Transmission of Carbapenem-Resistant Klebsiella pneumoniae in US Hospitals.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKp) is the most prevalent carbapenem-resistant Enterobacterales in the United States. We evaluated CRKp clustering in patients in US hospitals. METHODS: From April 2016 to August 2017, 350 patients with clonal group 258 CRKp were enrolled in the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae, a prospective, multicenter, cohort study. A maximum likelihood tree was constructed using RAxML. Static clusters shared ≤21 single-nucleotide polymorphisms (SNP) and a most recent common ancestor. Dynamic clusters incorporated SNP distance, culture timing, and rates of SNP accumulation and transmission using the R program TransCluster. RESULTS: Most patients were admitted from home (n = 150, 43%) or long-term care facilities (n = 115, 33%). Urine (n = 149, 43%) was the most common isolation site. Overall, 55 static and 47 dynamics clusters were identified involving 210 of 350 (60%) and 194 of 350 (55%) patients, respectively. Approximately half of static clusters were identical to dynamic clusters. Static clusters consisted of 33 (60%) intrasystem and 22 (40%) intersystem clusters. Dynamic clusters consisted of 32 (68%) intrasystem and 15 (32%) intersystem clusters and had fewer SNP differences than static clusters (8 vs 9; P = .045; 95% confidence interval [CI]: -4 to 0). Dynamic intersystem clusters contained more patients than dynamic intrasystem clusters (median [interquartile range], 4 [2, 7] vs 2 [2, 2]; P = .007; 95% CI: -3 to 0). CONCLUSIONS: Widespread intrasystem and intersystem transmission of CRKp was identified in hospitalized US patients. Use of different methods for assessing genetic similarity resulted in only minor differences in interpretation.

Use of pharmacokinetic/pharmacodynamic approaches for dose optimization: a case study of plazomicin.

With limited treatment options available for multidrug-resistant bacteria, dose optimization is critical for achieving effective drug concentrations at the site of infection. Yet, selecting an appropriate dose and appropriate time to administer the dose with dosing frequency requires extensive understanding of the interplay between drug pharmacokinetics/pharmacodynamics (PK/PD), the host immune system, and bacterial-resistant mechanisms. Model-informed dose optimization (MIDO) uses PK/PD models (e.g. population PK, mechanism-based models, etc.) that incorporate preclinical and clinical data to simulate/predict performance of treatment regimens in appropriate patient populations and/or infection types that may not be well-represented in clinical trials. Here, we highlight the stages of a MIDO approach for designing optimized regimens by reviewing current clinical, preclinical, and PK/PD modeling data available for plazomicin. Plazomicin is an aminoglycoside approved in 2018 for the treatment of complicated urinary tract infections in adults. Applying knowledge gained by PK/PD modeling can guide therapeutic drug monitoring to ensure that drug exposure is appropriate for clinical efficacy while limiting drug-related toxicity.

A Systems-Based Analysis of Mono- and Combination Therapy for Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections.

Antimicrobial resistance is a global threat. As "proof-of-concept," we employed a system-based approach to identify patient, bacterial, and drug variables contributing to mortality in patients with carbapenem-resistant Klebsiella pneumoniae (CRKp) bloodstream infections exposed to colistin (COL) and ceftazidime-avibactam (CAZ/AVI) as mono- or combination therapies. Patients (n = 49) and CRKp isolates (n = 22) were part of the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE-1), a multicenter, observational, prospective study of patients with carbapenem-resistant Enterobacterales (CRE) conducted between 2011 and 2016. Pharmacodynamic activity of mono- and combination drug concentrations was evaluated over 24 h using in vitro static time-kill assays. Bacterial growth and killing dynamics were estimated with a mechanism-based model. Random Forest was used to rank variables important for predicting 30-day mortality. Isolates exposed to COL+CAZ/AVI had enhanced early bacterial killing compared to CAZ/AVI alone and fewer incidences of regrowth compared to COL and CAZ/AVI. The mean coefficient of determination (R2) for the observed versus predicted bacterial counts was 0.86 (range: 0.75 - 0.95). Bacterial subpopulation susceptibilities and drug mechanistic synergy were essential to describe bacterial killing and growth dynamics. The combination of clinical (hypotension), bacterial (IncR plasmid, aadA2, and sul3) and drug (KC50) variables were most predictive of 30-day mortality. This proof-of-concept study combined clinical, bacterial, and drug variables in a unified model to evaluate clinical outcomes.

Characteristics of community-acquired carbapenem-resistant Enterobacterales.

BACKGROUND: Community-acquired carbapenem-resistant Enterobacterales (CA-CRE) are an important threat. METHODS: In CRACKLE-2, we defined patients with CA-CRE as admitted from home, without pre-existing conditions, and a positive culture within 48 h of admission. Healthcare-associated CRE (HA-CRE) were those with the lowest likelihood of community acquisition, not admitted from home and cultured >48 h after admission. Specific genetic markers in carbapenemase-producing Klebsiella pneumoniae were evaluated through random forest modelling. RESULTS: CA-CRE and HA-CRE were detected in 83 (10%) and 208 (26%) of 807 patients. No significant differences were observed in bacterial species or strain type distribution. K. pneumoniae (204/291, 70%) was the most common CRE species, of these 184/204 (90%) were carbapenemase producers (CPKP). The top three genetic markers in random forest models were kpi_SA15, fimE, and kpfC. Of these, kpi_SA15 (which encodes a chaperone/usher system) was positively associated (OR 3.14, 95% CI 1.13-8.87, P = 0.026), and kpfC negatively associated (OR 0.21, 95% CI 0.05-0.72, P = 0.015) with CA-CPKP. CONCLUSIONS: Ten percent of CDC-defined CRE were CA. The true proportion of CA-CRE in hospitalized patients is likely lower as patients may have had unrecorded prior healthcare exposure. The kpi_SA15 operon was associated with the CA phenotype.

Carbapenem-Resistant Acinetobacter baumannii in U.S. Hospitals: Diversification of Circulating Lineages and Antimicrobial Resistance.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is a major cause of health care-associated infections. CRAb is typically multidrug resistant, and infection is difficult to treat. Despite the urgent threat that CRAb poses, few systematic studies of CRAb clinical and molecular epidemiology have been conducted. The Study Network of Acinetobacter as a Carbapenem-Resistant Pathogen (SNAP) is designed to investigate the clinical characteristics and contemporary population structure of CRAb circulating in U.S. hospital systems using whole-genome sequencing (WGS). Analysis of the initial 120 SNAP patients from four U.S. centers revealed that CRAb remains a significant threat to hospitalized patients, affecting the most vulnerable patients and resulting in 24% all-cause 30-day mortality. The majority of currently circulating isolates belonged to ST2Pas, a part of clonal complex 2 (CC2), which is the dominant drug-resistant lineage in the United States and Europe. We identified three distinct sublineages within CC2, which differed in their antibiotic resistance phenotypes and geographic distribution. Most concerning, colistin resistance (38%) and cefiderocol resistance (10%) were common within CC2 sublineage C (CC2C), where the majority of isolates belonged to ST2Pas/ST281Ox. Additionally, we identified ST499Pas as the most common non-CC2 lineage in our study. Our findings suggest a shift within the CRAb population in the United States during the past 10 years and emphasize the importance of real-time surveillance and molecular epidemiology in studying CRAb dissemination and clinical impact. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAb) constitutes a major threat to public health. To elucidate the molecular and clinical epidemiology of CRAb in the United States, clinical CRAb isolates were collected along with data on patient characteristics and outcomes, and bacterial isolates underwent whole-genome sequencing and antibiotic susceptibility phenotyping. Key findings included emergence of new sublineages within the globally predominant clonal complex 2 (CC2), increased colistin and cefiderocol resistance within one of the CC2 sublineages, and emergence of ST499Pas as the dominant non-CC2 CRAb lineage in U.S. hospitals.

Accessory Genomes Drive Independent Spread of Carbapenem-Resistant Klebsiella pneumoniae Clonal Groups 258 and 307 in Houston, TX.

Carbapenem-resistant Klebsiella pneumoniae (CRKp) is an urgent public health threat. Worldwide dissemination of CRKp has been largely attributed to clonal group (CG) 258. However, recent evidence indicates the global emergence of a CRKp CG307 lineage. Houston, TX, is the first large city in the United States with detected cocirculation of both CRKp CG307 and CG258. We sought to characterize the genomic and clinical factors contributing to the parallel endemic spread of CG258 and CG307. CRKp isolates were collected as part of the prospective, Consortium on Resistance against Carbapenems in Klebsiella and other Enterobacterales 2 (CRACKLE-2) study. Hybrid short-read and long-read genome assemblies were generated from 119 CRKp isolates (95 originated from Houston hospitals). A comprehensive characterization of phylogenies, gene transfer, and plasmid content with pan-genome analysis was performed on all CRKp isolates. Plasmid mating experiments were performed with CG307 and CG258 isolates of interest. Dissection of the accessory genomes suggested independent evolution and limited horizontal gene transfer between CG307 and CG258 lineages. CG307 contained a diverse repertoire of mobile genetic elements, which were shared with other non-CG258 K. pneumoniae isolates. Three unique clades of Houston CG307 isolates clustered distinctly from other global CG307 isolates, indicating potential selective adaptation of particular CG307 lineages to their respective geographical niches. CG307 strains were often isolated from the urine of hospitalized patients, likely serving as important reservoirs for genes encoding carbapenemases and extended-spectrum ß-lactamases. Our findings suggest parallel cocirculation of high-risk lineages with potentially divergent evolution. IMPORTANCE The prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKp) infections in nosocomial settings remains a public health challenge. High-risk clones such as clonal group 258 (CG258) are particularly concerning due to their association with blaKPC carriage, which can severely complicate antimicrobial treatments. There is a recent emergence of clonal group 307 (CG307) worldwide with little understanding of how this successful clone has been able to adapt while cocirculating with CG258. We provide the first evidence of potentially divergent evolution between CG258 and CG307 with limited sharing of adaptive genes. Houston, TX, is home to the largest medical center in the world, with a large influx of domestic and international patients. Thus, our unique geographical setting, where two pandemic strains of CRKp are circulating, provides an indication of how differential accessory genome content can drive stable, endemic populations of CRKp. Pan-genomic analyses such as these can reveal unique signatures of successful CRKp dissemination, such as the CG307-associated plasmid (pCG307_HTX), and provide invaluable insights into the surveillance of local carbapenem-resistant Enterobacterales (CRE) epidemiology.

Contemporary Clinical and Molecular Epidemiology of Vancomycin-Resistant Enterococcal Bacteremia: A Prospective Multicenter Cohort Study (VENOUS I).

BACKGROUND: Vancomycin-resistant enterococci (VRE) are major therapeutic challenges. Prospective contemporary data characterizing the clinical and molecular epidemiology of VRE bloodstream infections (BSIs) are lacking. METHODS: The Vancomycin-Resistant Enterococcal BSI Outcomes Study (VENOUS I) is a prospective observational cohort of adult patients with enterococcal BSI in 11 US hospitals. We included patients with Enterococcus faecalis or Enterococcus faecium BSI with ≥1 follow-up blood culture(s) within 7 days and availability of isolate(s) for further characterization. The primary study outcome was in-hospital mortality. Secondary outcomes were mortality at days 4, 7, 10, 12, and 15 after index blood culture. A desirability of outcome ranking was constructed to assess the association of vancomycin resistance with outcomes. All index isolates were subjected to whole genome sequencing. RESULTS: Forty-two of 232 (18%) patients died in hospital and 39 (17%) exhibited microbiological failure (lack of clearance in the first 4 days). Neutropenia (hazard ratio [HR], 3.13), microbiological failure (HR, 2.4), VRE BSI (HR, 2.13), use of urinary catheter (HR, 1.85), and Pitt BSI score ≥2 (HR, 1.83) were significant predictors of in-hospital mortality. Microbiological failure was the strongest predictor of in-hospital mortality in patients with E faecium bacteremia (HR, 5.03). The impact of vancomycin resistance on mortality in our cohort changed throughout the course of hospitalization. Enterococcus faecalis sequence type 6 was a predominant multidrug-resistant lineage, whereas a heterogeneous genomic population of E faecium was identified. CONCLUSIONS: Failure of early eradication of VRE from the bloodstream is a major factor associated with poor outcomes.

Clinical outcomes and bacterial characteristics of carbapenem-resistant Klebsiella pneumoniae complex among patients from different global regions (CRACKLE-2): a prospective, multicentre, cohort study.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a global threat. We therefore analysed the bacterial characteristics of CRKP infections and the clinical outcomes of patients with CRKP infections across different countries. METHODS: In this prospective, multicentre, cohort study (CRACKLE-2), hospitalised patients with cultures positive for CRKP were recruited from 71 hospitals in Argentina, Australia, Chile, China, Colombia, Lebanon, Singapore, and the USA. The first culture positive for CRKP was included for each unique patient. Clinical data on post-hospitalisation death and readmission were collected from health records, and whole genome sequencing was done on all isolates. The primary outcome was a desirability of outcome ranking at 30 days after the index culture, and, along with bacterial characteristics and 30-day all-cause mortality (a key secondary outcome), was compared between patients from China, South America, and the USA. The desirability of outcome ranking was adjusted for location before admission, Charlson comorbidity index, age at culture, Pitt bacteremia score, and anatomical culture source through inverse probability weighting; mortality was adjusted for the same confounders, plus region where relevant, through multivariable logistic regression. This study is registered at ClinicalTrials.gov, NCT03646227, and is complete. FINDINGS: Between June 13, 2017, and Nov 30, 2018, 991 patients were enrolled, of whom 502 (51%) met the criteria for CRKP infection and 489 (49%) had positive cultures that were considered colonisation. We observed little intra-country genetic variation in CRKP. Infected patients from the USA were more acutely ill than were patients from China or South America (median Pitt bacteremia score 3 [IQR 2-6] vs 2 [0-4] vs 2 [0-4]) and had more comorbidities (median Charlson comorbidity index 3 [IQR 2-5] vs 1 [0-3] vs 1 [0-2]). Adjusted desirability of outcome ranking outcomes were similar in infected patients from China (n=246), South America (n=109), and the USA (n=130); the estimates were 53% (95% CI 42-65) for China versus South America, 50% (41-61) for the USA versus China, and 53% (41-66) for the USA versus South America. In patients with CRKP infections, unadjusted 30-day mortality was lower in China (12%, 95% CI 8-16; 29 of 246) than in the USA (23%, 16-30; 30 of 130) and South America (28%, 20-37; 31 of 109). Adjusted 30-day all-cause mortality was higher in South America than in China (adjusted odds ratio [aOR] 4·82, 95% CI 2·22-10·50) and the USA (aOR 3·34, 1·50-7·47), with the mortality difference between the USA and China no longer being significant (aOR 1·44, 0·70-2·96). INTERPRETATION: Global CRKP epidemics have important regional differences in patients' baseline characteristics and clinical outcomes, and in bacterial characteristics. Research findings from one region might not be generalisable to other regions. FUNDING: The National Institutes of Health.

Molecular and clinical epidemiology of carbapenem-resistant Enterobacterales in the USA (CRACKLE-2): a prospective cohort study.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are a global threat. We aimed to describe the clinical and molecular characteristics of Centers for Disease Control and Prevention (CDC)-defined CRE in the USA. METHODS: CRACKLE-2 is a prospective, multicentre, cohort study. Patients hospitalised in 49 US hospitals, with clinical cultures positive for CDC-defined CRE between April 30, 2016, and Aug 31, 2017, were included. There was no age exclusion. The primary outcome was desirability of outcome ranking (DOOR) at 30 days after index culture. Clinical data and bacteria were collected, and whole genome sequencing was done. This trial is registered with ClinicalTrials.gov, number NCT03646227. FINDINGS: 1040 patients with unique isolates were included, 449 (43%) with infection and 591 (57%) with colonisation. The CDC-defined CRE admission rate was 57 per 100 000 admissions (95% CI 45-71). Three subsets of CDC-defined CRE were identified: carbapenemase-producing Enterobacterales (618 [59%] of 1040), non-carbapenemase-producing Enterobacterales (194 [19%]), and unconfirmed CRE (228 [22%]; initially reported as CRE, but susceptible to carbapenems in two central laboratories). Klebsiella pneumoniae carbapenemase-producing clonal group 258 K pneumoniae was the most common carbapenemase-producing Enterobacterales. In 449 patients with CDC-defined CRE infections, DOOR outcomes were not significantly different in patients with carbapenemase-producing Enterobacterales, non-carbapenemase-producing Enterobacterales, and unconfirmed CRE. At 30 days 107 (24%, 95% CI 20-28) of these patients had died. INTERPRETATION: Among patients with CDC-defined CRE, similar outcomes were observed among three subgroups, including the novel unconfirmed CRE group. CDC-defined CRE represent diverse bacteria, whose spread might not respond to interventions directed to carbapenemase-producing Enterobacterales. FUNDING: National Institutes of Health.

The Pitt Bacteremia Score Predicts Mortality in Nonbacteremic Infections.

BACKGROUND: Predicting mortality risk in patients is important in research settings. The Pitt bacteremia score (PBS) is commonly used as a predictor of early mortality risk in patients with bloodstream infections (BSIs). We determined whether the PBS predicts 14-day inpatient mortality in nonbacteremia carbapenem-resistant Enterobacteriaceae (CRE) infections. METHODS: Patients were selected from the Consortium on Resistance Against Carbapenems in Klebsiella and Other Enterobacteriaceae, a prospective, multicenter, observational study. We estimated risk ratios to analyze the predictive ability of the PBS overall and each of its components individually. We analyzed each component of the PBS in the prediction of mortality, assessed the appropriate cutoff value for the dichotomized score, and compared the predictive ability of the qPitt score to that of the PBS. RESULTS: In a cohort of 475 patients with CRE infections, a PBS ≥4 was associated with mortality in patients with nonbacteremia infections (risk ratio [RR], 21.9; 95% confidence interval [CI], 7.0, 68.8) and with BSIs (RR, 6.0; 95% CI, 2.5, 14.4). In multivariable analysis, the hypotension, mechanical ventilation, mental status, and cardiac arrest parameters of the PBS were independent risk factors for 14-day all-cause inpatient mortality. The temperature parameter as originally calculated for the PBS was not independently associated with mortality. However, a temperature <36.0°C vs ≥36°C was independently associated with mortality. A qPitt score ≥2 had similar discrimination as a PBS ≥4 in nonbacteremia infections. CONCLUSIONS: Here, we validated that the PBS and qPitt score can be used as reliable predictors of mortality in nonbacteremia CRE infections.

The Role of Trimethoprim/Sulfamethoxazole in the Treatment of Infections Caused by Carbapenem-Resistant Enterobacteriaceae.

In the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE), trimethoprim-sulfamethoxazole (TMP-SMX) had a limited role in the treatment of less severe carbapenem-resistant Enterobacteriaceae (CRE) infections, especially urinary tract infections. Of tested CRE, only 29% were susceptible to TMP-SMX. Development of resistance further limits the use of TMP-SMX in CRE infections.

Cross Talk between MarR-Like Transcription Factors Coordinates the Regulation of Motility in Uropathogenic Escherichia coli.

The MarR-like protein PapX represses the transcription of the flagellar master regulator genes flhDC in uropathogenic Escherichia coli (UPEC), the primary cause of uncomplicated urinary tract infections (UTIs). PapX is encoded by the pap operon, which also encodes the adherence factors termed P fimbriae. Both adherence and motility are critical for productive colonization of the urinary tract. However, the mechanisms involved in coordinating the transition between adherence and motility are not well characterized. UPEC strain CFT073 carries both papX and a homolog, focX, located in the foc operon encoding F1C fimbriae. In this study, we characterized the dose effects of "X" genes on flagellar gene expression and cross talk between focX and papX We found that both FocX and PapX repress flhD transcription. However, we determined that the ΔpapX mutant was hypermotile, while the loss of focX did not affect motility. We further investigated this phenotype and found that FocX functions as a repressor of papX Additionally, we identified a proximal independent promoter upstream of both focX and papX and assessed the expression of focX and papX during culture in human urine and on LB agar plates compared to LB medium. Finally, we characterized the contributions of PapX and FocX to fitness in the ascending murine model of UTI and observed a subtle, but not statistically significant, fitness defect in colonization of the kidneys. Altogether, these results expand our understanding of the impact of carrying multiple X genes on the coordinated regulation of motility and adherence in UPEC.

TosR-Mediated Regulation of Adhesins and Biofilm Formation in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli strains utilize a variety of adherence factors that assist in colonization of the host urinary tract. TosA (type one secretion A) is a nonfimbrial adhesin that is predominately expressed during murine urinary tract infection (UTI), binds to kidney epithelial cells, and promotes survival during invasive infections. The tosRCBDAEF operon encodes the secretory machinery necessary for TosA localization to the E. coli cell surface, as well as the transcriptional regulator TosR. TosR binds upstream of the tos operon and in a concentration-dependent manner either induces or represses tosA expression. TosR is a member of the PapB family of fimbrial regulators that can participate in cross talk between fimbrial operons. TosR also binds upstream of the pap operon and suppresses PapA production. However, the scope of TosR-mediated cross talk is understudied and may be underestimated. To quantify the global effects of TosR-mediated regulation on the E. coli CFT073 genome, we induced expression of tosR, collected mRNA, and performed high-throughput RNA sequencing (RNA-Seq). These findings show that production of TosR affected the expression of genes involved with adhesins, including P, F1C, and Auf fimbriae, nitrate-nitrite transport, microcin secretion, and biofilm formation.IMPORTANCE Uropathogenic E. coli strains cause the majority of UTIs, which are the second most common bacterial infection in humans. During a UTI, bacteria adhere to cells within the urinary tract, using a number of different fimbrial and nonfimbrial adhesins. Biofilms can also develop on the surfaces of catheters, resulting in complications such as blockage. In this work, we further characterized the regulator TosR, which links both adhesin production and biofilm formation and likely plays a crucial function during UTI and disseminated infection.

Signature-tagged mutagenesis and co-infection studies demonstrate the importance of P fimbriae in a murine model of urinary tract infection.

Escherichia coli is the leading cause of urinary tract infections (UTIs), one of the most common infections in humans. P fimbria was arguably the first proposed virulence factor for uropathogenic E. coli, based on the capacity of E. coli isolated from UTIs to adhere to exfoliated epithelial cells in higher numbers than fecal strains of E. coli. Overwhelming epidemiologic evidence has been presented for involvement of P fimbriae in colonization. It has been difficult, however, to demonstrate this requirement for uropathogenic strains in animal models of infections or in humans. In this study, a signature-tagged mutagenesis screen identified a P-fimbrial gene (papC) and 18 other genes as being among those required for full fitness of cystitis isolate E. coli F11. A P-fimbrial mutant was outcompeted by the wild-type strain in cochallenge in the murine model of ascending UTI, and this colonization defect could be complemented with the cloned pap operon. To our knowledge, this study is the first to fulfill molecular Koch's postulates in which a pathogenic strain was attenuated by mutation of pap genes and then complemented to restore fitness, confirming P fimbria as a virulence factor in a pathogenic clinical isolate.

Bacterial colonization of host cells in the absence of cholesterol.

Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/-) mouse embryonic fibroblasts (MEFs). DHCR24(-/-) MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/-) MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/-) MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(V)ß(3) integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/-) MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

Salmonella - at home in the host cell.

The Gram-negative bacterium Salmonella enterica has developed an array of sophisticated tools to manipulate the host cell and establish an intracellular niche, for successful propagation as a facultative intracellular pathogen. While Salmonella exerts diverse effects on its host cell, only the cell biology of the classic "trigger"-mediated invasion process and the subsequent development of the Salmonella-containing vacuole have been investigated extensively. These processes are dependent on cohorts of effector proteins translocated into host cells by two type III secretion systems (T3SS), although T3SS-independent mechanisms of entry may be important for invasion of certain host cell types. Recent studies into the intracellular lifestyle of Salmonella have provided new insights into the mechanisms used by this pathogen to modulate its intracellular environment. Here we discuss current knowledge of Salmonella-host interactions including invasion and establishment of an intracellular niche within the host.