Derivation and validation of clinical prediction rules for reduced vancomycin susceptibility in Staphylococcus aureus bacteraemia.

Reduced vancomycin susceptibility (RVS) may lead to poor clinical outcomes in Staphylococcus aureus bacteraemia. We conducted a cohort study of 392 patients with S. aureus bacteraemia within a university health system. The association between RVS, as defined by both Etest [vancomycin minimum inhibitory concentration (MIC) >1·0 µg/ml] and broth microdilution (vancomycin MIC ≥1·0 µg/ml), and patient and clinical variables were evaluated to create separate predictive models for RVS. In total, 134 (34·2%) and 73 (18·6%) patients had S. aureus isolates with RVS by Etest and broth microdilution, respectively. The final model for RVS by Etest included methicillin resistance [odds ratio (OR) 1·51, 95% confidence interval (CI) 0·97-2·34], non-white race (OR 0·67, 95% CI 0·42-1·07), healthcare-associated infection (OR 0·56, 95% CI 0·32-0·96), and receipt of any antimicrobial therapy ≤30 days prior to the culture date (OR 3·06, 95% CI 1·72-5·44). The final model for RVS by broth microdilution included methicillin resistance (OR 2·45, 95% CI 1·42-4·24), admission through the emergency department (OR 0·54, 95% CI 0·32-0·92), presence of an intravascular device (OR 2·24, 95% CI 1·30-3·86), and malignancy (OR 0·51, 95% CI 0·26-1·00). The availability of an easy and rapid clinical prediction rule for early identification of RVS can be used to help guide the timely and individualized management of these serious infections.

Potential for pet animals to harbour methicillin-resistant Staphylococcus aureus when residing with human MRSA patients.

Colonization by methicillin-resistant Staphylococcus aureus (MRSA) may be persistent in people and is horizontally transmissible. The scientific literature suggests that domestic pets may also participate in cross-transmission of MRSA within households. The objectives of this study were to evaluate the prevalence of and risk factors for MRSA carriage by pets residing in households with an MRSA-infected person. From 66 households in which an MRSA-infected patient resided, we screened 47 dogs and 52 cats using a swab protocol. Isolates from pets and humans were genotyped using two techniques and compared for concordance. Human participants completed a 22-question survey of demographic and epidemiologic data relevant to staphylococcal transmission. Eleven of 99 pets (11.5%) representing 9 (13.6%) of households were MRSA-positive, but in only six of these households were the human and animal-source strains genetically concordant. Human infection by strain USA 100 was significantly associated with pet carriage [OR = 11.4 (95% CI 1.7, 76.9); P = 0.013]. Yet, for each day of delay in sampling the pet after the person's MRSA diagnosis, the odds of isolating any type of MRSA from the pet decreased by 13.9% [(95% CI 2.6, 23.8); P = 0.017)]. It may be concluded that pets can harbour pandemic strains of MRSA while residing in a household with an infected person. However, the source of MRSA to the pet cannot always be attributed to the human patient. Moreover, the rapid attrition of the odds of obtaining a positive culture from pets over time suggests that MRSA carriage may be fleeting.

Socioeconomic risk factors for bacteraemic pneumococcal pneumonia in adults.

Demographic and clinical risk factors are important in guiding vaccination policy for pneumococcal pneumonia. We present data on these variables from a population-based surveillance network covering adult bacteraemic pneumococcal pneumonia (BPP) in the Delaware Valley region from 2002 to 2004. Surveillance data were used with U.S. Census data and a community health survey to calculate stratified incidence rates. Missing data were handled using multiple imputation. Overall rates of adult BPP were 10.6 cases/100 000 person-years. Elevated rates were seen in the elderly (>65 years), Native Americans, African Americans, the less-educated (less than high-school education), the poor, smokers, and individuals with histories of asthma, cancer, or diabetes. Multivariable modelling suggested that income was more robustly associated with risk than African American race. Of methodological interest, this association was not apparent if census block-group median income was used as a proxy for self-reported income. Further research on socioeconomic risk factors for BPP is needed.

BMS-284756 (T-3811ME) a new fluoroquinolone: in vitro activity against Legionella, efficacy in a guinea pig model of L. pneumophila pneumonia and pharmacokinetics in guinea pigs.

The activity of BMS-284756 was studied against extracellular Legionella spp. and intracellular Legionella pneumophila, and for the treatment of guinea pigs with L. pneumophila pneumonia. The BMS-284756 MIC(50) of 22 different Legionella spp. strains was 0.008 mg/L, compared with 0.016 and 0.125 mg/L for levofloxacin and azithromycin, respectively. BMS-284756 (1 mg/L) reduced the intracellular concentrations of two L. pneumophila strains grown in guinea pig alveolar macrophages by c. 1.5 log(10 )cfu/mL, and was more active than erythromycin, but less active than azithromycin or levofloxacin at the same drug concentrations. Efficacy studies of BMS-284756, levofloxacin and azithromycin were performed in guinea pigs with L. pneumophila pneumonia. In infected guinea pigs given BMS-284756 10 mg/kg ip, mean peak plasma levels were 1.8 mg/L at 0.5 h and 0.7 mg/L at 1 h post-dose. The elimination half-life in plasma was 0.5 h, and the AUC(0-24 )was 1.7 mg*h/L, about 2% of the AUC(0-24 )for a single 400 mg oral dose in man. Sixteen of 18 L. pneumophila-infected guinea pigs treated with BMS-284756 10 mg/kg ip once daily for 5 days survived for 7 days post-antimicrobial therapy, as did 11 of 12 guinea pigs treated with azithromycin 15 mg/kg ip once daily for 2 days. All 12 animals that were treated with levofloxacin 10 mg/kg ip once daily for 5 days survived. None of 12 control animals treated with saline survived. Animals treated with BMS-284756 had significantly higher residual lung counts of L. pneumophila at the end of therapy than did animals treated with levofloxacin or azithromycin, which may be attributable to the very low drug concentrations that were obtained. BMS-284756 was more active than erythromycin against L. pneumophila in infected macrophages, and effectively treated animals with experimental L. pneumophila pneumonia. These data support further studies of BMS-284756 for the treatment of Legionnaires' disease.

In vitro activity of ABT-773 against Legionella pneumophila, its pharmacokinetics in guinea pigs, and its use to treat guinea pigs with L. pneumophila pneumonia.

The activity of ABT-773 was studied against extracellular and intracellular Legionella pneumophila and for the treatment of guinea pigs with L. pneumophila pneumonia. The ABT-773 MIC at which 50% of isolates are inhibited (MIC(50)) for 20 different Legionella sp. strains was 0.016 microg/ml, whereas the MIC(50)s of clarithromycin and erythromycin were 0.032 and 0.125 microg/ml, respectively. ABT-773 (1 microg/ml) was bactericidal for two L. pneumophila strains grown in guinea pig alveolar macrophages. In contrast, erythromycin and clarithromycin had easily reversible static activity only. Therapy studies of ABT-773 and erythromycin were performed with guinea pigs with L. pneumophila pneumonia. When ABT-773 was given to infected guinea pigs by the intraperitoneal route (10 mg/kg of body weight), mean peak levels in plasma were 0.49 microg/ml at 0.5 h and 0.30 microg/ml at 1 h postinjection. The terminal half-life phase of elimination from plasma was 0.55 h, and the area under the concentration-time curve from 0 to 24 h (AUC(0-24)) was 0.65 microg. h/ml. For the same drug dose, mean levels in the lung were 15.9 and 13.2 microg/g at 0.5 and 1 h, respectively, with a half-life of 0.68 h and an AUC(0-24) of 37.0 microg. h/ml. Ten of 15 L. pneumophila-infected guinea pigs treated with ABT-773 (15 mg/kg/dose given intraperitoneally once daily) for 5 days survived for 9 days post-antimicrobial therapy, as did 14 of 15 guinea pigs treated with erythromycin (30 mg/kg given intraperitoneally twice daily) for 5 days. All of the ABT-773-treated animals that died appeared to do so because of drug-induced peritonitis rather than overwhelming pneumonia. None of 12 animals treated with saline survived. ABT-773 is as effective as erythromycin against L. pneumophila in infected macrophages and in a guinea pig model of Legionnaires' disease. These data support studies of the clinical effectiveness of ABT-773 for the treatment of Legionnaires' disease.

Epidemiological investigation of fluoroquinolone resistance in infections due to extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

The incidence of infections due to extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EK) has increased markedly in recent years. Treatment is difficult because of frequent multidrug resistance. Although fluoroquinolones offer effective therapy for ESBL-EK infections, their usefulness is threatened by increasing fluoroquinolone resistance. To identify risk factors for fluoroquinolone resistance in ESBL-EK infections, a case-control study of all patients with ESBL-EK infections from 1 June 1997 through 30 September 1998 was conducted. Of 77 ESBL-EK infections, 43 (55.8%) were resistant to fluoroquinolones. Independent risk factors for fluoroquinolone resistance were fluoroquinolone use (odds ratio [OR], 11.20; 95% confidence interval [CI], 1.99-63.19), aminoglycoside use (OR, 5.83; 95% CI, 1.12-30.43), and long-term care facility residence (OR, 3.39; 95% CI, 1.06-10.83). The genotypes of fluoroquinolone-resistant ESBL-EK isolates were closely related. Efforts should be directed at modification of these risk factors to preserve the utility of fluoroquinolones in the treatment of ESBL-EK infections.

Potential virulence role of the Legionella pneumophila ptsP ortholog.

We previously identified the Legionella pneumophila ptsP (phosphoenolpyruvate phosphotransferase) ortholog gene as a putative virulence factor in a study of signature-tagged mutagenesis using a guinea pig pneumonia model. In this study, we further defined the phenotypic properties of L. pneumophila ptsP and its complete sequence. The L. pneumophila ptsP was 2,295 bases in length. Its deduced amino acid sequence had high similarity with ptsP orthologs of Pseudomonas aeruginosa, Azotobacter vinelandii, and Escherichia coli, with nearly identical lengths. Here we show that while the mutant grew well in laboratory media, it was defective in both lung and spleen multiplication in guinea pigs. It grew slowly in guinea pig alveolar macrophages despite good uptake into the cells. Furthermore, there was minimal growth in a human alveolar epithelial cell line (A549). Transcomplementation of the L. pneumophila ptsP mutant almost completely rescued its growth in alveolar macrophages, in A549 cells, and in guinea pig lung and spleen. The L. pneumophila ptsP mutant was capable of evasion of phagosome-lysosome fusion and resided in ribosome-studded phagosomes. Pore formation activity of the mutant was normal. The L. pneumophila ptsP mutant expressed DotA and IcmX in apparently normal amounts, suggesting that the ptsP mutation did not affect dotA and icmX regulation. In addition, the mutant was resistant to serum and neutrophil killing. Taken together, these findings show that L. pneumophila ptsP is required for full in vivo virulence of L. pneumophila, most probably by affecting intracellular growth.

In vitro activity of gemifloxacin (SB-265805, LB20304a) against Legionella pneumophila and its pharmacokinetics in guinea pigs with L. pneumophila pneumonia.

The activity of gemifloxacin against intracellular Legionella pneumophila and for the treatment of guinea pigs with L. pneumophila pneumonia was studied. Gemifloxacin, azithromycin, and levofloxacin (1 microg/ml) reduced bacterial counts of two L. pneumophila strains grown in guinea pig alveolar macrophages by 2 to 3 log(10) units. Gemifloxacin and levofloxacin had roughly equivalent intracellular activities. In contrast, erythromycin had static activity only. Therapy studies of gemifloxacin, azithromycin, and levofloxacin were performed in guinea pigs with L. pneumophila pneumonia. When gemifloxacin (10 mg/kg) was given by the intraperitoneal (i.p.) route to infected guinea pigs, mean peak levels in plasma were 1.3 microg/ml at 0.5 h and 1.2 microg/ml at 1 h postinjection. The terminal half-life phase of elimination from plasma was 1.3 h, and the area under the concentration-time curve from 0 to 24 h (AUC(0--24)) was 2.1 microg. h/ml. For the same drug dose, mean levels in lungs were 3.4 microg/g at both 0.5 and 1 h, with a half-life of 1.5 h and an AUC(0--24) of 6.0 microg. h/ml. All 15 L. pneumophila-infected guinea pigs treated with gemifloxacin (10 mg/kg/dose given i.p. once daily) for 2 days survived for 9 days after antimicrobial therapy, as did 13 of 14 guinea pigs treated with the same dose of gemifloxacin given for 5 days. All 12 azithromycin-treated animals (15 mg/kg/dose given i.p. once daily for 2 days) survived, as did 11 of 12 animals treated with levofloxacin (10 mg/kg/dose given i.p. once daily for 5 days). None of 12 animals treated with saline survived. Gemifloxacin is effective against L. pneumophila in infected macrophages and in a guinea pig model of Legionnaires' disease, even with an abbreviated course of therapy. These data support studies of the clinical effectiveness of gemifloxacin for the treatment of Legionnaires' disease.

Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: risk factors for infection and impact of resistance on outcomes.

The prevalence of antibiotic resistance among extended-spectrum beta-lactamase (ESBL)--producing Escherichia coli and Klebsiella pneumoniae has increased markedly in recent years. Thirty-three patients with infection due to ESBL-producing E. coli or K. pneumoniae (case patients) were compared with 66 matched controls. Total prior antibiotic use was the only independent risk factor for ESBL-producing E. coli or K. pneumoniae infection (odds ratio, 1.10; 95% confidence interval, 1.03--1.18; P=.006). Case patients were treated with an effective antibiotic a median of 72 hours after infection was suspected, compared with a median of 11.5 hours after infection was suspected for controls (P<.001). ESBL-producing E. coli or K. pneumoniae infection was associated with a significantly longer duration of hospital stay and greater hospital charges (P=.01 and P<.001, respectively). Finally, many ESBL-producing E. coli and K. pneumoniae isolates were closely related. ESBL-producing E. coli and K. pneumoniae infections have a significant impact on several important clinical outcomes, and efforts to control outbreaks of infection with ESBL-producing E. coli and K. pneumoniae should emphasize judicious use of all antibiotics as well as barrier precautions to reduce spread.

In vitro activity of quinupristin/dalfopristin (Synercid, RP 59500) against Legionella spp.

The activities of quinupristin/dalfopristin (Synercid), erythromycin and azithromycin against 22 Legionella spp. isolates were measured by a microbroth dilution method. The MICs that inhibited 90% of strains tested were 0.5, 0.35, and 0.5 microg/mL for quinupristin/dalfopristin, erythromycin, and azithromycin, respectively. Quinupristin/dalfopristin was only partially active against intracellular L. pneumophila at high (2 microg/mL), but not low (1 microg/mL) concentration. Activity of the drug in a guinea pig model of Legionnaires' disease could not be accurately determined because of drug toxicity for the guinea pig, although there was evidence that the drug has in vivo activity.

The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence.

Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that the iraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionella species. A newly derived mutant containing a targeted disruption of iraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest that iraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.

Discovery of virulence genes of Legionella pneumophila by using signature tagged mutagenesis in a guinea pig pneumonia model.

Legionella pneumophila is the cause of Legionnaires' disease, which is a form of potentially fatal pneumonia. To identify genes required for virulence of the bacterium, a library of 1,386 L. pneumophila signature tagged transposon mutants was studied for guinea pig virulence. The mutants were screened in pools of 96 each in a guinea pig model of L. pneumophila pneumonia. Sixteen unique mutant clones were determined to have attenuated virulence after being screened twice in the animal model. All 16 mutants failed to multiply in both lungs and spleens. Four of the sixteen had no apparent defect for intracellular multiplication in macrophages. Partial DNA sequences of the interrupted genes adjacent to the transposon insertions showed that six of them had mutations in five known L. pneumophila virulence genes: dotB, dotF/icmG, dotO/icmB, icmX, and proA. Three of the sequenced clones contained mutations in genes without known homology to other published bacterial genes, and seven clones appeared to be homologous to five different known bacterial genes but are still being characterized. With this methodology, we demonstrate the existence of L. pneumophila genes responsible for non-macrophage-related virulence. The discovery of L. pneumophila virulence genes indicates the utility of the signature tagged mutagenesis technique for pulmonary pathogens.

In vitro activity of SCH 27899 (Ziracin) against Legionella species.

The activities of Sch 27899 (Ziracin), erythromycin, and ofloxacin against 102 Legionella spp. isolates were measured by a microbroth dilution method. The MICs that inhibited 90% of strains tested were 0.25, 0.5, and 0.06 microgram/mL for Sch 27899, erythromycin, and ofloxacin, respectively. The activity of Sch 27899 against intracellular Legionella pneumophila could not be determined because of complete inactivation of the drug by tissue culture medium components.

The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila.

Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.

In vitro activity of the ketolide HMR 3647 (RU 6647) for Legionella spp., its pharmacokinetics in guinea pigs, and use of the drug to treat guinea pigs with Legionella pneumophila pneumonia.

The activities of HMR 3647, HMR 3004, erythromycin, clarithromycin, and levofloxacin for 97 Legionella spp. isolates were determined by microbroth dilution susceptibility testing. Growth inhibition of two Legionella pneumophila strains grown in guinea pig alveolar macrophages was also determined. The concentrations required to inhibit 50% of strains tested were 0.06, 0.02, 0.25, 0.03, and 0.02 microg/ml for HMR 3647, HMR 3004, erythromycin, clarithromycin, and levofloxacin, respectively. BYEalpha broth did not significantly inhibit the activities of the drugs tested, as judged by the susceptibility of the control Staphylococcus aureus strain; however, when Escherichia coli was used as the test strain, levofloxacin activity tested in BYEalpha broth was fourfold lower. HMR 3647, HMR 3004, erythromycin, and clarithromycin (0.25 and 1 microg/ml) reduced bacterial counts of two L. pneumophila strains grown in guinea pig alveolar macrophages by 0.5 to 1 log10, but regrowth occurred over a 2-day period. HMR 3647, erythromycin, and clarithromycin appeared to have equivalent intracellular activities which were solely static in nature. HMR 3004 was more active than all drugs tested except levofloxacin. In contrast, levofloxacin (1 microg/ml) was bactericidal against intracellular L. pneumophila and significantly more active than the other drugs tested. Therapy studies with HMR 3647 and erythromycin were performed in guinea pigs with L. pneumophila pneumonia. When HMR 3647 was given (10 mg/kg of body weight) by the intraperitoneal route to infected guinea pigs, mean peak plasma levels were 1.4 microg/ml at 0.5 h and 1.0 microg/ml at 1 h postinjection. The terminal half-life phase of elimination from plasma was 1.4 h. All 16 L. pneumophila-infected guinea pigs treated with HMR 3647 (10 mg/kg/dose given intraperitoneally once daily) for 5 days survived for 9 days after antimicrobial therapy, as did all 16 guinea pigs treated with the same dose of HMR 3647 given twice daily. Fourteen of 16 erythromycin-treated (30 mg/kg/dose given intraperitoneally twice daily) animals survived, whereas 0 of 12 animals treated with saline survived. HMR 3647 is effective against L. pneumophila in vitro, in infected macrophages, and in a guinea pig model of Legionnaires' disease. HMR 3647 given once daily should be evaluated as a treatment for Legionnaires' disease in humans.

Epidemic Legionnaires' disease two decades later: old sources, new diagnostic methods.

In July 1995 we investigated a pneumonia outbreak in a Pennsylvania town. We conducted epidemiological and molecular microbiological studies to determine the outbreak source and interrupt transmission of disease. Legionnaires' disease (LD) was quickly identified by urine antigen testing, and a newly developed immunohistochemical stain confirmed nosocomial transmission to a hospital inpatient. LD was confirmed in 22 patients. Case-patients were more likely than controls to have been within 1,000 feet of the hospital (matched odds ratio, 21.0; 95% confidence interval, 2.9-368) during the 2 weeks prior to illness. Legionella pneumophila serogroup 1 (Lp-1) was isolated from hospital cooling towers (CTs) and rooftop air samples but not from hospital potable water or community CTs. Hospital CT and air Lp-1 isolates matched all five patient isolates by monoclonal antibody, arbitrarily primed polymerase chain reaction, and pulsed-field gel electrophoresis subtyping. Strategies to prevent LD must include minimizing transmission from CTs.