Journal of Applied Microbiology

Cutler, S.J.

doi: 10.1111/j.1365-2672.2009.04598.xpmid: 19886891

Summary Borrelial relapsing fever was once a major worldwide epidemic disease that made a significant impact on Livingstone during his epic travels through Africa and throughout Europe. Indeed, the term ‘relapsing fever’ was first used to describe clinical cases of this disease in Edinburgh. During the last century, we have witnessed the demise of the louse‐borne infection, largely through improving standards of living resulting in a reduction in body lice, the vector for Borrelia recurrentis (louse‐borne relapsing fever (LBRF)). The tick‐borne zoonotic form of the disease persists in endemic foci around the world (tick‐borne relapsing fever (TBRF)). Indeed, TBRF is reportedly the most common bacterial infection from Senegal and listed within the top ten causes of mortality in children under five in Tanzania. In Ethiopia, LBRF is again within the top ten causes of hospital admission, associated with significant morbidity and mortality. Despite these figures, many now regard relapsing fever as an unusual tropical disease. Certainly, recent cases have been imported following travel from endemic zones. More surprisingly, cases have been reported following family reunions in Colorado, USA. A further case was reported from the Mt Wilson observatory in Los Angeles, USA. In many regions, the infection is zoonotic with natural reservoirs in several vertebrate species. In West Africa, infection is again primarily zoonotic. Whether those species found predominantly in East Africa are zoonoses or are infections of humans alone is still debated, however, the life cycle may be determined by the feeding preferences of their arthropod vectors.

Van Derlinden, E.; Lule, I.; Bernaerts, K.; Van Impe, J.F.

doi: 10.1111/j.1365-2672.2009.04512.xpmid: 19735318

Aims: Non‐sigmoid growth curves of Escherichia coli obtained at constant temperatures near the maximum growth temperature (Tmax) were previously explained by the coexistence of two subpopulations, i.e. a stress‐sensitive and a stress‐resistant subpopulation. Mathematical simulations with a heterogeneous model support this hypothesis for static experiments at 45°C. In this article, the behaviour of E. coli, when subjected to a linearly increasing temperature crossing Tmax, is studied. Methods and Results: Subpopulation dynamics are studied by culturing E. coli K12 MG1655 in brain heart infusion broth in a bioreactor. The slowly increasing temperature (°C h−1) starting from 42°C results in growth up to 60°C, a temperature significantly higher than the known Tmax. Given some additional presumptions, mathematical simulations with the heterogeneous model can describe the dynamic experiments rather well. Conclusions: This study further confirms the existence of a stress‐resistant subpopulation and reveals the unexpected growth of E. coli at temperatures significantly higher than Tmax. Significance and Impact of the Study: The growth of the small stress‐resistant subpopulation at unexpectedly high temperatures asks for a revision of currently applied models in food safety and food quality strategies.

Golan, A.; Kerem, Z.; Tun, O.M.; Luzzatto, T.; Lipsky, A.; Yedidia, I.

doi: 10.1111/j.1365-2672.2009.04517.xpmid: 19732215

Aims: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. Methods and Results: Several calibration steps were required to distinctly gate the GFP‐labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. Conclusions: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. Significance and Impact of the Study: The combination of time‐ and cost‐saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.

Gu, H.; Qi, X.; Li, X.; Jiang, H.; Wang, Y.; Liu, F.; Lu, S.; Yang, Y.; Liu, F.

doi: 10.1111/j.1365-2672.2009.04520.xpmid: 19732212

Aim: The main objective of our study is to develop a reverse transcriptase loop‐mediated isothermal amplification (RT‐LAMP)‐based system for rapid and specific detection of H3 swine influenza virus (SIV). Methods and Results: The system, H3 RT‐LAMP, contained a set of six novel primers that targeted eight distinct regions of the viral haemagglutinin (HA) gene that are highly conserved among H3 influenza A viruses but not between H3 and other subtypes. H3 RT‐LAMP accurately and specifically detected H3 SIV of different isolates from culture and from swine lung samples. The system is at least 10‐fold more sensitive than the conventional RT‐PCR assay and even comparable to the real‐time RT‐PCR method, with the detection limit of about one plaque‐forming unit per reaction. Of 27 swine lung samples tested, 11 samples were positive in reactions with the RT‐LAMP and real‐time RT‐PCR methods, while only 7 were positive with the conventional RT‐PCR assay. Importantly, the assay can be completed within 45 min and is faster than the conventional RT‐PCR and real‐time RT‐PCR approaches. Conclusions: Our results provide the first direct evidence that RT‐LAMP is highly specific and sensitive for detecting H3 SIV. Significance and Impact of the Study: These results suggest that LAMP offers a promising alternative tool for rapid, inexpensive and specific diagnosis of influenza virus infection of swine and other animals in frontline settings.

Nijhuis, E.H.; Pastoor, R.; Postma, J.

doi: 10.1111/j.1365-2672.2009.04519.xpmid: 19732213

Aims: To develop a strain‐specific TaqMan® PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. Methods and Results: A primer–probe combination was designed on the basis of a strain‐specific sequence selected using REP‐PCR (repetitive extragenic palindromic‐polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer–probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10‐fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. Conclusions: The primer–probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan® PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. Significance and Impact of the Study: The developed TaqMan® PCR method is a strain‐specific real‐time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.

Egert, M.; Schmidt, I.; Bussey, K.; Breves, R.

doi: 10.1111/j.1365-2672.2009.04510.xpmid: 19735319

Aim: To determine the microbial composition of biofilms in domestic toilets by molecular means. Methods and Results: Genomic DNA was extracted from six biofilm samples originating from households around Düsseldorf, Germany. While no archaeal 16S rRNA or fungal ITS genes were detected by PCR, fingerprinting of bacterial 16S rRNA genes revealed a diverse community in all samples. These communities also differed considerably between the six biofilms. Using the Ribosomal Database Project (RDP) classifier tool, 275 cloned 16S rRNA gene sequences were assigned to 11 bacterial phyla and 104 bacterial genera. Only 15 genera (representing 121 sequences affiliated with Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes and Proteobacteria) occurred in at least half of the samples or contributed at least 10% of the sequences in a single biofilm. These sequences were defined as ‘typical’ for toilet biofilms, and they were examined in more detail. On a 97% sequence similarity level, these sequences represented 56 species. Twelve of these were closely related to well‐described bacterial species, and only two of them were categorized as belonging to risk group 2. No 16S rRNA genes of typical faecal bacteria were detected in any sample. Virtually all ‘typical’ clones were found to be closely related to bacteria or to sequences obtained from environmental sources, implicating that the flushing water is the main source of recruitment. Conclusion: In view of the great diversity of mostly yet‐uncultured bacteria and the considerable differences between individual toilets, very general strategies appear to be most suited for the removal and prevention of toilet biofilms. Significance and Impact of the Study: For the first time, a molecular fingerprinting and cloning approach was used to monitor the species composition in biofilm samples taken from domestic toilets. Knowledge about the microbial composition of biofilms in domestic toilets is a prerequisite for developing and evaluating strategies for their removal and prevention.

Sillankorva, S.; Pleteneva, E.; Shaburova, O.; Santos, S.; Carvalho, C.; Azeredo, J.; Krylov, V.

doi: 10.1111/j.1365-2672.2009.04549.xpmid: 19796092

Aims: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. Methods and Results: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. Conclusions: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage‐related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. Significance and Impact of the Study: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.

Rubini, M.R.; Dillon, A.J.P.; Kyaw, C.M.; Faria, F.P.; Poças‐Fonseca, M.J.; Silva‐Pereira, I.

doi: 10.1111/j.1365-2672.2009.04528.xpmid: 19793137

Aims: Penicillium echinulatum is effective for bioconversion processes. However, nothing is known about the molecular biology of its cellulolytic system. We describe for the first time the isolation, cloning and expression of a P. echinulatum cellulase cDNA (Pe‐egl1) encoding a putative endoglucanase. Methods and Results: Pe‐egl1 cDNA was identified from random sequencing of a P. echinulatum cDNA library. The deduced EGL1 protein possibly belongs to the glycosyl hydrolase family 5A, with 387 amino acid residues and strong similarity with other fungal endoglucanases. The cDNA was heterologously expressed in Pichia pastoris. The recombinant EGL1 secreted by a Pic. pastoris recombinant strain revealed the characteristics of particular interest: an optimal activity over a broad pH range (5·0–9·0), and an optimal temperature of 60°C. The recombinant EGL1 also showed high thermostability (84% of residual activity after 1 h of pre‐incubation at 70°C). Calcium exerted a strong stimulatory effect over EGL1 activity. Conclusions: Altogether, these results point to the potential application of this P. echinulatum endoglucanase in cellulose processing industries, particularly the textile one because of its biochemical properties. Significance and Impact of the Study: The characterization and heterologous expression of the first P. echinulatun cDNA inaugurates the exploitation of this potential industrial micro‐organism.

DeNittis, M.; Querol, A.; Zanoni, B.; Minati, J.L.; Ambrosoli, R.

doi: 10.1111/j.1365-2672.2009.04547.xpmid: 19796093

Aims: A research was undertaken to explore the possibility to use Biolog system of microbial metabolic characterization for the monitoring of yeast population evolution during alcoholic fermentation for wine production. Methods and Results: An application of Biolog system was employed for the characterization of yeasts of oenological interest, in pure cultures and mixed consortia, in various cell concentrations. The system’s capacity to discriminate among different cell concentrations of the same yeast strain was ascertained, along with the capacity to discriminate between mixed and pure populations. Conclusions: The tested application of Biolog system resulted suitable for a quick recognition (24 h) of the presence of starter cultures within mixed populations of autochthonous yeasts. Such discrimination was confirmed with the one resulting from molecular techniques. Significance and Impact of the Study: The study suggests the possibility to employ Biolog system for an early monitoring of yeast evolution in modern wine‐making fermentations, where specialized yeasts are more and more frequently used as starters and their ability to overcome autochthonous yeast populations is crucial.

Qiao, N.; Shao, Z.

doi: 10.1111/j.1365-2672.2009.04513.xpmid: 19732216

Aims: Our goal was to identify a novel biosurfactant produced by a marine oil‐degrading bacterium. Methods and Results: Biosurfactants were produced by Alcanivorax dieselolei strain B‐5T growing with diesel oil as the sole carbon and energy source. Culture supernatant was first extracted with chloroform/methanol (1 : 1, v/v), then further purified step by step with a normal phase silica gel column, a Sephadex LH20 gel column and a preparative thin layer plate. The main component was determined to be a lipopeptide; it was chemically characterized with nuclear magnetic resonance, liquid chromatography‐quadrupole ion‐trap mass spectrometry, amino acid analysis and GC–MS and was found to be a mixture of proline lipids. The monomers of the proline lipids were composed of a proline residue and a fatty acid (C14:0, C16:0 or C18:0). The critical micelle concentration of the mixed proline lipids was determined to be 40 mg l−1. Moreover, activity variations in ranges of pH, temperature and salinity were also detected and showed reasonable stability. Conclusions: Alcanivorax dieselolei B‐5 produced a novel linear lipoamino biosurfactant, characterized as a proline lipid. Significance and Impact of the Study: A proline lipid was characterized for the first time as a bacterial biosurfactant. This product has potential in both environmental and industrial applications.