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Persons with cystic fibrosis, starting in early life, have intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid (SCFA) propionate. We demonstrate here that CFTR-/- Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1β-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. Bacteroides isolates also produce low levels of butyrate; this SCFA is positively correlated with inhibition of the inflammatory response. Finally, the introduction of Bacteroides-supplemented stool from infants with CF into the gut of CftrF508del mice results in an increase in propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of E. coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. Together, our data indicate the important role of Bacteroides and Bacteroides-derived propionate in the context of the developing microbiome in infants and children with CF, which could help explain the observed gut-lung axis in CF.

A recent workshop titled “Developing Models to Study Polymicrobial Infections,” sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of bacteria with members responsible for causing lung infections in cystic fibrosis (CF) patients. The most severe outcome of Bcc infection in CF patients is cepacia syndrome, a disease characterized by necrotizing pneumonia with bacteremia and sepsis. B. cenocepacia is strongly associated with cepacia syndrome, making it one of the most virulent members of the Bcc. Mechanisms underlying the pathogenesis of B. cenocepacia in lung infections and cepacia syndrome remain to be uncovered. B. cenocepacia is primarily an intracellular pathogen and encodes the type VI secretion system (T6SS) effector TecA, which is translocated into host phagocytes. TecA is a deamidase that inactivates multiple Rho GTPases, including RhoA. Inactivation of RhoA by TecA triggers assembly of the pyrin inflammasome, leading to secretion of proinflammatory cytokines, such as interleukin-1β, from macrophages. Previous work with the B. cenocepacia clinical isolate J2315 showed that TecA increases immunopathology during acute lung infection in C57BL/6 mice and suggested that this effector acts as a virulence factor by triggering assembly of the pyrin inflammasome. Here, we extend these results using a second B. cenocepacia clinical isolate, AU1054, to demonstrate that TecA exacerbates weight loss and lethality during lung infection in C57BL/6 mice and mice engineered to have a CF genotype. Unexpectedly, pyrin was dispensable for TecA virulence activity in both mouse infection models. Our findings establish that TecA is a B. cenocepacia virulence factor that exacerbates lung inflammation, weight loss, and lethality in mouse infection models. IMPORTANCE B. cenocepacia is often considered the most virulent species in the Bcc because of its close association with cepacia syndrome in addition to its capacity to cause chronic lung infections in CF patients (1). Prior to the current study, virulence factors of B. cenocepacia important for causing lethal disease had not been identified in a CF animal model of lung infection. Results of this study describe a CF mouse model and its use in demonstrating that the T6SS effector TecA of B. cenocepacia exacerbates inflammatory cell recruitment and weight loss and is required for lethality and, thus, acts as a key virulence factor during lung infection. This model will be important in further studies to better understand TecA's role as a virulence factor and in investigating ways to prevent or treat B. cenocepacia infections in CF patients. Additionally, TecA may be the founding member of a family of virulence factors in opportunistic pathogens.

Burkholderia cenocepacia (Bc) is a member of the Burkholderia cepacia complex (Bcc), a group of bacteria with members responsible for causing lung infections in cystic fibrosis (CF) patients. The most severe outcome of Bcc infection in CF patients is cepacia syndrome, a disease characterized by necrotizing pneumonia with bacteremia and sepsis. Bc is strongly associated with cepacia syndrome making it one of the most virulent members of the Bcc. Mechanisms underlying the pathogenesis of Bc in lung infections and cepacia syndrome remain to be uncovered. Bc is primarily an intracellular pathogen, and encodes the type VI secretion system (T6SS) anti-host effector TecA, which is translocated into host cells. TecA is a deamidase that inactivates multiple Rho GTPases, including RhoA. Inactivation of RhoA by TecA triggers assembly of the pyrin inflammasome, leading to secretion of proinflammatory cytokines such as IL-1β from macrophages. Previous work with the Bc clinical isolate J2315 showed that TecA increases immunopathology during acute lung infection in C57BL/6 mice and suggested that this effector acts as a virulence factor by triggering assembly of the pyrin inflammasome. Here, we extend these results using a second Bc clinical isolate, AU1054, to demonstrate that TecA exacerbates weight loss and lethality during lung infection in C57BL/6 mice and CF mice. Unexpectedly, pyrin was dispensable for TecA virulence activity in both mouse infection models. Our findings establish that TecA is a Bc virulence factor that exacerbates lung inflammation, weight loss, and lethality in a mouse lung infection model.ImportanceBc is often considered the most virulent species in the Bcc because of its close association with cepacia syndrome in addition to its capacity to cause chronic lung infections in CF patients (Loutet and Valvano 2010). Prior to this study virulence factors of Bc important for causing lethal disease had not been identified in a CF animal model of lung infection. Results of this study describe a CF mouse model and its use in demonstrating that the T6SS effector TecA of Bc exacerbates inflammatory cell recruitment and weight loss and is required for lethality and thus acts as a key virulence factor during lung infection. This model will be important in further studies to better understand TecA’s role as a virulence factor and in investigating ways to prevent or treat Bc infections in CF patients. Additionally, TecA may be the founding member of a family of virulence factors in opportunistic pathogens.

Cystic fibrosis (CF) is a genetic disease that causes patients to accumulate thick, dehydrated mucus in the lung and develop chronic, polymicrobial infections due to reduced mucociliary clearance. These chronic polymicrobial infections and subsequent decline in lung function are significant factors in the morbidity and mortality of CF. Pseudomonas aeruginosa and Streptococcus spp. are among the most prevalent organisms in the CF lung; the presence of P. aeruginosa correlates with lung function decline, and the Streptococcus milleri group (SMG), a subgroup of the viridans streptococci, is associated with exacerbations in patients with CF. Here we characterized the interspecies interactions that occur between these two genera. We demonstrated that multiple P. aeruginosa laboratory strains and clinical CF isolates promote the growth of multiple SMG strains and oral streptococci in an in vitro coculture system. We investigated the mechanism by which P. aeruginosa enhances growth of streptococci by screening for mutants of P. aeruginosa PA14 that are unable to enhance Streptococcus growth, and we identified the P. aeruginosapqsL::TnM mutant, which failed to promote growth of Streptococcus constellatus and S. sanguinis. Characterization of the P. aeruginosa ΔpqsL mutant revealed that this strain cannot promote Streptococcus growth. Our genetic data and growth studies support a model whereby the P. aeruginosa ΔpqsL mutant overproduces siderophores and thus likely outcompetes Streptococcus sanguinis for limited iron. We propose a model whereby competition for iron represents one important means of interaction between P. aeruginosa and Streptococcus spp. IMPORTANCE Cystic fibrosis (CF) lung infections are increasingly recognized for their polymicrobial nature. These polymicrobial infections may alter the biology of the organisms involved in CF-related infections, leading to changes in growth, virulence, and/or antibiotic tolerance, and could thereby affect patient health and response to treatment. In this study, we demonstrate interactions between P. aeruginosa and streptococci using a coculture model and show that one interaction between these microbes is likely competition for iron. Thus, these data indicate that one CF pathogen may influence the growth of another, and they add to our limited knowledge of polymicrobial interactions in the CF airway.

Here we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities., Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect 42 bacterial and fungal genera or species common in cystic fibrosis (CF) sputum and demonstrated the taxon specificity of these probes, as well as a linear response over more than 3 logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString, and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. Compared at the genus level, the relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and with further development, may provide quick and easily interpreted diagnostic information on diverse bacteria and fungi at the genus or species level. IMPORTANCE Here we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.

Cystic Fibrosis (CF) is a genetic disease that causes patients to accumulate thick, dehydrated mucus in the lung and develop chronic, polymicrobial infections due to reduced mucociliary clearance. These chronic polymicrobial infections and subsequent decline in lung function are significant factors in the morbidity and mortality of CF.Pseudomonas aeruginosaandStreptococcusspp. are among the most prevalent organisms in the CF lung; the presence ofP. aeruginosacorrelates with lung function decline and theStreptococcus millerigroup (SMG), a subgroup of the viridans streptococci, is associated with exacerbations in patients with CF. Here we characterize the interspecies interactions that occur between these two genera. We demonstrated that multipleP. aeruginosalaboratory strains and clinical CF isolates promote the growth of multiple SMG strains and oral streptococci in anin vitrococulture system. We investigated the mechanism by whichP. aeruginosaenhances growth of streptococci by screening for mutants ofP. aeruginosaPA14 unable to enhanceStreptococcusgrowth, and we identified theP. aeruginosa pqsL::TnMmutant, which failed to promote growth ofS. constellatusandS. sanguinis. Characterization of theP. aeruginosaΔpqsLmutant revealed that this strain cannot promoteStreptococcusgrowth. Our genetic data and growth studies support a model whereby theP. aeruginosaΔpqsLmutant overproduces siderophores, and thus likely outcompetesStreptococcus sanguinisfor limited iron. We propose a model whereby competition for iron represents one important means of interaction betweenP. aeruginosaandStreptococcusspp.ImportanceCystic fibrosis (CF) lung infections are increasingly recognized for their polymicrobial nature. These polymicrobial infections may alter the biology of the organisms involved in CF-related infections, leading to changes in growth, virulence and/or antibiotic tolerance, and could thereby affect patient health and response to treatment. In this study, we demonstrate interactions betweenP. aeruginosaand streptococci using a coculture model, and show that one interaction between these microbes is likely competition for iron. Thus, these data indicate that one CF pathogen may influence the growth of another and add to our limited knowledge of polymicrobial interactions in the CF airway.

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

Cokeromyces recurvatus is a zygomycetes yeast form that is very rarely detected in Papanicolaou (Pap) tests, in which it typically represents an innocuous colonizer. Its morphology closely resembles that of the better known Paracoccidioides brasiliensis, which can disseminate widely and cause clinically significant disease. We present a case of C. recurvatus detected in a cervical liquid-based preparation obtained from a 38-year old healthy woman. Careful cytomorphologic evaluation, in combination with culture and molecular techniques, was utilized to make a diagnosis and prevent the misdiagnosis of P. brasiliensis.

In a loss-of-viability screen using small molecules against methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 with a sub-MIC of a β-lactam, we found a small molecule, designated DNAC-1, which potentiated the effect of oxacillin (i.e., the MIC of oxacillin decreased from 64 to 0.25 μg/ml). Fluorescence microscopy indicated a disruption in the membrane structures within 15 min of exposure to DNAC-1 at 2× MIC. This permeabilization was accompanied by a rapid loss of membrane potential, as monitored by use of the DiOC 2 (3,3′-diethyloxacarbocyanine iodide) dye. Macromolecular analysis showed the inhibition of staphylococcal cell wall synthesis by DNAC-1. Transmission electron microscopy of treated MRSA USA300 cells revealed a slightly thicker cell wall, together with mesosome-like projections into the cytosol. The exposure of USA300 cells to DNAC-1 was associated with the mislocalization of FtsZ accompanied by the localization of penicillin-binding protein 2 (PBP2) and PBP4 away from the septum, as well as mild activation of the vraRS -mediated cell wall stress response. However, DNAC-1 does not have any generalized toxicity toward mammalian host cells. DNAC-1 in combination with ceftriaxone is also effective against an assortment of Gram-negative pathogens. Using a murine subcutaneous coinjection model with 10 8 CFU of USA300 as a challenge inoculum, DNAC-1 alone or DNAC-1 with a sub-MIC of oxacillin resulted in a 6-log reduction in bacterial load and decreased abscess formation compared to the untreated control. We propose that DNAC-1, by exerting a bimodal effect on the cell membrane and cell wall, is a viable candidate in the development of combination therapy against many common bacterial pathogens.

The identification of pathogen-specific volatile metabolic “fingerprints” could lead to the rapid identification of disease-causing organisms either directly from ex vivo patient bio-specimens or from in vitro cultures. In the present study, we have evaluated the volatile metabolites produced by 100 clinical isolates belonging to ten distinct pathogen groups that, in aggregate, account for 90 % of bloodstream infections, 90 % of urinary tract infections, and 80 % of infections encountered in the intensive care unit setting. Headspace volatile metabolites produced in vitro were concentrated using headspace solid-phase microextraction and analyzed via two-dimensional gas chromatography time-of-flight mass spectrometry (HS-SPME-GC×GC-TOFMS). A total of 811 volatile metabolites were detected across all samples, of which 203 were: 1) detected in 9 or 10 (of 10) isolates belonging to one or more pathogen groups, and 2) significantly more abundant in cultures relative to sterile media. Network analysis revealed a distinct metabolic fingerprint associated with each pathogen group, and analysis via Random Forest (RF) using leave-one-out cross-validation (LOOCV) resulted in a 95 % accuracy for the differentiation between groups. The present findings support the results of prior studies that have reported on the differential production of volatile metabolites across pathogenic bacteria and fungi, and provide additional insight through the inclusion of pathogen groups that have seldom been studied previously, including Acinetobacter spp., coagulase-negative Staphylococcus, and Proteus mirabilis, as well as the utilization of HS-SPME-GC×GC-TOFMS for improved sensitivity and resolution relative to traditional gas chromatography-based techniques.

OBJECTIVES Aztreonam for inhalation solution (AZLI) was recently approved by the FDA for treating cystic fibrosis (CF) patients infected with Pseudomonas aeruginosa. Here we investigated the effect of aztreonam alone or in combination with tobramycin on P. aeruginosa biofilms grown on CF airway epithelial cells. METHODS P. aeruginosa biofilms, produced by laboratory strains or clinical isolates, were formed on confluent CF airway cells before treatment overnight with aztreonam or tobramycin alone or in combination. Alternatively, antibiotics were added 1 h after bacterial inoculation to assess their ability to impair biofilm formation at 5 h. Bacterial cfu remaining after treatment were then determined by plate counting. RESULTS In the absence of antibiotics, all strains developed biofilms that disrupted CF airway epithelial monolayers overnight. Tobramycin reduced the cfu of all strains grown as biofilms. Aztreonam reduced the cfu of some strains by ∼1 log unit without preserving the integrity of cystic fibrosis airway cell monolayers, while decreasing the biofilms of other clinical isolates by ∼4 log units and protecting the monolayers from being compromised. The combination of aztreonam and tobramycin reduced the cfu of two strains by an additional 0.5 and 2 log units, respectively. Of all the mechanisms explored, Psl exopolysaccharide production might explain the variations in biofilm tolerance to aztreonam in some of the strains. CONCLUSIONS Effects of aztreonam on P. aeruginosa biofilms in the in vitro co-culture model are strain-dependent. The simultaneous application of aztreonam and tobramycin may be beneficial for a subset of CF patients by eliminating susceptible P. aeruginosa strains.

The expression of virulence factors in Staphylococcus aureus is tightly coordinated by a vast network of regulatory molecules. In this report, we characterize a genetic locus unique to staphylococci called rsr that has a role in repressing two key virulence regulators, sarR and agr . Using strain SH1000, we showed that the transcription of virulence effectors, such as hla , sspA , and spa , is altered in an rsr mutant in a way consistent with agr upregulation. Analysis of RNAIII expression of the agr locus in rsr and rsr-sarR mutants indicated that rsr likely contributes to agr expression independently of SarR. We also provide evidence using a murine model of S. aureu s skin infection that the effects mediated by rsr reduce disease progression.

The regulation of cellular processes by eukaryote-like serine/threonine kinases is widespread in bacteria. In the last 2 years, several studies have examined the role of serine/threonine kinases in Staphylococcus aureus on cell wall metabolism, autolysis, and virulence, mostly in S. aureus laboratory isolates in the 8325-4 lineage. In this study, we showed that the pknB gene (also called stk1 ) of methicillin-resistant S. aureus (MRSA) strain COL and the community-acquired MRSA (CA-MRSA) strain USA300 is involved in cell wall metabolism, with the pknB mutant exhibiting enhanced sensitivity to β-lactam antibiotics but not to other classes of antibiotics, including aminoglycosides, ciprofloxacin, bactrim, and other types of cell wall-active agents (e.g., vancomycin and bacitracin). Additionally, the pknB mutant of USA300 was found to be more resistant to Triton X-100-induced autolysis and also to lysis by lysostaphin. We also showed that pknB is a positive regulator of sigB activity, resulting in compromise in its response to heat and oxidative stresses. In association with reduced sigB activity, the expression levels of RNAII and RNAIII of agr and the downstream effector hla are upregulated while spa expression is downmodulated in the pknB mutant compared to the level in the parent. Consistent with an enhanced agr response in vitro , virulence studies of the pknB mutant of USA300 in a murine cutaneous model of infection showed that the mutant was more virulent than the parental strain. Collectively, our results have linked the pknB gene in CA-MRSA to antibiotic resistance, sigB activity, and virulence and have highlighted important differences in pknB phenotypes (virulence and sigB activity) between laboratory isolates and the prototypic CA-MRSA strain USA300.

Background The current prevalence of hospital-acquired infections and evolving amplification of bacterial resistance are major public health concerns. A heightened awareness of intraoperative transmission of potentially pathogenic bacterial organisms may lead to implementation of effective preventative measures. Methods Sixty-one operative suites were randomly selected for analysis. Sterile intravenous stopcock sets and two sites on the anesthesia machine were decontaminated and cultured aseptically at baseline and at case completion. The primary outcome was the presence of a positive culture on the previously sterile patient stopcock set. Secondary outcomes were the number of colonies per surface area sampled on the anesthesia machine, species identification, and antibiotic susceptibility of isolated organisms. Results Bacterial contamination of the anesthesia work area increased significantly at the case conclusion, with a mean difference of 115 colonies per surface area sampled (95% confidence interval [CI], 62-169; P < 0.001). Transmission of bacterial organisms, including vancomycin-resistant enterococcus, to intravenous stopcock sets occurred in 32% (95% CI, 20.6-44.9%) of cases. Highly contaminated work areas increased the odds of stopcock contamination by 4.7 (95% CI, 1.42-15.42; P = 0.011). Contaminated intravenous tubing was associated with a trend toward increased nosocomial infection rates (odds ratio, 3.08; 95% CI, 0.56-17.5; P = 0.11) and with an increase in mortality (95% CI odds ratio, 1.11-infinity; P = 0.0395). Conclusion Potentially pathogenic, multidrug-resistant bacterial organisms are transmitted during the practice of general anesthesia to both the anesthesia work area and intravenous stopcock sets. Implementation of infection control measures in this area may help to reduce both the evolving problem of increasing bacterial resistance and the development of life-threatening infectious complications.

IFN-γ-producing CD4+T cells, although important for protection against acuteToxoplasma gondiiinfection, can cause gut pathology, which may prove to be detrimental for host survival. Here we show that mice lacking IL-15 gene develop a down-regulated IFN-γ-producing CD4+T cell response against the parasite, which leads to a reduction in gut necrosis and increased level of survival against infection. Moreover, transfer of immune CD4+T cells from WT to IL-15−/−mice reversed inhibition of gut pathology and caused mortality equivalent to levels of parental WT mice. Down-regulated CD4+T cell response in the absence of IL-15, manifested as reduced antigen-specific proliferation, was due to defective priming of the T cell subset by dendritic cells (DCs) of these animals. When stimulated with antigen-pulsed DCs from WT mice, CD4+T cells from IL-15−/−mice were primed optimally, and robust proliferation of these cells was observed. A defect in the DCs of knockout mice was further confirmed by their reduced ability to produce IL-12 upon stimulation withToxoplasmalysate antigen. Addition of exogenous IL-15 to DC cultures from knockout mice led to increased IL-12 production by these cells and restored their ability to prime an optimal parasite-specific CD4+T cell response. To our knowledge, this is the first demonstration of the role of IL-15 in the development of CD4+T cell immunity against an intracellular pathogen. Furthermore, based on these observations, targeting of IL-15 should have a beneficial effect on individuals suffering from CD4+T cell-mediated autoimmune diseases.

We investigated the role of interleukin-17 (IL-17)/IL-17 receptor (IL-17R)-mediated signaling in the protective immunity againstToxoplasma gondii. IL-17R−/−mice developed a normal adaptive immunity against the parasite. However, increased mortality in the knockout animals can be attributed to a defect in the migration of polymorphonuclear leukocytes to infected sites during early infection.

We report the identification of a virulent Streptococcus organism associated with fulminant endocarditis, using 16S rRNA gene amplification, sequencing and assembly from formalin-fixed, paraffin-embedded archival heart valve tissue, years after the autopsy of a patient.

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC ≤ 2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC ≥ 8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.

Gamma(delta) T cells have been reported to play an essential effector role during the early immune response against a wide variety of infectious agents. Recent studies have suggested that the gamma(delta) T cell subtype may also be important for the induction of adaptive immune response against certain microbial pathogens. In the present study, an early increase of gamma(delta) T cells during murine infection with Encephalitozoon cuniculi, an intracellular parasite, was observed. The role of gamma(delta) T cells against E. cuniculi infection was further evaluated by using gene-knockout mice. Mice lacking gamma(delta) T cells were susceptible to E. cuniculi infection at high challenge doses. The reduced resistance of delta(-/-) mice was attributed to a down-regulated CD8+ immune response. Compared with parental wild-type animals, suboptimal Ag-specific CD8+ T cell immunity against E. cuniculi infection was noted in delta(-/-) mice. The splenocytes from infected knockout mice exhibited a lower frequency of Ag-specific CD8+ T cells. Moreover, adoptive transfer of immune TCR(alpha)beta+ CD8+ cells from the delta(-/-) mice failed to protect naive CD8(-/-) mice against a lethal E. cuniculi challenge. Our studies suggest that gamma(delta) T cells, due to their ability to produce cytokines, are important for the optimal priming of CD8+ T cell immunity against E. cuniculi infection. This is the first evidence of a parasitic infection in which down-regulation of CD8+ T cell immune response in the absence of gamma(delta) T cells has been demonstrated.

Background & Aims: Acute inflammatory ileitis occurs in susceptible (C57BL/6) mice after oral infection with Toxoplasma gondii . Overproduction of interferon (IFN)-γ and synthesis of nitric oxide mediate the inflammation. We evaluated the role of transforming growth factor (TGF)-β produced by intraepithelial lymphocytes (IELs) in this process. Methods: We analyzed the histologic and immunologic consequences of adoptive transfer of antigen-primed IELs into susceptible mice treated with anti–TGF-β before oral challenge with T. gondii cysts. An in vitro coculture of enterocytes and IELs assessed the production of chemokines and cytokines in the presence of anti–TGF-β. Results: Antigen-primed IELs prevent acute ileitis in susceptible mice that is reversed with anti–TGF-β. Resistant mice (CBA/J) develop ileitis after treatment with anti–TGF-β. Antigen-primed IELs can induce systemic immunosuppression as measured by depressed IFN-γ production. In vitro, primed IELs reduce the production of inflammatory chemokines by infected enterocytes and IFN-γ by splenocytes. Conclusions: Regulation of the ileal inflammatory process resulting from T. gondii is dependent on TGF-β–producing IELs. The IELs are an essential component in gut homeostasis after oral infection with this parasite. GASTROENTEROLOGY 2001;120:914-924

Chemokines are critical for the recruitment of effector immune cells to sites of infection. Mice lacking the chemokine receptor CCR1 have defects in neutrophil trafficking and proliferation. In the present study, we tested the susceptibility of CCR1 knockout mice to infection with the obligate intracellular protozoan parasite Toxoplasma gondii. In comparison with parental wild-type mice, CCR1−/− mice exhibited dramatically increased mortality to T. gondii in association with an increased tissue parasite load. No differences were observed in Ag-specific T cell proliferation or in cytokine responses between mutant and wild-type mice. However, the influx of PMNs to the peripheral blood and to the liver were reduced in CCR1−/− mice during early infection. Our results suggest that CCR1-dependent migration of neutrophils to the blood and tissues may have a significant impact in controlling parasite replication.

In this study, we report that infection with Plasmodium yoelii 17XL, a lethal strain of rodent malaria, does not result in death in the DBA/2 strain of mice. In contrast to BALB/c mice, DBA/2 mice developed significantly less parasitemia and never manifested symptoms of cerebral malaria (CM) on infection with this parasite. Moreover, the histological changes evident in the brain of susceptible BALB/c were absent in DBA/2 mice. Interestingly, the resistant DBA/2 mice when treated with recombinant interleukin (IL)-2, were found to develop CM symptoms and the infection became fatal by 6 to 8 days after infection. This condition was associated with an augmented interferon-γ and nitric oxide production. Unexpectedly, IL-10 levels were also elevated in IL-2-treated DBA/2 mice during late stage of infection (at day 6 of infection) whereas the inverse relationship between IL-10 and interferon-γ or nitric oxide was maintained in the early stage of infection (at day 3 after infection). The level of tumor necrosis factor-α production was moderately increased in the late phase of infection in these mice. Histology of brain from IL-2-treated mice demonstrated the presence of parasitized erythrocytes and infiltration of lymphocytes in cerebral vessels, and also displayed some signs of endothelial degeneration. Confocal microscopical studies demonstrated preferential accumulation of γδ T cells in the cerebral vessels of IL-2-treated and -infected mice but not in mice treated with IL-2 alone. The cells recruited in the brain were activated because they demonstrated expression of CD25 (IL-2R) and CD54 (intercellular adhesion molecule 1) molecules. Administration of anti-γδ mAb prevented development of CM in IL-2-treated mice until day 18 after infection whereas mice treated with control antibody showed CM symptoms by day 6 after infection. The information concerning creating pathological sequelae and death in an otherwise resistant mouse strain provides an interesting focus for the burden of pathological attributes on death in an infectious disease.

We report a skin and soft-tissue infection outbreak among football team members due to a USA300 methicillin-susceptible Staphylococcus aureus (MRSA) strain with genes coding for Panton-Valentine leukocidin and the arginine catabolic mobile element. We postulate that the strain is a community-associated USA300 MRSA strain that lost methicillin resistance but retained important virulence factors.

Neospora caninum is a coccidial protozoan parasite that infects a large range of mammals including dogs, cats, mice, and cattle. Morphologically, N. caninum appears indistinguishable from Toxoplasma gondii, although they are genetically distinct. To date there have been no reported cases of this infection in humans, although nonhuman primates may be susceptible to infection. Inbred A/J mice develop no clinical and little histologic evidence of infection in spite of a high-dose inoculum of N. caninum. Splenocytes obtained from infected mice proliferate in vitro in response to both N. caninum and T. gondii-soluble antigen. A transient state of T cell hyporesponsiveness to parasite antigen and mitogen was observed at Day 7 p.i. This downregulatory response could be partially reversed by the addition of the nitric oxide antagonist LNMMA, but not antibody to IL-10. Mice infected with N. caninum produce significant quantities of IL-12 and IFN gamma, most evident shortly after infection. In vivo, antibody to IL-12 is able to neutralize immune resistance to the parasite. Moreover, in vivo depletion of IFN gamma with antibody renders the mice susceptible to infection. These observations suggest that N. caninum induces a T cell immune response in the infected host that is at least partially mediated by IL-12 and IFN gamma.

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

The critical role of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by both laboratory and clinical experts, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including Tickborne Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

The anticipated eradication of poliovirus emphasizes the need to identify other enteroviral causes of severe central nervous system disease. Enterovirus 68 has been implicated only in cases of respiratory illness. We therefore report a case of fatal meningomyeloencephalitis caused by enterovirus 68 in a 5-year-old boy, which required neuropathology, microbiology, and molecular techniques to diagnose.

Staphylococcus aureus bacteremia (SAB) is a lethal and increasingly common infection in hospitalized patients. We assessed the impact of infectious diseases consultation (IDC) on clinical management and hospital mortality of SAB in 240 hospitalized patients in a retrospective cohort study. Patients who received IDC were older than those who did not (57.9 vs. 51.7 yr; p = 0.05), and were more likely to have a health care-associated infection (63% vs. 45%; p < 0.01). In patients who received IDC, there was a higher prevalence of severe complications of SAB such as central nervous system involvement (5% vs. 0%, p = 0.01), endocarditis (20% vs. 2%; p < 0.01), or osteomyelitis (15.6% vs. 3.4%; p < 0.01). Patients who received IDC had closer blood culture follow-up and better antibiotic selection, and were more likely to have pus or prosthetic material removed. Hospital mortality from SAB was lower in patients who received IDC than in those who did not (13.9% vs. 23.7%; p = 0.05). In multivariate survival analysis, IDC was associated with substantially lower hazard of hospital mortality during SAB (hazard 0.46; p = 0.03). This mortality benefit accrued predominantly in patients with methicillin-resistant SAB (hazard 0.3; p < 0.01), and in patients who did not require ICU admission (hazard 0.15; p = 0.01). In conclusion, IDC is associated with reduced mortality in patients with staphylococcal bacteremia.

PURPOSE An outbreak of pneumococcal conjunctivitis occurred at Dartmouth College in 2002. We describe the clinical features, outcomes, and costs associated with this outbreak. METHODS Six hundred ninety-eight students were diagnosed with conjunctivitis; culture of conjunctival discharge was obtained for 254. A screening protocol was used to evaluate 67 patients. A retrospective survey was offered to all 698 cases and follow-up clinical examination to all patients with culture-confirmed infection (n = 110). Local ophthalmology offices were contacted to develop a cost analysis. The college health service provided conjunctivitis data for nonoutbreak years. RESULTS Of 67 patients evaluated using the screening protocol, findings associated with culture-confirmed Streptococcus pneumoniae conjunctivitis (P < 0.01) were red eye visible from 2 feet, any type of conjunctival discharge, obscuration of tarsal conjunctival blood vessels, and chemosis. Two hundred thirty-two students responded to our retrospective survey; 89% reported bilateral eye involvement; 96% received topical antibiotics and noted symptom improvement within 3 days of treatment. No ocular sequelae were identified as a result of this infection. No recurrent outbreaks have occurred at Dartmouth since the initial event. The estimated cost of this outbreak including evaluations, cultures, and antibiotics ranged from $66,468 to $120,583. CONCLUSIONS The ST448 strain of S. pneumoniae caused a disruptive outbreak of conjunctivitis at Dartmouth College. A screening protocol was effective at identifying culture-positive cases. Although most culture-positive patients experienced bilateral conjunctivitis, the clinical course was mild with quick resolution of symptoms after initiating antibiotics and no ocular sequelae.

Background Hand hygiene is a vital intervention to reduce health-care associated infections, but compliance remains low. The authors hypothesized that improvements in intraoperative hand hygiene compliance would reduce transmission of bacteria to surgical patients and reduce the incidence of postsurgical healthcare-associated infections. Methods The authors performed a controlled before-and-after study over 2 consecutive months. One hundred fourteen operative cases were enrolled. Two predesignated sites on the anesthesia machine were selected, decontaminated, and cultured via aseptic technique. These sites and the peripheral intravenous stopcock were cultured again after completion of the surgery. The treatment phase used a novel personal hand-decontamination device capable of recording hand-decontamination events. Results There were no significant differences in patient location, age, or case duration and procedure type between groups. Use of the Sprixx GJ device (Harbor Medical Inc., Santa Barbara, CA) increased hourly hand decontamination events by 27-fold as compared with baseline rates (P < 0.002; 95% confidence interval, 3.3-13.4). Use of the device was associated with a reduction in contamination in the anesthesia work area and peripheral intravenous tubing. Intravenous tubing contamination was identified in 32.8% of cases in the control group versus 7.5% in the treatment group (odds ratio, 0.17; 95% confidence interval, 0.06-0.51; P < 0.01). Healthcare-associated infections rates were reduced in the device group (3.8%) as compared with the control group (17.2%) (odds ratio, 0.19; 95% confidence interval, 0.00-0.81; P = 0.02). Conclusions Improved hand hygiene compliance through the use of a novel hand sanitation strategy reduces the risk of intraoperative bacterial transmission. The intervention was associated with a reduction in healthcare-associated infections.

Minocycline exerts beneficial immune modulatory effects in several noninfectious neurodegenerative disease models; however, its potential to influence the host immune response during central nervous system bacterial infections, such as brain abscess, has not yet been investigated. Using a minocycline-resistant strain of Staphylococcus aureus to dissect the antibiotic's bacteriostatic versus immune modulatory effects in a mouse experimental brain abscess model, we found that minocycline significantly reduced mortality rates within the first 24 hours following bacterial exposure. This protection was associated with a transient decrease in the expression of several proinflammatory mediators, including interleukin-1beta and CCL2 (MCP-1). Minocycline was also capable of protecting the brain parenchyma from necrotic damage as evident by significantly smaller abscesses in minocycline-treated mice. In addition, minocycline exerted anti-inflammatory effects when administered as late as 3 days following S. aureus infection, which correlated with a significant decrease in brain abscess size. Finally, minocycline was capable of partially attenuating S. aureus-dependent microglial and astrocyte activation. Therefore, minocycline may afford additional therapeutic benefits extending beyond its antimicrobial activity for the treatment of central nervous system infectious diseases typified by a pathogenic inflammatory component through its ability to balance beneficial versus detrimental inflammation.

We report a case of multi-drug resistant Helcococcus-like organism isolated in pure culture from a chest abscess in a 31-year-old pregnant woman.

Interferon (IFN)-gamma-producing CD8+ T cells are important for the successful resolution of the obligate intracellular parasite Toxoplasma gondii by preventing the reactivation or controlling a repeat infection. Previous reports from our laboratory have shown that exogenous interleukin (IL)-15 treatment augments the CD8+ T cell response against the parasite. However, the role of endogenous IL-15 in the proliferation of activated/memory CD8+ T cells during toxoplasma or any other infection is unknown. In this study, we treated T. gondii immune mice with soluble IL-15 receptor alpha (sIL-15Ralpha) to block the host endogenous IL-15. The treatment markedly reduced the ability of the immune animals to control a lethal infection. CD8+ T cell activities in the sIL-15Ralpha-administered mice were severely reduced as determined by IFN-gamma release and target cell lysis assays. The loss of CD8+ T cell immunity due to sIL-15Ralpha treatment was further demonstrated by adoptive transfer experiments. Naive recipients transferred with CD44(hi) activated/memory CD8+ T cells and treated with sIL-15Ralpha failed to resist a lethal T. gondii infection. Moreover, sIL-15Ralpha treatment of the recipients blocked the ability of donor CD44(hi) activated/memory CD8+ T cells to replicate in response to T. gondii challenge. To our knowledge, this is the first demonstration of the important role of host IL-15 in the development of antigen-specific memory CD8+ T cells against an intracellular infection.

Summary: Purpose: Cryptogenic epilepsy, the group of epilepsy syndromes for which an etiology is unknown, comprises ∼20% of all epilepsy syndromes. We selected patients in this subgroup of epilepsy and tested them for evidence of Toxoplasma gondii IgG antibodies by the enzyme-linked immunosorbent assay. T. gondii is found in up to 20% of the U.S. population forming dormant brain cysts in the latent bradyzoite form. We investigated the hypothesis that dormant T. gondii infection might be associated with cryptogenic epilepsy. Methods: We selected patients with cryptogenic epilepsies and tested them for evidence of T. gondii IgG antibodies by the enzyme-linked immunosorbent assay. A control group was also tested for comparison. Results: We have found a statistically-significant elevation of T. gondii antibodies among cryptogenic epilepsy patients as compared to controls [59% increase in optical density (OD), p = 0.013]. This association persisted after adjustment for subjects' gender and age in a multiple logistic regression model; however, it was no longer as statistically significant. Conclusions: Our results suggest that chronic T. gondii infection with brain cysts may be a cause of cryptogenic epilepsy.

A polymerase chain reaction (PCR) screen was used to examine the diversity of myosins in 7 Apicomplexan parasites: Toxoplasma gondii, Plasmodium falciparum, Neospora caninum, Eimeria tenella, Sarcocystis muris, Babesia bovis, and Cryptosporidium parvum. Using degenerate PCR primers compatible with the majority of known myosin classes, putative myosin sequences were obtained from all of these species. All of the sequences obtained showed greatest similarity to previously identified apicomplexan myosins, suggesting that the diversity of myosins in these parasites is limited. Myosin classes that are known to be widespread across the phylogenetic spectrum, e.g., the myosins I, II, and V, were not seen in the Apicomplexa. Thus, like the plants, the Apicomplexa may have evolved their own unique cohort of myosins that are responsible for the myosin-driven cellular functions observed in these parasites.

Toxoplasma gondii remains a serious cause of morbidity and mortality in individuals that are immunosuppressed, patients with AIDS in particular. The cellular immune response, especially by gamma interferon (IFN-γ)-producing CD8 + T cells, is an essential component of protective immunity against the parasite. In the present study the role of CD8 + T cells during the reactivation of Toxoplasma infection in an immunocompromised murine model was evaluated. Chronically infected mice were challenged with LP-BM5 virus, and the kinetics of CD8 + T-cell function was studied. At 10 weeks after viral infection, mice showed obvious signs of systemic illness and began to die. At this stage, CD8 + T cells were unresponsive to antigenic stimulation and unable to kill Toxoplasma -infected targets. IFN-γ production by the CD8 + T cells from dual-infected animals reached background levels, and a dramatic fall in the frequency of precursor cytotoxic T lymphocytes was observed. Histopathological analysis of the tissues demonstrated signs of disseminated toxoplasmosis as a result of reactivation of infection. However, treatment of the dual-infected animals with immune CD8 + T cells at 5 weeks post-LP-BM5 challenge prevented the reactivation of toxoplasmosis, and mice continued to live. Our study for the first time demonstrates a therapeutic role for CD8 + T cells against an opportunistic infection in an immunocompromised state.

Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation of Toxoplasma gondii . Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8 + and CD4 + T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6- lpr ) and FasL-deficient (B6- gld ) mice. Inoculation of 5,000 T. gondii tachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8 + than CD4 + T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6- lpr and B6- gld mice than in wild-type B6 mice ( P < 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.

Production of nitric oxide by macrophages is believed to be an important microbicidal mechanism for a variety of intracellular pathogens, includingToxoplasma gondii. Mice with a targeted disruption of the inducible nitric oxide synthase gene (iNOS) were infected orally withT. gondiitissue cysts. Time to death was prolonged compared with parental controls. Histologic analysis of tissue from infected mice showed scattered small foci of inflammation with parasites in various tissues of iNOS−/− mice, whereas tissue from the parental C57BL/6 mice had more extensive tissue inflammation with few visible parasites. In particular, extensive ulceration and necrosis of distal small intestine and fatty degeneration of the liver was seen in the parental mice at day 7 postinfection, as compared with the iNOS−/− mice where these tissues appeared normal. Serum interferon γ and tumor necrosis factor α levels postinfection were equally elevated in both mouse strains. Treatment of the parental mice with a NO synthase inhibitor, aminoguanidine, prevented early death in these mice as well as the hepatic degeneration and small bowel necrosis seen in acutely infected control parentals. These findings indicate that NO production during acute infection withT. gondiican kill intracellular parasites but can be detrimental, even lethal, to the host.

We report the isolation of Corynebacterium macginleyi from the corneal ulcer culture of a patient, later enrolled in the Steroids for Corneal Ulcer Trial (SCUT). To our knowledge this is the first published report from North America of the recovery of C. macginleyi from a serious ocular infection.

The host response to intracellular pathogens requires the coordinated action of both the innate and acquired immune systems. Chemokines play a critical role in the trafficking of immune cells and transitioning an innate immune response into an acquired response. We analyzed the host response of mice deficient in the chemokine receptor CCR5 following infection with the intracellular protozoan parasite Toxoplasma gondii. We found that CCR5 controls recruitment of natural killer (NK) cells into infected tissues. Without this influx of NK cells, tissues from CCR5-deficient (CCR5−/−) mice were less able to generate an inflammatory response, had decreased chemokine and interferon γ production, and had higher parasite burden. As a result, CCR5−/− mice were more susceptible to infection with T. gondii but were less susceptible to the immune-mediated tissue injury seen in certain inbred strains. Adoptive transfer of CCR5+/+ NK cells into CCR5−/− mice restored their ability to survive lethal T. gondii infection and demonstrated that CCR5 is required for NK cell homing into infected liver and spleen. This study establishes CCR5 as a critical receptor guiding NK cell trafficking in host defense., Synopsis Toxoplasma gondii, an obligate intracellular parasite, evokes a strong cellular immune response, which is critical for host protection. Chemokines and their receptors control the trafficking of immune cells to sites of infection. In this study, the authors examined the immune response to T. gondii in mice deficient in the chemokine receptor CCR5. CCR5-deficient mice exhibited a decreased inflammatory response, which led to a higher parasite burden and ultimate mortality. The deficiency in the immune response in CCR5-deficient mice was the result of decreased migration of natural killer (NK) cells into infected tissues. Transfer of normal NK cells into CCR5-deficient mice restored NK cell homing into infected tissue and the normal immune response capable of controlling T. gondii infection. These findings demonstrate an important role for CCR5 in trafficking the NK cells during acute T. gondii infection. Furthermore, since CCR5 is the main co-receptor for HIV-1 entry into cells, the results of this study raise a concern about the potential complication of impaired NK cell function in the clinical use of CCR5 antagonists, currently being developed as therapeutics for HIV-1 and inflammatory diseases. This may be of particular concern for HIV-1-infected patients coinfected with T. gondii or for those who acquire an acute T. gondii infection while taking a CCR5 antagonist.

Here, we describe the complete deduced amino acid sequence of three unconventional myosins identified in the protozoan parasite Toxoplasma gondii. Phylogenetic analysis reveals that the three myosins represent a novel, highly-divergent class addition to the myosin superfamily. Toxoplasma gondii myosin-A (TgM-A) is a remarkably small approximately 93 kDa myosin that shows a striking departure from typical myosin heavy chain structure in having a head and tail domain but no discernible neck domain. In other myosins, the neck is defined by one or more IQ motifs that serve as potential light chain binding domains. No IQ motifs are apparent in TgM-A. The tail domain of TgM-A encompasses only 57 amino acid residues and is characterized by its highly basic charge (pI = 10.8). The other two Toxoplasma myosins, TgM-B and TgM-C appear to be the product of differential RNA splicing with TgM-B yielding a protein of approximately 114 kDa and TgM-C a protein of approximately 125 kDa. These two myosins are identical throughout their head domain and neck domain which contains a single IQ motif. TgM-B and C share the proximal 245 residues of their tail domain and then diverge in their tail structure distally. The tails, like that of TgM-A, share no homology to any other myosin tails apart from a highly basic charge. The identification of yet another class of unconventional myosins, including a myosin as novel in structure as the 93 kDa TgM-A, continues to underscore the diversity of this family of molecular motors.

Piglet tracheal organ cultures were infected with respiratory syncytial virus (RSV) and observed for 21 days. Light and immunofluorescence microscopy demonstrated destruction of the ciliated epithelial cells and the presence of viral antigens in the epithelium. Virus was shed in high titer for 12--19 days. Ciliostasis could be quantitated, and it was shown that several strains of RSV grew and damaged tracheal organ cultures in a similar fashion. A temperature-sensitive mutant of RSV, ts-1, was examined at permissive (33 C) and restrictive (37 C) temperatures. This mutant, although somewhat attenuated at 37 C, was still found to cause damage to the ciliated epithelium and to replicate at both temperatures. THIS BEHAVIOR IS SIMILAR TO THAT AFTER INOCULATION OF TS-1 INTO VOLUNTEERS. This in vitro model may prove useful in the study of RSV disease and in the evaluation of candidate live virus vaccines.