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Evolution of bacteria to selective chemical pressure (e.g. antibiotics) is well studied in contrast to the influence of physical stressors. Here we show that instantaneous physical stress in a homogeneous environment (without concentration gradient) induces fast adaptation of Escherichia coli. We exposed E. coli to a large number of collisions of around 105 per bacterium per second with sharp ZnO nanorods. The pressure exerted on the bacterial cell wall was up to 10 GPa and induced phenotype changes. The bacteria’s shape became more spherical, the density of their periplasm increased by around 15% and the average thickness of the cell wall by 30%. Such E. coli cells appeared almost as Gram-positive bacteria in the standard Gram staining. Additionally, we observed a combination of changes occurring at the genomic level (mutations identified in form of single nucleotide polymorphisms) and down-regulation of expression of 61 genes encoding proteins involved in β-oxidation of fatty acids, glycolysis, the citric acid cycle, as well as uptake of amino acids and enzyme cofactors. Thus, we show that bacteria undergo phenotypic changes upon instantaneous, acute physical stress without any obviously available time for gradual adaptation.

Arctic terrestrial ecosystems are particularly vulnerable to the effects of ongoing and predicted climate changes. The current states of environmental biodiversity and ecological networks in the Arctic need to be known and understood to monitor how they change and how these changes may influence the particular components of the ecosystem. Despite the fact that the Arctic tundra is generally poor in nutrients, it has a surprisingly high biodiversity, especially of invertebrates and microorganisms. Besides macroclimatic features, there may be local factors influencing biodiversity, such as microclimate, water availability, or large seabird colonies depositing guano. This last can have a substantial impact on the soil’s physicochemical features, and consequently the distribution, number, and diversity of tundra-associated plants and animals in the vicinity of the colony. Changes in the Arctic biodiversity and the functioning of the ecosystem at all trophic levels are difficult to investigate using traditional methods. In this review, we discuss how modern molecular techniques, including next generation sequencing, influence our ability to investigate and understand this ecosystem at both the micro- and the macroscale and how they can complement the more traditional approaches to studying ecological networks in the Arctic.

In the study presented here, we tested, how large a fraction of lysogenic culture was undergoing filamentation, which could indicate triggering of the SOS response or SOS-independent prophage induction that is also known to cause cell filamentation. Here, antibiotic stress was triggered by adding mitomycin C and oxidative stress was induced by hydrogen peroxide. Observation of bacterial cells under an optical microscope revealed more filamenting cells for lysogenic Escherichia coli than for strains not carrying a prophage. Moreover, the amount of filamenting cells depended not only on the stress agents used and the type of the prophage, but also on the host. During induction of the 933W prophage, the resulting phage titer and the amount of elongating cells were different when using E. coli O157:H7 EDL933 clinical isolate and the E. coli MG1655 laboratory strain. The amount of filamenting cells correlates well with the observed phage titers.

Only in USA the nosocomial infections cause around 100,000 deaths per year. Majority of them could have been avoided if the pathogens would be identified shortly after infection. However, classical approaches are laborious; can take up to even 72 h. Here we demonstrate a step towards a sensor for fast detection of bacteria. The sensor is based on layer of oriented T4 bacteriophages. The sensitivity of biosensors increases four times for ordered over disordered layer of bacteriophages. This results in the limit of detection of Escherichia coli in the range of 10 2 –10 3 cfu/mL. Such value is much lower when compared to similar sensors based on physisorbed layer of phages and is of the same order of magnitude as probes with chemically immobilized bacteriophages described in the literature. Proper orientation of bacteriophages prevents receptor binding proteins from being blocked. For the purpose we utilized the electrostatic interactions upon application of electric potential to the surface on which phages are deposited.

Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its' 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host's transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the Siphoviridae family. Thus a taxonomic proposal of a new Tp84virus genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in Escherichia coli, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.

Metagenomics is a powerful tool to better understand the microbial niches, especially these from extreme habitats like oceans and seas, hot springs or deserts. However, one who is going to face the metagenomic studies should realize the challenges which might occur in the course of experiments. This manuscript indicates common problems in function-driven metagenomics, especially factors that influence gene expression are taken into account. Codon usage bias, internal cell accumulation, correct protein folding or presence of proper initiation factors are discussed and possible ways to overcome these problems are proposed. Finally, the annotation process is described, including possible limitations that one should take under consideration. What is more, the most popular databases for metagenomic data are mentioned and discussed.

Metagenomic studies have become increasingly popular. They allow for the estimation of biodiversity in complex populations. This diversity presents an enormous but largely unexpected genetic and biological pool and can be exploited for the recovery of novel genes, entire metabolic pathways and their products. Generally metagenomic study is a genomic analysis of organisms by direct extraction and cloning of DNA from their natural environment. The most common problems of modern metagenomics are as follows: majority of the microorganisms present in the environment cannot be cultivated by standard techniques, DNA extraction methods are not very effective, isolated DNA is contaminated with various compounds, a choice for a screening method is not obvious.

The aim of this study was to investigate the soil microbial communities of a phosphogypsum waste heap. The soil microbial community structures can differ over time, as they are affected by the changing environmental conditions caused by a long-term exposure to different kinds of pollutions, like is the case of soil in the post-production waste area in Wiślinka (in the northern part of Poland) currently undergoing restoration. Our analyses indicated that the most abundant phyla were Proteobacteria, Acidobacteria, and Actinobacteria, and generally such an abundance is common for most of the studied soils. The most dominant class were Alphaproteobacteria, with their participation in 33.46% of the total reads. Among this class, the most numbered order was Sphingomonadales, whereas among this order the Sphingomonadaceae family was the most abundant one. The Sphingomonadaceae family is currently in the center of interest of many researchers, due to the ability of some of its members to utilize a wide range of naturally occurring organic compounds and many types of environmental contaminants. This kind of knowledge about microbial populations can support efforts in bioremediation and can improve monitoring changes in the contaminated environments.

The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage ϕ24Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and ϕ24Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo-xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo-xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.

Due to deposition of birds' guano, eggshells or feathers, the vicinity of a large seabirds' breeding colony is expected to have a substantial impact on the soil's physicochemical features as well as on diversity of vegetation and the soil invertebrates. Consequently, due to changing physicochemical features the structure of bacterial communities might fluctuate in different soil environments. The aim of this study was to investigate the bacterial assemblages in the Arctic soil within the area of a birds' colony and in a control sample from a topographically similar location but situated away from the colony's impact area. A high number of OTUs found in both areas indicates a highly complex microbial populations structure. The most abundant phyla in both of the tested samples were: Proteobacteria, Acidobacteria, Actinobacteria, and Chloroflexi, with different proportions in the total share. Despite differences in the physicochemical soil characteristics, the soil microbial community structures at the phylum level were similar to some extent in the two samples. The only share that was significantly higher in the control area when compared to the sample obtained within the birds' colony, belonged to the Actinobacteria phylum. Moreover, when analyzing the class level for each phylum, several differences between the samples were observed. Furthermore, lower proportions of Proteobacteria and Acidobacteria were observed in the soil sample under the influence of the bird's colony, which most probably could be linked to higher nitrogen concentrations in that sample.

Svalbard reindeer (Rangifer tarandus platyrhynchus) is a non-migratory subspecies of reindeer inhabiting the high-arctic archipelago of Svalbard. In contrast to other Rangifer tarandus subspecies, Svalbard reindeer graze exclusively on natural sources of food and have no chance of ingestion of any crops. We report the use of a non-invasive method for analysis of fecal microbiome by means of sequencing the 16S rDNA extracted from the fecal microbiota of Rengifer tarandus platyrhynchus from a small, isolated population in Hornsund, South Spitsbergen National Park. Analyses of all samples showed that 99% of the total reads were represented by Bacteria. Taxonomy-based analysis showed that fecal bacterial communities consisted of 14 phyla. The most abundant phyla across the population were Firmicutes and Bacteroidetes, and those phyla jointly accounted for more than 95% of total bacterial sequences (ranging between 90.14% and 98.19%). Specifically, Firmicutes comprised 56.53% (42.98% - 63.64%) and Bacteroidetes comprised 39.17% (34.56% - 47.16%) of the total reads. The remaining 5% of the population reads comprised of Tenericutes, Cyanobacteria, TM7, Actinobacteria, Proteobacteria, Verrucomicrobia, Elusimicrobia, Planctomycetes, Fibrobacteres, Spirochaetes, Chloroflexi and Deferribacteres. Differences in the fecal bacteria composition between particular reindeer were not statistically significant which may reflect the restricted location and similar diet of all members of the local population.

A universal and effective method for long-term storage of bacteriophages has not yet been described. We show that randomly selected tailed phages could be stored inside the infected cells at −80 °C without a major loss of phage and host viability. Our results suggest the suitability of this method as a standard for phage preservation.

The use of bacteriophages, instead of antibodies, in the ELISA-based detection of bacterial strains was tested. This procedure appeared to be efficient, and specific strains of Salmonella enterica and Escherichia coli could be detected. The sensitivity of the assay was about 10(5) bacterial cells/well (10(6)/ml), which is comparable with or outperforms other ELISA tests detecting intact bacterial cells without an enrichment step. The specificity of the assay depends on the kind of bacteriophage used. We conclude that the use of bacteriophages in the detection and identification of bacteria by an ELISA-based method can be an alternative to the use of specific antibodies. The advantages of the use of bacteriophages are their environmental abundance (and, thus, a possibility to isolate various phages with different specificities) and the availability of methods for obtaining large amounts of phage lysates, which are simple, rapid, cheap, and easy.

Lysis inhibition (LIN) is a known feature of the T-even family of bacteriophages. Despite its historical role in the development of modern molecular genetics, many aspects of this phenomenon remain mostly unexplained. The key element of LIN is an interaction between two phage-encoded proteins, the T holin and the RI antiholin. This interaction is stabilized by RIII. In this report, we demonstrate the results of genetic experiments which suggest a synergistic action of two accessory proteins of bacteriophage T4, RI.-1, and RI.1 with RIII in the regulation of LIN.

Shiga toxin-producing Escherichia coli (STEC) may cause bloody diarrhea and hemorrhagic colitis, with sometimes severe complications. Because genes coding for Shiga toxins are located on lambdoid prophages, effective toxin production occurs only after prophage induction. However, although agents that effectively induce prophage lambda (a paradigm of the family of lambdoid phages) under laboratory conditions, such as UV irradiation or DNA replication inhibitors, are well known, it is unlikely that such factors are present in human intestine infected with STEC. In this report, we demonstrate that induction of a Shiga toxin-converting prophage in its host (E. coli O157:H7) occurs not only in the presence of DNA-interfering antibiotics (mitomycin C and norfloxacin) but also under conditions of oxidative stress [following treatment with hydrogen peroxide (H(2)O(2))]. Under these conditions, we observed not only effective prophage induction but also expression of the reporter gene (replacing the original stx2 gene). In the light of previously published reports, indicating that oxidative stress conditions might occur during colonization of human intestine by enteric bacteria, and that neutrophil-produced H(2)O(2) can increase production of the Shiga toxin in a clinical isolate of STEC, these results suggest that oxidative stress may be one of the agents responsible for stimulating the pathogenicity determinants of STEC, leading to induction of Shiga toxin-converting prophages in these bacteria.

Background: Genes encoding Shiga toxins in pathogenic Escherichia coli strains (Shiga toxin-producing E. coli, STEC) are located on lambdoid prophages. However, studies on the replication of these phages were not reported to date. Methods: Plasmids derived from Shiga toxin-converting phages were constructed, introduced into wild-type E. coli strain and studied using methods of molecular genetics. Results: The studied replicons behaved similarly to plasmids derived from bacteriophage λ, and DNA sequence analysis revealed their high level of homology to the λ replication region. Nevertheless, contrary to λ and two of six tested plasmids, four tested replicons were able to replicate in the E. colidnaA46 mutant. Specific nucleotide differences, causing amino acid substitutions relative to wild-type λ O and P proteins, appeared to be responsible for this phenotype. Conclusions: We conclude that replicons of phages bearing Shiga toxin genes are functionally similar to that of bacteriophage λ, however, some of them have important differences which influence replication regulation. Furthermore, our results may suggest that there are newly discovered molecular interactions in the DNA replication regulation of λ and lambdoid phages. Hence, plasmids derived from lambdoid phages appear to be convenient models to study the replication of Shiga toxin-converting bacteriophages.

Various processes of bacteriophage λ development in Escherichia coli cells bearing either the whole λ exo-xis region (with truncated, thus nonfunctional, exo and xis genes) or particular genes from this region were investigated. The presence of either the exo-xis region or the ea8.5 gene on a plasmid resulted in formation of fuzzy plaques by infecting phage. Both efficiency of plating and efficiency of lysogenization were decreased in such hosts. On the other hand, neither the efficiency of adsorption nor intracellular lytic development of the infecting phage (measured in one-step-growth experiments) was affected while significantly more host cells survived the infection, when containing the exo-xis region. Although no effects of the exo-xis region on the activity of the p L promoter was detected, this region contributed to a decreased transcription from the cII-stimulated promoters p I, p aQ and p E. These results, together with the results of measurement of efficiency of plating of phages bearing mutations in cI, cII and cIII genes on hosts containing the exo-xis region, strongly suggest that genes from this region (especially ea8.5) are involved in the regulation of bacteriophage λ development at the stage of the lysis-vs.-lysogenization decision.

The use of low concentrations (optimally 2.5 to 3.5 μg/ml, depending on top agar thickness) of ampicillin in the bottom agar of the plate allows for formation of highly visible plaques of bacteriophages which otherwise form extremely small plaques or no plaques on Escherichia coli lawns. Using this method, we were able to obtain plaques of newly isolated bacteriophages, propagated after induction of prophages present in six E. coli O157:H − strains which did not form plaques when standard plating procedures were employed.

Metagenomics approaches and recent improvements in the next‐generation sequencing methods, have become a method of choice in establishing a microbial population structure. Many commercial soil DNA extraction kits are available and due to their efficiency they are replacing traditional extraction protocols. However, differences in the physicochemical properties of soil samples require optimization of DNA extraction techniques for each sample separately. The aim of this study was to compare the efficiency, quality, and diversity of genetic material extracted with the use of commonly used kits. The comparative analysis of microbial community composition, displayed differences in microbial community structure depending on which kit was used. Statistical analysis indicated significant differences in recovery of the genetic material for 24 out of 32 analyzed phyla, and the most pronounced differences were seen for Actinobacteria. Also, diversity indexes and reproducibility of DNA extraction with the use of a given kit, varied among the tested methods. As the extraction protocol may influence the apparent structure of a microbial population, at the beginning of each project many extraction kits should be tested in order to choose one that would yield the most representative results and present the closest view to the actual structure of microbial population.

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC50 = 20 μM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.

Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as they are carriers of genes coding for Shiga toxins. Efficient expression of these genes requires lambdoid prophage induction and multiplication of the phage genome. Therefore, understanding the mechanisms regulating these processes appears essential for both basic knowledge and potential anti-EHEC applications. The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. Recent report indicated that the Ea8.5 protein, encoded in this region, contains a newly discovered fused homeodomain/zinc-finger fold, suggesting its plausible regulatory role. Moreover, subsequent studies demonstrated that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24B prophages. In this report, we demonstrate that after prophage induction, the increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region, while survival rate of such bacteria is lower, which corroborated previous observations. Importantly, by using quantitative real-time reverse transcription PCR, we have determined patterns of expressions of particular genes from this region. Unexpectedly, in both phages λ and Ф24B, these patterns were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents (mitomycin C and hydrogen peroxide). This may shed a new light on our understanding of regulation of lambdoid phage development, depending on the mode of lytic cycle initiation.

Shiga toxin-producing Escherichia coli (STEC) may cause bloody diarrhea and hemorrhagic colitis (HC), with subsequent systemic disease. Since genes coding for Shiga toxins (stx genes) are located on lambdoid prophages, their effective production occurs only after prophage induction. Such induction and subsequent lytic development of Shiga toxin-converting bacteriophages results not only in production of toxic proteins, but also in the lysis (and thus, the death) of the host cell. Therefore, one may ask the question: what is the benefit for bacteria to produce the toxin if they die due to phage production and subsequent cell lysis? Recently, a hypothesis was proposed (simultaneously but independently by two research groups) that STEC may benefit from Shiga toxin production as a result of toxin-dependent killing of eukaryotic cells such as unicellular predators or human leukocytes. This hypothesis could make sense only if we assume that prophage induction (and production of the toxin) occurs only in a small fraction of bacterial cells, thus, a few members of the population are sacrificed for the benefit of the rest, providing an example of "bacterial altruism." However, various reports indicating that the frequency of spontaneous induction of Shiga toxin-converting prophages is higher than that of other lambdoid prophages might seem to contradict the for-mentioned model. On the other hand, analysis of recently published results, discussed here, indicated that the efficiency of prophage excision under conditions that may likely occur in the natural habitat of STEC is sufficiently low to ensure survival of a large fraction of the bacterial host. A molecular mechanism by which partial prophage induction may occur is proposed. We conclude that the published data supports the proposed model of bacterial "altruism" where prophage induction occurs at a low enough frequency to render toxin production a positive selective force on the general STEC population.

Bacteriophage T4 is able to adjust its development to the growth parameters of the host cell. Here, we present evidence for the production of two different subpopulations of phage particles, which differ in their ability to infect starved Escherichia coli cells. The ability of phage T4 to produce a fraction of virions unable to infect starved cells is linked to the functions of genes rI and rIII, as well as rIIA. This may represent the adaptation of phage T4 in order to persist in unfavourable environmental conditions.

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of lambda prophage. Here, we demonstrate that H2O2-mediated lambda prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a lambda lysogen in the presence of H2O2. On the other hand, stimulation of the p(M) promoter by cI857 overproduced from a multicopy plasmid was decreased in the DeltaoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the p(M) promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p(M) promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced lambda prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating lambda prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.

Shiga toxin-producing Escherichia coli (STEC) strains are pathogens that cause severe infections, especially in children. Although early data suggested that the STEC phenotype was linked to the O157 serotype, subsequent studies indicated no such strict phenotype-serotype correlation. Thus, it seems that only a test detecting stx1 and stx2 genes (responsible for Shiga toxin production) can provide unambiguous results in STEC identification. Here, we present a system for the rapid detection of the presence of o157, stx1, and stx2 genes, on the basis of an asymmetric polymerase chain reaction and subsequent detection of the amplification products on electric biochips. The whole test lasts less than 100 minutes, and allows for the accurate identification of the stx1 and stx2 genes and the presence of pO157 plasmid in a bacterial colony or in a liquid bacterial culture. The procedure is fully automated, and the only required manual activity is a chip exchange.

Bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful. Here, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, λ, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats. Bacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively.

DNA extracted from faeces may be a valuable source of information about the animal itself, as well as its microflora. An isolated reindeer population from Svalbard (Rangifer tarandus platyrhynchus) was tested for the presence of Shiga toxin encoding genes in the collected faecal samples. Even though the reindeers were not interacting with any other ruminants, which are considered to be a major reservoir of Shiga toxin containing bacteria, the stx1 gene was detected in 9 out of 10 tested samples, and the stx2 subtype c was found in five tested samples. This is an exceptionally high proportion, especially in the case of stx1, when compared to those observed in semi-domesticated or wild cervid populations in less remote locations. Distribution of the investigated genes in a small, local population indicates a different pattern of transmission, which seems to favour bacteria carrying the stx1 genes over those carrying the stx2 genes. The overall high percentage of animals with microbiota containing the stx genes suggests an important role of these genes in such an extreme environment for either hosts or their gut bacteria. Additionally, male-specific DNA found in the faeces was isolated in order to establish a given animal’s sex. PCR based on two pairs of primers, DBY7 and DBY8 gave the expected length of product characteristic for Y chromosome. The results of molecular sexing were consistent with field observations.