Journal of Applied Microbiology

Perrier‐Cornet, J. ‐M.; Hayert, M.; Gervais, P.

doi: 10.1046/j.1365-2672.1999.00779.xpmid: 10432582

The shrinkage of yeast cells caused by high‐pressure treatment (250 MPa, 15 min) was investigated using direct microscopic observation. A viable staining method after treatment allowed the volume variation of two populations to be distinguished: an irreversible volume decrease (about 35% of the initial volume) of pressure‐inactivated cells during pressure holding time, and viable cells, which were less affected. A mass transfer was then induced during high‐pressure treatment. Causes of this transfer seem to be related to a pressure‐induced membrane permeabilization, allowing a subsequent leakage of internal solutes, where three ions (Na+, K+ and Ca2+), plus endogenous glycerol, were verified. This glycerol leakage was found to occur after yeast pressurization in a medium having low water activity, although the yeast was not inactivated. All these observations lead to the hypothesis that pressure‐induced cell permeabilization could be the cause of yeast inactivation under pressure.

Loshon, C. A.; Genest, P. C.; Setlow, B.; Setlow, P.

doi: 10.1046/j.1365-2672.1999.00783.xpmid: 10432583

Killing of wild‐type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α−β−) lacking the two major α/β‐type small, acid‐soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild‐type and α−β− spores to formaldehyde treatment, which caused significant expression of a recA‐lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross‐linking in both wild‐type and α−β− spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β‐type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.

Arnaut‐Rollier, I.; Vauterin, L.; De Vos, P.; Massart, D. L.; Devriese, L. A.; De Zutter, L.; Van Hoof, J.

doi: 10.1046/j.1365-2672.1999.00785.xpmid: 10432584

Pseudomonas strains were isolated from both fresh and cold‐stored broiler skin. Phenotypically‐based numerical taxonomic techniques were used to characterize the isolates and 36 reference strains. For this purpose, Biolog GN Microplates, API 20NE and a number of other biochemical tests were used. Jaccard clustering revealed the predominance of four major Pseudomonas groups: Ps. fragi, Ps. lundensis, strains belonging to Ps. fluorescens biovars and an unidentified group of strains displaying a high degree of similarity to Ps. fluorescens biovars. Within Ps. fluorescens, biovar A was best represented. The marked proteolytic character of members of Ps. fluorescens biovars A, B and C, as well as of members of the unidentified cluster, supports their possible role in the origin of organoleptic defects. In the Ps. lundensis cluster, a distinct group of Ps. lundensis‐like species was found. Further genotypic studies should be carried out to clarify the taxonomic status of the Ps. lundensis‐like strains and that of the unidentified group resembling Ps. fluorescens biovars A and B.

Hayford, A. E.; Jakobsen, M.

doi: 10.1046/j.1365-2672.1999.00786.xpmid: 10432585

Several isolates of Candida krusei from indigenous spontaneously fermented maize dough have been characterized for the purpose of selecting appropriate starter cultures and methods for their subspecifies typing. The present work describes the occurrence of C. krusei in Ghanaian fermented maize dough. For detailed pheno‐ and genotyping, 48 representative isolates were selected and comparison was made with clinical isolates of C. krusei and reference cultures. The techniques applied included the assimilation of carbon compounds by the API ID 32 C kit, determination of chromosome profile by pulse field gel electrophoresis, polymerase chain reaction (PCR) profiles, restriction endonuclease analysis (REA) and Southern blot hybridization. For the 48 isolates tested, 82% had the same assimilation profiles, being able to assimilate N‐acetyl‐glucosamine, dl‐lactate, glycerol and to ferment glucose. Chromosome and PCR profiles, REA and Southern blot hybridization techniques all had a high discriminatory power and revealed DNA polymorphism, which allowed for discrimination among the strains and hence subspecific typing. On the basis of PCR and REA profiles, isolates were grouped into clusters. Southern blot hybridization appeared to be the most sensitive with respect to strain specificity. Our results demonstrated that the three methods, PCR, REA and Southern blot hybridization, were suitable tools, easy to analyse, fast (with regard to PCR) and reliable methods for the typing of C. krusei isolates to species and below species level. Based on the use of these techniques, we demonstrated that several strains of C. krusei were involved in the fermentation of maize dough from the onset and remain dominant throughout the fermentation.

Gibson, H.; Taylor, J. H.; Hall, K. E.; Holah, J. T.

doi: 10.1046/j.1365-2672.1999.00790.xpmid: 10432586

The effectiveness of cleaning was investigated through food factory trials and laboratory experiments using a naturally occurring biofilm from a food factory environment and generated biofilms. The efficacy of factory cleaning and disinfection programmes was assessed by swabbing and total viable count (TVC) analysis of surfaces before cleaning, after cleaning and after disinfection. Cleaning produced a 0·91 log reduction in the attached population. Investigation of the effectiveness of a variety of cleaning methods in the removal of a naturally occurring food factory biofilm showed that the high pressure spray and the mechanical floor scrubber, which use a high degree of mechanical action, were most effective. Cleaning trials with biofilms of Pseudomonas aeruginosa or Staphylococcus aureus showed that spraying with water at pressures of 34·5, 51·7 and 68·9 bar did not significantly increase the removal, as assessed by direct epifluorescent microscopy (DEM) and swabbing and TVC analysis, beyond the three log reduction observed at 17·2 bar. The effect of spray time at 17·2 bar showed that increasing spray time from 1 to 10 s did not significantly increase removal of Ps. aeruginosa biofilm. Investigation of the optimum distance of the spray lance from the surface at 17·2 bar was found to be between 125 and 250 mm. The use of an alkaline, acidic or neutral detergent prior to spraying with water at 17·2 bar did not significantly increase the removal of Ps. aeruginosa or Staph. aureus. However, the acidic and alkaline products significantly (P= 0·05) affected the viability of Staph. aureus and Ps. aeruginosa, respectively, thereby minimizing the potential for the spread of contamination.

Veyrat, A.; Miralles, M. C.; Pérez‐Martínez, G.

doi: 10.1046/j.1365-2672.1999.00795.xpmid: 10432587

A random amplified polymorphic DNA (RAPD) assay coupled to a fast and reproducible cell lysis method from Lactobacillus colonies were developed to type lactobacilli of different strains and species, with the aim of precisely enumerating each of the different Lactobacillus strains inoculated in a nutrient‐rich environment, such as sausage meat batter. Colonization assays were carried out in an aseptic meat fermentation system for up to 14 d and the inoculated strains were challenged with mixtures of wild lactobacilli. The proportion of inoculated strains remaining at different times was compared with the total number of lactobacilli grown on MRS agar by RAPD. The colonization rate of the different strains tested was very different. The RAPD‐fast lysis method developed is simple and, with a low cost per assay, could also be applied to other food fermentations.

Fiksdal, L.; Tryland, I.

doi: 10.1046/j.1365-2672.1999.00796.xpmid: 10432588

The effect of u.v. light irradiation and two other types of stress (heat and starvation) on cellular functions of Escherichia coli have been studied. The severe reduction of the culturable cell number (cfu) and the direct viable count (DVC) after exposure to moderate u.v. light doses (48 mWs cm–2), was not reflected by the dehydrogenase activity (5‐cyano‐2,3‐ditolyl tetrazolium chloride (CTC)‐positive cells), the membrane integrity (SYTOX Green‐negative cells), the membrane potential (bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) (OXONOL)‐negative cells), and the β‐ d‐galactosidase activity. All parameters were affected by high u.v. light doses. Cellular activities (CTC, SYTOX, OXONOL, β‐ d‐galactosidase activity) were intact in non‐culturable cells with presumably severe damage to DNA, and the activities seemed not to be appropriate for detection of viable E. coli after u.v. light irradiation. Heating for 20–30 min at 63 °C was required to cause a severe loss of the β‐ d‐galactosidase activity and the numbers of CTC‐ positive, SYTOX Green‐negative or OXONOL‐negative cells. A large portion (≥38%) of pre‐ irradiated (190 mWs cm–2) cells maintained their ability to reduce CTC and exclude SYTOX Green and OXONOL after 51 d of starvation (dark, 7 °C) in phosphate‐buffered saline.

Ko, S. H.; Lebeault, J. M.

doi: 10.1046/j.1365-2672.1999.00797.xpmid: 10432589

Two bacterial strains, Pseudomonas aeruginosa K1 and Rhodococcus equi P 1, were used to degrade cyclo‐alkanes (such as decalin) by a co‐oxidation mechanism. Both strains possessed the capacity to degrade a broad range of n‐alkane mixtures (C7 to C28) within 24 h of incubation. Strain P1 rapidly degraded 10 g l−1 pristane within 24 h of incubation (μ= 0·36 h−1 and Yx/s = 0·6). The addition of hexadecane as a growth substrate (above 0·5%, v/v) resulted in complete degradation of 1% (v/v) decalin by strain P1 via a co‐oxidation mechanism. Co‐oxidation to degrade decalin or pristane by strain K1 proved unsuccessful. Strain P1 was able to degrade decalin totally in a saturated hydrocarbon mixture. Strain K1 was only able to degrade hexadecane from the hydrocarbon mixture, but its degradation rate was higher than that of strain P1. Therefore, there was competition for the hexadecane needed to co‐oxidize decalin. As a result, degradation of the hydrocarbon mixture, especially decalin, was incomplete in a mixed culture of strain P1 and K1. Serial addition of hexadecane (twice) allowed complete degradation of the remaining decalin by strain P1. Also, the biodegradation rate of the hydrocarbon mixture by a microbial population from gasoline‐contaminated soil was delayed by addition of strain K1 to the population, while the addition of strain P1 resulted in an increase in the biodegradation rate.

Nielsen, T. H.; Christophersen, C.; Anthoni, U.; Sørensen, J.

doi: 10.1046/j.1365-2672.1999.00798.xpmid: 10432590

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi‐preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H‐, 13C‐NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.

Edghill, L. A.; Russell, A. D.; Day, M. J.; Furr, J. R.

doi: 10.1046/j.1365-2672.1999.00801.xpmid: 10432591

Pseudomonas putida (UWC1), containing a genetically‐engineered plasmid (pQM899), that encodes for the production of catechol 2,3‐dioxygenase (C230), was used as a potential means of rapidly estimating bactericidal activity of chlorhexidine diacetate (CHA), phenol, cetylpyridinium chloride (CPC) and phenylmercuric nitrate (PMN). Enzyme C230 converts catechol to 2‐hydroxymuconic semialdehyde (2‐HMS), which is yellow in colour, via a meta cleavage pathway. Ideal conditions for production and measurement spectrophotometrically of 2‐HMS were determined. However, the correlation between this method and viable plate counts was not sufficiently accurate to enable 2‐HMS production to provide a sufficiently sensitive determination of biocidal activity. An alternative method, synchronous scanning fluorimetry, in which the decrease in catechol concentration was measured under standardized conditions, provided a good dose–response histogram for all the biocides tested. Although, in comparison with plate counts, there was an underestimation of the bactericidal effects of phenol and PMN, the results of this study suggest that this method has potential in determining the bactericidal efficacy of agents such as CHA and CPC