Journal of Medical Microbiology - Volume 54, Issue 11, 2005
Volume 54, Issue 11, 2005
Pathogenicity And Virulence
Prostaglandin production during growth of Candida albicans biofilms
Mohammed AS Alem and L Julia Douglas
https://doi.org/10.1099/jmm.0.46172-0
Both biofilms and planktonic (suspended) cells of Candida albicans synthesized extracellular prostaglandin(s) during growth at 37 °C, but biofilm cells secreted significantly more prostaglandin(s) when production was determined on the basis of cell dry weight. Prostaglandin synthesis by both cell types was sensitive to the cyclooxygenase inhibitors aspirin, diclofenac and etodolac. A morphological mutant blocked in two signalling pathways (cph1/cph1 efg1/efg1) produced prostaglandin levels similar to those of the parent strain, but formed yeast-only biofilms. These results suggest that prostaglandin production could be a significant virulence factor in biofilm-associated infections, although its role in C. albicans morphogenesis remains unclear.
Rhodococcus equi can survive a phagolysosomal environment in macrophages by suppressing acidification of the phagolysosome
Kiminori Toyooka, Shinji Takai and Teruo Kirikae
Rhodococcus equi is one of the most important causes of pneumonia in foals and has emerged as a significant opportunistic pathogen of immunosuppressed hosts such as human immunodeficiency virus-infected patients. Virulent R. equi harbouring an 85 kb plasmid, but not the avirulent form lacking the plasmid, has the ability to survive in macrophages. However, the survival mechanism is not known. In the present study, morphological interactions were observed between virulent or plasmid-cured avirulent R. equi and phagolysosomes in murine macrophage-like J774.1 cells by immunocytological methods. The J774.1 cells phagocytosed virulent and avirulent bacteria to a similar extent, and both bacteria replicated in single membrane vacuoles at similar rates up to 6 h after infection. Thereafter, the virulent bacteria continued to grow, whereas the avirulent bacteria stopped growing. When the infected cells were stained with phagosomal and lysosomal markers and observed with a confocal fluorescence microscope, the majority of phagosomes containing these bacteria were fused with lysosomes. Neither R. equi organism has the ability to hinder phagosome-lysosome fusion. The acidity in phagolysosomes containing R. equi was examined by staining with LysoTracker Red DND-99, an acidotropic probe. The phagolysosomes containing virulent organisms were not acidic as compared with avirulent organisms. Over 90 % of the phagolysosomes containing avirulent R. equi were stained with LysoTracker 6 h after infection, whereas less than 50 % of those containing virulent R. equi were stained. Furthermore, when the supernatant obtained from a virulent R. equi culture was added to the cell cultures, the acidity of acidic compartments in macrophages was reduced. The authors conclude that some substance(s) produced by virulent R. equi suppress acidification in phagolysosomes, and help R. equi survival and replication in the bactericidal environment.
Host Response
Invasive fungal infections are associated with severe depletion of circulating RANTES
Michael Ellis, Basel al-Ramadi, Ulla Hedström, Hussain Alizadeh, Victor Shammas and Jörgen Kristensen
Serum RANTES (regulated on activation, normal T-cell expressed and secreted) concentrations were measured in 14 patients who had haematological malignancies and developed invasive fungal infections (three of them definite, eight probable and three possible). RANTES levels fell substantially from pre-chemotherapy values at the start of and throughout the fungal infection, and recovered in patients who survived the fungal infection. However, in patients who died from the invasive fungal infection, RANTES levels did not recover. For survivors the mean ± sd levels for RANTES were 7656 ± 877 pg ml−1 on the day prior to chemotherapy, 3723 ± 2443 pg ml−1 on the first day of fungal infection diagnosis (significantly different from baseline; P = 0.001) and 9078 ± 2256 pg ml−1 at recovery from the fungal infection (significantly different from lowest value; P < 0.0001). Platelet counts were closely correlated with the RANTES levels (r = 0.63, P < 0.001). The RANTES concentrations for the three patients who died were similar to those who survived at all equivalent timepoints, but were significantly lower at the time of death (792 ± 877) compared to the values at recovery for survivors (P = 0.005). The finding that patients who died from an invasive fungal infection had very low platelet counts and RANTES concentrations suggests that these could play a role in host response to such infections.
Diagnostics, Typing And Identification
A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis
Luyi Zhao and David B Haslam
Inhibition of protein synthesis is a common mechanism by which bacterial and plant toxins injure human cells. Examples of toxins that inhibit protein synthesis include shiga toxins of Escherichia coli, diphtheria toxin, Pseudomonas exotoxin A and the plant toxin ricin. In order to facilitate studies on toxin pathogenesis and to enable screening for inhibitors of toxin action, a quantitative and highly sensitive assay for the action of these toxins on mammalian cells was developed. The cDNA encoding destabilized luciferase was cloned into an adenoviral expression plasmid and a high-titre viral stock was prepared. Following transduction of Vero cells, luciferase expression was found to be linear with respect to viral multiplicity of infection. Luciferase expression by as few as 10 cells was readily detected. Treatment of transduced cells with either cycloheximide or shiga toxin resulted in a decrease in luciferase activity, with a half-life ranging from 1 to 2 h. Inhibition of luciferase expression was evident at toxin concentrations as low as 1 pg ml−1. The assay was adapted for use in 24-, 96- and 384-well plates, enabling rapid processing of large numbers of samples. Using this approach, susceptibility of Vero, Hep2, Chang, A549, COS-1 and HeLa cells to three different toxins was determined. These results demonstrate that the luciferase-based assay is applicable to the study of numerous cell types, is quantitative, highly sensitive and reproducible. These features will facilitate studies on pathophysiology of toxin-mediated diseases and allow high-throughput screening for inhibitors of cytotoxicity.
Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis
Niclas Grahn, Mounira Hmani-Aifa, Karin Fransén, Peter Söderkvist and Hans-Jürg Monstein
Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27 %). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes' classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens.
Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae
Ryoichi Saito, Yoshiki Misawa, Kyoji Moriya, Kazuhiko Koike, Kimiko Ubukata and Noboru Okamura
A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 102 copies, corresponding to 2–20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.
Multiplex PCR for identification of Campylobacter coli and Campylobacter jejuni from pure cultures and directly on stool samples
Søren Persson and Katharina EP Olsen
A multiplex-PCR method, specifically designed for application in routine diagnostic laboratories, was developed for the detection of Campylobacter coli and Campylobacter jejuni. Primers were directed towards the following loci: the hippuricase gene (hipO) characteristic of C. jejuni, a sequence partly covering an aspartokinase gene characteristic of C. coli, and a universal 16S rDNA gene sequence serving as an internal positive control for the PCR. The method was tested on 47 C. coli strains and 88 C. jejuni strains, and found to be almost 100 % in concordance with biochemical analyses (all except for one C. coli strain), regardless of whether the DNA was prepared from colonies by a simple boiling procedure or by DNeasy Tissue Kit. Pure cultures of C. coli and C. jejuni were identified at 10–100 cells per PCR. When the multiplex-PCR method was used on spiked human stool samples, both strains were identified at 105 cells per ml stool. This sensitivity limit was the same whether the DNA was purified by the method of KingFisher mL or QIAamp DNA Stool Kit. When the same spiked stools were grown on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates before PCR, the sensitivity was 100 cells per ml stool, indicating that culturing of campylobacters on mCCDA plates is superior to direct DNA extraction at least when fresh stool samples are analysed by PCR.
Potential antimicrobial effects of human lactoferrin against oral infection with Listeria monocytogenes in mice
Hui-Young Lee, Jong-Hwan Park, Seung-Hyeok Seok, Min-Won Baek, Dong-Jae Kim, Byoung-Hee Lee, Pil-Don Kang, Yong-Soon Kim and Jae-Hak Park
Listeria monocytogenes is a food-borne pathogen that causes serious listeriosis in humans. Antimicrobial effects of human lactoferrin (hLF) against L. monocytogenes have been clearly demonstrated in in vitro studies. However, in vivo studies have not been reported yet. This study investigated whether the oral administration of hLF could inhibit oral infection of listeria in BALB/c mice. The MICs for several strains of L. monocytogenes were determined, and the most sensitive strain was used for the animal work. hLF was administered to BALB/c mice for 7 days, commencing 4 days before oral infection. The effect of hLF was determined by bacterial enumeration and histopathological analysis of the liver and spleen, which are well-known as the major targets of oral listeria infection in mice. In bacterial enumeration, hLF decreased the number of L. monocytogenes cells in the liver. Histopathologically, the size and frequency of necrotic foci in the liver samples decreased with hLF administration. However, these changes were not observed in the spleen samples. The mRNA levels of inflammatory cytokines, such as interleukin (IL)-1β, tumour necrosis factor (TNF)-α and interferon (IFN)-γ, decreased in the liver of mice receiving hLF. This study has shown that hLF decreases the hepatic colonization of L. monocytogenes, hepatic necrosis and expression of inflammatory cytokines. It revealed that perorally given hLF could mediate antimicrobial and anti-inflammatory activities remote from the gut (i.e. in the liver) of mice challenged with L. monocytogenes.
Penicillin-binding proteins of Bacteroides fragilis and their role in the resistance to imipenem of clinical isolates
Juan Ayala, Alberto Quesada, Santiago Vadillo, Jerónimo Criado and Segundo Píriz
In this study penicillin-binding proteins (PBPs) of Bacteroides fragilis and the resistance mechanisms of this micro-organism to 11 β-lactam antibiotics were analysed. The study focused on the role of PBP2Bfr and metallo-β-lactamase in the mechanism of resistance to imipenem. The mechanism of β-lactam resistance in B. fragilis was strain dependent. The gene encoding the orthologue of Escherichia coli PBP3 gene (pbpBBfr, which encodes the protein PBP2Bfr) was sequenced in five of the eight strains studied, along with the ccrA (cfiA) gene in strain 119, and their implications for resistance were examined. Differences were found in the amino-acid sequence of PBP2Bfr in strains AK-2 and 119, and the production of β-lactamases indicated that these differences may be involved in the mechanism of resistance to imipenem. In vitro binding competition assays with membrane extracts using imipenem indicated that the PBP that bound imipenem with the highest affinity was PBP2Bfr, and that increased affinity in strain 7160 may be responsible for the moderate susceptibility of this strain to imipenem. In the same way, the importance of the chromosomal class A β-lactamase CepA in the resistance mechanism of the B. fragilis strains NCTC 9344, 7160, 2013E, AK-4, 0423 and R-212 was studied. In these strains this is the principal resistance mechanism to antimicrobial agents studied other than imipenem.
Metallo-β-lactamase IMP-1 in Providencia rettgeri from two different hospitals in Japan
Katsuaki Shiroto, Yoshikazu Ishii, Soichiro Kimura, Jimena Alba, Kiwao Watanabe, Yoshiko Matsushima and Keizo Yamaguchi
In 2002, 495 indole-positive proteae strains were isolated from patients at 60 hospitals in Japan. Nine indole-positive proteae strains had reduced susceptibility to imipenem (MIC ⩾ 8 μg ml−1) and were identified as Providencia rettgeri by BD Phoenix. Eight of the nine Prov. rettgeri isolates were confirmed as metallo-β-lactamase producers by the double-disc synergy test. All the metallo-β-lactamases were classified as IMP-1 by PCR and DNA sequence analysis. These bla IMP−1 genes were encoded in the integron structure on conjugative plasmids. These plasmids could transfer from Prov. rettgeri clinical isolates to Escherichia coli ML4903 at a frequency between 1.5 × 10−5 and 5.5 × 10−7. The eight bla IMP-positive strains were isolated from two hospitals, and showed two different PFGE patterns, two different integron structures and two different incompatibility groups, which corresponded to the two hospitals. These results strongly suggest the possibility of nosocomial infections by bla IMP−1-producing Prov. rettgeri isolates.
Clinical Microbiology And Virology
Identification and antimicrobial susceptibility of micro-organisms recovered from cutaneous lesions of human American tegumentary leishmaniasis in Minas Gerais, Brazil
Claúdia O Fontes, Maria Auxiliadora R Carvalho, Jacques R Nicoli, Junia S Hamdan, Wilson Mayrink, Odair Genaro, Luiz S Carmo and Luiz M Farias
An evaluation of the microbiota present in cutaneous ulcers from 31 patients with a clinical and parasitological diagnosis of American tegumentary leishmaniasis (ATL) was carried out by the standard filter paper disc technique, including antimicrobial susceptibility of the bacterial isolates. Microbial examination indicated that 21 patients (67.7 %) were contaminated with one to four bacteria and some of them also with yeast. A total of 142 micro-organisms were isolated. Staphylococcus aureus was the most frequently recovered bacterium (95.2 % of positive patients) and was found to produce type B (70 % of the staphylococcal isolates) and type C (50 %) enterotoxins as well as toxic shock syndrome toxin (60 %). Proteus mirabilis (33.3 % of the positive patients), Streptococcus pyogenes (19.0 %), H2S-negative Proteus species (19.0 %), Klebsiella oxytoca (14.3 %), Enterobacter species (9.5 %), Peptostreptococcus species (9.5 %), Pseudomonas species (4.8 %), Prevotella bivia (4.8 %), Escherichia coli (4.8 %), Streptococcus agalactiae (4.8 %), Bacteroides fragilis (4.8 %), Candida albicans (9.5 %) and Candida tropicalis (4.8 %) were also isolated. Surprisingly, Staph. aureus isolates were susceptible to almost all tested drugs, although some of them were resistant to penicillin (69 %) and ampicillin + sulbactam (68 %). Concerning obligate anaerobes, all the Gram-negative isolates (25 % of the total) were resistant to metronidazole. The results of the present study show that microbial secondary contaminants, particularly Staph. aureus, should be considered in the diagnosis and treatment of ATL lesions.
Importance of Providencia species as a major cause of travellers' diarrhoea
Myonsun Yoh, Junko Matsuyama, Motoki Ohnishi, Kazuhiro Takagi, Hirozane Miyagi, Kazuhiro Mori, Kwon-Sam Park, Takahiro Ono and Takeshi Honda
In this study the importance of Providencia species as a cause of travellers' diarrhoea was examined using a selective medium developed by the authors. Providencia species could easily be distinguished from other enteric pathogens by the colour of the colonies obtained. Nine strains of Providencia alcalifaciens, nine of Providencia rettgeri and five of Providencia stuartii were isolated from 130 specimens, representing a surprisingly high incidence of infection compared with other pathogens isolated on SS agar and TCBS agar. Patients infected with P. rettgeri complained of abdominal pain, as for other Providencia species, but also of vomiting, which is rather characteristic of P. rettgeri infection. To analyse the pathogenicity of these isolates, their invasiveness was examined using Caco-2 cells. Most of the P. rettgeri strains invaded Caco-2 cells. Random amplified polymorphic DNA (RAPD) fingerprinting showed the same profile for two P. rettgeri isolates from individuals travelling in the same tour group. The results show that Providencia species, especially P. rettgeri, might cause diarrhoea, and that these species are important pathogens.
Veterinary Microbiology
Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis
Chris Dupont, Keith Thompson, Cord Heuer, Brigitte Gicquel and Alan Murray
An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75 % identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.
Human And Animal Microbial Ecology
Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism
Hidenori Hayashi, Rei Takahashi, Takahiro Nishi, Mitsuo Sakamoto and Yoshimi Benno
Microbiota in gut contents of jejunum, ileum, caecum and recto-sigmoid colon obtained from three elderly individuals at autopsy were compared using 16S rRNA gene libraries and terminal restriction fragment length polymorphism (T-RFLP). Random clones of 16S rRNA gene sequences were isolated after PCR amplification with universal primer sets of total genomic DNA extracted from each sample of gut contents. An average of 90 randomly selected clones were partially sequenced (about 500 bp). T-RFLP analysis was performed using the 16S rRNA gene amplified from each sample. The lengths of the terminal restriction fragments were analysed after digestion with HhaI and MspI. The jejunal and ileal microbiota consisted of simple microbial communities of streptococci, lactobacilli, 'Gammaproteobacteria', the Enterococcus group and the Bacteroides group. Most of the species were facultative anaerobes or aerobes. The Clostridium coccoides group and the Clostridium leptum subgroup, which are the most predominant groups in human faeces, were not detected in samples from the upper gastrointestinal tract. The caecal microbiota was more complex than the jejunal and ileal microbiota. The C. coccoides group, the C. leptum subgroup and the Bacteroides group were detected in the caecum. The recto-sigmoidal colonic microbiota consisted of complex microbial communities, with numerous species that belonged to the C. coccoides group, the C. leptum subgroup, the Bacteroides group, 'Gammaproteobacteria', the Bifidobacterium group, streptococci and lactobacilli, and included more than 26 operational taxonomic units. The results showed marked individual differences in the composition of microbiota in each region.
Three cases of vertebral osteomyelitis caused by Streptococcus dysgalactiae subsp. equisimilis
Aravind Kumar, Jonathan Sandoe and Naresh Kumar
Three cases of vertebral osteomyelitis caused by Streptococcus dysgalactiae subsp. equisimilis (Strep. equisimilis) are presented here. All three cases presented with fever, back pain, general malaise and weight loss for at least 4 weeks. Diagnosis was established by culture of a spinal biopsy and/or positive blood cultures together with radiological findings. In all three cases, 6–12 weeks of antibiotics were curative without recourse to surgery. The ability of Strep. equisimilis to cause vertebral osteomyelitis is highlighted. The need is emphasized for biopsy and microbiological investigation in patients presenting with back pain, fever, weight loss and evidence of a spinal lesion on imaging, even if neoplastic disease is suspected. Prolonged antibiotic therapy (at least 6 weeks) seems to be indicated.
Disseminated Nocardia farcinica infection in a uraemia patient with idiopathic thrombocytopenia purpura receiving steroid therapy
Chih-Cheng Lai, Li-Na Lee, Lee-Jene Teng, Ming Shiou Wu, Jui-Chang Tsai and Po-Ren Hsueh
Nocardia farcinica has been reported as an increasingly frequent cause of localized and disseminated infections in immunocompromised patients in recent years, but N. farcinica bacteraemia remains a rare finding. Here, the case is described of a 68-year-old man with end-stage renal disease and idiopathic thrombocytopenia purpura treated with steroid therapy who developed disseminated infection (bacteraemia, multilobar pneumonia and brain abscesses) due to N. farcinica. The isolate was confirmed by partial sequencing analysis of the 16S rRNA gene. The patient recovered after prolonged trimethoprim-sulfamethoxazole therapy with no recurrence in over 1 year.
Investigation of the putative virulence gene magA in a worldwide collection of 495 Klebsiella isolates: magA is restricted to the gene cluster of Klebsiella pneumoniae capsule serotype K1
Carsten Struve, Martin Bojer, Eva Møller Nielsen, Dennis Schrøder Hansen and Karen A Krogfelt
Invalid result of Plasmodium falciparum malaria detection with the BinaxNOW Malaria rapid diagnostic test
François Durand, Odile Faure, Jean-Paul Brion and Hervé Pelloux
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