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Antimicrobial susceptibility in Pseudomonas aeruginosa is dependent on a complex combination of host and pathogen-specific factors. Through the profiling of 971 clinical P. aeruginosa isolates from 590 patients and collection of paired patient metadata, we show that antimicrobial resistance is associated with not only patient-centric factors (e.g., cystic fibrosis and antipseudomonal prescription history) but also microbe-specific phenotypes (e.g., mucoid colony morphology). Additionally, isolates from different sources (e.g., respiratory tract, urinary tract) displayed rates of antimicrobial resistance that were correlated with source-specific antimicrobial prescription strategies. Furthermore, isolates from the same patient often displayed a high degree of heterogeneity, highlighting a key challenge facing personalized treatment of infectious diseases. Our findings support novel relationships between isolate and patient-level data sets, providing a potential guide for future antimicrobial treatment strategies. IMPORTANCEP. aeruginosa is a leading cause of nosocomial infection and infection in patients with cystic fibrosis. While P. aeruginosa infection and treatment can be complicated by a variety of antimicrobial resistance and virulence mechanisms, pathogen virulence is rarely recorded in a clinical setting. In this study, we discovered novel relationships between antimicrobial resistance, virulence-linked morphologies, and isolate source in a large and variable collection of clinical P. aeruginosa isolates. Our work motivates the clinical surveillance of virulence-linked P. aeruginosa morphologies as well as the tracking of source-specific antimicrobial prescription and resistance patterns.

As antimicrobial resistance becomes an increasing threat, bringing significant economic and health burdens, innovative antimicrobial treatments are urgently needed. While antimicrobial peptides (AMPs) are promising therapeutics, exhibiting high activity against resistant bacterial strains, limited stability and toxicity to mammalian cells has hindered clinical development. Attaching AMPs to polymers provides opportunities to present AMPs in a way that maximizes bacterial killing while enhancing compatibility with mammalian cells, stability, and solubility. Conjugation of an AMP to a linear hydrophilic polymer yields the desired improvements in stability, mammalian cell compatibility, and solubility, yet often markedly reduces bactericidal effects. Non-linear polymer architectures and supramolecular assemblies that accommodate multiple AMPs per polymer chain afford AMP-polymer conjugates that strike a superior balance of antimicrobial activity, mammalian cell compatibility, stability, and solubility. Therefore, we review the design criteria, building blocks, and synthetic strategies for engineering AMP-polymer conjugates, emphasizing the connection between molecular architecture and antimicrobial performance to inspire and enable further innovation to advance this emerging class of biomaterials.

Background Stunting affects up to one-third of the children in low-to-middle income countries (LMICs) and has been correlated with decline in cognitive capacity and vaccine immunogenicity. Early identification of infants at risk is critical for early intervention and prevention of morbidity. The aim of this study was to investigate patterns of growth in infants up through 48 months of age to assess whether the growth of infants with stunting eventually improved as well as the potential predictors of growth. Methods Height-for-age z-scores (HAZ) of children from Matiari (rural site, Pakistan) at birth, 18 months, and 48 months were obtained. Results of serum-based biomarkers collected at 6 and 9 months were recorded. A descriptive analysis of the population was followed by assessment of growth predictors via traditional machine learning random forest models. Results Of the 107 children who were followed up till 48 months of age, 51% were stunted (HAZ Conclusion We demonstrated that children most children with stunting at birth remained stunted at 48 months of age. Value was added for predicting growth outcomes with the use of traditional machine learning random forest models. HAZ at birth was found to be a strong predictor of subsequent growth in infants up through 48 months of age. Biomarkers of systemic inflammation, AGP, CRP, IL1, were also strong predictors of growth outcomes. These findings provide support for continued focus on interventions prenatally, at birth, and early infancy in children at risk for stunting who live in resource-constrained regions of the world.

Infections in immunocompromised patients that are caused by extensively drug-resistant (XDR) Acinetobacter baumannii strains have been increasingly reported worldwide. In particular, carbapenem-resistant A. baumannii strains are a prominent cause of health care-associated infections. Here, we report draft genome assemblies for two clinical XDR A. baumannii isolates obtained from hospitalized patients in Pakistan.

Chemokines are a family of small proteins best known for their ability to orchestrate immune cell trafficking and recruitment to sites of infection. Their role in promoting host defense is multiplied by a number of additional receptor-dependent biological activities, and most, but not all, chemokines have been found to mediate direct antimicrobial effects against a broad range of microorganisms. The molecular mechanism(s) by which antimicrobial chemokines kill bacteria remains unknown; however, recent observations have expanded our fundamental understanding of chemokine-mediated bactericidal activity to reveal increasingly diverse and complex actions. In the current review, we present and consider mechanistic insights of chemokine-mediated antimicrobial activity against bacteria. We also discuss how contemporary advances are reshaping traditional paradigms and opening up new and innovative avenues of research with translational implications. Towards this end, we highlight a developing framework for leveraging chemokine-mediated bactericidal and immunomodulatory effects to advance pioneering therapeutic approaches for treating bacterial infections, including those caused by multidrug-resistant pathogens.

Background. In modern academic medicine, especially in the fields of infectious diseases and global health, aspiring physician-scientists often wait years before achieving independence as basic, translational, and clinical investigators. This study employed mixed methods to evaluate the success of the Burroughs Wellcome Fund/American Society for Tropical Medicine and Hygiene (BWF/ASTMH) global health postdoctoral fellowship in promoting scientific independence. Methods. We examined quantitative data obtained from the National Institutes of Health (NIH) and qualitative data provided by the ASTMH and program participants to assess BWF/ASTMH trainees' success in earning NIH grants, publishing manuscripts, and gaining faculty positions. We also calculated the return on investment (ROI) associated with the training program by dividing direct costs of NIH research grants awarded to trainees by the direct costs invested by the BWF/ASTMH fellowship. Results. Forty-one trainees received fellowships between 2001 and 2015. Within 3 years of completing their fellowships, 21 of 35 (60%) had received career development awards, and within 5 years, 12 of 26 (46%) had received independent research awards. Overall, 22 of 35 (63%) received 1 or more research awards. BWF/ASTMH recipients with at least 3 years of follow-up data had coauthored a mean of 36 publications (range, 2-151) and 29 of 35 (82%) held academic positions. The return on investment was 11.9 overall and 31.8 for fellowships awarded between 2001 and 2004. Conclusions. Between 2001 and 2015, the BWF/ASTMH postdoctoral training program successfully facilitated progress to scientific independence. This program model underscores the importance of custom-designed postdoctoral training as a bridge to NIH awards and professional autonomy.

Shigella spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) Shigella spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR Shigella isolates from Pakistan, two Shigella flexneri isolates and one Shigella sonnei isolate.

Shigella spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) Shigella spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR Shigella isolates from Pakistan, two Shigella flexneri isolates and one Shigella sonnei isolate.

Environmental Enteric Dysfunction (EED) is an acquired small intestinal inflammatory condition underlying high rates of stunting in children

The objective of this study was to determine the incidence of respiratory viruses associated with severe pneumonia among children less than 2 years of age in the rural district of Matiari in Sindh, Pakistan. This study was a community-based prospective cohort active surveillance of infants enrolled at birth and followed for 2 years. Cases were identified using the World Health Organization's Integrated Management of Childhood Illnesses' definition of severe pneumonia. Nasopharyngeal swabs were obtained for assessment by multiplex RT-PCR for eight viruses and their subtypes, including RSV, influenza virus, human metapneumovirus, enterovirus/rhinovirus, coronavirus, parainfluenza virus, adenovirus, and human bocavirus. Blood cultures were collected from febrile participants. A total of 817 newborns were enrolled and followed with fortnightly surveillance for 2 years, accounting for a total of 1,501 child-years of follow-up. Of the nasopharyngeal swabs collected, 77.8% (179/230) were positive for one or more of the above mentioned respiratory viruses. The incidence of laboratory confirmed viral-associated pneumonia was 11.9 per 100 child-years of follow-up. Enterovirus/rhinovirus was detected in 51.7% patients, followed by parainfluenza virus type III (8.3%), and RSV (5.7%). Of the uncontaminated blood cultures, 1.4% (5/356) were positive. Respiratory viruses are frequently detected during acute respiratory infection episodes in children under 2 years old in a rural community in Pakistan. However, causal association is yet to be established and the concomitant role of bacteria as a co-infection or super-infection needs further investigation. J. Med. Virol. 88:1882-1890, 2016. © 2016 Wiley Periodicals, Inc.

We analysed susceptibility of multidrug-resistant organisms (MDROs) including New Delhi metallo-beta-lactamase-1 positive Enterobacteriaceae to chlorhexidine and compared results to their susceptible counterparts. Susceptibilities of chlorhexidine digluconate in a standard (CHX-S) preparation and two commercial disinfectants containing different CHX concentrations (2% w/v and 4% w/w) were performed. MDROs had narrower range of higher CHX-S minimum inhibitory concentrations (MICs) as compared to pan-sensitive organisms. The MIC values for commercial disinfectants products for MDROs were many folds higher (20-600 times), than CHX-S for in vitro use. Increasing antibiotic resistance among bacterial isolates can be an indirect marker of reduced susceptibility to chlorhexidine in hospital setting.

Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE . Resistance to CXCL10 also occurred following deletion of either aceF or lpdA , genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine.

The antimicrobial activity of the chemokine CXCL10 against vegetative cells of Bacillus anthracis occurs via both bacterial FtsE/X-dependent and-independent pathways. Previous studies established that the FtsE/X-dependent pathway was mediated through interaction of the N-terminal region(s) of CXCL10 with a functional FtsE/X complex, while the FtsE/X-independent pathway was mediated through the C-terminal α-helix of CXCL10. Both pathways result in cell lysis and death of B. anthracis. In other bacterial species, it has been shown that FtsE/X is involved in cellular elongation though activation of complex-associated peptidoglycan hydrolases. Thus, we hypothesized that the CXCL10-mediated killing of vegetative cells of B. anthracis through the FtsE/X-dependent pathway resulted from the disruption of peptidoglycan processing. Immunofluorescence microscopy studies using fluorescent peptidoglycan probes revealed that incubation of B. anthracis Sterne (parent) strain with CXCL10 or a C-terminal truncated CXCL10 (CTTC) affected peptidoglycan processing and/or incorporation of precursors into the cell wall. B. anthracis ΔftsX or ftsE(K123A/D481N) mutant strains, which lacked a functional FtsE/X complex, exhibited little to no evidence of disruption in peptidoglycan processing by either CXCL10 or CTTC. Additional studies demonstrated that the B. anthracis parent strain exhibited a statistically significant increase in peptidoglycan release in the presence of either CXCL10 or CTTC. While B. anthracis ΔftsX strain showed increased peptidoglycan release in the presence of CXCL10, no increase was observed with CTTC, suggesting that the FtsE/X-independent pathway was responsible for the activity observed with CXCL10. These results indicate that FtsE/X-dependent killing of vegetative cells of B. anthracis results from a loss of cell wall integrity due to disruption of peptidoglycan processing and suggest that FtsE/X may be an important antimicrobial target to study in the search for alternative microbial therapeutics.

Background The health of mothers and their newborns is intricately related. The weight of the infant at birth is a powerful predictor of infant growth and survival, and is considered to be partly dependent on maternal health and nutrition during pregnancy. We conducted a longitudinal study in an urban community within Karachi to determine maternal predictors of newborn birth weight. Methods Four hundred pregnant women were enrolled in the study during the period 2011–2013. Data related to symptoms of acute respiratory illness (fever, cough, difficulty breathing, runny nose, sore throat, headache, chills, and myalgia/lethargy) in the pregnant women were collected weekly until delivery. Birth weight of the newborn was recorded within 14 days of delivery and the weight of

Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT's capacity to suppress this response.

The emergence and spread of colistin resistance among multidrug-resistant (MDR) Klebsiella pneumoniae represent a critical threat to global health. Here, we report the complete genome sequences of 10 MDR, colistin-susceptible and -resistant K. pneumoniae clinical isolates obtained in Pakistan between 2010 and 2013.

The primary goal of biosafety education is to ensure safe practices among workers in biomedical laboratories. Despite several educational workshops by the Pakistan Biological Safety Association (PBSA), compliance with safe practices among laboratory workers remains low. To determine barriers to implementation of recommended biosafety practices among biomedical laboratory workers in Pakistan, we conducted a questionnaire-based survey of participants attending 2 workshops focusing on biosafety practices in Karachi and Lahore in February 2015. Questionnaires were developed by modifying the BARRIERS scale in which respondents are required to rate barriers on a 1-4 scale. Nineteen of the original 29 barriers were included and subcategorized into 4 groups: awareness, material quality, presentation, and workplace barriers. Workshops were attended by 64 participants. Among barriers that were rated as moderate to great barriers by at least 50% of respondents were: lack of time to read biosafety guidelines (workplace subscale), lack of staff authorization to change/improve practice (workplace subscale), no career or self-improvement advantages to the staff for implementing optimal practices (workplace subscale), and unclear practice implications (presentation subscale). A lack of recognition for employees' rights and benefits in the workplace was found to be a predominant reason for a lack of compliance. Based on perceived barriers, substantial improvement in work environment, worker facilitation, and enabling are needed for achieving improved or optimal biosafety practices in Pakistan.

Bacillus anthracis is killed by the interferon-inducible, ELR(−) CXC chemokine CXCL10. Previous studies showed that disruption of the gene encoding FtsX, a conserved membrane component of the ATP-binding cassette transporter-like complex FtsE/X, resulted in resistance to CXCL10. FtsX exhibits some sequence similarity to the mammalian CXCL10 receptor, CXCR3, suggesting that the CXCL10 N-terminal region that interacts with CXCR3 may also interact with FtsX. A C-terminal truncated CXCL10 was tested to determine if the FtsX-dependent antimicrobial activity is associated with the CXCR3-interacting N terminus. The truncated CXCL10 exhibited antimicrobial activity against the B. anthracis parent strain but not the ΔftsX mutant, which supports a key role for the CXCL10 N terminus. Mutations in FtsE, the conserved ATP-binding protein of the FtsE/X complex, resulted in resistance to both CXCL10 and truncated CXCL10, indicating that both FtsX and FtsE are important. Higher concentrations of CXCL10 overcame the resistance of the ΔftsX mutant to CXCL10, suggesting an FtsX-independent killing mechanism, likely involving its C-terminal α-helix, which resembles a cationic antimicrobial peptide. Membrane depolarization studies revealed that CXCL10 disrupted membranes of the B. anthracis parent strain and the ΔftsX mutant, but only the parent strain underwent depolarization with truncated CXCL10. These findings suggest that CXCL10 is a bifunctional molecule that kills B. anthracis by two mechanisms. FtsE/X-dependent killing is mediated through an N-terminal portion of CXCL10 and is not reliant upon the C-terminal α-helix. The FtsE/X-independent mechanism involves membrane depolarization by CXCL10, likely because of its α-helix. These findings present a new paradigm for understanding mechanisms by which CXCL10 and related chemokines kill bacteria., IMPORTANCE Chemokines are a class of molecules known for their chemoattractant properties but more recently have been shown to possess antimicrobial activity against a wide range of Gram-positive and Gram-negative bacterial pathogens. The mechanism(s) by which these chemokines kill bacteria is not well understood, but it is generally thought to be due to the conserved amphipathic C-terminal α-helix that resembles cationic antimicrobial peptides in charge and secondary structure. Our present study indicates that the interferon-inducible, ELR(−) chemokine CXCL10 kills the Gram-positive pathogen Bacillus anthracis through multiple molecular mechanisms. One mechanism is mediated by interaction of CXCL10 with the bacterial FtsE/X complex and does not require the presence of the CXCL10 C-terminal α-helix. The second mechanism is FtsE/X receptor independent and kills through membrane disruption due to the C-terminal α-helix. This study represents a new paradigm for understanding how chemokines exert an antimicrobial effect that may prove applicable to other bacterial species.

Chemokines are a family of chemotactic cytokines that function in host defense by orchestrating cellular movement during infection. In addition to this function, many chemokines have also been found to mediate the direct killing of a range of pathogenic microorganisms through an as-yet-undefined mechanism. As an understanding of the molecular mechanism and microbial targets of chemokine-mediated antimicrobial activity is likely to lead to the identification of unique, broad-spectrum therapeutic targets for effectively treating infection, we sought to investigate the mechanism by which the chemokine CXCL10 mediates bactericidal activity against the Gram-positive bacterium Bacillus anthracis , the causative agent of anthrax. Here, we report that disruption of the gene ftsX , which encodes the transmembrane domain of a putative ATP-binding cassette transporter, affords resistance to CXCL10-mediated antimicrobial effects against vegetative B. anthracis bacilli. Furthermore, we demonstrate that in the absence of FtsX, CXCL10 is unable to localize to its presumed site of action at the bacterial cell membrane, suggesting that chemokines interact with specific, identifiable bacterial components to mediate direct microbial killing. These findings provide unique insight into the mechanism of CXCL10-mediated bactericidal activity and establish, to our knowledge, the first description of a bacterial component critically involved in the ability of host chemokines to target and kill a bacterial pathogen. These observations also support the notion of chemokine-mediated antimicrobial activity as an important foundation for the development of innovative therapeutic strategies for treating infections caused by pathogenic, potentially multidrug-resistant microorganisms.

A cross-sectional survey using structured questionnaire was conducted to assess practices of microbiological laboratories working with pathogens. Forty-eight laboratory workers (50%) agreed that laboratory methods to detect antimicrobial resistance are not standardized in Pakistan, and 6% of the laboratory workers were not aware of the standardization of antimicrobial susceptibility testing in Pakistan. Reported rates of awareness regarding the role of waste disposal, disinfection, and handwashing in limiting the spread of antimicrobial resistance were 75%, 42%, and 81%, respectively. Our results provide baseline data for planning programs to train, supervise, and improve the operational quality of microbiological laboratories nationwide to prevent the spread of superbugs.

The emergence and dissemination of carbapenemases, bacterial enzymes able to inactivate most β-lactam antibiotics, in Enterobacteriaceae is of increasing concern. The concurrent spread of resistance against colistin, an antibiotic of last resort, further compounds this challenge further. Whole-genome sequencing (WGS) can play a significant role in the rapid and accurate detection/characterization of existing and emergent resistance determinants, an essential aspect of public health surveillance and response activities to combat the spread of antimicrobial resistant bacteria. In the current study, WGS data was used to characterize the genomic content of antimicrobial resistance genes, including those encoding carbapenemases, in 10 multidrug-resistant Klebsiella pneumoniae isolates from Pakistan. These clinical isolates represented five sequence types: ST11 (n = 3 isolates), ST14 (n = 3), ST15 (n = 1), ST101 (n = 2), and ST307 (n = 1). Resistance profiles against 25 clinically-relevant antimicrobials were determined by broth microdilution; resistant phenotypes were observed for at least 15 of the 25 antibiotics tested in all isolates except one. Specifically, 8/10 isolates were carbapenem-resistant and 7/10 isolates were colistin-resistant. The blaNDM-1 and blaOXA-48 carbapenemase genes were present in 7/10 and 5/10 isolates, respectively; including 2 isolates carrying both genes. No plasmid-mediated determinants for colistin resistance (e.g. mcr) were detected, but disruptions and mutations in chromosomal loci (i.e. mgrB and pmrB) previously reported to confer colistin resistance were observed. A blaOXA-48-carrying IncL/M-type plasmid was found in all blaOXA-48-positive isolates. The application of WGS to molecular epidemiology and surveillance studies, as exemplified here, will provide both a more complete understanding of the global distribution of MDR isolates and a robust surveillance tool useful for detecting emerging threats to public health.

Bone marrow-derived fibrocytes review as key cellular players in the promotion of the pathogenesis of variety of fibroproliferative disorders, including pulmonary fibrosis. Pulmonary fibrosis is associated with a number of disorders that affect the lung. Although there are several cellular types that are involved in the pathogenesis pulmonary fibrosis, the resident lung fibroblast has been viewed traditionally as the primary cell involved in promoting the deposition of ECM that culminates in pulmonary fibrosis. However, recent findings demonstrate that a circulating cell (i.e., the fibrocyte) can contribute to the evolution of pulmonary fibrosis. Fibrocytes are bone marrow-derived mesenchymal progenitor cells that express a variety of cell-surface markers related to leukocytes, hematopoietic progenitor cells, and fibroblasts. Fibrocytes are unique in that they are capable of differentiating into fibroblasts and myofibroblasts, as well as adipocytes. In this review, we present data supporting the critical role these cells play in the pathogenesis of pulmonary fibrosis.

Invasive aspergillosis is among the most common human fungal infections and occurs in patients with severe and complex defects in immune responses. NK cells have previously been found to be important in host defense against this infection, but the mechanism of this effect is not known. We hypothesized that NK cells mediate their protective effect in invasive aspergillosis by acting as the major source of IFN-γ during early infection. We found that, in the lungs of neutropenic mice with invasive aspergillosis, NK cells were the major population of cells capable of generating IFN-γ during early infection. Depletion of NK cells resulted in reduced lung IFN-γ levels and increased lung fungal load that was independent of T and B cell subsets. Depletion of NK cells and absence of IFN-γ resulted in a similar increase in susceptibility to the infection, but depletion of NK cells in IFN-γ-deficient hosts did not result in further increase in severity of the infection. NK cell-derived IFN-γ caused enhanced macrophage antimicrobial effects in vitro and also resulted in greater expression of IFN-inducible chemokines in the lungs. Finally, transfer of activated NK cells from wild-type, but not IFN-γ-deficient hosts, resulted in greater pathogen clearance from the lungs of both IFN-γ-deficient and wild-type recipients. Taken together, these data indicate that NK cells are the main source of early IFN-γ in the lungs in neutropenic invasive aspergillosis, and this is an important mechanism in the defense against this infection.

Although best managed by surgical resection, we present a case of Listeria monocytogenes endovascular graft infection alternatively treated with graft retention and antibiotic induction followed by a lifelong suppressive course. The epidemiological, pathological, and clinical features of this unique entity are reviewed.

Cryptococcus neoformansis an opportunistic yeast present in the environment. Practitioners are familiar with the presentation and management of the most common manifestation of cryptococcal infection, meningoencephalitis, in patients with AIDS or other conditions of immunocompromise. There is less awareness, however, of uncommon presentations where experience rather than evidence guides therapy. We report a case of primary cutaneous cryptococcosis (PCC) in a patient who had been immunosuppressed by chronic high-dose corticosteroid for the treatment of severe asthma. This case highlights the importance of early recognition of aggressive cellulitis that fails standard empiric antibiotic treatment in an immunocompromised patient. It also demonstrates successful treatment of PCC with a multispecialty approach including local debridement and fluconazole monotherapy.

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-α) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-α response was preserved in TLR2 −/− but not in MyD88 −/− macrophages. In vivo studies revealed that TLR2 −/− mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88 −/− mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.

Entamoeba histolytica is a human intestinal parasite that causes amebic dysentery. A cell surface amebic adhesin, the galactose and N-acetyl-D-galactosamine inhibitable (GalNAc) lectin mediates amebic adherence to and contact-dependent killing of host cells. Previous work has suggested that the GalNAc lectin transduces signals via protein interactions with its short cytoplasmic domain. We used a yeast two-hybrid system to screen an E. histolytica cDNA library for proteins that interact with the GalNAc lectin cytoplasmic domain. One isolate was the E. histolytica thiol-specific antioxidant (TSA). TSA is an enzyme that detoxifies hydrogen peroxide. TSA did not interact in yeast two-hybrid experiments with a mutant version of the lectin cytoplasmic domain, confirming the specificity of the lectin-TSA interaction. Furthermore, mutational analyses of the TSA isolate demonstrated that an in-frame five amino acid sequence introduced between amino acids 61-62 yielded a TSA mutant that did not interact with the lectin cytoplasmic domain upon expression in the yeast two-hybrid system. The association of TSA and GalNAc lectin was further supported by co-immunoaffinity purification. Confocal microscopy demonstrated co-localization of TSA and GalNAc lectin at sites of ameba:host cell contact. Recruitment of TSA by the GalNAc lectin suggests a novel mechanism of parasite defense against reactive oxygen intermediates generated by host peripheral mononuclear cells.

Killing by Entamoeba histolytica requires parasite adherence to host galactose- and N -acetyl- d -galactosamine (Gal/GalNAc)-containing cell surface receptors. A 260-kDa heterodimeric E. histolytica Gal/GalNAc lectin composed of heavy (Hgl) and light (Lgl) subunits has been previously described. Here we present the cloning and characterization of Igl, a 150-kDa intermediate subunit of the Gal/GalNAc lectin. Igl, Hgl, and Lgl colocalized on the surface membrane of trophozoites. Two unlinked copies of genes encoding Igl shared 81% amino acid sequence identity (GenBank accession no. AF337950 and AF337951 ). They encoded cysteine-rich proteins with amino- and carboxy-terminal hydrophobic signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored membrane proteins. The igl genes lacked carbohydrate recognition domains but were members of a large family of amebic genes containing CXXC and CXC motifs. These data indicate that Igl is part of the parasite's multimolecular Gal/GalNAc adhesin required for host interaction.

The hgl5 gene of Entamoeba histolytica is negatively regulated through the upstream regulatory element 3 (URE3) DNA motif TATTCTATT. This motif is also present and significant in the function of the E. histolytica fdx gene promoter. A yeast one-hybrid screen was used to identify an E. histolytica cDNA encoding a protein (URE3-BP) that recognized this DNA motif. Analysis of the predicted amino acid sequence demonstrated the presence of two EF-hand motifs but identified no canonical DNA binding motifs. URE3-BP, expressed in bacteria, demonstrated Ca2+-dependent and sequence-specific recognition of the URE3 DNA sequence as assessed by electrophoretic mobility shift assays. Antibodies raised against URE3-BP blocked the formation of the URE3 DNA-protein complex by native nuclear extracts. The URE3-BP protein was present in theE. histolytica nucleus and cytoplasm with an apparent molecular mass of 22.6 kDa. Our results represent the first use of a yeast genetic screen to identify, on the basis of function, a DNA-binding protein of an early branching eukaryote. Since the URE3 DNA can modulate gene expression in both a positive and negative manner, this protein may have more than one mechanism of interaction with transcriptional machinery. Characterization of URE3-BP should provide insight into transcription regulation and virulence control in this parasite.

Chemokines have been found to exert direct, defensin-like antimicrobial activity in vitro, suggesting that, in addition to orchestrating cellular accumulation and activation, chemokines may contribute directly to the innate host response against infection. No observations have been made, however, demonstrating direct chemokine-mediated promotion of host defense in vivo. Here, we show that the murine interferon-inducible CXC chemokines CXCL9, CXCL10, and CXCL11 each exert direct antimicrobial effects in vitro against Bacillus anthracis Sterne strain spores and bacilli including disruptions in spore germination and marked reductions in spore and bacilli viability as assessed using CFU determination and a fluorometric assay of metabolic activity. Similar chemokine-mediated antimicrobial activity was also observed against fully virulent Ames strain spores and encapsulated bacilli. Moreover, antibody-mediated neutralization of these CXC chemokines in vivo was found to significantly increase host susceptibility to pulmonary B. anthracis infection in a murine model of inhalational anthrax with disease progression characterized by systemic bacterial dissemination, toxemia, and host death. Neutralization of the shared chemokine receptor CXCR3, responsible for mediating cellular recruitment in response to CXCL9, CXCL10, and CXCL11, was not found to increase host susceptibility to inhalational anthrax. Taken together, our data demonstrate a novel, receptor-independent antimicrobial role for the interferon-inducible CXC chemokines in pulmonary innate immunity in vivo. These data also support an immunomodulatory approach for effectively treating and/or preventing pulmonary B. anthracis infection, as well as infections caused by pathogenic and potentially, multi-drug resistant bacteria including other spore-forming organisms., Author Summary Innate immunity is critical to host defense and plays a central role in protecting the lungs from respiratory pathogens. Among the mediators important in the innate host response to pulmonary infection are chemokines, proteins originally described for their ability to regulate immune cell trafficking during an inflammatory response. More recently, chemokines have been found to exert direct antimicrobial activity against a broad range of bacteria and fungi in vitro. While these observations suggest chemokines may contribute to host defense by killing microorganisms at local sites of infection through activities not associated with cellular chemokine receptors, the biological relevance of direct chemokine-mediated antimicrobial activity in vivo has not been established. Here we show that the murine chemokines CXCL9, CXCL10, and CXCL11 exert direct antimicrobial effects against B. anthracis in vitro and that neutralization of these CXC chemokines, but not their shared receptor CXCR3, increases host susceptibility to pulmonary B. anthracis infection in vivo. These data provide unique insight into the host mediators important in host-pathogen interaction and pathogenesis of disease and support the emerging concept that host chemokines mediate efficient, pleiotropic roles that include receptor-independent promotion of host defense in vivo.

Communication between research laboratories within a given field is often an important key to rapid successes within that field. We propose that consensus standards may be a useful tool to help facilitate such communication by providing a “common language” for laboratories that utilize similar methodologies within a field. The existence of consensus standards is well known in other fields, and through this commentary we hope to (i) introduce the concept of consensus standards to investigators in the field of microbial pathogenesis/host response who may not be familiar with them and (ii) provoke thought and discussion by others in the field regarding the possible usefulness of additional consensus standards for their own work.

Toll-like receptors and Nod-like receptors (NLR) play an important role in sensing invading microorganisms for pathogen clearance and eliciting adaptive immunity for protection against rechallenge. Nod1 and Nod2, members of the NLR family, are capable of detecting bacterial peptidoglycan motifs in the host cytosol for triggering proinflammatory cytokine production. In the current study, we sought to determine if Nod1/Nod2 are involved in sensing Bacillus anthracis infection and eliciting protective immune responses. Using mice deficient in both Nod1 and Nod2 proteins, we showed that Nod1/Nod2 are involved in detecting B. anthracis for production of tumor necrosis factor alpha, interleukin-1α (IL-1α), IL-1β, CCL5, IL-6, and KC. Proinflammatory responses were higher when cells were exposed to viable spores than when they were exposed to irradiated spores, indicating that recognition of vegetative bacilli through Nod1/Nod2 is significant. We also identify a critical role for Nod1/Nod2 in priming responses after B. anthracis aerosol exposure, as mice deficient in Nod1/Nod2 were impaired in their ability to mount an anamnestic antibody response and were more susceptible to secondary lethal challenge than wild-type mice.

Based on previous studies showing that host chemokines exert antimicrobial activities against bacteria, we sought to determine whether the interferon-inducible Glu-Leu-Arg-negative CXC chemokines CXCL9, CXCL10, and CXCL11 exhibit antimicrobial activities againstBacillus anthracis. In vitro analysis demonstrated that all three CXC chemokines exerted direct antimicrobial effects againstB. anthracisspores and bacilli including marked reductions in spore and bacillus viability as determined using a fluorometric assay of bacterial viability and CFU determinations. Electron microscopy studies revealed that CXCL10-treated spores failed to undergo germination as judged by an absence of cytological changes in spore structure that occur during the process of germination. Immunogold labeling of CXCL10-treated spores demonstrated that the chemokine was located internal to the exosporium in association primarily with the spore coat and its interface with the cortex. To begin examining the potential biological relevance of chemokine-mediated antimicrobial activity, we used a murine model of inhalational anthrax. Upon spore challenge, the lungs of C57BL/6 mice (resistant to inhalationalB. anthracisinfection) had significantly higher levels of CXCL9, CXCL10, and CXCL11 than did the lungs of A/J mice (highly susceptible to infection). Increased CXC chemokine levels were associated with significantly reduced levels of spore germination within the lungs as determined by in vivo imaging. Taken together, our data demonstrate a novel antimicrobial role for host chemokines againstB. anthracisthat provides unique insight into host defense against inhalational anthrax; these data also support the notion for an innovative approach in treatingB. anthracisinfection as well as infections caused by other spore-forming organisms.

Bacillus anthracis edema toxin (ET), composed of protective antigen and an adenylate cyclase edema factor (EF), elicits edema in host tissues, but the target cells and events leading from EF-mediated cyclic-AMP production to edema are unknown. We evaluated the direct effect of ET on several cell types in vitro and tested the possibility that mediators of vascular leakage, such as histamine, contribute to edema in rabbits given intradermal ET. ET increased the transendothelial electrical resistance of endothelial monolayers, a response that is mechanistically inconsistent with the in vivo vascular leakage induced by ET. Screening of several drugs by intradermal treatment prior to toxin injection demonstrated reduced ET-induced vascular leakage with a cyclo-oxygenase inhibitor (indomethacin), agents that interfere with histamine (pyrilamine or cromolyn), or a neurokinin antagonist (spantide). Systemic administration of indomethacin or celecoxib (cyclo-oxygenase inhibitors), pyrilamine, aprepitant (a neurokinin 1 receptor antagonist), or indomethacin with pyrilamine significantly reduced vascular leakage associated with ET. Although the effects of pyrilamine, cromolyn, or aprepitant on ET-induced vascular leakage suggest a possible role for mast cells (MC) and sensory neurons in ET-induced edema, ET did not elicit degranulation of human skin MC or substance P release from NT2N cells in vitro. Our results indicate that ET, acting indirectly or directly on a target yet to be identified, stimulates the production/release of multiple inflammatory mediators, specifically neurokinins, prostanoids, and histamine. These mediators, individually and through complex interactions, increase vascular permeability, and interventions directed at these mediators may benefit hosts infected with B. anthracis .