CpsA,a LytR-CpsA-Psr Family Protein in Mycobacterium marinum,Is Required for Cell Wall Integrity and Virulence

LytR-CpsA-Psr family proteins play an important role in bacterial cell wall integrity. Although the pathogenic relevance of LytR-CpsA-Psr family proteins has been studied in a few bacterial pathogens, their function in mycobacteria remains uncharacterized. In this work, a transposon insertion mutant (cpsA::Tn) of Mycobacterium marinum was studied. We found that inactivation of CpsA altered bacterial colony morphology, sliding motility, cell surface hydrophobicity, and cell wall permeability. Besides, the cpsA mutant exhibited a decreased arabinogalactan content, indicating that CpsA plays a role in cell wall assembly. Moreover, the mutant shows impaired growth within macrophage cell lines and is severely attenuated in zebrafish larvae and adult zebrafish. Taken together, our results indicated that CpsA, a previously uncharacterized protein, is important for mycobacterial cell wall integrity and is required for mycobacterial virulence.     

Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933

Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.Enterohemorrhagic Escherichia coli O157:H7 was first recognized as the probable cause of hemorrhagic colitis in humans in 1982 (50). Since then, this organism has emerged as a major cause of food-borne illness in countries around the world, including the United States (49), Europe (17, 18, 24, 36), Japan (39), and Australia (28). Outbreaks have been associated with a variety of food sources, including ground beef (50), green leafy vegetables (1, 41), and nonpasteurized milk (26), and environments such as municipal water and lakes (27, 55). Symptoms in infected humans range from mild diarrhea to severe, hemorrhagic colitis, with 5 to 10% of patients developing hemolytic uremic syndrome (HUS), making E. coli O157:H7 one of the leading causes of acute renal failure in children and the elderly (38).Early studies have shown that some strains of E. coli O157:H7 form biofilms on both abiotic and biotic surfaces outside the host (15, 51, 58). Biofilms are exopolymeric matrix-enclosed bacterial populations that are firmly adherent to each other and/or to surfaces (9). Biofilms have been shown to be responsible for protection from a variety of environmental stresses, such as acidification, high temperatures, and desiccation (52). Moreover, microbes in biofilms are highly resistant to other adverse conditions, such as sanitizers and household cleaners (44, 45) as well as antibiotics (23, 34). The ability of this pathogen to form biofilms on a wide range of food surfaces as well as food processing surfaces makes E. coli O157:H7 problematic in both the health and food industries (29).Biofilm formation is a dynamic and complex process and includes initial attachment of cells to the substratum, physiological changes within the organism, multiplication of the cells to form microcolonies, and eventually maturation of the biofilm (42). Because of this complexity, the process of biofilm formation and its regulation is poorly understood. Previous studies in E. coli O157:H7 have focused on individual genes and the specific genetic pathways that are responsible for biofilm formation (10, 51, 58, 60). In contrast, few studies have focused on studying genetic factors that control E. coli O157:H7 biofilm formation on a global scale, although studies of this type have been performed with other bacterial pathogens (43, 47, 59).The goal of this study was to gain additional insights into biofilm formation in E. coli O157:H7. A global mutational approach with a mini-Tn5 transposon was used to study the process of biofilm formation in strain EDL933, a strong biofilm-forming strain. This strain was first isolated during a multistate outbreak involving contaminated hamburgers (50). A library of >11,000 mutants was generated and screened for a biofilm-negative phenotype. Our results reinforced the fact that biofilm formation is a complex process involving a large number of genes and genetic pathways. This study discovered several pO157 genes that were not previously known to be linked to biofilm formation.     

Ferrochelatase Is Present in Brucella abortus and Is Critical for Its Intracellular Survival and Virulence

Brucella spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established, Brucella spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of Brucella abortus. During a B. abortus genomic sequencing project, a clone coding a putative gene homologous to hemH was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type hemH. These data demonstrate that B. abortus synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of B. abortus and thus for the establishment of chronic disease as well.     

A Mycobacterium marinum mel2 mutant is defective for growth in macrophages that produce reactive oxygen and reactive nitrogen species

Macrophages produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to bacterial infections. Mycobacteria are relatively resistant to ROS, but RNS inhibit growth of, and possibly even kill, mycobacteria in activated macrophages. We recently constructed a Mycobacterium marinum mel2 locus mutant, which is known to affect macrophage infection. We found previously that the mel2 locus confers resistance to ROS and RNS in laboratory medium, suggesting that this locus might play a similar role during growth in macrophages. Since J774A.1 murine macrophages produce high levels of ROS and RNS upon activation with gamma interferon (IFN-gamma), we examined the effects of IFN-gamma on ROS and RNS production by these cells as well as the effects on growth of M. marinum in these cells. We found that an M. marinum mutant with mutation of the first gene in the mel2 locus, melF, is defective for growth in IFN-gamma-plus-lipopolysaccharide-treated J774A.1 cells and that this defect is abrogated by the presence of either inhibitors of nitric oxide synthase or ROS scavengers. Furthermore, the M. marinum melF mutant displays a defect at late stages in the mouse footpad model of infection. These phenotypic characteristics could be complemented fully by the entire mel2 locus but only partially by the presence of melF alone, supporting data suggesting that this insertion mutation has polar effects on downstream genes in the mel2 locus. These observations demonstrate that the M. marinum mel2 locus plays a role in resistance to ROS and RNS produced by activated macrophages.     

Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen

Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nal^r strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis.     

Site-Directed Mutagenesis of the 19-Kilodalton Lipoprotein Antigen Reveals No Essential Role for the Protein in the Growth and Virulence of Mycobacterium intracellulare

The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the Mycobacterium intracellulare 19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in M. intracellulare FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth of M. intracellulare. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of M. intracellulare in vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of M. intracellulare; however, the exact nature of its biological function remains unknown.     

Origins of a 350-Kilobase Genomic Duplication in Mycobacterium tuberculosis and Its Impact on Virulence

In the present study, we have investigated the evolution and impact on virulence of a 350-kb genomic duplication present in the most recently evolved members of the Mycobacterium tuberculosis East Asian lineage. In a mouse model of infection, comparing HN878 subclones HN878-27 (no duplication) and HN878-45 (with the 350-kb duplication) revealed that the latter is impaired for in vivo growth during the initial 3 weeks of infection. Furthermore, the median survival time of mice infected with isolate HN878-45 is significantly longer (77 days) than that of mice infected with HN878-27. Whole-genome sequencing of both isolates failed to reveal any mutational events other than the duplication that could account for such a substantial difference in virulence. Although we and others had previously speculated that the 350-kb duplication arose in response to some form of host-applied selective pressure (P. Domenech, G. S. Kolly, L. Leon-Solis, A. Fallow, M. B. Reed, J. Bacteriol. 192:4562–4570, 2010, and B. Weiner, J. Gomez, T. C. Victor, R. M. Warren, A. Sloutsky, B. B. Plikaytis, J. E. Posey, P. D. van Helden, N. C. Gey van Pittius, M. Koehrsen, P. Sisk, C. Stolte, J. White, S. Gagneux, B. Birren, D. Hung, M. Murray, J. Galagan, PLoS One 7:e26038, 2012), here we show that this large chromosomal amplification event is very rapidly selected within standard in vitro broth cultures in a range of isolates. Indeed, subclones harboring the duplication were detectable after just five rounds of in vitro passage. In contrast, the duplication appears to be highly unstable in vivo and is negatively selected during the later stages of infection in mice. We believe that the rapid in vitro evolution of M. tuberculosis is an underappreciated aspect of its biology that is often ignored, despite the fact that it has the potential to confound the data and conclusions arising from comparative studies of isolates at both the genotypic and phenotypic levels.     

Branched-Chain Amino Acids Are Required for the Survival and Virulence of Actinobacillus pleuropneumoniae in Swine

In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 μmol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM−BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens.Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a disease of significant economic importance throughout the swine-raising areas of the world (6, 48). This pathogen possesses several well-studied virulence factors, including Apx toxins (20), capsular polysaccharides (57, 58), lipopolysaccharide (1, 17, 41), fimbriae (63), and iron-scavenging proteins (13, 50), which aid in the pathogenesis of acute pleuropneumonia marked by edema, hemorrhage, and necrosis (6, 26). In a search for additional virulence factors of this pathogen, we developed an in vivo expression technology (IVET) system and used this genetic tool to identify A. pleuropneumoniae gene promoters that are upregulated in vivo in the swine lung during infection compared to growth on laboratory media (22, 55).One of the A. pleuropneumoniae in vivo-induced (ivi) promoters that we identified drives the ilvIH operon, which encodes both large and small subunits of acetohydroxy acid synthase isozyme III (AHAS) (55). AHAS enzymes catalyze pivotal steps in the biosynthesis of the branched-chain amino acids (BCAA) isoleucine, leucine, and valine (31). In a survey of IVET, signature-tagged mutagenesis, and microarray studies of other pathogens, we observed that genes involved in BCAA biosynthesis were frequently identified in studies of pathogens that cause pneumonia, meningitis, or septicemia but not in pathogens of the gastrointestinal tract (55). This observation suggests that the ability to synthesize BCAA is critical for pathogens of the respiratory tract but not for gastrointestinal pathogens. BCAA are essential amino acids that must be acquired from ingested food for most mammals, including humans and pigs, and it is possible that fluids in “clean” body sites such as the lungs have only limited supplies of BCAA compared to the digestive tract.To test whether limitation of BCAA affects the expression of A. pleuropneumoniae genes that are induced in vivo, we compared expression from the A. pleuropneumoniae ivi promoters in a chemically defined medium (CDM) containing or lacking BCAA (55). We found that 25% (8 of 32) of the ivi promoters were upregulated during growth in CDM lacking BCAA compared to complete CDM. These included the ilvI promoter, as well as promoters for other genes potentially involved in survival within the host and virulence, such as hfq, a global regulator that binds sRNAs and mRNA and affects expression of virulence-associated genes in many pathogens (9, 49). These results strongly suggest that the environmental conditions encountered by A. pleuropneumoniae during infection of the swine lung include limitation of BCAA.The goals of the present study were to quantify free BCAA in porcine pulmonary secretions, to evaluate the effect of these concentrations of BCAA on expression of genes required for BCAA biosynthesis, and to test whether A. pleuropneumoniae mutants that cannot synthesize BCAA were attenuated. A. pleuropneumoniae deletion-disruption mutants of the ilvI biosynthetic gene and the lrp gene, which encodes a global regulator required for expression of several genes involved in BCAA biosynthesis, were constructed and shown to be attenuated in a porcine infection model. The low levels of available BCAA in pulmonary secretions and the attenuation of these mutants led us to examine the effect of small molecule inhibitors of AHAS on growth of A. pleuropneumoniae in vitro. Several AHAS inhibitors were shown to prevent growth in CDM lacking BCAA but not complete CDM. These results demonstrate that A. pleuropneumoniae, and likely other bacterial pathogens of the respiratory tracts of other mammals, encounter conditions where BCAA are available only in limited supply during infection, that these low levels of BCAA can affect bacterial gene expression, and that these pathogens must be able to synthesize BCAA to survive and cause disease in the lung.     

Role of the Popliteal Lymph Node in Infection with Mycobacterium marinum of the Hind Footpad of the Mouse, and Source of the Cells that Characterize the Process

We examined the possibility that the popliteal lymph node serves as the source of the lymphocytes that, together with macrophages, characterize Ihc lesion produced by infection with Mycobacterium marinum in the hind footpad of Ihc mouse. Naive mice were partially protected against challenge with M. marinum in the hind footpad by intravenous infusion of lymphoc) its harvested from the popliteal nodes of donor mice infected with M. marinum 7 days earlier. Lymphocytes harvested from the popliteal nodes of infected donors, labelled in vitro with ³H-uridine, and infused intravenously into naive mice that were immediately challenged in the hind footpads with M. marinum, localized in the popliteal nodes of the recipient mice but nol in the footpad lesions. Lymphocytes harvested from the spleens of naive donors and labelled in vitro appeared to home to the popliteal nude draining the M. marinum-infected footpad. Thus, the primary rule of the popliteal lymph node appeared to be passive trapping of the lymphocytes brought to it by the circulairon or afferent lymphatics. We then tried lo locate the sources of bolh lymphocytes and macrophages that characterize the lesion. Temporary occlusion of the abdominal aorta prevented labelling by intravenously infused ³H-thymidine (³H-TdR) of the mononuclear cells of both footpad lesion and popliteal node. Temporary occlusion of the left common iliac artery during ³H-TdR infusion prevented immediate labelling on the ipsilateral side. After 24 and 48 h, however, small numbers of labelled lymphocyles were found in the left hind footpad lesion. Amputation of the right leg at the hip joint, but run right poplitial lymphadenectomy, performed immediately after re-estublishment of patency of the left common iliac artery, prevented the late influx of labelled lymphocytes into the lesion of the left hind footpad. Thus, the chief source of both the lymphocytes and the macrophages of the footpad lesion appeared lo be the lesion ilself.     

Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole

The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores ~4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.     

Global Study of IS6110 in a Successful Mycobacterium tuberculosis Strain: Clues for Deciphering Its Behavior and for Its Rapid Detection

The Mycobacterium tuberculosis insertion sequence IS6110, besides being a very useful tool in molecular epidemiology, seems to have an impact on the biology of bacilli. In the present work, we mapped the 12 points of insertion of IS6110 in the genome of a successful strain named M. tuberculosis Zaragoza (which has been referred to as the MTZ strain). This strain, belonging to principal genetic group 3, caused a large unsuspected tuberculosis outbreak involving 85 patients in Zaragoza, Spain, in 2001 to 2004. The mapping of the points of insertion of IS6110 in the genome of the Zaragoza strain offers clues for a better understanding of the adaptability and virulence of M. tuberculosis. Surprisingly, the presence of one copy of IS6110 was found in Rv2286c, as was recently described for a successful Beijing sublineage. As a result of this analysis, a rapid method for detecting this particular M. tuberculosis strain has been designed.     

Relationship between IS901 in the Mycobacterium avium Complex Strains Isolated from Birds, Animals, Humans, and the Environment and Virulence for Poultry

A total of 738 strains of Mycobacterium avium complex (MAC) were examined in biological experiments on poultry by use of PCR methods with primers for detection of the insertion sequence IS901. Serotype strains of MAC from all known 28 serotypes were examined. Further strains were isolated from human immunodeficiency virus (HIV)-negative and HIV-positive patients, 6 animal species, 17 bird species, and the environment. Of 165 strains virulent for poultry, characterized by generalized tuberculosis, 164 strains contained IS901, a result which is statistically highly significant (P, 0.01). The remaining 573 strains were nonvirulent; however, IS901 was present in 24 strains. From among 20 strains of serotypes 1, 2, and 3, IS901 was found in 15 strains, only 5 of which were virulent for poultry. The remaining 111 strains, of serotypes 4 to 28, were nonvirulent and did not incorporate IS901. None of the 152 strains isolated from humans was virulent for poultry, including 12 strains which were IS901 positive.     

Rapid, radiolabeled-microculture method that uses macrophages for in vitro evaluation of Mycobacterium leprae viability and drug susceptibility

This paper describes a microculture rapid assay using radiolabeling and mouse macrophages to determine the viability and the drug susceptibility or resistance of Mycobacterium leprae. Comparison of M. leprae resident macrophage cultures maintained in 96-well flat-bottomed plates showed results for viability and susceptibility or resistance to dapsone that were similar to results for concurrent cultures in Leighton tubes with greater numbers of bacilli and macrophages.     

Fish Rhabdovirus Replication in Non-Piscine Cell Culture: New System for the Study of Rhabdovirus-Cell Interaction in Which the Virus and Cell Have Different Temperature Optima

The replication of three rhabdoviruses associated with diseases of fish has been demonstrated in cells of continuously cultivated non-piscine cell lines. Spring viremia of carp (SVC) virus and the salmonid fish viruses, Egtved and infectious hematopoietic necrosis virus, all replicated in mammalian WI-38 (human diploid cell strain) and BHK/21 cells and in cells of one or more reptilian cell lines at the temperatures commonly used to propagate these viruses in fish cells. The infections were cytopathic: SVC virus plaque assays may be performed in several types of mammalian cell culture. "Autointerference" apparently mediated by abortive "T" particle formation was observed during serial nondiluted passages of SVC virus in BHK/21 and TH1 cells, but not in RTG-2 or WI-38 cells. Optimal temperatures for replication of SVC and Egtved viruses in BHK/21 cells were identical to those determined in poikilothermic vertebrate cell cultures. However, these viruses replicated relatively more efficiently at suboptimal temperatures in "cold-blooded" vertebrate cells than in the hamster cells. Studies of [(3)H]uridine incorporation into uninfected BHK/21 cells incubated at different temperatures revealed that [(3)H]uridine uptake is sharply reduced at temperatures below 24.5 C. Growth curve studies of SVC virus in BHK/21 cells incubated at 23 C revealed that a clear-cut large excess of virus-induced [(3)H]uridine incorporation could be demonstrated in the absence of actinomycin D. Actinomycin D treatment (1 mug/ml) led to efficient inhibition of control cell [(3)H]uridine uptake, but also markedly reduced the total counts per minute of virus-induced [(3)H]uridine uptake, without depressing the yield of released infectious virus. Actinomycin D added to SVC virus-infected BHK/21 cell cultures at concentrations as low as 0.01 mug/ml caused a significant decrease in the level of virus-induced [(3)H]uridine uptake, despite the fact that this concentration is insufficient to efficiently suppress "background" cellular [(3)H]uridine incorporation.     

Impact of Free-Living Amoebae on Presence of Parachlamydia acanthamoebae in the Hospital Environment and Its Survival In Vitro without Requirement for Amoebae

Parachlamydia acanthamoebae is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen in hospital-acquired pneumonia. We examined whether the presence of P. acanthamoebae is related to the presence of Acanthamoeba in an actual hospital environment and assessed the in vitro survival of P. acanthamoebae. Ninety smear samples were collected between November 2007 and March 2008 (trial 1, n = 52) and between October 2008 and February 2009 (trial 2, n = 38) from the floor (dry conditions, n = 56) and sink outlets (moist conditions, n = 34) of a hospital. The prevalences of P. acanthamoebae DNA in the first and second trials were 64.3% and 76%, respectively. The prevalences of Acanthamoeba DNA in the first and second trials were 48% and 63.1%, respectively. A statistical correlation between the prevalence of P. acanthamoebae and that of Acanthamoeba was found (trial 1, P = 0.011; trial 2, P = 0.022), and that correlation increased when samples from just the dry area (floor smear samples, P = 0.002) were analyzed but decreased when samples from a moist area were analyzed (P = 0.273). The in vitro experiment showed that, without Acanthamoeba, P. acanthamoebae could not survive in dry conditions for 3 days at 30°C or 15 days at 15°C. Thus, both organisms were coincidentally found in an actual hospital environment, with the presence of Acanthamoeba having a significant effect on the long-term survival of P. acanthamoebae, suggesting that this potential human pathogen could spread through a hospital environment via Acanthamoeba.Chlamydiae, which are obligate intracellular bacterial pathogens, have been reclassified as the order Chlamydiales, which includes four families: Chlamydiaceae, Parachlamydiaceae, Waddliaceae, and Simkaniaceae (5). The family Chlamydiaceae is well known to have a broad range of distribution in animals and humans and to be the causative agents of human diseases (4, 20, 24, 31, 36). This family includes two major human pathogens: Chlamydophila pneumoniae, a causal agent of common respiratory infection and also suspected of being involved in some chronic diseases, such as asthma and atherosclerosis (6), and Chlamydia trachomatis, responsible for sexually transmitted disease and preventable blindness (33).Parachlamydiaceae, Waddliaceae, and Simkaniaceae have recently been recognized as chlamydiae that exhibit a wide range of distribution in the natural environment, such as in rivers and in soil (9). All these species can grow and survive dependently within the free-living amoeba Acanthamoeba, the most abundant genus of amoeba (1, 9, 11, 12). Parachlamydia acanthamoebae and Simkania negevensis have been associated with lower respiratory tract infections (3, 27), and Waddlia chondrophila, which was originally isolated from an aborted bovine fetus, is considered a potential abortogenic agent (9). There is accumulating evidence supporting the pathogenic role of P. acanthamoebae in humans (3, 9, 13). Several studies have reported that parachlamydial DNA was detected by PCR in mononuclear cells of sputa and in bronchoalveolar lavage samples from a patient with bronchitis (10). Other studies have suggested that P. acanthamoebae may cause inhalation pneumonia and be responsible for hospital-acquired pneumonia in HIV-infected patients and organ transplant recipients receiving immunosuppressive therapy (7, 8, 14). Thus, P. acanthamoebae, presumably spreading through amoebae, is emerging as a potential etiological agent of hospital-acquired pneumonia. However, the overlap between the distribution of P. acanthamoebae and that of Acanthamoeba in hospitals and the survival of the bacteria in harsh conditions without Acanthamoeba remain unknown. We examined whether the prevalence of P. acanthamoebae correlates with that of Acanthamoeba in a hospital in Sapporo, Japan. We also examined the in vitro survival of P. acanthamoebae and its requirement for Acanthamoeba.P. acanthamoebae Bn₉ (ATCC VR-1476) was purchased from the American Type Culture Collection. The bacteria were propagated in an amoeba cell culture system according to methods described previously (17). The numbers of infective progeny were determined according to the procedure described below. Free-living amoebae, Acanthamoeba castellanii C3 (ATCC 50739), were purchased from the American Type Culture Collection. Amoebae were maintained in PYG broth (0.75% [wt/vol] peptone, 0.75% [wt/vol] yeast extract, and 1.5% [wt/vol] glucose) at 30°C (35). The numbers of infective progeny of P. acanthamoebae were determined by a procedure known as the amoeba infectious unit (AIU) assay using coculture with amoebae as described previously (28).Ninety smear samples were obtained from the floor (n = 56) and sink outlets (n = 34) of a hospital (Hokkaido University Hospital) containing approximately 900 beds (number of outpatients, approximately 3,000 per day; number of hospital patients, approximately 1,000 per day) in a 12-story building located in the central area of Sapporo, Japan, from November 2007 to March 2008 (trial 1, n = 52) and from October 2008 to February 2009 (trial 2, n = 38). The samples were collected by wiping an approximately 1-m² area on the floor or sink outlet with sterilized gauze (approximately 25 cm²) moistened with Page''s amoeba saline (PAS) (30). Each piece of gauze was then vortexed for 60 s in 20 ml sterilized PAS containing 0.05% (vol/vol) Tween 80, and the suspension was then centrifuged at 2,100 × g for 20 min. Pellets were resuspended in 200 μl PAS and then used for P. acanthamoebae culture and DNA extraction.One-half of the resuspended smear pellet (100 μl) was used for P. acanthamoebae culture. Culture detection was performed by a method based on amoeba lysis described previously (18). In brief, serially diluted sample solution was added to 100 μl of amoeba suspension containing 1 × 10⁵ A. castellanii C3 cells in 1 well of a 96-well microplate and incubated for up to 10 days at 30°C in a normal atmosphere. The microplate was read daily to determine the highest dilution of bacteria that led to amoeba lysis.The DNA extraction was performed using the UltraClean soil DNA extraction kit (MBL, Carlsbad, CA) according to the manufacturer''s instructions. The primers used in PCR amplification were as follows: PacI (5′-GAG GTG AAG CAA ATC CCA AA-3′) and Pac2 (5′-CTC CTT GCG GTT AAG TCA GC-3′) for amplification of P. acanthamoebae 16S rRNA (191 bp), JDP1 (5′-GGC CCA GAT CGT TTA CCG TGA A-3′) and JDP2 (5′-TCT CAC AAG CTG CTA GGG AGT CA-3′) for amplification of 18S rRNA from Acanthamoeba species (423 to 551 bp) (34), and Bac11 (5′-GAG GAA GGT GGG GAT GAC GT-3′) and Bac12 (5′-AGG CCC GGG AAC GTA TTC AC-3′) for amplification of bacterial 16S rRNA excluding the order Chlamydiales (216 bp) (38). The primers for amplification of P. acanthamoebae 16S rRNA were designed based on GenBank cDNA sequences (accession number NR026357) using the program Primer 3 (http://frodo.wi.mit.edu/primer3/input.htm). To overcome inhibition of PCR amplification by humic acid, bovine serum albumin (BSA) was added to each reaction mixture according to methods described previously (23, 39). The quality of extracted DNA was confirmed by PCR amplification using universal primers that target bacterial 16S rRNA, which is conserved across a broad spectrum of bacteria. All smear samples (n = 90) yielded PCR amplicons of the expected size and were then used in specific P. acanthamoebae and Acanthamoeba PCRs. Search results returned by the BLAST program showed that the primers used for each PCR were specific for P. acanthamoebae and Acanthamoeba detection. The amount of template DNA used in each PCR was 2 μl. The total reaction volume was 25 μl and consisted of 200 μM each deoxynucleoside triphosphate (dNTP), 10 μM BSA, 1× commercial reaction buffer, and 0.625 U Taq DNA polymerase (New England Biolabs, Herts, United Kingdom). The thermal cycling profile involved an initial denaturation step at 94°C for 10 min; 35 cycles, each consisting of 30 s of denaturation at 94°C and 30 s of annealing at 60°C for P. acanthamoebae 16S rRNA, 60°C for Acanthamoeba species 18S rRNA, and 52°C for bacterial 16S rRNA; and a 45-s extension at 72°C. The amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The presence of amplified target genes in randomly selected positive specimens was confirmed by direct oligonucleotide sequencing of the PCR products (Macrogen, Seoul, South Korea). As a quality control for each PCR, diluted DNA extracted from the P. acanthamoebae Bn₉ or A. castellanii C3 strain was used in each amplification. As a negative control, DNA-free water (Sigma) was also used in amplification. To prevent contamination, the preparation of the PCR mixture was performed in a separate room. Alignment analysis and the construction of a phylogenetic tree for P. acanthamoebae amplicons (n = 4) with previously reported Parachlamydiaceae sequences were performed using Genetyx-Mac software (version 10.1) and the neighbor-joining method in MEGA software (version 4). The detection limits of the PCR for P. acanthamoebae 16S rRNA and Acanthamoeba 18S rRNA were examined by using DNA extracted from the sterilized gauze moistened with PAS that had been spiked with defined numbers of P. acanthamoebae AIU and Acanthamoeba cells, respectively. The detection limits of the PCR targeted to P. acanthamoebae and Acanthamoeba were 10² AIU and 10 cells, respectively.The procedure for monitoring bacterial viability was as follows. A bacterial solution of 100 μl containing approximately 10⁷ to 10⁸ AIU prepared with PAS was placed into wells of a 24-well plate with (moist conditions) or without (dry conditions) 900 μl PAS, and the plates incubated for up to 28 days at 15°C or 30°C in a normal atmosphere. At 0, 3, 7, 15, and 28 days after inoculation, the supernatant in each well was collected and centrifuged at 3,500 × g for 30 min. In the case of the samples for moist conditions, supernatants were directly transferred to a centrifuge tube. For the dry condition samples, the wells were washed with 900 μl PAS and this was then transferred to a centrifuge tube. The resulting bacterial pellet was resuspended in 100 μl PAS. The numbers of infective progeny as a marker of bacterial viability in the solution were determined by the AIU assay as described above. The bacterial membrane integrity as a possible indicator for bacterial viability was also confirmed with fluorescence microscopy by using a Live/Dead reduced biohazard viability/cytotoxicity kit (Molecular Probes, Eugene, OR), according to the manufacturer''s instructions.The correlation between the frequency of P. acanthamoebae and that of Acanthamoeba spp. was analyzed by Fisher''s exact test. The influence of floor level on the prevalence of both organisms was also analyzed by a two-way analysis of variance (ANOVA) test. Comparison of bacterial numbers in the in vitro experiment was assessed by an unpaired t test. A P value of less than 0.05 was considered significant.     

Incorporation of Different Bridge Length Linkers in Enzyme and Its Use in the Preparation of Enzyme Conjugates for Immunoassay

Abstract

An enzyme horseradish peroxidase (HRP), as a starting material, has been used to introduce different bridge length linkers, and its use in the preparation of enzyme conjugates for immunoassay is described. HRP was conjugated to adipic acid dihydrazide (ADH), gamma amino butyric acid (GABA), followed by ADH and 6‐amino caproic acid (6ACA) followed by ADH. The different bridge length linkers‐incorporated enzyme was coupled to a carboxylic derivative of cortisol. Four enzyme conjugates with different bridge length were prepared, such as cortisol‐21‐hemisuccinate–HRP (cortisol‐21‐HS–HRP), cortisol‐21‐HS–ADH–HRP, cortisol‐21‐HS–ADH–GABA–HRP, and cortisol‐21‐HS–ADH–6ACA–HRP. The influence of linker on sensitivity and specificity of the cortisol assay was studied. The study revealed that incorporation of a linker between hapten and enzyme increases the sensitivity and specificity of the assay.     

Assessment of an Oral Mycobacterium bovis BCG Vaccine and an Inactivated M. bovis Preparation for Wild Boar in Terms of Adverse Reactions,Vaccine Strain Survival,and Uptake by Nontarget Species

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment.