Amplification of Oral Streptococcal DNA from Human Incisors and Bite Marks

Challenges to the evidentiary value of morphometric determinations have led to a requirement for scientifically substantiated approaches to the forensic analysis of bite marks. Human teeth support genotypically distinctive populations of bacteria that could be exploited for forensic purposes. This study explored the feasibility of directly amplifying bacterial DNA from bite marks for comparison with that from teeth. Samples from self-inflicted experimental bite marks (n = 24) and human incisors were amplified by PCR using primers specific for streptococcal 16S ribosomal DNA. Amplicon profiles (resolved by denaturing gradient gel electrophoresis) from bite mark samples aligned significantly more closely with profiles generated from the teeth responsible than with those from other teeth. Streptococcal amplicons were generated from dental samples applied to excised porcine skin for up to 48 h. These findings indicate that streptococcal DNA can be amplified directly from bite marks, and have potential application in bite mark analysis.     

Microbial Analysis of Bite Marks by Sequence Comparison of Streptococcal DNA

Bite mark injuries often feature in violent crimes. Conventional morphometric methods for the forensic analysis of bite marks involve elements of subjective interpretation that threaten the credibility of this field. Human DNA recovered from bite marks has the highest evidentiary value, however recovery can be compromised by salivary components. This study assessed the feasibility of matching bacterial DNA sequences amplified from experimental bite marks to those obtained from the teeth responsible, with the aim of evaluating the capability of three genomic regions of streptococcal DNA to discriminate between participant samples. Bite mark and teeth swabs were collected from 16 participants. Bacterial DNA was extracted to provide the template for PCR primers specific for streptococcal 16S ribosomal RNA (16S rRNA) gene, 16S–23S intergenic spacer (ITS) and RNA polymerase beta subunit (rpoB). High throughput sequencing (GS FLX 454), followed by stringent quality filtering, generated reads from bite marks for comparison to those generated from teeth samples. For all three regions, the greatest overlaps of identical reads were between bite mark samples and the corresponding teeth samples. The average proportions of reads identical between bite mark and corresponding teeth samples were 0.31, 0.41 and 0.31, and for non-corresponding samples were 0.11, 0.20 and 0.016, for 16S rRNA, ITS and rpoB, respectively. The probabilities of correctly distinguishing matching and non-matching teeth samples were 0.92 for ITS, 0.99 for 16S rRNA and 1.0 for rpoB. These findings strongly support the tenet that bacterial DNA amplified from bite marks and teeth can provide corroborating information in the identification of assailants.     

Genetic diversity in Swiss cheese starter cultures assessed by pulsed field gel electrophoresis and arbitrarily primed PCR

AIMS: To assess intraspecific genetic heterogeneity among commercial Swiss cheese starter culture strains of Lactobacillus helveticus, Streptococcus thermophilus and Propionibacterium freudenreichii and to compare the efficacy of two genetic typing methods. METHODS AND RESULTS: Two genetic typing methods, pulsed field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR), were used. Nine Strep. thermophilus strains revealed eight PFGE and five AP-PCR genotypes. Seventeen Lactobacillus strains yielded 16 and five genotypes by PFGE and AP-PCR, respectively. Eleven Propionibacterium strains yielded 10 PFGE genotypes. Cluster analysis of PFGE profiles generated similarity coefficients for Strep. thermophilus, Lact. helveticus and Prop. freudenreichii strains of 29.5%, 60.3%, and 30.5%, respectively. Milk acidification rates for Strep. thermophilus and Lact. helveticus were determined. CONCLUSIONS: Pulsed field gel electrophoresis is more discriminatory than AP-PCR. The Lact. helveticus group is more homogeneous than the other species examined. Strains with > 87% similarity by PFGE consistently had the same acidification rate and AP-PCR profile. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial strains sold for Swiss cheese manufacture in the United States are genetically diverse. Clustering of genetically related bacteria may be useful in identifying new strains with industrially relevant traits.     

Comparison of arbitrarily primed-polymerase chain reaction and pulse-field gel electrophoresis for characterizing methicillin-resistant Staphylococcus aureus

AIMS: The aim of this study was to analyse genotypes for clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including hetero-vancomycin-resistant Staph. aureus (VRSA), at a Japanese university hospital. METHODS AND RESULTS: Seventy-eight clinical isolates of MRSA were analysed by arbitrarily primed-polymerase chain reaction (AP-PCR) using ERIC2 primer and by pulse-field gel electrophoresis (PFGE) following SmaI digestion. Analyses of the nine genotypes and 28 subtypes defined by PFGE, and of the three genotypes and 22 subtypes defined by AP-PCR, both facilitated epidemiological tracing. Used in combination, AP-PCR and PFGE provided more precise classification than the use of a single genotyping method. The six hetero-VRSA isolates were classified into four genotypes defined by the combination of both methods, but these genotypes contained non-VRSA isolates. CONCLUSIONS: The results suggest that both PFGE and AP-PCR are useful in discriminating MRSA, but not hetero-VRSA, isolates for epidemiological analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining the results of PFGE with the results of AP-PCR can provide more detail differentiation of MRSA and hetero-MRSA isolates than either method alone.     

A clone of Leptospira interrogans sensu stricto is the major cause of leptospirosis in the archipelago of Andaman and Nicobar Islands, India

AIMS: Andaman and Nicobar Islands in India has a century long history of human leptospirosis. Several isolates have been recovered over the years from different locations. The present study was undertaken to understand the clonal relationship between all these pathogenic leptospires recovered from these islands. METHODS AND RESULTS: Arbitrarily primed polymerase chain reaction (AP-PCR) was employed to genetically characterize 40 isolates recovered during 1995--2001 and their fingerprints were compared with those of 26 reference strains of known genetic and serological affinities. Sequences of PCR-amplified products from representative isolates were compared with those of different strains belonging to seven genospecies. AP-PCR fingerprints revealed that 32 of the 40 isolates were clonal in nature and fingerprints of all the isolates matched with known reference strains of pathogenic Leptospira interrogans sensu stricto. Comparison of sequence data of PCR amplified products of reference strains and isolates also corroborated these findings. CONCLUSIONS: The study revealed that 80% of the isolates recovered from these islands were clonal in nature and all the isolates taken in the study belonged to Leptospira interrogans sensu stricto. SIGNIFICANCE AND IMPACT OF THE study: An extension of the study in animal population would help in understanding the transmission dynamics of this commonly circulating clone in these islands, which in turn might help in effective control of this public health problem.     

The Effects of Nisin on the Microbial Flora of the Dental Plaque of Monkeys (Macaca fascicularis)

Nisin was incorporated in a soft diet and fed to eight monkeys for five months. At the end of this period dental plaque was removed from one site in the mouths of these animals and from a similar site in eight control animals. Twelve different selective media were used for the cultivation of the bacteria in the plaque. Streptococci, Haemophilus spp. and Gram negative anaerobes were the organisms most frequently isolated. There was some evidence that streptococci constituted a lesser proportion of the flora in the nisin-fed animals.     

The occurrence of Aeromonas and Streptococcus in Rainbow Trout, Salmo gairdneri Richardson

Smears were taken from the eyes, spleen and blood from rainbow trout collected from a fish farm and classified as clinically sick, under treatment, recovered and apparently healthy. The smears were examined microscopically and the bacteria present identified as Aeromonads or Streptococci. The results indicated a high incidence of Aeromonads in the eye and the spleen but not the blood in all groups including those apparently healthy while streptococci were present in the blood as well as the eye and the spleen.     

Predominant Catalase-negative Soil Bacteria. I. Streptococcal Population Indigenous to Soil

Streptococci related to Streptococcus sanguis were shown to represent a major segment of the bacterial flora of certain soils. Upon initial isolation, these streptococci were quite pleomorphic and displayed morphological characteristics common to several other genera of bacteria, but after prolonged cultivation on laboratory media the isolates demonstrated a typical streptococcal morphology. It was proposed that a requirement for correction of the control mechanisms affecting growth rate and cell wall synthesis in part might explain the difficulty encountered in isolation of these organisms from soil and their initial pleomorphism and cultural instability. The possible role of these soil organisms as etiological agents of disease and the occurrence of other groups of streptococci in soil were discussed.     

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.     

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.     

A Diagnosis of Alligator mississippiensis Bite Marks with Comparisons to Existing Crocodylian Datasets

Crocodylians are known to consume and modify bones, but actualistic observations of their bite marks have been limited to forensic case studies and surveys of two taxa: Crocodylus niloticus and Crocodylus porosus. To further explore patterns of crocodylian bite mark expression, we conducted a survey of traces left by Alligator mississippiensis. We compared the results to pre-existing crocodylian datasets regarding the potentially diagnostic traits of bisected marks, hook scores, and a lack of furrows. Mark type did not correlate with vital statistics of the sampled animals or collections protocol. Bisected marks were found in rates similar to those seen in one previous survey of C. niloticus, and rates of hook scoring and bone breakage were higher. These traces were all present in higher rates than those reported in C. porosus. Unlike results seen in Crocodylus, furrows were identified in the A. mississippiensis samples. Hook scores were also identified, but recent surveys of non-crocodylian taxa have shown that these features are not unique to crocodylians and instead are related to inertial feeding strategies. The presence and rate of bisected marks found in this study bolster the interpretation that these traces are a clade-wide phenomenon and a useful diagnostic indicator for Crocodylia.     

Normal vaginal flora in chimpanzees (Pan troglodytes): qualitative and quantitative study

Lactobacilli are the predominant microorganisms in the vaginal flora of human beings, and are known to play an important role in protecting them from genital infections. On the other hand, the composition of the vaginal flora differs among laboratory animal species, and lactobacilli are not the predominant vaginal microorganism in many laboratory animals. We speculated that the vaginal flora of chimpanzees would be more similar to those of human beings than to those of other animal species, because chimpanzees are phylogenetically close to human beings, and their reproductive physiology is similar to that of human beings. To clarify our speculation, we examined the development of the vaginal flora in chimpanzees (Pan troglodytes). Streptococci, lactobacilli, and members of the family Bacteroidaceae were the most predominant bacteria in the vagina of mature chimpanzees (9 to 22 years old). During development of the vaginal flora of chimpanzees, the total number of bacteria increased with age and reached a plateau just before sexual maturity (5 to 7 years of age; juvenile period). Lactobacilli were already one of the predominant bacteria before sexual maturity. In mature chimpanzees, the total number of bacteria (aerobes and anaerobes) in the vagina was highest during the swelling phase of the menstrual cycle. During the swelling phase in mature chimpanzees, streptococci, lactobacilli, and Bacteroidaceae were the most frequently isolated (100%) organisms, and the total number of organisms recovered from vaginal specimens from these three groups was the highest. In mature chimpanzees in which the number of bacteria was the highest, lactobacilli were the predominant bacteria. Taken together, these results suggest that these three bacterial groups (streptococci, lactobacilli, and Bacteroidaceae) are indigenous to the vagina of chimpanzees, and chimpanzees would be the most suitable laboratory animals for studying the role of lactobacilli in the vagina of human beings.     

Prevention and treatment of tail biting in weaned piglets

The aims of this study were to evaluate four preventive measures and two curative treatments of tail biting. The preventive measures were: chain, rubber hose, straw rack (5 g/pig/day) and the provision of straw on the floor twice daily by hand (2 × 10 g/pig/day). The two curative treatments, which were applied following the onset of tail biting in a pen were: straw twice daily (as in the fourth preventive measure) and the removal of the biter. In total, 960 undocked weaned piglets (10 piglets per pen) were observed during 5 weeks. Tail lesions (none, bite marks and wounds) were recorded daily. The incidence of pens with wounded pig tails was significantly lower when straw was provided twice daily (8% of pens) compared to the chain (58% of pens) and rubber hose (54% of pens) treatment, but did not differ significantly from the straw rack treatment (29% of pens). Tails with bite marks were significantly less common in pens with twice daily straw (16% of pens) compared to chain (88% of pens), rubber hose (79% of pens) and straw rack (75% of pens). No significant difference was found between the curative treatments. Both treatments showed a reduced incidence of red fresh blood on the tails at days 1–9 following curative treatment, compared to day 0. However, neither curative treatment eliminated tail biting entirely. In conclusion, this study indicates that tail biting is best prevented with a small amount of straw, provided twice daily, and to a lesser extent with a straw rack, compared to providing a chain or a rubber hose. Once tail biting has occurred, providing a small amount of straw twice daily and removing the biter appears to be equally effective.     

Coaggregation-mediated interactions of streptococci and actinomyces detected in initial human dental plaque

Streptococci and actinomyces that initiate colonization of the tooth surface frequently coaggregate with each other as well as with other oral bacteria. These observations have led to the hypothesis that interbacterial adhesion influences spatiotemporal development of plaque. To assess the role of such interactions in oral biofilm formation in vivo, antibodies directed against bacterial surface components that mediate coaggregation interactions were used as direct immunofluorescent probes in conjunction with laser confocal microscopy to determine the distribution and spatial arrangement of bacteria within intact human plaque formed on retrievable enamel chips. In intrageneric coaggregation, streptococci such as Streptococcus gordonii DL1 recognize receptor polysaccharides (RPS) borne on other streptococci such as Streptococcus oralis 34. To define potentially interactive subsets of streptococci in the developing plaque, an antibody against RPS (anti-RPS) was used together with an antibody against S. gordonii DL1 (anti-DL1). These antibodies reacted primarily with single cells in 4-h-old plaque and with mixed-species microcolonies in 8-h-old plaque. Anti-RPS-reactive bacteria frequently formed microcolonies with anti-DL1-reactive bacteria and with other bacteria distinguished by general nucleic acid stains. In intergeneric coaggregation between streptococci and actinomyces, type 2 fimbriae of actinomyces recognize RPS on the streptococci. Cells reactive with antibody against type 2 fimbriae of Actinomyces naeslundii T14V (anti-type-2) were much less frequent than either subset of streptococci. However, bacteria reactive with anti-type-2 were seen in intimate association with anti-RPS-reactive cells. These results are the first direct demonstration of coaggregation-mediated interactions during initial plaque accumulation in vivo. Further, these results demonstrate the spatiotemporal development and prevalence of mixed-species communities in early dental plaque.     

Ventral bite marks in Mesozoic ammonoids

Reports on the predators of ammonoids are rare, although ammonoids were abundant and diverse invertebrates in many Paleozoic and Mesozoic marine ecosystems. Most previous work on lethal ammonoid predation has focused on (sub)circular tooth marks which resulted from fish and mosasaur attacks. In the present study we discuss a relatively common type of bite mark in ammonoid shells, the ‘ventral bite mark’. This typically occurs in a restricted position on the ventral side of the outer body chamber whorl and does not affect either the aperture or the phragmocone. Ammonoid specimens revealing ventral bite marks used in this study were collected from a wide range of strata which range in age from the Lower Jurassic to the uppermost Cretaceous (close to the Cretaceous–Paleogene boundary). These ventral bite marks are absent in the Paleozoic collections studied. The vast majority of ventral bite marks are situated at the end of the body chamber, close to the phragmocone. This is interpreted as the result of predatory attacks on the back or blind side of ammonoids in their living position. The predators aimed for the vital parts and muscle attachments to obtain the edible soft tissues. The agents for most of the ventral bite marks to ammonoids are probably coleoid cephalopods (especially teuthoids) and predatory fishes to a lesser extent.     

Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments

Permafrost represents a unique ecosystem that has allowed the prolonged survival of certain bacterial lineages at subzero temperatures. To better understand the permafrost microbial community, it is important to identify isolation protocols that optimize the recovery of genetically diverse bacterial lineages. We have investigated the impact of different low-temperature isolation protocols on recovery of aerobic bacteria from northeast Siberian permafrost of variable geologic origin and frozen for 5000 to 3 million years. Low-nutrient media enhanced the quantitative recovery of bacteria, whereas the isolation of diverse morphotypes was maximized on rich media. Cold enrichments done directly in natural, undisturbed permafrost led not only to recovery of increased numbers of bacteria but also to isolation of genotypes not recovered by means of liquid low-temperature enrichments. On the other hand, direct plating and growth at 4°C also led to recovery of diverse genotypes, some of which were not recovered following enrichment. Strains recovered from different permafrost samples were predominantly oligotrophic and non-spore-forming but were otherwise variable from each other in terms of a number of bacteriological characteristics. Our data suggest that a combination of isolation protocols from different permafrost samples should be used to establish a culture-based survey of the different bacterial lineages in permafrost. Received: December 2, 1999 / Accepted: January 18, 2000     

Seed-breaking forces exerted by orang-utans with their teeth in captivity and a new technique for estimating forces produced in the Wild

Orang-utans (Pongo pygmaeus) at the Singapore Zoological Gardens were presented with two thick-shelled edible seeds, Mezzettia parviflora (Annonaceae) and Macadamia ternifolia (Proteaceae) in order to estimate their maximum bite forces. The orang-utans could break the Macadamia seeds in one bite, while those of Mezzettia required repeated attempts. Examination of shell fragments showed that many had scratches and some had clear, but small (ca. 1–2 mm diameter), impressions on them. Building upon this information, semi-imitative tests were performed on the seeds in a universal testing machine by loading them in compression with either flat plates or metal casts of orang-utan cheek teeth. The maximum forces required to break the seeds were similar with both the flat plates and the metal teeth; the average for the Macadamia seeds being about 2,000 N (which forms a minimum estimate for the maximum bite forces in orang-utans) and for the Mezzettia seeds, 6,000 N. The work done with the metal teeth was much greater than with the plates. A mechanical analysis showed that this extra work went into producing permanent impressions (“bite marks”) in the shell with the tooth cusps. These impressions were larger than those found on the shells of seeds bitten by the orang-utans. Nevertheless, it is shown theoretically that the size of these indentations can give an estimate of the bite forces used. The maximum force developed in the machine tests with the metal teeth was correlated with the force calculated from analysis of the bite marks. The method is suitable for use in field studies where the marks left on remnants of hard foods eaten by primates may be used to estimate, very roughly, the forces used to produce them. © 1994 Wiley-Liss, Inc.     

Killing in the Pliocene: shark attack on a dolphin from Italy

Abstract: Shark bite marks, including striae, sulci and abrasions, in a well‐preserved fossil dolphin skeleton referred to Astadelphis gastaldii (Cetacea, Delphinidae) from Pliocene sediments of Piedmont (northern Italy), are described in detail. The exceptional combination of a fossil dolphin having a significant part of the skeleton preserved and a large number of bite marks on the bones represents one of the few detailed documentations of shark attack in the past. Most bite marks have been referred to a shark about 4 m long with unserrated teeth, belonging to Cosmopolitodus hastalis, on the basis of their shape and their general disposition on the dolphin skeleton. According to our hypothesis, the shark attacked the dolphin with an initial mortal bite to the abdomen from the rear and right, in a similar way as observed for the living white shark when attacking pinnipeds. A second, less strong, bite was given on the dorsal area when the dolphin, mortally injured, probably rolled to the left. The shark probably released the prey, dead or dying, and other sharks or fishes probably scavenged the torn body of the dolphin.     

Genetic analysis of adherence by oral streptococci

Streptococci are one of the most successful bacterial colonizers of the human body and are major components of oral biofilms. The bacterial cells express multiple cell-surface adhesins that are responsible for the ability of streptococci to adhere to a wide range of substrates which include salivary and serous proteins, epithelial cells and other bacterial cells. Analysis of adherence-defective mutants has indicated the importance of high molecular mass wall-associated polypeptides and of enzymes catalyzing extracellular glucan polysaccharide synthesis to the adherence and accumulation of oral streptococci. The analysis of isogenic mutants of streptococci, generated through insertional inactivation (or allelic exchange), has confirmed the essential roles of specific surface polypeptides both to adhesive processes and to correct assembly of the cell wall layers.     

Comparison of culture media and chairside assays for enumerating mutans streptococci

AIM: This study compared several traditional culture-based media and chairside cultural assays for ability to recover mutans streptococci (MS) from pure cultures and from saliva samples. METHODS AND RESULTS: When pure cultures were used with traditional culture-based media, mitis-salivarius bacitracin (MSB) agar demonstrated less support for bacterial recovery than trypticase-yeast extract-cysteine sucrose-bacitracin (TYCSB) agar and the modified medium of Ritz (HLR-S). One species of MS, Streptococcus ferus (c), was not recovered on MSB medium. Chairside cultural tests displayed considerable disparity between tests in recovering bacteria from pure cultures. On the glass adherence assay (Mucount), S. ferus was not detected and Streptococcus criceti was not detected on the dipslide assay (Cariescreen SM) or on the plastic adherence assay (Dentocult SM Strip mutans). The frequency of isolation of pure strains of bacteria other than MS was common. From saliva samples, the frequency of isolation of MS on HLR-S and TYCSB media and the glass adherence assay was 91-97%. The frequency of isolation on MSB medium and on the dip-slide and plastic adherence assays was significantly decreased (37, 47 and 69%, respectively). Recovery scores varied considerably among the culture methods studied and tended to be highest on the HLR-S medium and on the glass adherence assay. CONCLUSIONS: Growth and recovery profiles of pure bacterial cultures and of saliva samples for the MS varied according to different media. SIGNIFICANCE AND IMPACT OF THE STUDY: Caution should be exercised in comparing results between studies that employ different cultural methods for MS enumeration.