Rare and Common Variants Conferring Risk of Tooth Agenesis.

We present association results from a large genome-wide association study of tooth agenesis (TA) as well as selective TA, including 1,944 subjects with congenitally missing teeth, excluding third molars, and 338,554 controls, all of European ancestry. We also tested the association of previously identified risk variants, for timing of tooth eruption and orofacial clefts, with TA. We report associations between TA and 9 novel risk variants. Five of these variants associate with selective TA, including a variant conferring risk of orofacial clefts. These results contribute to a deeper understanding of the genetic architecture of tooth development and disease. The few variants previously associated with TA were uncovered through candidate gene studies guided by mouse knockouts. Knowing the etiology and clinical features of TA is important for planning oral rehabilitation that often involves an interdisciplinary approach.

Rehabilitation of a cystic mixed dentition mandible following marsupialization with a multipurpose acrylic splint acting as a space maintainer and an obturator.

Radicular cysts are the most common odontogenic cystic lesions of inflammatory origin which can be managed by marsupialisation specially if the cyst is large and is in relation to the vital structures. This article presents a case in which a radicular cyst was present in association with grossly carious deciduous molars and has been treated by marsupialisation. Postoperatively a surgical splint was inserted to maintain the patency of the bone cavity. This obturator splint also acts as a space maintainer to prevent space loss and ensure unimpeded eruption of permanent premolars.

Identification and characterization of Class 1 integron resistance gene cassettes among Salmonella strains isolated from imported seafood.

A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport [strain 62], Typhimurium var. Copenhagen [strain 629], and Lansing [strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics.

Isolation and molecular characterization of fluoroquinolone-resistant Escherichia coli from poultry litter.

Nineteen fluoroquinolone-resistant Escherichia coli strains were isolated from poultry litter. Sixteen of the 19 strains were serotyped to groups 6, 8, 53, 56, 153, and 174. Three strains were not serotyped to any known group. All isolates were resistant to multiple antibiotics. Most strains were resistant to gentamicin, kanamycin, chloramphenicol, and streptomycin. Ribotyping of the multidrug-resistant isolates with restriction enzyme PvuII showed 5 different ribogroups, suggesting independent development of resistance instead of clonal spread. Quinolone resistance was associated with mutations of the quinolone resistance-determining region (QRDR) of the gyr A gene in all cases. To determine the incidence of gyr A mutations in fluoroquinolone-resistant E. coli isolates, a rapid PCR-based assay was used by amplifying a 164-bp region of the gyr A gene containing the mutation sites followed by digestion of the PCR product with restriction enzyme HinfI. A higher level of resistance to ciprofloxacin [minimum inhibitory concentration (MIC) >4 microg] was associated with double mutations, but the mutants with a low level of resistance (MIC <2 microg) had only a single mutation. Those strains that were ciprofloxacin-resistant (MIC <2 microg) had a single mutation of a C-to-T transition at position 248 (Ser 83-->Leu) or a G-to-A transition at position 259 (Asp 87-->Asn). The ciprofloxacin-resistant (MIC >4 microg) isolates had mutations at both positions. Fluoroquinolone resistance was present among different serotypes and ribotypes, and drug resistance profiles suggest that the incidence of resistance does not indicate a clonal population in avian E. coli.

Molecular characterization of fluoroquinolone-resistant Campylobacter spp. isolated from poultry.

Campylobacteriosis, an infectious disease caused by Campylobacter jejuni and Campylobacter coli, is treated by fluoroquinolone antibiotics in clinical practices. However, use of these drugs in animal husbandry may select for fluoroquinolone-resistant campylobacters and, thereby, compromise the clinical treatment of infection. In this study, 21 fluoroquinolone-resistant campylobacters were isolated from poultry samples. Morphological and biochemical characteristics indicated that 19 isolates were C. jejuni and two were C. coli. All isolates were resistant to multiple antibiotics but sensitive to chloramphenicol and gentamicin. These isolates were characterized at the molecular level by amplifying the flagellin gene (flaA) by PCR. The PCR protocol amplified a 1.7-kb flaA gene from all isolates. RFLP analysis of the 1.7-kb amplicons after digestion with DdeI yielded four distinct patterns. The 21 fluoroquinolone-resistant campylobacter isolates were further characterized by pulsed-field gel electrophoresis (PFGE) and compared with the PFGE patterns of nine fluoroquinolone-sensitive campylobacter strains. Four of the 21 fluoroquinolone-resistant isolates were untypable by the PFGE protocol. The PFGE analysis with SalI or SmaI indicated that seven or five, respectively, of the 17 resistant isolates had identical macrorestriction profiles (mrps). However, PFGE analysis with a combination of SalI and SmaI indicated that four of the 17 isolates had similar macrorestriction profiles. The PFGE patterns of the 17 fluoroquinolone-resistant isolates were different from the nine sensitive campylobacter strains.

Detection and characterization of erythromycin-resistant methylase genes in Gram-positive bacteria isolated from poultry litter.

The epidemiology of four erythromycin-resistant methylase ( erm) genes, ermA, ermB, ermC and msrA, was determined in erythromycin-resistant staphylococci, enterococci and streptococci isolated from poultry litter. All isolates were resistant to multiple antibiotics. Southern hybridization indicated that 4 of the 20 staphylococci contained the ermC gene on plasmids: on a 2.2 kb plasmid in Staphylococcus hominis and S. sciuri, on a 6.0 kb plasmid in S. xylosus, and on a 7.0 kb plasmid in S. lentus. In 16 of the 20 staphylococci, the ermA gene was harbored exclusively on the chromosome, as a double chromosomal insert on 8.0 and 6.2 kb EcoRI fragments. None of the staphylococci harbored the msrA gene. Dot-blot analysis indicated that all enterococci and streptococci hybridized with a biotinylated ermB gene probe. Southern hybridization indicated that only 2 of the 19 erythromycin-resistant enterococci contained the ermB gene on plasmids. The gene was localized on 4.0 kb and 5.9 kb plasmids, respectively, in two Enterococcus faecium isolates. Results from our studies indicate that the patterns of occurrence of erm genes, the sizes of the plasmids and the copy numbers of the inserts were different from the existing information on the presence of erm genes in clinical strains of Staphylococcus spp.

DNA packaging and developmental intermediates of a broad host range Vibrio vulnificus bacteriophage 71A-6.

The structural intermediates in the capsid assembly and DNA packaging pathway of Vibrio vulnificus bacteriophage 71A-6, a rod-shaped double-stranded DNA podovirus, were isolated by ultracentrifugation and studied by electron microscopy, SDS-PAGE and pulsed-field gel electrophoretic analysis. Bacteriophage 71A-6 synthesized rod-shaped capsids (mean length=200+/-8 nm; mean width=47+/-3 nm n=50) during its development. Several host proteins that probably help in the assembly and maturation of the capsids were attached to these capsids as spherical structures. A capsid-DNA or DNA packaging complex that consisted of the mature capsids, DNA and a 42.5-kDa protein was also isolated. The size of the capsids increased in length and decreased in width (mean length=220+/-8 nm; mean width=45+/-3 nm n=50) either during or after the DNA packaging. The capsid fractions contained about 12 phage structural proteins and eight host proteins. At least three proteins were tentatively identified: a 38.5-kDa major capsid protein, a 35.2-kDa tail protein and 42.5-kDa packaging initiator or terminator protein. The size of the bacteriophage 71A-6 genome was determined to be 143.0-kb by pulsed-field gel electrophoresis. The total mass of all the mature phage proteins corresponded to only 14.0% of the coding capacity of phage genome.

Identification of predominant human and animal anaerobic intestinal bacterial species by terminal restriction fragment patterns (TRFPs): a rapid, PCR-based method.

Identification of predominant human and animal intestinal tract anaerobes by conventional methods is cumbersome, time-consuming and less sensitive as compared to molecular methods. We have developed a molecular technique to identify most of the abundant intestinal microflora by polyermase chain reaction (PCR) amplification of a 16S rRNA gene fragment using a pair of universal PCR primers. The forward PCR primer was labelled with 6-carboxyfluorescein amino hexy (6-FAM) fluorescent dye to detect the terminal fragment of the PCR products after digestion with restriction enzymes. The PCR products were purified and digested with restriction enzymes and were analysed by capillary electrophoresis using an automated DNA sequencer. The data was analysed with GeneScan software 2.1. Eleven bacteria (Eubacterium biforme, E. limosum, Peptostreptococcus productus, Lactobacillus acidophilus, Bacteroides thetaiotaomicron, B. vulgatus, B. distasonis, Clostridium clostridiiforme, C. leptum, C. perfringens and Escherichia coli) that are predominant in human and animal intestinal tract were successfully identified by this rapid molecular technique. This protocol is rapid, accurate, sensitive and capable of identifying multiple organisms in a single sample.

Comparative molecular analysis of erythromycin-resistance determinants in staphylococcal isolates of poultry and human origin.

The ermA, ermB, ermC and msrA/msrB genes were detected in multidrug-resistant Staphylococcus spp. strains by PCR. Among 25 human clinical staphylococcal isolates the ermA, ermB, ermC and the msrA/msrB genes were detected in 88, 72, 4 and 100% of the strains, respectively. Among 24 poultry isolates the ermA, ermB, ermC and the msrA/msrB genes were detected in 100, 16.6, 50 and 12.5% of the strains, respectively. The ermA gene was found exclusively on the chromosome, whereas the ermC gene was found on 2.4-4.2 kb plasmids. Restriction fragment length polymorphism (RFLP) analysis of the ermA gene with Eco RI revealed five patterns (25.0, 21.0, 10.5, 6.2 and 4. 8 kb) for the clinical strains and two (8.0 and 6.2 kb) for the poultry strains. The 6.2 kb RFLP pattern, in both the poultry and human clinical isolates, indicates a common lineage for the ermA gene.

Characterization of erythromycin-resistant methylase genes from multiple antibiotic resistant Staphylococcus spp isolated from milk samples of lactating cows.

OBJECTIVE: To isolate and characterize erythromycin-resistant methylase genes in multiple-antibiotic resistant staphylococci isolated from milk samples. ANIMALS: 300 lactating cows. PROCEDURE: 23 erythromycin-resistant staphylococci were isolated from milk samples of 300 lactating cows. The prevalence of erythromycin-resistant methylase (erm) genes, ermC and ermA genes, and the multicomponent macrolide efflux pump in staphylococci msrA genes were identified and characterized by use of multiplex polymerase chain reaction (PCR), Southern hybridization, restricted fragment length polymorphism (RFLP) analysis, and dot-blot hybridization. RESULTS: Biochemical characterization indicated that 3 of 23 (13%) isolates were coagulase-positive Staphylococcus aureus, and the rest were coagulase-negative. Multiplex PCR resulted in amplification of a 520-base pair (bp) region of the ermC gene from the cell lysates of a strain of S simulans M-21 and S sciuri M-28. The ermC gene in both isolates was found on a 3-kilobase plasmid. The ermA gene was found on the chromosome of 21 isolates, and 6 RFLP patterns were observed. None of the isolates harbored the msrA gene. CONCLUSIONS: Erythromycin-resistant Staphylococcus spp isolated from milk samples of lactating cows may serve as reservoirs of erm genes homologous to those described in human isolates. However, the chromosomal insert patterns and prevalence of these genes, the sizes of plasmids harboring the genes, and the number of inserts of the genes (copy number) may differ from that of human isolates.

Transfer of erythromycin resistance from poultry to human clinical strains of Staphylococcus aureus.

The transfer of ermA and ermC genes, the two most common resistance determinants of erythromycin resistance, was studied with Luria-Bertani broth in the absence of additional Ca(2+) or Mg(2+) ions. Fifteen human and five poultry isolates of Staphylococcus aureus, which were resistant to erythromycin but carried different genetic markers for erythromycin resistance, were used for conjugation. Since both the donors (Amp(s)-Tet(r)) and recipients (Amp(r)-Tet(s)) were resistant to erythromycin, the transconjugants were initially picked up as ampicillin- and tetracycline-resistant colonies. The resistance transfer mechanisms of the chromosomally located erythromycin rRNA methylase gene ermA and the plasmid-borne ermC gene were monitored by a multiplex PCR and gene-specific internal probing assay. Four groups of transconjugants, based upon the transfer of the ermA and/or ermC gene, were distinguished from each other by the use of this method. Selective antibiotic screening revealed only one type of transconjugant that was resistant to ampicillin and tetracycline. A high frequency of transfer (4.5 x 10(-3)) was observed in all of the 23 transconjugants obtained, and the direction of tetracycline and erythromycin resistance marker transfer was determined to be from poultry to clinical isolates. The transfers of the ermA and ermC genes were via transposition and transformation, respectively.

Detection of multidrug-resistant Salmonella typhimurium DT104 by multiplex polymerase chain reaction.

Salmonella typhimurium definitive type 104 (DT104) is a virulent pathogen for humans and animals with many strains having multiple drug resistance characteristics. The organism typically carries resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT-resistant). A multiplex PCR method was developed to simultaneously amplify four genes, florfenicol (flo(st)), virulence (spvC), invasion (invA), and integron (int) from S. typhimurium DT104 (ACSSuT-type). Twenty-two ACSSuT-resistant DT104 isolates in our collection gave 100% positive reactions to this PCR assay by amplifying 584-, 392-, 321- and 265-bp PCR products, using primers specific to the respective target genes. One Salmonella strain DT23, ACSSuT-resistant, phage type 711 failed to amplify the 584-bp fragment, indicating that this method is specific for DT104-type ACSSuT-resistant S. typhimurium strains. One clinical and one bovine ASSuT-resistant strains that were sensitive to chloramphenicol and florfenicol did not yield a 584-bp fragment, indicating the absence of the flo(st) gene. This method will be useful for rapid identification of ACSSuT-type DT104 strains from clinical, food and environmental samples.

Simultaneous detection of erythromycin-resistant methylase genes ermA and ermC from Staphylococcus spp. by multiplex-PCR.

A comparative analysis of the two most dominant erythromycin-resistance determinant genes in Staphylococcus sppnamely, the ermA and ermC genes, was carried out. Sixty erythromycin-resistant strains of Staphylococcus spp. were tested, of which 24 were avian and 36 were clinical isolates. Our results indicated the prevalence of ermA over the ermC gene as opposed to the widely held opinion of the ermC gene being the most dominant resistance determinant gene. A multiplex-PCR assay was developed to detect the presence of ermA and ermC genes. Two pairs of primers, specific for the detection of ermA and ermC genes, were used in a multiplex-polymerase chain reaction (PCR) assay to yield amplified DNA products of 610 and 520 bp, respectively. Their digestion with restriction enzyme FokI that yielded a 477 bp and a 132 bp digestion product for ermA and a 333 bp and a 187 bp digestion product for ermC confirmed the authenticity of PCR products. The method could be used to amplify the ermA and ermC genes with as little as 5 pg of template DNA. The use of excess primers or the template DNA resulted in gene-specific amplification and no non-specific amplification was observed by changing the primer to primer or template to primer ratios. Furthermore, no amplification from erythromycin-sensitive S. aureus strain was observed. Using this assay, the poultry strains were found to contain either ermA alone (50%) or a combination of ermA (100%) and ermC (50%) both. The clinical strains contained either ermA (94.5%) or ermC (5.5%) but never both. The gene-specific internal probes were also used to verify the above findings and a high degree of correlation between the multiplex PCR and Southern hybridization data was observed.

Biochemical and molecular characterization of erthromycin-resistant avian Staphylococcus spp. isolated from chickens.

The epidemiology of the two common erythromycin-resistant methylase (erm) genes ermC and ermA was analyzed in 12 coagulase-negative Staphylococcus spp. and 34 coagulase-positive Staphylococcus spp. isolated from chicken. Southern hybridization indicated that only 2 of the 12 coagulase-negative Staphylococcus spp. strains contained the ermC gene on the plasmid; 1 strain of Staphylococcus xylosus harbored the ermC gene on a 2.5-kb plasmid, and 1 strain of Staphylococcus cohnii harbored the gene on a 4.0-kb plasmid. Twelve of the 34 strains of Staphylococcus aureus contained the ermC gene. Eleven of these strains had the ermC gene on a 2.5-kb plasmid, and 1 strain had the gene on a 4.0-kb plasmid. Ten of the 12 coagulase-negative Staphylococcus spp. and 22 of the 34 coagulase-positive Staphylococcus spp. harbored the ermA gene exclusively on the chromosome. Two different ermA EcoRI restriction fragment length polymorphisms (RFLP) were identified. A majority of the isolates was found to have two chromosomal inserts (8.0- and 6.2-kb EcoRI fragments) of ermA. One strain of S. aureus had different chromosomal inserts (6.4- and 5.8-kb EcoRI fragments) of ermA. Our results indicate that either the ermC or ermA gene, homologous to those described in human isolates, was present in all avian Staphylococcus spp. and that ermA was the predominant gene in coagulase-negative and coagulase-positive avian Staphylococcus spp. The size and copy numbers of the ermA gene were different from its human counterpart.

Direct in-gel hybridization of digoxigenin-labelled non-radioactive probes.

An improved, simple, cost-effective and non-radioactive procedure for in-gel hybridization is described for the detection of signal in dried agarose gels. Large and small digoxigenin-labelled DNA and oligonucleotide probes hybridized efficiently and specifically with the complementary DNA sequences in the gel. The signal-to-noise ratios for the gels dried at 55 degrees C at 1 atmospheric pressure were 3-3.5-fold higher than the gels dried at 25 degrees C under vacuum. The method shows an increased sensitivity over currently available non-radioactive methods for in-gel hybridization. A single copy of a gene insert could be detected by the use of this procedure.

Identification of Aeromonas trota (hybridization group 13) by amplification of the aerolysin gene using polymerase chain reaction.

Aeromonas trota is recognized as an important enteropathogen, and its haemolysin (aerolysin) is purported to be one of the virulence factors. Rapid detection and identification of A. trota is important for early and specific diagnosis of the infectious diseases that it causes. Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to amplify a species-specific sequence of the aerA gene, which encodes the aerolysin of A. trota. A DNA fragment of 622 bp was amplified from both lysed cells and isolated DNA from A. trota. The identity of the amplified 622 bp fragment was confirmed by digestion with BamH I restriction endonuclease, which produced the predicted 557 and 65 bp fragments. The lower limit for detection of the aerA gene by PCR amplification was 10 pg of total DNA or 10-15 cells ml-1. Primer specificity for A. trota was determined by the PCR assay with cells of 55 strains of Aeromonas sppincluding all of the 14 currently recognized DNA hybridization groups. A strain of Aeromonas enteropelogenes that had been reclassified as A. trota was also PCR positive. The method described here can be used to detect aerolysin-producing A. trota (hybridization group 13) strains from environmental and clinical samples without the use of selective media or additional biochemical tests.

Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine.

A gram-negative, rod-shaped bacterium capable of utilizing L-asparagine as its sole source of carbon and nitrogen was isolated from soil and identified as Enterobacter cloacae. An intracellularly expressed L-asparaginase was detected and it deaminated L-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction mixture indicated that 2.8 mM L-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase activity was found in cells cultured on L-fructose, D-galactose, saccharose, or maltose, and in cells cultured on L-asparagine as the sole nitrogen source. The pH and temperature optimum of L-asparaginase was 8.5 and 37-42 degrees C, respectively. The half-life of the enzyme at 30 degrees C and 37 degrees C was 10 and 8 h, respectively.

Influence of selected physical parameters on the biodegradation of acrylamide by immobilized cells of Rhodococcus sp.

The influences of concentration of acrylamide, pH, temperature, duration of storage of encapsulated cells and presence of different metals and chelators on the ability of immobilized cells of a Rhodococcus sp. to degrade acrylamide were evaluated. Immobilized cells (3 g) rapidly degraded 64 and 128 mM acrylamide in 3 and 5 h, respectively, whereas free cells took more than 24 h to degrade 64 mM acrylamide. An acrylamide concentration of 128 mM inhibited the growth of the free cells. Immobilized bacteria were slow to degrade acrylamide at 10 degrees C. Less than 60% of acrylamide was degraded in 4 h. However, 100% of the compound was degraded in less than 3 h at 28 degrees C and 45 degrees C. The optimum pH for the degradation of acrylamide by encapsulated cells was pH 7.0. Less than 10% of acrylamide was degraded at pH 6.0, while ca. 60% of acrylamide was degraded at pH 8.0 and 8.5. Copper and nickel inhibited the degradation, suggesting the presence of sulfhydryl (-SH) groups in the active sites of the acrylamide degrading amidase. Iron enhanced the rates of degradation and chelators (EDTA and 1,10 phenanthroline) reduced the rates of degradation suggesting the involvement of iron in its active site(s) of the acrylamide-degrading-amidase. Immobilized cells could be stored up to 10 days without any detectable loss of acrylamide-degrading activity.