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1.  Candida albicans HWP1 gene expression and host antibody responses in colonization and disease 
Journal of medical microbiology  2006;55(Pt 10):1323-1327.
In vivo expression of the developmentally regulated Candida albicans hyphal wall protein 1 (HWP1) gene was analysed in human subjects who were culture positive for C. albicans and had oral symptoms (n=40) or were asymptomatic (n=29), or had vaginal symptoms (n=40) or were asymptomatic (n=29). HWP1 mRNA was present regardless of symptoms, implicating hyphal and possibly pseudohyphal forms in mucosal carriage as well as disease. As expected, in control subjects without oral symptoms (n=10) and without vaginal symptoms (n=10) who were culture negative in oral and vaginal samples, HWP1 mRNA was not detected. However, exposure to Hwp1 in healthy culture-negative controls, as well as in oral candidiasis and asymptomatic mucosal infections, was shown by the existence of local salivary and systemic adaptive antibody responses to Hwp1. The results are consistent with a role for Hwp1 in gastrointestinal colonization as well as in mucosal symptomatic and asymptomatic infections. Overall, Hwp1 and hyphal growth forms appear to be important factors in benign and invasive interactions of C. albicans with human hosts.
doi:10.1099/jmm.0.46737-0
PMCID: PMC3244616  PMID: 17005778
2.  Swarming motility, secretion of type 3 effectors and biofilm formation phenotypes exhibited within a large cohort of Pseudomonas aeruginosa clinical isolates 
Journal of medical microbiology  2010;59(Pt 5):511-520.
Summary
Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable of acutely infecting or persistently colonizing susceptible hosts. P. aeruginosa colonizes surfaces in vitro by either biofilm formation or swarming motility. The choice of behavior is influenced by the physical properties of the surface and specific nutrient availability, and subject to regulatory networks that also govern Type 2 and Type 3 protein secretion. Biofilm formation by clinical isolates has been well-studied. However, the swarming behavior of human isolates has not been extensively analyzed. We collected isolates from 237 hospitalized patients without cystic fibrosis and analyzed motility and secretion phenotypes of each isolate. We found biofilm formation and swarming to be negatively associated, while swarming was positively associated with the secretion of both proteases and Type 3 exoenzymes. Most isolates were capable of Type 3 secretion and biofilm formation, even though these traits are considered to favor distinct modes of pathogenesis. Our data demonstrate that while clinical isolates display diverse motility, biofilm, and secretion phenotypes, many of the predicted relationships between swarming motility and other phenotypes observed in laboratory strains also hold true for bacteria isolated from human patients.
doi:10.1099/jmm.0.017715-0
PMCID: PMC2855384  PMID: 20093376
3.  Swarming motility, secretion of type 3 effectors and biofilm formation phenotypes exhibited within a large cohort of Pseudomonas aeruginosa clinical isolates 
Journal of Medical Microbiology  2010;59(Pt 5):511-520.
Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable of acutely infecting or persistently colonizing susceptible hosts. P. aeruginosa colonizes surfaces in vitro by either biofilm formation or swarming motility. The choice of behaviour is influenced by the physical properties of the surface and specific nutrient availability, and subject to regulatory networks that also govern type 2 and type 3 protein secretion. Biofilm formation by clinical isolates has been well-studied. However, the swarming behaviour of human isolates has not been extensively analysed. We collected isolates from 237 hospitalized patients without cystic fibrosis and analysed motility and secretion phenotypes of each isolate. We found biofilm formation and swarming to be negatively associated, while swarming was positively associated with the secretion of both proteases and type 3 exoenzymes. Most isolates were capable of type 3 secretion and biofilm formation, even though these traits are considered to favour distinct modes of pathogenesis. Our data demonstrate that while clinical isolates display diverse motility, biofilm and secretion phenotypes, many of the predicted relationships between swarming motility and other phenotypes observed in laboratory strains also hold true for bacteria isolated from human patients.
doi:10.1099/jmm.0.017715-0
PMCID: PMC2855384  PMID: 20093376
6.  Diagnosis of a Trypanosoma lewisi-like (Herpetosoma) infection in a sick infant from Thailand 
Journal of medical microbiology  2007;56(Pt 8):1118-1121.
Trypanosomes were observed in a peripheral blood smear from a 45-day-old Thai infant displaying fever, anaemia, cough and anorexia. Human trypanosomiasis is not endemic to Thailand, so parasite identification was undertaken to determine likely sources of the infection. Several morphological parameters of the trypanosomes were similar to those of Trypanosoma evansi and statistically different from those of Trypanosoma lewisi-like parasites from a naturally infected indigenous rat. However, duplicate PCR assays with primers flanking trypanosome rRNA internal transcribed spacer 1 (ITS1) resulted in amplicons of ~623 bp that corresponded to the expected size for T. lewisi-like parasites. The ITS1 sequence from the infant’s blood was 98 and 49% identical to T. lewisi and T. evansi sequences, respectively. Based on molecular results, it was concluded that the infant was infected with a T. lewisi-like (Herpetosoma) species.
doi:10.1099/jmm.0.47222-0
PMCID: PMC3066167  PMID: 17644723
7.  Experimental infection of dairy calves with Ehrlichia chaffeensis 
Journal of medical microbiology  2007;56(Pt 12):1660-1668.
Human monocytic ehrlichiosis (HME) is a zoonotic emerging tick-borne disease with clinical signs that range from mild symptoms to multiple organ failure and death. Ehrlichia chaffeensis, the aetiologic agent of HME, is reported to infect a divergent range of mammals. Although cattle are common hosts of the primary vector of this pathogen, the susceptibility of this host to E. chaffeensis has not been reported to date. This study was undertaken to determine if cattle could provide a useful infection model of E. chaffeensis. Dairy calves were injected with DH82 cells infected with the Arkansas, St Vincent or 91HE17 strain of E. chaffeensis, and monitored for signs of clinical ehrlichiosis and for infection of peripheral blood and ticks by PCR assay. Splenectomized and spleen-intact calves were injected with cryopreserved stabilates of E. chaffeensis-infected DH82 cells for the first experiment. Mild clinical signs were occasionally observed among these calves, and only two blood samples were PCR-positive, while several ticks fed on each calf tested PCR-positive. The second experiment involved injection of normal calves with active cultures of the same E. chaffeensis strains. Interestingly, three of six calves inoculated with active cultures became recumbent and died or had to be euthanized. All of the surviving calves in this experiment tested PCR-positive on multiple dates, but fewer ticks fed on these calves were PCR-positive. These results suggest that a bovine disease model could facilitate the understanding of factors that affect the severity of HME.
doi:10.1099/jmm.0.47427-0
PMCID: PMC3066168  PMID: 18033836
8.  Allele variability of critical virulence genes (eae, bfpA and perA) in typical and atypical EPEC in Peruvian children 
Journal of medical microbiology  2010;59(Pt 1):25-31.
Summary
Enteropathogenic Escherichia coli (EPEC) are a leading cause of infantile diarrhea in developing countries. The aim of this study was to describe the allelic diversity of critical virulence EPEC genes and their association with clinical characteristics. 120 EPEC strains isolated from a cohort diarrhea study in Peruvian children were characterized for the allele type of eae (intimin), bfpA (bundlin pilin protein of bundle-forming pilus) and perA (plasmid encoded regulator) genes by PCR-restriction fragment length polymorphism. Atypical EPEC strains (eae+, bfp-) were the most common pathotype in diarrhea (54/74, 73%) and control samples from children without diarrhea (40/46, 87%). Overall there were 13 eae alleles; the most common were beta (34/120, 28%), theta (24/120, 20%), kappa (14/120, 12%) and mu (8/120, 7%). There were 5 bfpA alleles; the most common were beta1/7 (10/26), alpha3 (7/26) and beta5 (3/26). There were 3 perA alleles: beta (8/16), alpha (7/16) and gamma (1/16). The strains belonged to 36 distinct serogroups; O55 was the most frequent. The gamma-intimin allele was more frequently found in diarrhea episodes of longer duration (>7 days) than shorter (3/26, 12% vs. 0/48, 0%, p<0.05). The kappa-intimin allele had the highest clinical severity score in comparison to other alleles (p<0.05). In Peruvian children the virulence genes of EPEC strains are highly variable. Further studies are needed to evaluate additional virulence markers to determine whether relationships exist between specific variants and clinical features of disease.
doi:10.1099/jmm.0.013706-0
PMCID: PMC2823808  PMID: 19797469
9.  Allelic variability of critical virulence genes (eae, bfpA and perA) in typical and atypical enteropathogenic Escherichia coli in Peruvian children 
Journal of Medical Microbiology  2010;59(Pt 1):25-31.
Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhoea in developing countries. The aim of this study was to describe the allelic diversity of critical EPEC virulence genes and their association with clinical characteristics. One hundred and twenty EPEC strains isolated from a cohort diarrhoea study in Peruvian children were characterized for the allele type of eae (intimin), bfpA (bundlin pilin protein of bundle-forming pilus) and perA (plasmid encoded regulator) genes by PCR-RFLP. Atypical EPEC strains (eae+, bfp−) were the most common pathotype in diarrhoea (54/74, 73 %) and control samples from children without diarrhoea (40/46, 87 %). Overall, there were 13 eae alleles; the most common were beta (34/120, 28 %), theta (24/120, 20 %), kappa (14/120, 12 %) and mu (8/120, 7 %). There were five bfpA alleles; the most common were beta1/7 (10/26), alpha3 (7/26) and beta5 (3/26). There were three perA alleles: beta (8/16), alpha (7/16) and gamma (1/16). The strains belonged to 36 distinct serogroups; O55 was the most frequent. The gamma-intimin allele was more frequently found in diarrhoea episodes of longer duration (>7 days) than those of shorter duration (3/26, 12 % vs 0/48, 0 %, P<0.05). The kappa-intimin allele had the highest clinical severity score in comparison with other alleles (P<0.05). In Peruvian children, the virulence genes of EPEC strains are highly variable. Further studies are needed to evaluate additional virulence markers to determine whether relationships exist between specific variants and clinical features of disease.
doi:10.1099/jmm.0.013706-0
PMCID: PMC2823808  PMID: 19797469
10.  Effect of Age on Susceptibility to Salmonella typhimurium Infection in C57BL/6 Mice 
Journal of medical microbiology  2009;58(Pt 12):1559-1567.
Aging is associated with a decline in immune function, which predisposes the elderly to higher incidence of infections. Information on the mechanism of age-related increase in susceptibility to Salmonella enterica serovar Typhimurium (S. Typhimurium) is limited. In particular, little is known regarding the involvement of the immune response in this age-related difference. We employed the streptomycin (STREP)-pretreated C57BL/6 mice to develop a mouse model that would demonstrate age-related difference in susceptibility and immune response to S. Typhimurium. In this model, old mice inoculated orally with 3×108 CFU or 1 ×106 CFU doses of S. Typhimurium had significantly greater S. Typhimurium colonization in ileum, colon, Peyer’s patches, spleen, and liver than those of young mice. Old mice had significantly higher weight loss than young mice on days 1 and 2 postinfection. In response to S. Typhimurium infection, the old mice failed to increase ex vivo production of IFN-γ and TNF-α in spleen and mesenteric lymph node cells to the same degree as those observed in the young mice, which was associated with their inability to maintain the presence of neutrophils and macrophages at a “youthful” level. These results indicate that STREP-pretreated C57BL/6 old mice are more susceptible to S. Typhimurium infection than young mice, which might be due to impaired IFN-γ and TNF-α production as well as the corresponding change in the number of neutrophils and macrophages in response to S. Typhimurium infection compared to young mice.
doi:10.1099/jmm.0.013250-0
PMCID: PMC2783761  PMID: 19729455
11.  Effect of age on susceptibility to Salmonella Typhimurium infection in C57BL/6 mice 
Journal of Medical Microbiology  2009;58(Pt 12):1559-1567.
Ageing is associated with a decline in immune function, which predisposes the elderly to a higher incidence of infections. Information on the mechanism of the age-related increase in susceptibility to Salmonella enterica serovar Typhimurium (S. Typhimurium) is limited. In particular, little is known regarding the involvement of the immune response in this age-related change. We employed streptomycin (Sm)-pretreated C57BL/6 mice to develop a mouse model that would demonstrate age-related differences in susceptibility and immune response to S. Typhimurium. In this model, old mice inoculated orally with doses of 3×108 or 1×106 c.f.u. S. Typhimurium had significantly greater S. Typhimurium colonization in the ileum, colon, Peyer's patches, spleen and liver than young mice. Old mice had significantly higher weight loss than young mice on days 1 and 2 post-infection. In response to S. Typhimurium infection, old mice failed to increase ex vivo production of IFN-γ and TNF-α in the spleen and mesenteric lymph node cells to the same degree as observed in young mice; this was associated with their inability to maintain the presence of neutrophils and macrophages at a ‘youthful’ level. These results indicate that Sm-pretreated C57BL/6 old mice are more susceptible to S. Typhimurium infection than young mice, which might be due to impaired IFN-γ and TNF-α production as well as a corresponding change in the number of neutrophils and macrophages in response to S. Typhimurium infection compared to young mice.
doi:10.1099/jmm.0.013250-0
PMCID: PMC2783761  PMID: 19729455
12.  Characterisation of CNF1-Producing Escherichia coli Strains from Faeces of Healthy Macaques 
Journal of medical microbiology  2009;58(Pt 10):1354-1358.
Summary
Twenty-five of 92 (27%) clinically normal macaques had beta-haemolytic E. coli isolated from their faeces. Five of the six isolates chosen for further characterisation were multiple antibiotic resistant and PCR-positive for cnf1 with a demonstrated cytopathic effect in vitro. By rep-PCR genotyping, genetic similarity was established for selected isolates. This is the first report of E. coli strains producing CNF1 in nonhuman primates.
doi:10.1099/jmm.0.012088-0
PMCID: PMC2743768  PMID: 19541782
13.  Characterization of cytotoxic necrotizing factor 1-producing Escherichia coli strains from faeces of healthy macaques 
Journal of Medical Microbiology  2009;58(Pt 10):1354-1358.
Twenty-five (27 %) of 92 clinically normal macaques were found to have β-haemolytic Escherichia coli isolated from their faeces. Five of six isolates chosen for further characterization had multiple antibiotic resistance and were PCR-positive for cytotoxic necrotizing factor 1 (cnf1) with a demonstrated cytopathic effect in vitro. By repetitive element sequence-based PCR genotyping, genetic similarity was established for selected isolates. We believe this to be the first report of E. coli strains producing CNF1 in non-human primates.
doi:10.1099/jmm.0.012088-0
PMCID: PMC2743768  PMID: 19541782
14.  Inhibitors of Cellular Signaling are Cytotoxic or Block the Budded-to-Hyphal Transition in the Pathogenic Yeast Candida albicans 
Journal of medical microbiology  2009;58(Pt 6):779-790.
Summary
Pathogenic yeast Candida albicans can grow in multiple morphological states including budded, pseudohyphal, and true hyphal forms. The ability to interconvert between budded and hyphal forms, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans virulence and is regulated by multiple environmental and cellular signals. To identify small molecule inhibitors of known cellular processes that can also block the BHT, a microplate-based morphological assay was used to screen the BIOMOL-ICCB Known Bioactives collection from the ICCB-Longwood Screening Facility. Of 480 molecules tested, 53 were cytotoxic to C. albicans and 16 were able to block the BHT without inhibiting budded growth. These 16 BHT inhibitors affect protein kinases, protein phosphatases, Ras signaling pathways, G-protein coupled receptors, calcium homeostasis, nitric oxide and guanylyl cyclase signaling, and apoptosis in mammalian cells. Several of these molecules were also able to inhibit filamentous growth in other Candida species as well as the pathogenic filamentous fungi Aspergillus fumigatus, suggesting a broad fungal host range for these inhibitory molecules. Results from secondary assays, including hyphal-specific transcription and septin localization, are consistent with the inhibitors affecting known BHT signaling pathways in C. albicans. Therefore, these molecules will not only be invaluable in deciphering the signaling pathways regulating the BHT, but also may serve as starting points for potential new antifungal therapeutics.
doi:10.1099/jmm.0.006841-0
PMCID: PMC2742683  PMID: 19429755
Candida albicans; budded-to-hyphal transition; cell signaling; small molecules
15.  Inhibitors of cellular signalling are cytotoxic or block the budded-to-hyphal transition in the pathogenic yeast Candida albicans 
Journal of Medical Microbiology  2009;58(Pt 6):779-790.
The pathogenic yeast Candida albicans can grow in multiple morphological states including budded, pseudohyphal and true hyphal forms. The ability to interconvert between budded and hyphal forms, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans virulence, and is regulated by multiple environmental and cellular signals. To identify small-molecule inhibitors of known cellular processes that can also block the BHT, a microplate-based morphological assay was used to screen the BIOMOL–Institute of Chemistry and Cell Biology (ICCB) Known Bioactives collection from the ICCB-Longwood Screening Facility (Harvard Medical School, Boston, MA, USA). Of 480 molecules tested, 53 were cytotoxic to C. albicans and 16 were able to block the BHT without inhibiting budded growth. These 16 BHT inhibitors affected protein kinases, protein phosphatases, Ras signalling pathways, G protein-coupled receptors, calcium homeostasis, nitric oxide and guanylate cyclase signalling, and apoptosis in mammalian cells. Several of these molecules were also able to inhibit filamentous growth in other Candida species, as well as the pathogenic filamentous fungus Aspergillus fumigatus, suggesting a broad fungal host range for these inhibitory molecules. Results from secondary assays, including hyphal-specific transcription and septin localization analysis, were consistent with the inhibitors affecting known BHT signalling pathways in C. albicans. Therefore, these molecules will not only be invaluable in deciphering the signalling pathways regulating the BHT, but also may serve as starting points for potential new antifungal therapeutics.
doi:10.1099/jmm.0.006841-0
PMCID: PMC2742683  PMID: 19429755
16.  The Etiology of Influenza-Like-Illness in Adults Includes Parainfluenzavirus Type 4 
Journal of medical microbiology  2009;58(Pt 4):408-413.
Influenza viruses cause significant morbidity and mortality in adults each winter. At the same time, other respiratory viruses circulate and cause respiratory illness with flu-like symptoms. Respiratory syncytial virus (RSV), the human parainfluenza viruses (HPIV), and human metapneumovirus (HMPV) have all been associated with morbidity and mortality in adults, including nosocomial infections. We evaluated 154 respiratory specimens collected from adults with influenza-like/acute respiratory illness (ILI) seen at the Edward Hines Veteran’s Affairs (VA) Hospital during two successive winters, 1998-1999 and 1999-2000. The samples were tested for 10 viruses in two nested multiplex RT-PCR reactions. One to three respiratory viruses were detected in 68% of the samples. As expected, influenza A virus (Flu-A) infections were most common (50% of the samples), followed by RSV-A (16%). Surprisingly, HPIV-4 infections (5.8%) were the third most prevalent. Mixed infections were also relatively common (11%). When present, HPIV infections were approximately three times more likely to be included in a mixed infection than Flu-A or RSV. Mixed infections and HPIV-4 are likely to be missed using rapid diagnostic tests. This study confirms that ILI in adults and the elderly can be caused by RSV and the HPIVs, including HPIV-4, which co-circulate with Flu-A.
doi:10.1099/jmm.0.006098-0
PMCID: PMC2778239  PMID: 19273634
17.  Neutrophil enhancement of Pseudomonas aeruginosa biofilm development: human F-actin and DNA as targets for therapy 
Journal of Medical Microbiology  2009;58(Pt 4):492-502.
In the cystic fibrosis (CF) airway, chronic infection by Pseudomonas aeruginosa results from biofilm formation in a neutrophil-rich environment. We tested the capacity of human neutrophils to modify early biofilm formation of P. aeruginosa strain PAO1, and an isogenic CF strain isolated early and years later in infection. In a static reactor, P. aeruginosa biofilm density of all strains was enhanced at 24 h in the presence of neutrophils, with the greatest relative increase associated with the lowest inoculum of P. aeruginosa tested. Previously, neutrophil-induced biofilm enhancement was shown to largely result from the incorporation of F-actin and DNA polymers into the bacterial biofilm. This finding was advanced by the comparison of biofilm enhancement from intact unstimulated neutrophils and from lysed or apoptotic neutrophils. Apoptotic neutrophils, with an intact cell membrane, were unable to contribute to biofilm enhancement, while lysed neutrophils evoked a similar response to that of intact cells. Using F-actin and DNA as targets, the capacity of negatively charged poly(amino acids) to disrupt, or prevent, early biofilm formation was tested. Anionic poly(aspartic acid) effectively prevented or disrupted biofilm formation. Combination of poly(aspartic acid) with DNase resulted in a synergistic increase in biofilm disruption. These results demonstrate that the presence of dying neutrophils can facilitate the initial stages of biofilm development by low inocula of P. aeruginosa. Neutrophil F-actin represents a potential new therapeutic target for disruption of pathogenic biofilms.
doi:10.1099/jmm.0.005728-0
PMCID: PMC2677169  PMID: 19273646
18.  Aetiology of influenza-like illness in adults includes parainfluenzavirus type 4 
Journal of Medical Microbiology  2009;58(Pt 4):408-413.
Influenza viruses cause significant morbidity and mortality in adults each winter. At the same time, other respiratory viruses circulate and cause respiratory illness with influenza-like symptoms. Human respiratory syncytial virus (HRSV), human parainfluenza viruses (HPIV) and human metapneumovirus have all been associated with morbidity and mortality in adults, including nosocomial infections. This study evaluated 154 respiratory specimens collected from adults with influenza-like/acute respiratory illness (ILI) seen at the Edward Hines Jr VA Hospital, Hines, IL, USA, during two successive winters, 1998–1999 and 1999–2000. The samples were tested for ten viruses in two nested multiplex RT-PCRs. One to three respiratory viruses were detected in 68 % of the samples. As expected, influenza A virus (FLU-A) infections were most common (50 % of the samples), followed by HRSV-A (16 %). Surprisingly, HPIV-4 infections (5.8 %) were the third most prevalent. Mixed infections were also relatively common (11 %). When present, HPIV infections were approximately three times more likely to be included in a mixed infection than FLU-A or HRSV. Mixed infections and HPIV-4 are likely to be missed using rapid diagnostic tests. This study confirms that ILI in adults and the elderly can be caused by HRSV and HPIVs, including HPIV-4, which co-circulate with FLU-A.
doi:10.1099/jmm.0.006098-0
PMCID: PMC2778239  PMID: 19273634
19.  Roles of the plasticity regions of Helicobacter pylori in gastroduodenal pathogenesis 
Journal of medical microbiology  2008;57(Pt 5):545-553.
Putative virulence genes of Helicobacter pylori are generally classified into three categories: strain-specific genes, phase-variable genes and genes with variable structures/genotypes. Among these, there has recently been considerable interest in strain-specific genes found outside of the cag pathogenicity island, especially genes in the plasticity regions. Nearly half of the strain-specific genes of H. pylori are located in the plasticity regions in strains 26695 and J99. Strain HPAG1, however, seems to lack a typical plasticity region; instead it has 43 HPAG1-specific genes which are either undetectable or incompletely represented in the genomes of strains 26695 and J99. Recent studies showed that certain genes or combination of genes in this region may play important roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. Most previous studies have focused on the plasticity region in strain J99 (jhp0914–jhp0961) and the jhp0947 gene and the duodenal ulcer promoting (dupA) gene are good candidate markers for gastroduodenal diseases although there are some paradoxical findings. The jhp0947 gene is reported to be associated with an increased risk of both duodenal ulcers and gastric cancers, whereas the dupA gene, which encompasses jhp0917 and jhp0918, is reported to be associated with an increased risk of duodenal ulcers and protection against gastric cancers. In addition, recent studies showed that approximately 10–30% of clinical isolates possess a 16.3 kb type IV secretion apparatus (tfs3) in the plasticity region. Studies on the plasticity region have only just begun, and further investigation is necessary to elucidate the roles of genes in this region in gastroduodenal pathogenesis.
doi:10.1099/jmm.0.2008/000570-0
PMCID: PMC2833349  PMID: 18436586
20.  Dichotomous Metabolism of Enterococcus faecalis Induced by Hematin Starvation Modulates Colonic Gene Expression 
Journal of medical microbiology  2008;57(Pt 10):1193-1204.
Summary
Enterococcus faecalis is an intestinal commensal that cannot synthesize porphyrins and only expresses a functional respiratory chain when provided exogenous hematin. In the absence of hematin, E. faecalis reverts to fermentative metabolism and produces extracellular superoxide that can damage epithelial cell DNA. The acute response of the colonic mucosa to hematin-starved E. faecalis was identified by gene array. E. faecalis was inoculated into murine colons using a surgical ligation model that preserved tissue architecture and homeostasis. The mucosa was exposed to hematin-starved E. faecalis and compared to a control consisting of the same strain grown with hematin. At 1 hour post-inoculation six mucosal genes were differentially regulated and this increased to 42 genes at 6 hours. At 6 hours a highly significant biological interaction network was identified with functions that included NF-κB signaling, apoptosis, and cell cycle regulation. Colon biopsies showed no histological abnormalities by hematoxylin and eosin staining. Immunohistochemical staining, however, detected NF-κB activation in tissue macrophages using antibodies to the nuclear localization sequence for p65 and the F4/80 marker for murine macrophages. Similarly, hematin-starved E. faecalis strongly activated NF-κB in murine macrophages in vitro. Furthermore, primary and transformed colonic epithelial cells activated the G2/M checkpoint in vitro following exposure to hematin-starved E. faecalis. Modulation of this cell cycle checkpoint was due to extracellular superoxide produced as a result of the respiratory block in hematin-starved E. faecalis. These results demonstrate that the uniquely dichotomous metabolism of E. faecalis can significantly modulate gene expression in the colonic mucosa for pathways associated with inflammation, apoptosis, and cell cycle regulation.
doi:10.1099/jmm.0.47798-0
PMCID: PMC2692828  PMID: 18809545
21.  Typing of Histoplasma capsulatum strains by fatty acid profile analysis 
Journal of medical microbiology  2007;56(Pt 6):788-797.
The performance of fatty acid profiling for strain differentiation of Histoplasma capsulatum was assessed. Total fatty acids were isolated from the yeast-phase cells of seven stock and two previously unreported clinical strains of H. capsulatum var. capsulatum, as well as from one unreported clinical strain and one stock strain of H. capsulatum var. duboisii, and one strain of each of three other dimorphic zoopathogenic fungal species, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Sporothrix schenckii. Different colony morphology and pigmentation types of the H. capsulatum strains were also included. The most frequently occurring fatty acids were oleic, palmitic, stearic and linoleic acids. There were variations in the relative percentage fatty acid contents of H. capsulatum strains that could be used for strain identification and discrimination. Differentiation between H. capsulatum strains was achieved by the comparison of detected fatty acids accompanied by principal component analysis using calculated Varimax-rotated principal component loadings. Statistical analysis yielded three major principal components that explained over 94% of total variance in the data. All the strains of H. capsulatum var. capsulatum RFLP classes II and III were grouped into two distinct clusters: the heterogenic RFLP class I formed a large, but also well-defined group, whereas the outgroup strains of H. capsulatum var. duboisii, B. dermatitidis, P. brasiliensis and S. schenckii were shifted away. These data suggest that fatty acid profiling can be used in H. capsulatum strain classification and epidemiological studies that require strain differentiation at the intraspecies level.
doi:10.1099/jmm.0.47067-0
PMCID: PMC2748824  PMID: 17510264
22.  Serum antibodies to West Nile virus in naturally exposed and vaccinated horses 
Journal of medical microbiology  2008;57(Pt 9):1087-1093.
A polyvalent enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutralization tests (PRNT) were used to measure serum antibodies to the West Nile virus (WNV) in horses naturally exposed to or vaccinated against this flavivirus in Connecticut and New York State, USA. Relying on a PRNT as a “gold standard”, the main objective was to validate a modified ELISA containing a recombinant WNV envelope protein antigen. It was also important to assess specificity by testing sera from horses that had other, undiagnosed illnesses. Sera for the latter study were obtained from 43 privately owned horses during 1995−1996. Analyses by an ELISA and PRNT confirmed the presence of WNV antibodies in 21 (91%) of 23 sera from naturally exposed horses and in 85% of the 20 vaccinated subjects; overall results for both study groups were highly concordant (91% agreement). Humoral responses of naturally exposed and immunized horses were similar. Both serologic tests were useful in confirming past infections of the WNV, but there was no evidence that horses with undiagnosed illnesses were exposed to the WNV prior to the 1999 outbreak.
doi:10.1099/jmm.0.47849-0
PMCID: PMC2562728  PMID: 18719177
23.  Evidence that the cytolethal distending toxin locus was once part of a genomic island in the periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans strain Y4 
Journal of medical microbiology  2007;56(Pt 11):1519-1527.
The authors have previously shown that the periodontal pathogen Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans Y4 contains an operon for a genotoxin known as the cytolethal distending toxin (Cdt). The cdt locus in strain Y4 is flanked by remnants of heterologous plasmid and integrase sequences. In this study, the DNA sequence immediately downstream from the cdt locus on the Y4 chromosome was examined. The extended sequence contained a region that had all the characteristics of a typical bacterial pathogenicity or genomic island. The genomic island (GIY4-1) was approximately 22 kb long, was flanked by a bacteriophage attachment (att) sequence and contained a full-length integrase/resolvase gene (xerD). A total of 22 complete and partial ORFs represented putative DNA replication/DNA binding/conjugation proteins as well as hypothetical proteins. GIY4-1 was most closely related to putative genomic islands in Haemophilus ducreyi 35000HP and Haemophilus influenzae 86-028NP and to a chromosomal region in Haemophilus somnus 129PT. GIY4-1 was not present in HK1651, which was used as the prototype strain for genomic sequencing of A. actinomycetemcomitans. Several sequences in GIY4-1 were homologous to ORFs found on the A. actinomycetemcomitans plasmid pVT745. None of the identified ORFs in GIY4-1 appeared to encode potential virulence genes. However, several unique observations supported the possibility that the cdt locus of A. actinomycetemcomitans Y4 was originally contained within the genomic island.
doi:10.1099/jmm.0.47273-0
PMCID: PMC2717017  PMID: 17965355
24.  Neutrophil enhancement of Pseudomonas aeruginosa biofilm development 
Journal of medical microbiology  2009;58(Pt 4):492-502.
In the cystic fibrosis (CF) airway, chronic infection by Pseudomonas aeruginosa results from biofilm formation in a neutrophil-rich environment. We tested the capacity of human neutrophils to modify early biofilm formation of P. aeruginosa strain PAO1, and an isogenic CF strain isolated early and years later in infection. In a static reactor, P. aeruginosa biofilm density of all strains was enhanced at 24 h in the presence of neutrophils, with the greatest relative increase associated with the lowest inoculum of P. aeruginosa tested. Previously, neutrophil-induced biofilm enhancement was shown to largely result from the incorporation of F-actin and DNA polymers into the bacterial biofilm. This finding was advanced by the comparison of biofilm enhancement from intact unstimulated neutrophils and from lysed or apoptotic neutrophils. Apoptotic neutrophils, with an intact cell membrane, were unable to contribute to biofilm enhancement, while lysed neutrophils evoked a similar response to that of intact cells. Using F-actin and DNA as targets, the capacity of negatively charged poly(amino acids) to disrupt, or prevent, early biofilm formation was tested. Anionic poly(aspartic acid) effectively prevented or disrupted biofilm formation. Combination of poly(aspartic acid) with DNase resulted in a synergistic increase in biofilm disruption. These results demonstrate that the presence of dying neutrophils can facilitate the initial stages of biofilm development by low inocula of P. aeruginosa. Neutrophil F-actin represents a potential new therapeutic target for disruption of pathogenic biofilms.
doi:10.1099/jmm.0.005728-0
PMCID: PMC2677169  PMID: 19273646
25.  Multilocus sequence typing analysis of Shigella flexneri isolates collected in Asian countries 
Journal of medical microbiology  2007;56(Pt 11):1460-1466.
The multilocus sequence typing scheme used previously for phylogenetic analysis of Escherichia coli was applied to 107 clinical isolates of Shigella flexneri. DNA sequencing of 3423 bp throughout seven housekeeping genes identified eight new allele types and ten new sequence types among the isolates. S. flexneri serotypes 1-5, X and Y were clustered together in a group containing many allelic variants while serotype 6 formed a distinct group, as previously established.
doi:10.1099/jmm.0.47322-0
PMCID: PMC2652033  PMID: 17965345

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