Epidemiology of Invasive Klebsiella pneumoniae with Hypermucoviscosity Phenotype in a Research Colony of Nonhuman Primates

Invasive Klebsiella pneumoniae with hypermucoviscosity phenotype (HMV K. pneumoniae) is an emerging human pathogen that, over the past 20 y, has resulted in a distinct clinical syndrome characterized by pyogenic liver abscesses sometimes complicated by bacteremia, meningitis, and endophthalmitis. Infections occur predominantly in Taiwan and other Asian countries, but HMV K. pneumoniae is considered an emerging infectious disease in the United States and other Western countries. In 2005, fatal multisystemic disease was attributed to HMV K. pneumoniae in African green monkeys (AGM) at our institution. After identification of a cluster of subclinically infected macaques in March and April 2008, screening of all colony nonhuman primates by oropharyngeal and rectal culture revealed 19 subclinically infected rhesus and cynomolgus macaques. PCR testing for 2 genes associated with HMV K. pneumoniae, rmpA and magA, suggested genetic variability in the samples. Random amplified polymorphic DNA analysis on a subset of clinical isolates confirmed a high degree of genetic diversity between the samples. Environmental testing did not reveal evidence of aerosol or droplet transmission of the organism in housing areas. Further research is needed to characterize HMV K. pneumoniae, particularly with regard to genetic differences among bacterial strains and their relationship to human disease and to the apparent susceptibility of AGM to this organism.Abbreviations: AGM, African green monkey; HMV K. pneumoniae, invasive Klebsiella pneumoniae with hypermucoviscosity phenotype; NHP, nonhuman primate; RAPD, random amplification of polymorphic DNAKlebsiella pneumoniae is an enteric, gram-negative, lactose-fermenting bacillus with a prominent capsule. This bacterium has been associated with peritonitis, septicemia, pneumonia, and meningitis in both Old and New World primates,^10,13,29 although it also is reported to constitute normal fecal and oral flora in many nonhuman primates (NHP).¹² Pathogenic strains associated with the upper respiratory tract typically are heavily encapsulated.¹² Over the past several decades, human medical literature indicates the emergence of an invasive K. pneumoniae disease in Taiwan and other Asian countries, in which community-acquired pyogenic liver abscesses have been attributed to strains of invasive K. pneumoniae with a unique hypermucoviscous phenotype (HMV K. pneumoniae).^{6,17-19,21,26,34} The hypermucoviscous phenotype has also been associated with other serious complications, including bacteremia, meningitis, and endophthalmitis. This strain of Klebsiella has become an emerging cause of pyogenic liver abscesses in some nonAsian countries, including the United States.^16,20,36,39 The majority of clinical cases of HMV K. pneumoniae are in the Asian population, particularly in patients with diabetes mellitus.^3,4,33 Determination of the HMV phenotype typically is based on a positive string test.^8,35,39Several virulence factors have been associated with HMV K. pneumoniae. Klebsiella spp. generally develop prominent polysaccharide capsules which increase virulence by protecting the bacteria from phagocytosis and preventing destruction by bactericidal serum factors. Capsular serotypes K1 or K2 have been reported as the major virulence determinants for human HMV K. pneumoniae liver abscesses.^5,8,37,38 In addition, the mucoviscosity-associated gene magA, which encodes a structural outer membrane protein of the K1 serotype, and rmpA (regulator of the mucoid phenotype gene; located on a plasmid) have been proposed as virulence factors.^{9,27,31,40,41} Recently, it was suggested that 2 clones, CC23 ^K1 and CC82^K1, are strongly associated with primary liver abscess and respiratory infection, respectively.²Over a period of several months in 2005 to 2006, 7 African green monkeys (AGM; Chlorocebus aethiops) in the US Army Medical Research Institute of Infectious Diseases research colony developed abscesses in multiple locations and either died or were euthanized when the abscesses were determined to be nonresectable.³⁵ HMV K. pneumoniae of the K2 serotype and carrying rmpA was determined to be the cause of the infection in 1 case, and the 6 other cases had similar clinical and pathologic features. This report³⁵ is the only documentation, to our knowledge, of natural infection with HMV K. pneumoniae in NHP. As a result of these cases, the US Army Medical Research Institute of Infectious Diseases instituted policies to exclude HMV K. pneumoniae from the colony. The organism was included as a specific pathogen-free requirement for vendors, and K. pneumoniae culture results were reported during quarantine periods and on routine semiannual examination for all colony NHP.     

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.     

HIV Transmission in a State Prison System, 1988–2005

Introduction

HIV prevalence among state prison inmates in the United States is more than five times higher than among nonincarcerated persons, but HIV transmission within U.S. prisons is sparsely documented. We investigated 88 HIV seroconversions reported from 1988–2005 among male Georgia prison inmates.

Methods

We analyzed medical and administrative data to describe seroconverters'' HIV testing histories and performed a case-crossover analysis of their risks before and after HIV diagnosis. We sequenced the gag, env, and pol genes of seroconverters'' HIV strains to identify genetically-related HIV transmission clusters and antiretroviral resistance. We combined risk, genetic, and administrative data to describe prison HIV transmission networks.

Results

Forty-one (47%) seroconverters were diagnosed with HIV from July 2003–June 2005 when voluntary annual testing was offered. Seroconverters were less likely to report sex (OR [odds ratio] = 0.02, 95% CI [confidence interval]: 0–0.10) and tattooing (OR = 0.03, 95% CI: <0.01–0.20) in prison after their HIV diagnosis than before. Of 67 seroconverters'' specimens tested, 33 (49%) fell into one of 10 genetically-related clusters; of these, 25 (76%) reported sex in prison before their HIV diagnosis. The HIV strains of 8 (61%) of 13 antiretroviral-naïve and 21 (40%) of 52 antiretroviral-treated seroconverters were antiretroviral-resistant.

Discussion

Half of all HIV seroconversions were identified when routine voluntary testing was offered, and seroconverters reduced their risks following their diagnosis. Most genetically-related seroconverters reported sex in prison, suggesting HIV transmission through sexual networks. Resistance testing before initiating antiretroviral therapy is important for newly-diagnosed inmates.     

Reversal of Small,Dense LDL Subclass Phenotype by Normalization of Adiposity

Excess adiposity and high‐carbohydrate diets have been associated with an atherogenic lipoprotein phenotype (ALP) characterized by increased concentrations of small, dense low‐density lipoprotein (LDL) particles (pattern B). We tested whether weight loss and normalization of adiposity could reverse ALP in overweight men with pattern B. After consuming a moderate‐carbohydrate, high‐fat diet for 3 weeks, pattern B and nonpattern B (pattern A) men were randomized to a weight loss (n = 60 and n = 36, respectively) or control weight‐stable arm (n = 20 and n = 17, respectively). Men in the weight loss arm consumed ～1,000 fewer calories per day over 9 weeks to induce an average ～9 kg weight loss. In the control group, weight stability was maintained for 4 weeks after randomization. Weight loss led to the conversion of pattern B to pattern A in 58% of baseline pattern B men. Among men who achieved BMIs of <25 kg/m² (62% of pattern B men vs. 83% of pattern A men), 81% of pattern B men converted to pattern A. Weight loss was associated with a significantly greater decrease in small, dense LDL subclass 3b in pattern B relative to pattern A men. The lipoprotein profiles of pattern A men who converted from pattern B were comparable to those of men with pattern A at baseline. Conversion of LDL subclass pattern B to pattern A and reversal of ALP can be achieved in a high proportion of overweight men by normalization of adiposity.     

Interaction between Plate Make and Protein in Protein Crystallisation Screening

Background

Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate.

Methodology/Principal Findings

We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised.

Conclusions/Significance

Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallise, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.     

Isolation, characterization and cross-species amplification of polymorphic microsatellite loci in Asclepias (Apocynaceae)

Ten polymorphic loci were isolated and characterized from the milkweed, Asclepias syriaca L., of North America. These loci successfully cross-amplified in A. exaltata L. Polymorphism ranged from two to 16 alleles per locus per species in 68 individuals of A. syriaca and 56 individuals of A. exaltata. Expected heterozygosities ranged from 0.017 to 0.851 and significant deviations from Hardy–Weinberg equilibrium were found for two and three loci in A. syriaca and A. exaltata, respectively. No linkage disequilibrium was detected. These markers should prove useful for assessing population genetic structure and interspecific gene flow in these and other species of Asclepias.     

A Combined CXCL10, CXCL8 and H-FABP Panel for the Staging of Human African Trypanosomiasis Patients

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic tropical disease. It progresses from the first, haemolymphatic stage to a neurological second stage due to invasion of parasites into the central nervous system (CNS). As treatment depends on the stage of disease, there is a critical need for tools that efficiently discriminate the two stages of HAT. We hypothesized that markers of brain damage discovered by proteomic strategies and inflammation-related proteins could individually or in combination indicate the CNS invasion by the parasite.

Methods

Cerebrospinal fluid (CSF) originated from parasitologically confirmed Trypanosoma brucei gambiense patients. Patients were staged on the basis of CSF white blood cell (WBC) count and presence of parasites in CSF. One hundred samples were analysed: 21 from stage 1 (no trypanosomes in CSF and ≤5 WBC/µL) and 79 from stage 2 (trypanosomes in CSF and/or >5 WBC/µL) patients. The concentration of H-FABP, GSTP-1 and S100β in CSF was measured by ELISA. The levels of thirteen inflammation-related proteins (IL-1ra, IL-1β, IL-6, IL-9, IL-10, G-CSF, VEGF, IFN-γ, TNF-α, CCL2, CCL4, CXCL8 and CXCL10) were determined by bead suspension arrays.

Results

CXCL10 most accurately distinguished stage 1 and stage 2 patients, with a sensitivity of 84% and specificity of 100%. Rule Induction Like (RIL) analysis defined a panel characterized by CXCL10, CXCL8 and H-FABP that improved the detection of stage 2 patients to 97% sensitivity and 100% specificity.

Conclusion

This study highlights the value of CXCL10 as a single biomarker for staging T. b. gambiense-infected HAT patients. Further combination of CXCL10 with H-FABP and CXCL8 results in a panel that efficiently rules in stage 2 HAT patients. As these molecules could potentially be markers of other CNS infections and disorders, these results should be validated in a larger multi-centric cohort including other inflammatory diseases such as cerebral malaria and active tuberculosis.     

Active Trachoma and Ocular Chlamydia trachomatis Infection in Two Gambian Regions: On Course for Elimination by 2020?

Background

Trachoma has been endemic in The Gambia for decades. National trachoma control activities have been in place since the mid-1980''s, but with no mass antibiotic treatment campaign. We aimed to assess the prevalence of active trachoma and of actual ocular Chlamydia trachomatis infection as measured by polymerase chain reaction (PCR) in the two Gambian regions that had had the highest prevalence of trachoma in the last national survey in 1996 prior to planned national mass antibiotic treatment distribution in 2006.

Methodology/Principal Findings

Two stage random sampling survey in 61 randomly selected Enumeration Areas (EAs) in North Bank Region (NBR) and Lower River Region (LRR). Fifty randomly selected children aged under 10 years were examined per EA for clinical signs of trachoma. In LRR, swabs were taken to test for ocular C. trachomatis infection. Unadjusted prevalences of active trachoma were calculated, as would be done in a trachoma control programme. The prevalence of trachomatous inflammation, follicular (TF) in the 2777 children aged 1–9 years was 12.3% (95% CI 8.8%–17.0%) in LRR and 10.0% (95% CI 7.7%–13.0%) in NBR, with significant variation within divisions (p<0.01), and a design effect of 3.474. Infection with C. trachomatis was found in only 0.3% (3/940) of children in LRR.

Conclusions/Significance

This study shows a large discrepancy between the prevalence of trachoma clinical signs and ocular C. trachomatis infection in two Gambian regions. Assessment of trachoma based on clinical signs alone may lead to unnecessary treatment, since the prevalence of active trachoma remains high but C. trachomatis infection has all but disappeared. Assuming that repeated infection is required for progression to blinding sequelae, blinding trachoma is on course for elimination by 2020 in The Gambia.