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.

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.

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.

Rapid purification of an active recombinant His-tagged 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of 2,3-dihydroxybiphenyl, the third step in the biphenyl degradation pathway. The nucleotide sequence of the Pseudomonas putida OU83 gene bphC, which encodes 2,3-DBPD, was cloned into a plasmid pQE31. The His-tagged 2,3-DBPD produced by a recombinant Escherichia coli strain, SG13009(pREP4)(pAKC1), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 2,3-DBPD construction, carrying a single 6 x His tail on the N-terminal of the polypeptide, was active. SDS-PAGE analysis of the purified active 2,3-DBPD gave a single band of 34 kDa; this is in agreement with the size of the bphC coding region. The K(m) for 2,3-dihydroxybiphenyl was 14.5 +/- 2 microM. The enzyme activity was enhanced by ferrous ion but inhibited by ferric ion. The enzyme activity was inhibited by thiol-blocking reagents and heavy metals HgCl2, CuSO4, NiSO4, and CdCl2. The yield was much higher and the time required to purify recombinant 2,3-DBPD from clone pAKCl was faster than by the conventional chromatography procedures.

Nucleotide sequence of the gene encoding cis-biphenyl dihydrodiol dehydrogenase (bphB) and the expression of an active recombinant His-tagged bphB gene product from a PCB degrading bacterium, Pseudomonas putida OU83.

The nucleotide sequence of the bphB gene of Pseudomonas putida strain OU83 was determined. The bphB gene, which encodes cis-biphenyl dihydrodiol dehydrogenase (BDDH), was composed of 834 base pairs with an ATG initiation codon and a TGA termination codon. It can encode a polypeptide of 28.91 kDa, containing 277 amino acids. Promoter-like and ribosome-binding sequences were identified upstream of the bphB gene. The bphB nucleotide sequence was used to produce His-tagged BDDH, in Escherichia coli. The His-tagged BDDH construction, carrying a single 6 x His tail on the N-terminal portion, was active. The molecular mass of the native enzyme was 128 kDa and on SDS-PAGE analysis the molecular mass was 31 kDa. This enzyme requires NAD+ for its activity and its optimum pH is 8.5. Nucleotide and the deduced amino acid sequence analyses revealed a high degree of homology between the bphB gene from Pseudomonas putida OU83 and the bphB genes from P. cepacia LB400 and P. pseudoalcaligenes KF707.

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.

Degradation of organic cyanides by Pseudomonas aeruginosa.

A bacterium capable of utilizing acetonitrile (methyl cyanide) as the sole source of carbon and nitrogen was isolated from soil and identified as Pseudomonas aeruginosa. This bacterium could also utilize and oxidize numerous lower-mol-wt nitrile compounds and their corresponding amides as growth substrates. A metabolite of acetonitrile in the culture medium was determined to be ammonia. The accumulation of ammonia in the culture medium was proportional to the concentration of the substrate and the inoculum. Cell extracts of the bacterium contained activities corresponding to nitrile aminohydrolase (E C 3.5.5.1) and amidase (E C 3.5.1.4), which regulate the degradation of acetonitrile. Both enzymes were inducible and hydrolyzed a wide range of substrates, and it was determined that the specific activity of amidase was far greater than the activity of nitrile aminohydrolase.

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.

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.

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.

Simultaneous degradation of acetonitrile and biphenyl by Pseudomonas aeruginosa.

A bacterium capable of utilizing either acetonitrile as the sole source of carbon and nitrogen or biphenyl as the sole source of carbon was isolated from soil and identified as Pseudomonas aeruginosa. The bacterium also utilized other nitriles, amides, and polychlorinated biphenyls (PCBs) as growth substrates. Acetonitrile- or biphenyl-grown cells oxidized these substrates without a lag. In studies with [14C]acetonitrile, nearly 74% of the carbon was recovered as 14CO2 and 8% was associated with the biomass. In studies with [14C]biphenyl, nearly 68% of the carbon was recovered as 14CO2 and nearly 6% was associated with the biomass. Although higher concentrations of acetonitrile as the sole sources of nitrogen inhibited the rates of [14C]biphenyl mineralization, lower concentrations (0.05%, w/v) gave a 77% stimulation in 14CO2 recovery. Pseudomonas aeruginosa metabolized acetonitrile to ammonia and acetic acid and biphenyl to benzoic acid. The bacterium also simultaneously utilized biphenyl as the sole carbon source and acetonitrile as the sole nitrogen source. However, biphenyl utilization increased only after the depletion of acetonitrile. Metabolites of the mixed substrate were ammonia and benzoic acid, which completely disappeared in the later stages of incubation. Nitrile hydratase and amidase were responsible for the transformation of acetonitrile to acetic acid and ammonia.

Degradation of Acetonitrile by Pseudomonas putida.

A bacterium capable of utilizing high concentrations of acetonitrile as the sole source of carbon and nitrogen was isolated from soil and identified as Pseudomonas putida. This bacterium could also utilize butyronitrile, glutaronitrile, isobutyronitrile, methacrylonitrile, propionitrile, succinonitrile, valeronitrile, and some of their corresponding amides, such as acetamide, butyramide, isobutyramide, methacrylamide, propionamide, and succinamide as growth substrates. Acetonitrile-grown cells oxidized acetonitrile with a K(m) of 40.61 mM. Mass balance studies with [C]acetonitrile indicated that nearly 66% of carbon of acetonitrile was released as CO(2) and 14% was associated with the biomass. Metabolites of acetonitrile in the culture medium were acetic acid and ammonia. The acetate formed in the early stages of growth completely disappeared in the later stages. Cell extracts of acetonitrile-grown cells contained activities corresponding to nitrile hydratase and amidase, which mediate the breakdown of actonitrile into acetic acid and ammonia. Both enzymes were intracellular and inducible and hydrolyzed a wide range of substrates. The specific activity of amidase was at least 150-fold higher than the activity of the enzyme nitrile hydratase.

Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.

Degradation of acrylamide by immobilized cells of a Pseudomonas sp. and Xanthomonas maltophilia.

Two bacterial isolates capable of utilizing acrylamide as the sole source of carbon and nitrogen were isolated from herbicide-contaminated soil samples and identified as Pseudomonas sp. and Xanthomonas maltophilia. Batch cultures of Pseudomonas sp. and X. maltophilia completely degraded 62.8 mM acrylamide to acrylic acid and ammonia in 24 and 48 h, respectively. Pseudomonas sp. and X. maltophilia, when immobilized in calcium alginate, markedly increased the rate of degradation of acrylamide over batch cultures. Cells of the isolates immobilized in calcium alginate degraded acrylamide to acrylic acid and ammonia in less than 6 h. Initial metabolism of acrylamide by immobilized cells of Pseudomonas sp. followed by inoculation with nonimmobilized cells after 6 h totally removed acrylamide and its metabolites in 72 h. A similar procedure with X. maltophilia resulted in the total metabolism of acrylamide in 96 h. An inducible, intracellular amidase was responsible for the hydrolysis of acrylamide to acrylic acid and ammonia. The specific activity of Pseudomonas sp. amidase was higher than the specific activity of X. maltophilia amidase.

Detection of erythromycin resistant methylase gene by the polymerase chain reaction.

A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 520-bp region of the erythromycin resistant methylase (ermC) gene sequence. The identity of the PCR-amplified 520-bp DNA was confirmed by HinCII endonuclease restriction digestion, which produced the predicted 440-bp and 80-bp DNA fragments. A 20-mer (alpha-32P) oligonucleotide probe specifically hybridized with these amplified products confirming the specificity and reliability of this diagnostic assay. The assay could detect the ermC gene in bacterial suspensions containing as few as 10(3) cells ml-1. The assay was used to detect the presence of the ermC gene in several Gram-positive bacterial strains identified as Streptococcus sp., Staphylococcus sp., Micrococcus sp., Lactobacillus sp. and Enterococcus sp., isolated from water samples maintained in experimental animal cages and clinical sources. Only bacteria identified as Staphylococcus sp. were resistant to the antibiotic. Although 17 strains of Staphylococcus sp. isolated from clinical samples were resistant to erythromycin, only seven of these isolates tested positive for the presence of the ermC gene. Of these strains, five were identified as coagulase-positive S. aureus and the rest were identified as coagulase-negative S. epidermidis. The erythromycin resistance in all seven ermC positive isolates was constitutive. The entire diagnostic assay, including template preparation, amplification and electrophoresis can be completed within 6 h.

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.

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.