Natural Bacterial Populations Research Articles

Genetic characterization of some lactic acid bacteria occurring in an artisanal protected denomination origin (PDO) Italian cheese, the Toma piemontese

We studied the natural bacterial population used in the production of artisanal protected denomination origin (PDO) Toma piemontese cheese in order to obtain preliminary information on the lactic acid bacteria involved in the traditional fermentation of this cheese. A total of 116 coccal isolates and 11 mesophilic lactobacilli were collected from different curd and cheese samples, and were identified by the combined use of PCR 16S–23S rDNA spacer analyses, species-specific probes and 16S rDNA sequencing. The results obtained were supported by DNA/DNA hybridization studies and by reference cultures used as markers. Lactococci constituted 67% of the coccal isolates. They were identified as Lactococcus lactis and L. garvieae, with an incidence of this latter species higher than expected. The majority of these lactococcal isolates displayed an atypical phenotype of growing in the presence of 6.5% NaCl and at 10°C. Enterococci were also isolated, principally from the cheese samples (16% of the coccal isolates), together with strains of Streptococcus macedonicus and S. thermophilus, the latter in a lower amount. Lactobacilli (principally Lactobacillus paracasei strains) were only detected in three samples and their incidence was very low ( 1, 3 and 7 cfu g –1, respectively). The presence of undesirable micro-organisms in some samples of curd emphasizes the necessity of improving the general hygienic conditions of cheese production. The results suggest the possibility of preserving the wild bacterial population in order to protect the typical organoleptic characteristics of this traditional raw milk cheese and to select new strains for the dairy industry.

Soil nitrifying enrichments as biofilter starters in intensive recirculating saline water aquaculture

Intensive recirculating aquaculture relies on biofilters to sustain satisfactory water quality in the ponds. Establishment of new biofilters in aquaculture ponds without a start-up culture requires a long period of time and may therefore cause significant losses and environmental harm due to discharge of nitrogen-rich effluents. A laboratory scale setup (7-l aquaria with shrimp and fish) demonstrated that an external start-up nitrifying enrichment culture performed similarly to the natural bacterial population of an established pond biofilter, and superior to the performance of similar biofilters without a start-up culture (control). Ammonia concentration in the control treatment increased daily and reached 18 mg l −1 during a 14-day experiment, whereas in the treated aquaria, it averaged less than 2 mg l −1 . Fish growth and survival were similar in the treated aquaria (average growth of 0.45 g/14 days, and 95% survival) and significantly higher than in the control (average growth of 0.0 g/14 days, and 80% survival). The source for the enrichment cultures was soil samples collected from the region where the farm is situated. This approach may lead to the development of bacterial amendments (probiotic products) that can be used as start-up cultures for new operations or damaged filters, and potentially enhance nitrification in established filters. As the cultures are collected from soils, it is unlikely that they will be contaminated with fish disease-causing agents. This will improve water quality and consequently aquatic animal production.

Comparing the effects of resource enrichment and grazing on viral production in a meso-eutrophic reservoir

As viral production depends on bacteria, factors which influence bacterial production should also impact viral production. Likewise, viruses and heterotrophic nanoflagellates (HNF) both exploit bacterial prey, so HNF grazing could influence interactions between viruses and bacteria. To examine these relationships, we examined samples from experiments in which natural bacterial populations were subjected to relaxation of nutrient limitation and different levels of grazing pressure from HNF. We observed that stimulation of bacterial production and abundance with the relaxation of nutrient limitation resulted in a higher standing stock of viruses, higher viral production and also a higher virus-induced lysis rate of bacterioplankton. These relationships suggest that the relative effect of virus-induced mortality is higher in more productive environments. We found that viral abundance, viral production and virus-induced mortality of bacteria was highest in the treatments in which grazing rates on bacteria by HNF were highest, and lowest in the treatments where no eukaryotic predators were present. Thus, high grazing rates were associated with high virus production rates. The resource enrichment had a stronger effect on viral production and infection of bacteria than grazing. Averaged over time for single treatments, viruses lysed a significant portion (range, 18 to 66%) of the bacterial production per day.

Use of Negative Air Ionization for Reducing Microbial Contamination on Stainless Steel Surfaces

Microbiological concerns in food plant sanitation that relies heavily on physical and chemical methods for removing and killing bacteria could be reduced by the use of non-chemical intervention methods. This initial work on the effects of electrostatic space charge on biofilms shows promise as a viable intervention option for reducing bacterial contamination on surfaces. Natural bacterial populations from a poultry processing facility were collected, allowed to multiply and form biofilms, and assessed for susceptibility to negative air ionization. A small chamber with an electrostatic space charge system was used to treat the mixed bacterial populations that were grown on stainless steel coupons (1 × 4 cm). The object of the system was to transfer a strong negative electrostatic charge to bacteria that were attached to coupons at the base of the chamber. The system effectively decreased the survival levels of bacteria on the stainless steel, with a reduction efficiency of 99.8%. All of the swab samples taken from coupons were culture positive for bacteria, and the bacterial counts from the ionized surfaces were significantly less than for the non-ionized surfaces (P < 0.01). These results demonstrate the potential efficacy of negative air ionization against bacterial contamination on surfaces in the poultry processing environment.

Breakpoints: current practice and future perspectives

Much of the discussion over the past decades on the value and setting of breakpoints has been due to the fact that the breakpoint was used in two ways; as an indicator to predict the probability of clinical success and also to detect resistant (sub) populations. It is apparent that these two meanings have lead to a different approach to setting, interpretation and use of breakpoints based on clinical efficacy on the one hand and breakpoints based on detection of resistance on the other. Nevertheless, several of the current guidelines make no perceptible distinction between these two meanings. A case is therefore strongly made to recognize that there is a difference between clinical and microbiological breakpoints. The microbiological breakpoint may be used to detect organisms that do not belong to the natural bacterial population, but somehow have acquired resistance and might be useful in recognizing emergence of resistant subpopulations and may lead to subsequent measures to be taken. Alternatively, the clinical breakpoint is of principal value to the clinician in that it results in a classification of S (susceptible), I (intermediate susceptible) and R (resistant) and is used in clinical practice and correlate with a measure of clinical efficacy. Methods developed during the last few years to arrive at meaningful clinical breakpoints are discussed, such as CART analysis and Monte Carlo simulation. In discussing future developments, it is suggested that current reports containing S, I, and R be at least supplemented with the MICs measured and, using current techniques available such as Monte Carlo simulation, provide the probability of successful eradication of the micro-organism and successful treatment based on population pharmacokinetics and Minimal Inhibitory Concentration (MIC) distributions.

Bioaugmentation and treatment of cephalexin drug-based pharmaceutical effluent in an upflow anaerobic fluidized bed system

Degradation of Aroclor 1242 was studied in granular biofilm reactors with limited aeration. An aerobic biphenyl degrader, Rhodococcus sp. M5, was used to supplement a natural bacterial population present in a “bioaugmented” reactor, while the “non-bioaugmented” reactor only contained natural granular sludge. The bioaugmentation, however appeared to have no effect on the reactor performance. Aroclor measurements showed its disappearance in both reactors with only 16–19% of Aroclor recovered from the reactor biomass and effluent. Simultaneously, a chlorine balance indicated that dechlorination occurred at a specific rate of 1.43 mg PCB (g volatile suspended solids)−1 d−1, which was comparable to the observed rate of Aroclor disappearance. Intermediates detected in both reactors were biphenyl, benzoic acid, and mono-hydroxybiphenyls. This suggests that a near-complete mineralization of Aroclor can be achieved in a single-stage anaerobic/aerobic system due to a combination of reductive and oxidative degradation mechanisms.

DEGRADATION OF AROCLOR 1242 IN A SINGLE-STAGE COUPLED ANAEROBIC/AEROBIC BIOREACTOR

Degradation of Aroclor 1242 was studied in granular biofilm reactors with limited aeration. An aerobic biphenyl degrader, Rhodococcus sp. M5, was used to supplement a natural bacterial population present in a “bioaugmented” reactor, while the “non-bioaugmented” reactor only contained natural granular sludge. The bioaugmentation, however appeared to have no effect on the reactor performance. Aroclor measurements showed its disappearance in both reactors with only 16–19% of Aroclor recovered from the reactor biomass and effluent. Simultaneously, a chlorine balance indicated that dechlorination occurred at a specific rate of 1.43 mg PCB (g volatile suspended solids) −1 d −1, which was comparable to the observed rate of Aroclor disappearance. Intermediates detected in both reactors were biphenyl, benzoic acid, and mono-hydroxybiphenyls. This suggests that a near-complete mineralization of Aroclor can be achieved in a single-stage anaerobic/aerobic system due to a combination of reductive and oxidative degradation mechanisms.

Ectopic transposition of a group II intron in natural bacterial populations.

Self-splicing group II introns are thought to be the evolutionary progenitors of eukaryotic spliceosomal introns. The invasion of novel (ectopic) sites by group II introns is considered to be a key mechanism by which spliceosomal introns may have become widely dispersed. However, the dynamics of these events in populations are unknown. In bacteria, only two group II introns have been shown to splice and to be mobile in vivo. One of these introns, RmInt1 from Sinorhizobium meliloti, which encodes a protein with no endonuclease domain, has been shown to invade the ectopic oxi1 site independently of recombinase. In this study, we analysed ectopic transposition of the RmInt1 intron in a natural population of S. meliloti. We characterized S. meliloti isolates by polymerase chain reaction amplification of a gene, dapB, which is found only on the pRmeGR4b plasmid diagnostic of GR4-type strains. The diversity within this specific field population of bacteria was analysed by restriction fragment length polymorphism using ISRm2011-2 (homing site of RmInt1) and RmInt1 as probes. We found that ectopic transposition of RmInt1 to the oxi1 site occurred in this natural bacterial population. This ectopic transposition was also the most frequent genetic event observed. This work provides further evidence that the ectopic transposition of group II introns is an important mechanism for their spread in natural bacterial populations.

Distribution and ecophysiology of the nitrifying bacteria emphasizing cultured species

Nitrification is an important factor in the global nitrogen cycle. Therefore, an increasing number of publications deal with in situ studies of natural bacterial populations participating in this process. However, some crucial points complicate suchlike investigations. At the time being, a total of 25 species of ammonia-oxidizers and eight species of nitrite-oxidizers are cultured but the existence of many more species has been indicated by molecular in situ investigations. With that, only a part of the existing nitrifiers has been defined via isolation and subsequent physiological and molecular characterization. Furthermore, the distribution patterns of the distinct species of nitrifiers depend on various environmental parameters. Hence the composition of nitrifying bacterial communities is complex and divers in heterogeneous habitats. In consequence of the above-mentioned problems, the representation of nitrifying community structures obtained from in situ investigations often has been incomplete and unbalanced in many respects. Polyphasic approaches, applying a combination of classical as well as molecular methods in parallel, could help to find the way for overcoming these problems in the future. Isolation and characterization of as many as possible new species seems to be one of the most important missing steps to advance at this way.

Distribution and ecophysiology of the nitrifying bacteria emphasizing cultured species

Nitrification is an important factor in the global nitrogen cycle. Therefore, an increasing number of publications deal with in situ studies of natural bacterial populations participating in this process. However, some crucial points complicate suchlike investigations. At the time being, a total of 25 species of ammonia-oxidizers and eight species of nitrite-oxidizers are cultured but the existence of many more species has been indicated by molecular in situ investigations. With that, only a part of the existing nitrifiers has been defined via isolation and subsequent physiological and molecular characterization. Furthermore, the distribution patterns of the distinct species of nitrifiers depend on various environmental parameters. Hence the composition of nitrifying bacterial communities is complex and divers in heterogeneous habitats. In consequence of the above-mentioned problems, the representation of nitrifying community structures obtained from in situ investigations often has been incomplete and unbalanced in many respects. Polyphasic approaches, applying a combination of classical as well as molecular methods in parallel, could help to find the way for overcoming these problems in the future. Isolation and characterization of as many as possible new species seems to be one of the most important missing steps to advance at this way.

Natural conjugative plasmids induce bacterial biofilm development.

Horizontal gene transfer is a principal source of evolution leading to change in the ecological character of bacterial species. Bacterial conjugation, which promotes the horizontal transfer of genetic material between donor and recipient cells by physical contact, is a phenomenon of fundamental evolutionary consequence. Although conjugation has been studied primarily in liquid, most natural bacterial populations are found associated with environmental surfaces in complex multispecies communities called biofilms. Biofilms are ideally suited to the exchange of genetic material of various origins, and it has been shown that bacterial conjugation occurs within biofilms. Here I investigate the direct contribution of conjugative plasmids themselves to the capacity of the bacterial host to form a biofilm. Natural conjugative plasmids expressed factors that induced planktonic bacteria to form or enter biofilm communities, which favour the infectious transfer of the plasmid. This general connection between conjugation and biofilms suggests that medically relevant plasmid-bearing strains are more likely to form a biofilm. This may influence both the chances of biofilm-related infection risks and of conjugational spread of virulence factors.

Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting

Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers.

An aerobic recA-, umuC-dependent pathway of spontaneous base-pair substitution mutagenesis in Escherichia coli

Antimutator alleles indentify genes whose normal products are involved in spontaneous mutagenesis pathways. Mutant alleles of the recA and umuC genes of Escherichia coli, whose wild-type alleles are components of the inducible SOS response, were shown to cause a decrease in the level of spontaneous mutagenesis. Using a series of chromosomal mutant trp alleles, which detect point mutations, as a reversion assay, it was shown that the reduction in mutagenesis is limited to base-pair substitutions. Within the limited number of sites than could be examined, transversions at AT sites were the favored substitutions. Frameshift mutagenesis was slightly enhanced by a mutant recA allele and unchanged by a mutant umuC allele. The wild-type recA and umuC genes are involved in the same mutagenic base-pair substitution pathway, designated “SOS-dependent spontaneous mutagenesis” (SDSM), since a recAumuC strain showed the same degree and specificity of antimutator activity as either single mutant strain. The SDSM pathway is active only in the presence of oxygen, since wild-type, recA, and umuC strains all show the same levels of reduced spontaneous mutagenesis anaerobically. The SDSM pathway can function in starving/stationary cells and may, or may not, be operative in actively dividing cultures. We suggest that, in wild-type cells, SDSM results from basal levels of SOS activity during DNA synthesis. Mutations may result from synthesis past cryptic DNA lesions (targeted mutagenesis) and/or from mispairings during synthesis with a normal DNA template (untargeted mutagenesis). Since it occurs in chromosomal genes of wild-type cells, SDSM may be biologically significant for isolates of natural enteric bacterial populations where extended starvation is often a common mode of existence.

Characterization of Shiga toxin producing E. coli and O157 serotype E. coli isolated in France from healthy domestic cattle

A study was carried out in France in collaboration with the meat industry to investigate the occurrence and characteristics of Shiga toxin-producing E. coli (STEC) and O157 E. coli in a population of healthy bovines representative of French livestock. A total of 851 animals belonging to three bovine classes (106 young bulls, 374 dairy cows and 371 meat cows) were included in the study. Samples of feces and of the corresponding carcasses were collected from March 97 to August 97 in seven abattoirs spread throughout the national territory. STEC cultures from the 1702 samples were screened using PCR for the presence of stx genes. Positive samples were further subjected to colony blot hybridization and to O157-specific immunomagnetic separation. Probe-positive colonies and O157 colonies were then analyzed for the presence of virulence genes and phenotypic characters (serotype, Stx production). In 154 (18.1%) feces and 91 (10.7%) carcass samples stx genes were detected. Two hundred and twenty-two STEC colonies were isolated from 67 (7.9%) feces and 16 (1.9%) carcass samples, with 183 STEC isolated from feces and 39 from carcasses. Only eight O157 isolates were collected from feces samples. None of these O157 E. coli isolates presented stx genes and thus could not be considered as pathogenic regarding hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). In 3.2% of STEC isolated from feces and in 10.2% of STEC from carcasses eae genes were detected. In 17% of STEC from feces and in 30.7% from carcasses ehx genes were detected. Using these data, the 222 STEC colonies could be classified in 11 different ‘virulence patterns’ (presence/absence of stx1, stx2, eae and ehx genes), showing that more than 77% of isolates presented only one virulence factor. Only three STEC on 222 colonies (1.3%) presented the three virulence factors stx, eae and ehx in association, none of them reacting with antisera specific for enterohemorrhagic E. coli. (EHEC). These data, together with the fact that only five isolates on the 222 (2.2%) reacted with such antisera (three O111 and two O26 isolates) demonstrated that the natural bacterial populations isolated during this study were clearly distinct from EHEC.

Isotope fractionation by sulfate-reducing natural populations and the isotopic composition of sulfide in marine sediments

Isotope fractionations during sulfate reduction by natural bacterial populations were measured in seven different marine sediments and compared with the isotopic composition of solid-phase sulfides in the same sediments. The measured fractionations during sulfate reduction could explain only between 41% and 85% of the 34S depletion in the sedimentary sulfides. This result directly demonstrates that the depletion of 34S in solid sulfides is an expression of the combined activity of sulfate-reducing organisms with additional fractionations accumulated during the oxidative part of the sulfur cycle. The only known process to significantly augment the fractionations created during sulfate reduction is the microbial disproportionation of the intermediate sulfur compounds: elemental sulfur, thiosulfate, and sulfite. In a simple model, we show how important each of these disproportionation processes could be in generating the isotopic composition of sedimentary sulfides. The sulfate reduction rates in the sediments studied varied by a factor of 1000 and did not correlate with the fractionation during sulfate reduction. By contrast, a correlation was observed between sulfate- reduction rate and the extent of 34S depletion into sedimentary sulfides, the most 34S-depleted sulfides found in sediments supporting the lowest rates of sulfate reduction. Thus, the fractionations imposed during the disproportionating processes are better expressed in sediments with low sulfate-reduction rates. The direct phototrophic oxidation of sulfide to sulfate, with minimal fractionation, may have been important in the sediments that had a high sulfate-reduction rate.

Population structure and evolutionary dynamics of pathogenic bacteria

Evidence concerning the significance of recombination within natural bacterial populations has historically come from two main sources: multilocus enzyme electrophoresis (MLEE) and nucleotide sequence data. Here we discuss evidence from a third method, multilocus sequence typing (MLST), which is a development of MLEE based on nucleotide sequencing that combines the advantages of both approaches. MLST has confirmed both the existence of clones and the high rates of recombination for several bacterial pathogens. The data are consistent with “epidemic” population structures, where clones are superimposed upon a backdrop of frequent recombination, thus, in the short term, resisting the homogenising effect of recombination. The nature of the selective advantage of clones, however, and how this advantage relates to virulence are unclear. The current evidence also has broader implications concerning bacterial species definition, the management of antibiotic-resistant bacteria and the assessment of the dangers of releasing genetically modified organisms into the environment. BioEssays 22:1115–1122, 2000. © 2000 John Wiley & Sons, Inc.

Surface finishes on stainless steel reduce bacterial attachment and early biofilm formation: scanning electron and atomic force microscopy study

Three common finishing treatments of stainless steel that are used for equipment during poultry processing were tested for resistance to bacterial contamination. Methods were developed to measure attached bacteria and to identify factors that make surface finishes susceptible or resistant to bacterial attachment and biofilm formation. Samples of the treated surfaces (sand-blasted, sanded, and electropolished) were exposed to natural bacterial populations from chicken carcass rinses to allow growth of bacteria and development of biofilms on the surfaces. The kinetics of bacterial growth during surface exposure was followed by UV-visible spectrophotometry, and counts of bacteria and early biofilm formation were measured following scanning electron microscopy (SEM). The surface morphology of the samples was analyzed by atomic force microscopy (AFM) with samples from each of the batches of treatments used in the SEM studies. Relative differences in the surface morphology, including fractal dimensions, Z ranges, roughness, and other measurements corresponded by treatment with the differences in reduction of bacterial counts shown by SEM. The surface types varied in affinity for bacteria, and both physical and electrochemical treatments improved resistance of stainless steel to bacterial attachment. Electropolished stainless steel was the least rough surface and showed significantly fewer bacterial cells and beginning biofilm formations than the other treated surfaces. Food safety could be improved if bacterial populations could be reduced during processing by increasing the use of materials that are resistant to bacterial contamination. These findings will aid equipment manufacturers and processors in selecting materials and finishes that are most resistant to bacteria and biofilm formation.

Analysis of long-side-chain alkylaromatics in crude oil for evaluation of their fate in the environment.

It has long been believed that the n-hexane fraction of crude oil only contains saturates. However, we found that one-third of its content was aromatics with long alkyl side chains and that these aromatics could be separated from the saturates by preparative thin-layer chromatography. The separated alkylaromatic fraction was characterized by UV-visible, NMR and mass spectrometries. A gas chromatographic-mass spectrometric analysis showed the presence of a homologous series of long-side-chain n-alkylaromatics, namely mono-, di-, and tri-n-alkylbenzenes in the C7-C27 range (the subscript to C indicates the total number of carbon atoms in the alkyl side chain) and di- and tri-n-alkylbenzothiophenes in the C3-C22 range. The biodegradation of these crude oil components by a natural bacterial population in seawater and their photooxidation by artificial sunlight were investigated. The n-alkylbenzenes were found to be quite susceptible to biodegradation but resistant to photooxidation, whereas the n-alkylbenzothiophenes were almost completely photooxidized and substantially biodegraded.

Population structure and evolutionary dynamics of pathogenic bacteria.

Evidence concerning the significance of recombination within natural bacterial populations has historically come from two main sources: multilocus enzyme electrophoresis (MLEE) and nucleotide sequence data. Here we discuss evidence from a third method, multilocus sequence typing (MLST), which is a development of MLEE based on nucleotide sequencing that combines the advantages of both approaches. MLST has confirmed both the existence of clones and the high rates of recombination for several bacterial pathogens. The data are consistent with "epidemic" population structures, where clones are superimposed upon a backdrop of frequent recombination, thus, in the short term, resisting the homogenising effect of recombination. The nature of the selective advantage of clones, however, and how this advantage relates to virulence are unclear. The current evidence also has broader implications concerning bacterial species definition, the management of antibiotic-resistant bacteria and the assessment of the dangers of releasing genetically modified organisms into the environment.

Apparent differences in antimicrobial susceptibility as a consequence of national guidelines

Apparent differences in antimicrobial susceptibility as a consequence of national guidelines