowns as well as gloves by personnel controlled the outbreak. These findings suggest that multidrug-resistant E. faecium strains with transferable vanB class vancomycin resistance will emerge as important nosocomial pathogens. Because extensive environmental contamination may occur when affected patients develop diarrhea, barrier precautions, including the use of both gowns and gloves, should be implemented as soon as these pathogens are encountered.

The epidemiology of two cases of pseudallescheriasis in organ transplant patients are described and the disease in that population is reviewed. Disseminated hospital‐acquired infection occurred in a liver transplant recipient and was fatal despite therapy with miconazole. A heart transplant recipient developed localized disease following soil contamination of soft tissue trauma which was cured with surgical resection and miconazole therapy. Itraconazole showed in vitroactivity against Pseudallescheria boydii andshould be evaluated in pseudallescheriasis. P. boydiiinfections are important complications of transplantation and should be considered in the differential diagnosis of community‐acquired as well as nosocomial fungal infections in this population. Es werden zwei Fälle von Pseudallescheriasis bei Transplantationspatienten beschrieben, und die Literatur hierüber wird zusammenfassend gewichtet. Eine disseminierte Hospitalinfektion wurde an einem Lebertransplantationspatienten beobachtet, die trotz Miconazol‐Therapie tödlich ausging. Ein Herztransplantationspatient entwickelte eine Lokalinfektion nach einer Erdbodenkontamination bei einem Weichteiltrauma, die durch Resektion und Miconazolgaben geheilt werden konnte. Itraconazol erwies sich in vitroals aktiv gegen P. boydiiund solite bei Pseudallescheriasis gepriift werden. P. boydii‐Infektionen stellen wichtige Komplikationen bei Organtransplantationen dar und sollten differentialdiagnostisch bei Umgebungsinfekten wie bei nosokomialen Infektionen dieser Patientengruppe in Erwägung gezogen werden.

Among 97 enterococci cultured from animals, gentamicin MICs were > or = 2,000 micrograms/ml for 9 isolates and between 250 and 1,024 micrograms/ml for 6 isolates. For two isolates tested (gentamicin MICs, 256 and 512 micrograms/ml, respectively), there was no in vitro synergy with penicillin plus gentamicin, resistance was transferable, and there was no hybridization with a probe specific for 6'-aminoglycoside acetyltransferase-2"-aminoglycoside phosphotransferase. The results of the study indicate the presence of a unique gentamicin resistance genotype in enterococci of animal origin.

Enterococcus faecalis SF350 is a clinical isolate from Winnipeg, Canada, with high-level (MIC > 2,000 micrograms/ml) gentamicin resistance. The genetic determinant for gentamicin resistance was located on the chromosome of SF350 and could be mobilized by a coresident conjugative plasmid, pYN120. Genetic and physical analyses showed that the gentamicin resistance determinant was located on a 27-kb transposable element which was designated Tn924.

A rabbit endocarditis model was utilized to evaluate the virulence conferred by the conjugative plasmid pAD1 with the following strains: Enterococcus faecalis plasmid-free FA2-2 and FA2-2 containing plasmids pAD1 (hemolysin and aggregation substance positive), pAM9058 (insertional inactivation of hemolysin), and pAM944 or pAM947 (insertional inactivation of aggregation substance). All isolates were similar in ability to produce endocarditis. Mean vegetation weight was greater in animals inoculated with strains that produced aggregation substance (P < 0.01). Mortality was significantly increased in animals given FA2-2 containing pAD1 compared with those given all other strains (P < 0.01). These results suggest that the combination of hemolysin and aggregation substance is associated with increased mortality and that vegetation weight is associated with production of aggregation substance in experimental E. faecalis endocarditis.

In this study, 113 Enterococcus faecium, 37 Enterococcus faecalis, 24 Enterococcus gallinarum, 15 Enterococcus raffinosus, and 13 Enterococcus casseliflavus clinical isolates and American Type Culture Collection (ATCC) strains were evaluated by contour-clamped homogeneous electric field electrophoresis. Thirty-one of the E. faecium, 22 of the E. faecalis, 24 of the E. gallinarum, 15 of the E. raffinosus, and 13 of the E. casseliflavus isolates were also evaluated by DNA-DNA hybridization. Genomic DNAs from type strains E. faecalis ATCC 19433, E. faecium ATCC 19434, E. gallinarum ATCC 49573, E. raffinosus ATCC 49427, and E. casseliflavus ATCC 25788 were labeled with biotin for use as probes. E. faecalis differed from all other species in always having a largest fragment of > 400 kb. E. gallinarum was different from all other species in having all SmaI fragments of < 200 kb. Biotin-labeled probes showed a high degree of hybridization with genomic DNA from the same species and a low degree of hybridization when hybridized to genomic DNA from different species for all isolates tested except for four isolates identified as E. faecium by conventional biochemical methods. The DNA from these four isolates hybridized strongly to DNA from E. gallinarum ATCC 49573 and weakly to E. faecium ATCC 19434 DNA and had all SmaI fragments of < 200 kb in size. These data suggest that these isolates are nonmotile E. gallinarum. DNA from each ATCC type strain hybridized strongly with itself and had only a low degree of hybridization with DNA from other ATCC type strains tested. These results suggest that contour-clamped homogeneous electric field electrophoresis patterns and DNA-DNA hybridization with biotin-labeled probes may be of use for species differentiation of some enterococci.

Between November 1981 and October 1984, 48 of 3,458 clinical isolates of Streptococcus faecalis at the University of Michigan Hospital showed high-level (greater than 2,000 micrograms/ml) resistance to gentamicin, as well as to all other clinically available aminoglycosides. Thirteen percent of clinical isolates in the University of Michigan Hospital currently demonstrate this level of resistance. Transfer of resistance to a plasmid-free streptococcal recipient was observed in filter matings for 44 of 48 such isolates. Analysis of transconjugants by agarose gel electrophoresis showed that gentamicin resistance was transferred alone in 8 isolates and was combined with other antimicrobial resistances on the same plasmid in the other 36 isolates. There were seven isolates which transferred the property of horse blood beta-hemolysis along with gentamicin resistance. Transfer of gentamicin resistance was either by conjugative plasmids or by a nonconjugative but mobilizable plasmid. Transfer frequencies on filters ranged from 4.7 X 10(-2) to 4.1 X 10(-8). Based on donor and transconjugant antibiotic resistance markers, agarose gel electrophoresis, transfer properties, and restriction enzyme analysis, the plasmid content of transconjugants was heterogeneous and could be classified into at least seven different patterns. These findings argue against clonal dissemination as the cause of the increased frequency of resistant strains and suggest that resistance to gentamicin appears on a variety of physically distinct conjugative and nonconjugative plasmids in S. faecalis.

Contour-clamped homogeneous gel electrophoresis has increasingly been used and has generally been considered the method of choice for the delineation of enterococcial strains. It has been suggested that the contour-clamped homogeneous electric field (CHEF) electrophoresis digestion patterns of genomic DNA indicate the relatedness of strains when SmaI patterns differ by six or fewer bands. To evaluate the potential reasons for the diversity of bands among clonally related isolates, we studied plasmid-free Enterococcus faecalis FA2-2 and derivatives with transposon Tn916 (encoding for tetracycline resistance) insertions on the chromosome. We obtained derivatives with seven different Tn916 insertion sites and up to two copies of Tn916 on the chromosome, resulting in differences of one to seven bands by CHEF electrophoresis; eight different patterns were observed. With Tn916 insertions, there was either (i) loss of a band(s) with the generation of a new band(s), (ii) the generation of a new band(s) only, or (iii) the loss of a band(s) with no new visible band(s). These results indicate that strains can have up to seven different SmaI bands by CHEF electrophoresis and still be closely related.

Candida species have recently emerged as important nosocomial pathogens. Because of the lack of a reliable system for detecting differences within the same species, little is known about the epidemiology of infection with Candida species. We describe a typing system for Torulopsis glabrata and the non-C. albicans Candida species that uses contour-clamped homogeneous electric field electrophoresis (CHEF), a version of pulsed-field gradient gel electrophoresis, and compared it with restriction enzyme analysis (REA) of genomic DNA. One hundred seventeen clinical isolates from 40 patients were evaluated. CHEF and REA were performed on each of the isolates, and the results of the two procedures were compared. The REA procedure revealed 8 different types of Candida lusitaniae, 20 of Torulopsis glabrata, 5 of Candida tropicalis, 3 of Candida parapsilosis, and 7 of Candida kefyr, whereas the CHEF method revealed 14 different types of C. lusitaniae, 16 of T. glabrata, 10 of C. tropicalis, 10 of C. parapsilosis, and 7 of C. kefyr. The CHEF technique yielded unique patterns of electrophoretic karyotypes that could be used to distinguish intraspecies variations. When compared with REA, CHEF demonstrated greater sensitivity in recognizing subtle strain-to-strain variations in most isolates and will be a useful epidemiologic tool for studying non-C. albicans Candida species and T. glabrata.

Molecular typing methods were compared by using 66 ampicillin-resistant, non-beta-lactamase-producing Enterococcus faecium clinical isolates from diverse geographic areas. Whole-plasmid analysis, restriction enzyme analysis of plasmid DNA with EcoRI and HindIII, and contour-clamped homogeneous electric field electrophoresis with digestion by SmaI and ApaI were performed on all isolates. Whole-plasmid analysis identified 47 different groups. Restriction enzyme analysis of plasmid DNA identified 50 groups when EcoRI was used and 51 groups when HindIII was used. Results with EcoRI and HindIII differed in 9 of 66 isolates. Grouping results with whole-plasmid analysis differed from results of restriction enzyme analysis of plasmid DNA (combining EcoRI and HindIII) in 20 of 66 isolates. Contour-clamped homogeneous electric field electrophoresis identified 46 groups when SmaI was used and 44 groups when ApaI was used. Results with SmaI and ApaI differed in 3 of 66 isolates. Grouping results with contour-clamped homogeneous electric field electrophoresis (combining SmaI and ApaI) differed from results of restriction enzyme analysis of plasmid DNA (combining EcoRI and HindIII) in 17 of 66 isolates. The combined use of whole-plasmid analysis, restriction enzyme analysis of plasmid DNA with two enzymes, and contour-clamped homogeneous electric field electrophoresis with two restriction enzymes should be considered when E. faecium is typed for epidemiologic investigation.

In a randomized prospective study ampicillin/sulbactam and cefamandole were compared in the therapy of patients hospitalized with community acquired pneumonia. Patients receiving ampicillin/sulbactam (n=37) and cefamandole (n=38) were similar with respect to age (mean age 70 vs. 76 years respectively), clinical characteristics, severity of illness and underlying disease. Pathogens isolated from patients in the cefamandole and ampicillin/sulbactam group, respectively, wereStreptococcus pneumoniae(7 vs. 7 patients),Haemophilus parainfluenzae(7 vs. 6 patients),Haemophilus influenzae(5 vs. 5 patients),Staphylococcus aureus(5 vs. 4 patients),Escherichia coli(4 vs. 4 patients),Klebsiella pneumoniae(3 vs. 3 patients),Enterobacterspp. (2 vs. 3 patients),Moraxella catarrhalis(1 vs. 2 patients), and organisms of the oral flora (4 vs. 3 patients). The rate of resistance to penicillin was 80 %, to clindamycin 76 %, to erythromycin 45 %, to ampicillin 43 %, and to cefazolin 18 %. Overall successful treatment rates of 81 % for cefamandole and 97 % for ampicillin/sulbactam (p=0.05) were observed. Both cefamandole and ampicillin/sulbactam were shown to be effective agents for therapy of community acquired pneumonia; however ampicillin/sulbactam demonstrated superior overall clinical efficacy.

ABSTRACTFrom 125 separate cloacal cultures from three turkey flocks fed virginiamycin, 104 Enterococcus faeciumand 186Enterococcus faecalisisolates were obtained. As the turkeys aged, there was a higher percentage of quinupristin-dalfopristin-resistant E. faeciumisolates, with isolates from the oldest flock being 100% resistant. There were no vancomycin-resistant enterococci. Results of pulsed-field gel electrophoresis (PFGE) indicated there were 11 PFGE types of E. faecalisand 7 PFGE types of E. faeciumthat were in more than one group of flock cultures.

Enterococcus gallinarum SF9117 is a veterinary isolate for which the MIC of gentamicin is 256 micrograms/ml. Time-kill studies with a combination of ampicillin plus gentamicin failed to show synergism against SF9117. A probe representing aac(6')-aph(2") did not hybridize to DNA from SF9117. A 3.2-kb fragment from plasmid pYN134 of SF9117 was cloned and conferred resistance to gentamicin in Escherichia coli DH5 alpha. Nucleotide sequence analysis revealed the presence of a 918-bp open reading frame whose deduced amino acid sequence had a region with homology to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2"). The gene is designated aph(2")-Ic, and its observed phosphotransferase activity is provisionally designated APH(2")-Ic. An intragenic probe hybridized to the genomic DNA from an Enterococcus faecium isolate from the peritoneal fluid of one patient and to the plasmid DNA of an Enterococcus faecalis isolate from the blood of another patient. An enterococcal isolate containing this novel resistance gene might not be readily detected in clinical laboratories that use gentamicin at 500 or 2,000 micrograms/ml for screening for high-level resistance.

Antimicrobial susceptibilities and DNA contents were analyzed for six clinical isolates of Enterococcus faecalis that had high-level resistance to gentamicin (MIC > 2,000 micrograms/ml) but not streptomycin and were obtained from patients in diverse geographic areas. Contour-clamped homogeneous electric field electrophoresis of genomic DNA showed all isolates to be different strains. Gentamicin resistance was transferred from four isolates to plasmid-free enterococcal recipients in filter matings. Restriction enzyme analysis of transconjugants showed distinct gentamicin resistance plasmids. A probe specific for the gentamicin resistance determinant hybridized to the plasmids of four isolates and to the chromosomes of two isolates. These findings suggest that clonal dissemination is not responsible for the spread of these resistant strains, that resistance determinants occur on different plasmids as well as on the chromosome of E. faecalis, and that the genetic determinants of resistance are related.

We identified two beta-lactamase-positive enterococci. One strain was high-level (MIC, greater than 2,000 microgram/ml) gentamicin resistant; the other was not (MIC, 12.5 microgram/ml). beta-Lactamase production was extrachromosomally mediated in both strains, and both strains showed an inoculum effect reversed by beta-lactamase inhibitors. The strain lacking high-level gentamicin resistance showed synergistic killing with a combination of penicillin, clavulanic acid, and gentamicin.

ABSTRACTFecal samples from 200 consecutive patients admitted to a community hospital yielded 107 enterococci. High-level gentamicin resistance occurred in 10 (14%) of the Enterococcus faecalisisolates. Ampicillin resistance occurred in two (3%) of the E. faecalisisolates and six (23%) of the Enterococcus faeciumisolates. There were no vancomycin-resistant enterococci. Risk factors for enterococci with high-level aminoglycoside (gentamicin) or ampicillin resistance included prior hospitalization and previous antibiotic use.

The DNAs of 3* vancomycin-resistant Enterococcus faecium isolates from five hospitals in three states were analyzed by contour-clamped homogeneous electric field electrophoresis and plasmid analysis. There were 22 strain types. One strain type was common to patients in three hospitals in two states. These results suggest the apparent intra- and interhospital spread of vancomycin-resistant E. faecium.

A prospective study identified 9 (32%) of 28 ampicillin-resistant (MIC greater than or equal to 16 micrograms/ml) enterococcus isolates as Enterococcus raffinosus. A case-control study found no significant differences with respect to underlying diseases, catheterization, or surgery between patients with ampicillin-resistant E. raffinosus and those with ampicillin-susceptible Enterococcus spp. Prior treatment with antibiotics and prolonged hospitalization were more frequent among patients with ampicillin-resistant E. raffinosus. Patients with the same strain (determined by plasmid analysis) were frequently hospitalized concurrently.

beta-Lactamase-producing (Bla+) enterococci are rare but have been reported from several areas. We report another Bla+ enterococcus with high-level gentamicin resistance. Susceptibility and bactericidal activity studies of four Bla+ enterococci against potential alternative antibiotics, including ampicillin-sulbactam, daptomycin, teicoplanin, and vancomycin, are presented.

Restriction endonuclease analysis (REA) was evaluated as an epidemiologic typing tool to distinguish Branhamella catarrhalis strains. Fourteen beta-lactamase-producing strains were collected over a 16-month period at a hospital where a nosocomial outbreak of this organism was previously documented by REA. REA produced 12 distinct patterns which correlated with epidemiologic data. Chromosomal REA appears to be a useful technique for distinguishing B. catarrhalis strains.

The in vitro susceptibilities of 21 clinical isolates of Streptococcus faecalis to trimethoprim (TMP) in combination with sulfamethoxazole (SMX) was evaluated in Mueller-Hinton broth (MHB) in the presence and absence of folinic acid as well as in urine. The mean MIC and MBC in MHB, expressed as the TMP concentration, was 0.13 and 0.32 micrograms of TMP-SMX per ml, respectively. In MHB supplemented with folinic acid, the mean MIC and MBC was 3.3 and 5.5 micrograms of TMP-SMX per ml, respectively. In urine the mean MIC of TMP-SMX for these isolates was 8.1 micrograms/ml (range, 1.6 to 50 micrograms/ml). All isolates were inhibited by less than 0.01 micrograms of TMP-SMX per ml when methotrexate was added to the urine.

Enterococcus faecalis SF4855 is a beta-lactamase-producing isolate resistant to high levels of gentamicin, with determinants for these resistances on the chromosome. SF4855 transferred both determinants into E. faecalis FA2-2 and UV202 at a frequency of 10(-9) in the presence of the MLS plasmid pYN120. beta-Lactamase and gentamicin resistance probes hybridized to three locations on the chromosome of FA2-2 transconjugants on contour-clamped homogeneous electric field electrophoresis. The study results suggest mobilization of the beta-lactamase determinant.

Growth in a single broth microdilution well containing gentamicin at a concentration of 500 micrograms/ml predicted high-level resistance to gentamicin (MIC, greater than or equal to 2,000 micrograms/ml) in 505 of 508 clinical isolates of enterococci. Failure to achieve synergistic killing with the combination of penicillin and an aminoglycoside was demonstrated with 100% specificity in 20 strains which showed resistance to 500 micrograms of gentamicin per ml. We recommend this procedure be adopted as a routine screening test to detect high-level aminoglycoside resistance in enterococci.

Enterococci are common pathogens that cause intra-abdominal, pelvic, urinary tract and nosocomial infection, including bacteraemia. They are now the second leading cause of nosocomial infections. They are also the third leading cause of nosocomial bacteraemia, after Staphylococcus aureusand coagulase-negative staphylococci. Antibiotic resistance is an ever-increasing problem in enterococci, to the extent that there are now enterococcal strains that are resistant to all available antibiotics, with serious infections, bacteraemia, and deaths related to infections caused by the organism. Important resistances in enterococci include penicillin resistance as a result of beta-lactamase or alterations of penicillin-binding proteins, high-level (MIC >2000 mg 1-1) gentamicin resistance, and glycopeptide resistance. Enterococci become resistant to antimicrobial agents by a variety of genetic routes including the acquisition of plasmids or transposons. Most resistant strains are transmitted between hospitalized patients as a result of clonal dissemination of strains. Isolates exhibiting multiple antimicrobial resistance pose a considerable therapeutic challenge.

Vaginitis due to Saccharomyces species is extremely rare. Nine patients with 20 vaginal isolates of Saccharomyces cerevisiae who presented with either asymptomatic vaginal colonization or symptomatic vaginitis indistinguishable from that caused by Candida albicans are described. All patients had a history of chronic or recurrent vaginitis, and all but two had systemic or local predisposing factors. In vitro tests of antimycotic sensitivity revealed reduced susceptibility of S. cerevisiae to the majority of available azole agents, with outright resistance to fluconazole. In accordance with these findings, the clinical response to conventional topical and oral antimycotic drugs was frequently suboptimal and incomplete. Electrophoretic karyotyping of strains revealed several distinct types of S. cerevisiae; this information permitted both longitudinal follow-up and differentiation of relapse from reinfection. In three patients with recurrent vaginitis, a unique epidemiological relationship was documented between S. cerevisiae and Torulopsis glabrata, another unusual and resistant vaginal pathogen. Isolation of S. cerevisiae from the vagina of symptomatic patients should not be ignored; treatment of vaginal infection with this yeast requires selected, often prolonged therapy.

Enterococci expressing resistance to antimicrobial agents are increasingly important nosocomial pathogens. Effective strategies to prevent or abort outbreaks of resistant enterococcal infection will rely on an accurate understanding of the mechanisms by which these organisms spread. A 1065-bp insertion-like sequence (156770) is present in varying copy numbers in > 90% of enterococcal strains thus far examined. Hybridization patterns resulting from hybridization of enterococcal genomic DNA with an internal 156770 probe vary considerably between unrelated strains and correlate well with results of pulsed-field gel electrophoresis and field-inversion gel electrophoresis in identifying clonal relationships among enterococcal isolates. 156770 analysis of several outbreaks of resistant enterococci has confirmed the spread of single resistant clones rather than the emergence of resistance within the resident flora. These results suggest that 156770 hybridization will be a useful tool for tracing the epidemiology of nosocomial enterococcal infections.