Globally dispersed mobile drug-resistance genes in gram-negative bacterial isolates from patients with bloodstream infections in a US urban general hospital.

Mobile drug-resistance genes with identical nucleic acid sequences carried by multidrug-resistant Escherichia coli strains that cause community-acquired infections are becomingly increasingly dispersed worldwide. Over a 2-year period, we analysed gram-negative bacterial (GNB) pathogens from the blood of inpatients at an urban public hospital to determine what proportion of these isolates carried such globally dispersed drug-resistance genes. Of 376 GNB isolates, 167 (44 %) were Escherichia coli, 50 (13 %) were Klebsiella pneumoniae, 25 (7 %) were Pseudomonas aeruginosa, 25 (7 %) were Proteus mirabilis and 20 (5 %) were Enterobacter cloacae; the remainder (24 %) comprised 26 different GNB species. Among E. coli isolates, class 1 integrons were detected in 64 (38 %). The most common integron gene cassette configuration was dfrA17-aadA5, found in 30 (25 %) of 119 drug-resistant E. coli isolates and in one isolate of Moraxella morganii. Extended-spectrum ß-lactamase (ESBL) genes were found in 16 E. coli isolates (10 %). These genes with identical sequences were found in nearly 40 % of bloodstream E. coli isolates in the study hospital, as well as in a variety of bacterial species from clinical and non-clinical sources worldwide. Thus, a substantial proportion of bloodstream infections among hospitalized patients were caused by E. coli strains carrying drug-resistance genes that are dispersed globally in a wide variety of bacterial species.

CCL3 induced migration occurs independently of intracellular calcium release.

The molecular signalling pathway of cell migration and whether it can occur independently of the release of intracellular calcium is still not completely understood. Therefore we investigated here the molecular mechanisms of CCL3 induced cell migration and the importance of intracellular calcium for chemotaxis in more detail. We show that CCL3 induced cell migration is dependent on activation of PLC. Several PKC inhibitors block the release of intracellular calcium independently of CCL3 activation and do not affect cell migration. This confirms that the release of intracellular calcium is not necessary for chemotaxis towards CCL3 and that PKC inhibitors should be used with caution in calcium release assays.

In vitro adherence and accumulation of Staphylococcus epidermidis RP 62 A and Staphylococcus epidermidis M7 on four different bone cements.

Bacterial resistance of Staphylococcus epidermidis, a serious pathogen of implant-related infections, to antibiotics is related to the production of a glycocalyx slime that impairs antibiotic access and the killing by host defense mechanisms. In vitro studies of different bone cements containing antibiotics, developed for the prevention of biomaterial-associated infection, could not always demonstrate complete eradication of biomaterial-adherent bacteria. We have investigated four different bone cements in regard to bacterial accumulation of a slime-producing strain RP 62 A and its isogenic mutant M7 lacking the ability to produce exopolysaccharide slime using a bacterial adhesion assay and modified Kirby-Bauer technique. A significant effect of exopolysaccharide production for the accumulation on bone cement could be demonstrated. The gentamicin/clindamycin bone cement was the only tested biomaterial that produced a large zone of bacterial inhibition in the inoculated area adjacent to the biomaterial. The bacterial adhesion was not reduced significantly and there was no correlation between zones of inhibition on blood agar plates and the quantitative adhesion assay. The clinical efficacy of the gentamicin/clindamycin bone cement must be proven in vivo.

A heterogeneous outbreak of Enterobacter cloacae and Serratia marcescens infections in a surgical intensive care unit.

OBJECTIVE: To investigate an outbreak of invasive disease due to Enterobacter cloacae and Serratia marcescens in a surgical intensive care unit (ICU). DESIGN: Pulsed-field gel electrophoresis (PFGE) analysis of restriction fragments was used to characterize the outbreak isolate genotypes. A retrospective cohort study of surgical ICU patients was conducted to identify risk factors associated with invasive disease. Unit staffing data were analyzed to compare staffing levels during the outbreak to those prior to and following the outbreak. SETTING: An urban hospital in San Francisco, California. PATIENTS: During the outbreak period, December 1997 through January 1998, there were 52 patients with a minimum ICU stay of > or = 72 hours. Of these, 10 patients fit our case definition of recovery of E. cloacae or S. marcescens from a sterile site. RESULTS: PFGE analysis revealed a highly heterogeneous population of isolates. Bivariate analysis of patient-related risk factors revealed duration of central lines, respiratory colonization, being a burn patient, and the use of gentamicin or nafcillin to be significantly associated with invasive disease. Both respiratory colonization and duration of central lines remained statistically significant in a multivariate analysis. Staffing data suggested a temporal correlation between understaffing and the outbreak period. CONCLUSIONS: Molecular epidemiological techniques provided a rapid means of ruling out a point source or significant cross-contamination as modes of transmission. In this setting, patient-related risk factors, such as respiratory colonization and duration of central lines, may provide a focus for heightened surveillance, infection control measures, and empirical therapy during outbreaks caused by common nosocomial pathogens. In addition, understaffing of nurses may have played a role in this outbreak, highlighting the importance of monitoring staffing levels.

Emergence of trimethoprim-sulfamethoxazole resistance in the AIDS era.

Trimethoprim-sulfamethoxazole (TMP-SMX) is widely used for Pneumocystis carinii pneumonia prophylaxis in human immunodeficiency virus (HIV)-infected patients, but little is known about the effects of this practice on the emergence of TMP-SMX-resistant bacteria. A serial cross-sectional study of resistance to TMP-SMX among all clinical isolates of Staphylococcus aureus and 7 genera of Enterobacteriaceae was performed at San Francisco General Hospital. Resistance among all isolates was <5.5% from 1979 to 1986 but then markedly increased, reaching 20.4% in 1995. This was most prominent in HIV-infected patients: resistance increased from 6.3% in 1988 to 53% in 1995. The largest increases in resistance were in Escherichia coli (24% in 1988 to 74% in 1995) and S. aureus (0% to 48%) obtained from HIV-infected patients. A rapid increase in the use of prophylactic TMP-SMX in HIV disease was also observed during this time in San Francisco and is likely responsible for the increase in TMP-SMX resistance.

Adherence to and accumulation of S. epidermidis on different biomaterials due to extracellular slime production. In vitro comparison of a slime-producing strain (Rp 62 A) and its isogenic slime negative mutant (M7).

In an in vitro study, the bacterial adherence of a slime-producing strain (RP 62 A) was compared with its isogenic slime-negative mutant (M7). Standardized biomaterial discs were incubated under growth conditions in tryptic soy broth containing either strain RP 62 A or M7. After 24 h of incubation, the attached bacteria were removed by sonication and the colony-forming units were counted after plating of serial dilutions. We observed a significantly increased adherence and accumulation of the slime-producing strain (RP 62 A). In contrast to the slime negative mutant (M7) (p = 0.0001). The highest colony counts were found for the slime-producing strain on polyethylene and polymethylmethacrylate. The slime-negative mutant lacked the ability of accumulation. Our in-vitro results show the relevance of slime production by S. epidermidis for in-vitro colonisation of biomaterials, with a preference for polyethylene and polymethylmethacrylate.

A randomized clinical trial of mupirocin in the eradication of Staphylococcus aureus nasal carriage in human immunodeficiency virus disease.

Seventy-six human immunodeficiency virus (HIV)-infected patients with Staphylococcus aureus nasal carriage were randomized to treatment groups receiving intranasal mupirocin or placebo twice daily for 5 days. Nasal cultures for S. aureus were obtained at 1, 2, 6, and 10 weeks after therapy. At 1 week, 88% of mupirocin-treated patients had negative nasal cultures compared with 8% in placebo patients (P<.001). The percentage of mupirocin-treated patients with persistently negative nasal cultures decreased over time (63%, 45%, and 29% at 2, 6, and 10 weeks, respectively) but remained significantly greater than the placebo group (3% at 2, 6, and 10 weeks). In mupirocin-treated patients, most (16/19) instances of nasal recolonization were with pretreatment strains (determined by means of by pulsed field gel electrophoresis); mupirocin resistance was not observed. Five days of treatment with mupirocin eliminated S. aureus nasal carriage in HIV-infected patients for several weeks; however, since the effect waned over time, intermittent dosing regimens should be considered for long-term eradication.

Characterization of antimicrobial resistance in Streptococcus pyogenes isolates from the San Francisco Bay area of northern California.

During 1994 and 1995, 157 isolates of Streptococcus pyogenes from patients with invasive disease were consecutively collected in the San Francisco Bay area to determine the frequency of antimicrobial resistance. Susceptibility testing was performed according to the guidelines of the National Committee for Clinical Laboratory Standards by the disk method and by broth microdilution. For comparison of susceptibility patterns, an additional 149 strains were randomly collected from patients with pharyngitis. For San Francisco County, 32% of the isolates from invasive-disease-related specimens but only 9% of the isolates from throat cultures from the same period were resistant to erythromycin (P = 0.0007). Alameda County and Contra Costa County had rates of resistance of

Slime production of Staphylococcus epidermidis: increased bacterial adherence and accumulation onto pure titanium.

In an in vitro study using Staphylococcus epidermidis RP 62 A, a slime-producing strain and its isogenic slime-negative mutant M7, we demonstrated that both strains adhere to pure titanium discs with significantly higher colony counts for the slime-producing strain. The colony count was dependent on temperature, time and strain. Prolonged incubation time (24 h) under growth conditions leads to higher colony counts for the slime-producing strain RP 62 A. As the slime-negative mutant M7 can adhere to, but not form multiple layers on metallic surfaces, increase of incubation time does not produce higher colony counts on the metallic surface. We conclude that slime production is important for adherence and subsequent accumulation of S. epidermidis onto pure titanium discs in vitro.

The abilities of a Staphylococcus epidermidis wild-type strain and its slime-negative mutant to induce endocarditis in rabbits are comparable.

The abilities of a parent and mutant pair of Staphylococcus epidermidis strains, the slime-producing parent RP62A and its slime-negative mutant, to establish endocarditis in a rabbit model of aortic valve endocarditis and to accumulate and adhere to surfaces in vitro were compared. Vegetation titer and infection rate depended on the presence or absence of a catheter (P = 0.020) and on inoculum size (P < 0.001) but not on the infecting strain. The ability of the parent strain vis-à-vis its mutant to accumulate in vitro on surfaces as demonstrated in a slime test did not correlate with any enhancement in the development of endocarditis in the rabbit model. In vitro initial adherence rates were identical. Both isolates accumulated to the same reduced extent in vitro in the presence of serum, albumin, or gelatin. Adhesion was equally promoted by addition of fibronectin. These data suggest that the in vitro phenomenon of accumulation described as slime production in the absence of serum may not be an important virulence determinant in vivo.

A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces.

Two distinct pathogenic mechanisms, adhesion to polymer surfaces and subsequent accumulation of sessile bacterial cells, are considered important pathogenic steps in foreign body infections caused by Staphylococcus epidermidis. By using mitomycin mutagenesis, we have recently generated a mutant, strain M7, from S. epidermidis RP62A which is unaffected in adhesion but deficient in accumulation on glass or polystyrene surfaces and lacks a 115-kDa extracellular protein (designated the 140-kDa antigen; F. Schumacher-Perdreau, C. Heilmann, G. Peters, F. Götz, and G. Pulverer, FEMS Microbiol. Lett. 117:71-78, 1994). To evaluate the role of this protein in accumulation, we harvested extracellular proteins from S. epidermidis RP62A grown on dialysis membranes placed over chemically defined medium, purified the protein by using ion-exchange chromatography, determined its N-terminal amino acid sequence, and raised antiserum in rabbits. The antibody recognized only a single band in a Western immunoblot of the crude extracellular extract. With the microtiter biofilm test, antiserum at a dilution of < or =1:1,000 blocked accumulation of RP62A up to 98% whereas preimmune serum did not. The 140-kDa antigen was found only in extracellular products from bacteria grown under sessile conditions. Of 58 coagulase-negative clinical isolates, 32 strains were 140-kDa antigen positive and produced significantly larger amounts of biofilm than the 26 strains that were 140-kDa antigen negative. The 140-kDa protein appears to be biochemically and functionally unrelated to any previously described factors associated with biofilm formation. Thus, the 140-kDa antigen, referred to as accumulation-associated protein, may be a factor essential in S. epidermidis accumulation and, due to its immunogenicity, may allow the development of novel immunotherapeutic strategies for prevention of foreign body infection.

Mucin-positive epithelial mesotheliomas: a histochemical, immunohistochemical, and ultrastructural comparison with mucin-producing pulmonary adenocarcinomas.

Pathologists routinely use histochemistry, immunohistochemistry, and electron microscopy to differentiate epithelial mesotheliomas from pulmonary adenocarcinomas. Epithelial mesotheliomas are usually mucicarmine-, PAS-diastase, and carcinoembryonic antigen-negative, whereas about 60-75% of pulmonary adenocarcinomas are mucicarmine- and PAS-diastase-positive, and about 90% express polyclonal carcinoembryonic antigen. During a pathologic evaluation of pleural neoplasms between 1975 and 1990, 10 epithelial mesotheliomas were identified that were mucicarmine- and in some instances PAS-diastase-positive (diagnosis of mesothelioma confirmed by ultrastructural examination), with four mesotheliomas focally expressing carcinoembryonic antigen. The mucicarmine, PAS-diastase, and carcinoembryonic antigen staining were usually eradicated or reduced in intensity by pretreatment of the tissue sections with hyaluronidase, suggesting that hyaluronic acid was responsible for the positive mucin reactions. In three cases the epithelial mesotheliomas showed focal regions of mucicarmine, PAS-d-, and Alcian blue-hyaluronidase-resistant staining. In contrast, 10 mucicarmine-, PAS-diastase-, Alcian blue-, and carcinoembryonic antigen-positive pulmonary adenocarcinomas were not affected by hyaluronidase pretreatment of the tissue. Besides the usual ultrastructural features of well- to moderately well-differentiated epithelial mesotheliomas, the mucin-positive epithelial mesotheliomas often showed medium-electron-dense secretory material covering the microvilli, aggregates of medium electron-dense material in association with the microvilli, producing an ultrastructural morphology that has been observed only in epithelial mesotheliomas.

In-vitro activity of penicillin G plus sulbactam in comparison with other beta-lactamase inhibitor combinations and oxacillin against staphylococci.

The in-vitro activity of penicillin G in combination with the beta-lactamase inhibitor, sulbactam, against penicillin-sensitive S. aureus (n = 10) and penicillin-resistant, methicillin-sensitive S. aureus (n = 69) and S. epidermidis (n = 20) was tested in comparison with ampicillin/sulbactam, amoxicillin/clavulanic acid, piperacillin/tazobactam and oxacillin. The combination of penicillin G plus sulbactam was found to lead to MIC values for beta-lactamase producing staphylococci comparable to those for penicillin-sensitive staphylococci, with MIC90 values between < or = 0,03 mg/L and 0.06 mg/L.

A four-year prospective study on microbial ecology of explanted prosthetic hips in 52 patients with "aseptic" prosthetic joint loosening.

The bacteriology of explanted prosthetic hips and surrounding soft tissue was studied in 52 patients undergoing surgical revision for joint loosening. In a prospective four-year study, positive bacterial cultures were recorded in 34 (76%) patients. Coagulase-negative staphylococci were the predominant isolates, and 11 patients (33%) had more than three organisms isolated, 7 (20%) had two only, and 11 (33%) had one species. Among the 23 patients from whom specimens from all 11 predetermined anatomic sites were cultured, the highest frequency of positive cultures (52% and 47%) came from the shaft and capsular tissue, respectively. Organisms were less frequently recovered from the cement and acetabulum (13% and 4%, respectively). Using molecular typing in eight patients with paired isolates of the same species, clonal identity was found in four. An additional patient underwent a second revision for loosening 17 months after the first revision and the same clone of Staphylococcus epidermidis was isolated on both occasions.

Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation.

Biofilm formation is thought to result from the concerted action of primary attachment to a specific surface and accumulation in multilayered cell clusters. Here we describe the isolation and characterization of transposon (Tn917) mutants of Staphylococcus epidermidis O-47 which were biofilm negative in the polystyrene microtiter plate assay. Among 5,000 Tn917 insertion mutants, 4 biofilm-negative mutants were isolated. Each mutant carried one copy of Tn917. The mutants were divided into two phenotypic classes: class A (mut1 and mut1a) and class B (mut2 and mut2a). Mutants of phenotypic class A lacked four cell surface proteins, were less hydrophobic, and were affected in primary attachment to polystyrene, but were still able to form multilayered cell clusters. They were able to form a biofilm on a glass surface, a trait that was even more pronounced than in the wild-type stain O-47. Loss of several surface proteins might have led to the reduced surface hydrophilic structures, thus favoring primary attachment to a glass surface and leading to subsequent biofilm formation. Mutants of phenotype class B were able to attach to polystyrene but were unable to form multilayered cell clusters, had unchanged cell surface proteins and hydrophobicity, and were unable to form a biofilm on a glass surface, mut1 and mut2 could be complemented by wild-type DNA fragments containing the Tn917 insertion sites of mut1 and mut2, respectively. The complemented biofilm-positive clone mut1 (pRC20) produced a 60-kDa protein which is postulated to function as the adhesin for binding to plastic. The traits of binding to polystyrene and the ability to form multilayered cell clusters are phenotypically and genetically distinct.

Methicillin-resistant Staphylococcus haemolyticus on the hands of health care workers: a route of transmission or a source?

We undertook a cross-sectional study of hand carriage and environmental contamination of methicillin-resistant coagulase-negative staphylococci on three wards of a single subspeciality surgical service. Sixteen hand cultures from 15 health care workers and 32 environmental cultures were obtained. Of 49 isolates, 35 (72%) were Staphylococcus haemolyticus. This species comprised 14 of the 16 (87%) hand isolates and 21 of the 32 (66%) environmental isolates. Using restriction length polymorphism of total DNA, we identified a single clone of S. haemolyticus on the hands of four health care workers and in the environment at seven locations on two wards. The widespread dissemination of a single clone suggests transmission of S. haemolyticus on the wards and prompts further prospective studies.

Micrococcus luteus endocarditis: case report and review of the literature.

This paper includes a report on a case of prosthetic valve endocarditis due to Micrococcus luteus and a review of the sixteen cases of endocarditis due to Micrococcus species reported in the literature. The patient was successfully treated with rifampicin combined for two weeks with gentamicin and vancomycin and for another four weeks with teicoplanin. The hospital course was uneventful and no surgery was required.

The forgotten ones. A story of street children and schooling in South Asia.

PIP: The number of "street children" in India and Nepal is increasing at an alarming rate because of urbanization. Each year an estimated 150,000 young Nepalese girls are sold as prostitutes in India where the number of working children under age 14 has grown from 13.59-18.17 million during 1981-90. Indeed, these figures may be unrealistically low, with actual numbers of street and working children in India at 44 million. Street children are at risk of exploitation by adults in hazardous, or even banned, jobs. The children recognize that the skills provided by learning centers offer them their only hope of an improved life. The experiences of 3 such nongovernmental organizations (NGOs) in India and Nepal shed some light on the special needs of these children. Alternative schools are ideally set up in the slums where the children live and work. The curriculum of the schools should include opportunities for enhancing self-esteem and learning income-producing and survival skills as well as basic literacy training. It may take several years to create a curriculum specific to the children of a particular area. NGOs would benefit from international assistance in using new technologies and strategies to help learning-disabled children. Donations from international, sources could also facilitate an exchange of information among NGOs, which now view each other as competitors for scarce funds. NGOs try to persuade parents that the education of their children is more beneficial for families in the longterm than putting the children out to work. The children come to school in various stages of cleanliness, but they find acceptance there; fun is incorporated into school time, and teachers assure the children of their worth and act as important role models for them. It is hard to find teachers trained in nonformal education to work for the minimal salaries NGOs can offer. Funding is a special problem, since governments tend to ignore these children, but international donations could provide much needed learning materials and teacher training. Governments must realize that it is less expensive to meet the current needs of these children than to incur the longterm cost of harboring millions of ignorant young people.^ieng