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1.  Urinary tuberculosis is associated with the development of urothelial carcinoma but not renal cell carcinoma: a nationwide cohort study in Taiwan 
British Journal of Cancer  2013;109(11):2933-2940.
Obstructive uropathy and chronic urinary tract infection increase the risk of urinary tract cancer. Urinary tuberculosis (UTB) can cause chronic urinary tract inflammation, lead to obstructive uropathy, and potentially contribute to the development of urinary tract cancer. However, the association between UTB and urinary tract cancer has not been studied.
This study enrolled 135 142 tuberculosis (TB) cases (male, 69%) from a nationwide health insurance research database in Taiwan and investigated the risk factors for urinary tract cancer, with emphasis on a history of UTB. The incidence of urinary tract cancer in the general population without TB was also calculated for comparison.
The TB patients had a mean age of 57.5±19.5 years. Of the 1287 UTB and 133 855 non-UTB patients, 15 (1.2%) and 396 (0.3%) developed urothelial carcinoma, respectively (P<0.001); and 2 (0.2%) and 96 (0.1%) developed renal cell carcinoma, respectively (P=0.240). Cox regression analysis revealed that age, male sex, end-stage renal disease, obstructive uropathy, arsenic intoxication, organ transplantation, and UTB (hazard ratio: 3.38 (2.01–5.69)) were independent risk factors for urothelial carcinoma. The hazard ratio of UTB was higher among female patients (5.26 (2.12–13.06)) than that among male patients (2.96 (1.57–5.60)).
Urinary tuberculosis had a strong association with urothelial carcinoma, but not with renal cell carcinoma. In TB endemic areas, the urinary tract of TB patients should be scrutinised. It is also imperative that these patients be followed-up carefully in the post-treatment period, and urinalysis, ultrasonography or endoscopy should be an integral part of the follow-up.
PMCID: PMC3844900  PMID: 24129236
cohort study; obstructive uropathy; Taiwan; tuberculosis; urinary tract cancer; National Health Insurance Research Database
2.  Empirical treatment with a fluoroquinolone delays the treatment for tuberculosis and is associated with a poor prognosis in endemic areas 
Thorax  2006;61(10):903-908.
A study was conducted to evaluate the effect of the empirical use of fluoroquinolones on the timing of antituberculous treatment and the outcome of patients with tuberculosis in an endemic area.
All patients with culture confirmed tuberculosis aged ⩾14 years diagnosed between July 2002 and December 2003 were included and their medical records were reviewed.
Seventy nine (14.4%) of the 548 tuberculosis patients identified received a fluoroquinolone (FQ group), 218 received a non‐fluoroquinolone antibiotic (AB group), and 251 received no antibiotics before antituberculous treatment. Fifty two (65.8%) experienced clinical improvement after fluoroquinolone use. In the FQ group the median interval from the initial visit to starting antituberculous treatment was longer than in the AB group and in those who received no antibiotics (41 v 16 v 7 days), and the prognosis was worse (hazard ratio 6.88 (95% CI 1.84 to 25.72)). More patients in the FQ and AB groups were aged >65 years (53.2% and 61.0% v 31.5%), had underlying disease (53.2% and 46.8% v 34.3%), and were hypoalbuminaemic (67.2% and 64.9% v 35.1%). Of the nine mycobacterial isolates obtained after fluoroquinolone use from nine patients whose initial isolates were susceptible to ofloxacin, one (11.1%) was resistant to ofloxacin (after fluoroquinolone use for 7 days). Independent factors for a poor prognosis included empirical fluoroquinolone use, age >65, underlying disease, hypoalbuminaemia, and lack of early antituberculous treatment.
14.4% of our patients with tuberculosis received a fluoroquinolone before the diagnosis. With a 34 day delay in antituberculous treatment and more frequent coexistence of underlying disease and hypoalbuminaemia, empirical fluoroquinolone treatment was associated with a poor outcome. Mycobacterium tuberculosis isolates could obtain ofloxacin resistance within 1 week.
PMCID: PMC2104756  PMID: 16809417
fluoroquinolone; antibiotics; tuberculosis; treatment; prognosis
3.  Sequence analysis of scrA and scrB from Streptococcus sobrinus 6715. 
Infection and Immunity  1993;61(6):2602-2610.
The complete nucleotide sequences of Streptococcus sobrinus 6715 scrA and scrB, which encode sucrose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-phosphate hydrolase, respectively, have been determined. These two genes were transcribed divergently, and the initiation codons of the two open reading frames were 192 bp apart. The transcriptional initiation sites were determined by primer extension analysis, and the putative promoter regions of these two genes overlapped partially. The gene encoding enzyme IIScr, scrA, contained 1,896 nucleotides, and the molecular mass of the predicted protein was 66,529 Da. The hydropathy plot of the predicted amino acid sequence indicated that enzyme IIScr was a relatively hydrophobic protein. The gene encoding sucrose-6-phosphate hydrolase, scrB, contained 1,437 nucleotides. The molecular mass of the predicted protein was 54,501 Da, and the encoded enzyme was hydrophilic. The predicted amino acid sequences of the two open reading frames exhibited approximately 45 and 70% identity with those encoded by scrA and scrB, respectively, from Streptococcus mutans GS5. Homology also was observed between the N-terminal region of the S. sobrinus 6715 enzyme IIScr and other enzyme IIs specific for the glucopyranoside molecule, all of which generate glucopyranoside-6-phosphate during translocation and phosphorylation of the respective substrates. The sequence of the C-terminal domain of the S. sobrinus 6715 enzyme IIScr shared significant homology with enzyme IIIGlc from Escherichia coli and Salmonella typhimurium and with the C-terminal domain of enzyme IIBgl from E. coli, indicating that the two functional domains, enzyme IIScr and enzyme IIIScr, were covalently linked as a single polypeptide in S. sobrinus 6715. The deduced amino acid sequence of the gene product of S. sobrinus scrB shared strong homology with sucrase from Bacillus subtilis, Klebsiella pneumoniae, and Vibrio alginolyticus, suggesting conservation based on the physiological roles of these proteins.
PMCID: PMC280890  PMID: 8500898
4.  Aspergillus laryngotracheobronchitis presenting as stridor in a patient with peripheral T cell lymphoma. 
Thorax  1996;51(8):869-870.
Invasive aspergillosis is a serious opportunistic infection in immunocompromised patients. The case history is described of a 44 year old patient with peripheral T cell lymphoma who developed hoarseness and stridor after chemotherapy. Aspergillus fumigatus was isolated repeatedly from the sputum. Bronchoscopic examination showed symmetrical creamy-white exophytic lesions involving both vocal cords and the supraglottic area. There was diffuse tracheobronchitis with multiple raised cream-coloured plaques in the trachea which histologically consisted of numerous septate branching hyphae consistent with Aspergillus species. The lesions responded to systemic treatment with amphotericin B.
PMCID: PMC472577  PMID: 8795683
5.  Effects of tetracycline on the streptococcal flora of periodontal pockets. 
The effects of tetracycline on the subgingival streptococcal flora of periodontal patients were examined. Before antibiotic treatment, tetracycline-resistant isolates were obtained from 24 to 25 patients. In most patients, the proportion of the subgingival flora resistant to tetracycline increased after 2 weeks of therapy (1,000 mg of tetracycline/day) and then decreased after the cessation of treatment. Cultural conditions used for primary isolation were designed to favor the growth of facultative streptococci. Consequently, the majority (99%) of resistant isolates were identified as streptococci. Among 407 tetracycline-resistant Streptococcus isolates chosen for further classification, 9 species were identified, with S. sanguis (63%) and S. mitis (19%) predominating. There were no significant differences in the distribution of species isolated before and after treatment and after the cessation of tetracycline treatment. Plasmids were isolated from only 23 of 121 resistant streptococcal strains examined, suggesting that tetracycline resistance is not plasmid mediated in the majority of these oral streptococci.
PMCID: PMC283793  PMID: 7425602
6.  Rapid screening procedure for detection of plasmids in streptococci. 
Journal of Bacteriology  1979;140(3):1112-1115.
An enrichment procedure, yielding plasmid deoxyribonucleic acid preparations normally containing less than 5% chromosomal contamination, has been devised for the isolation of plasmids from virtually all species of streptococci. The procedure is rapid, reproducible, and inexpensive, requiring no radioisotopes or density gradient centrifugation. The procedure can be used for routine screening of several hundred isolates in a short period of time, and plasmids obtained from 10- to 20-ml cultures can readily be visualized in agarose gels.
PMCID: PMC216761  PMID: 533764
7.  Ultrasound guided pericardial drainage and intrapericardial instillation of mitomycin C for malignant pericardial effusion. 
Thorax  1994;49(6):594-595.
BACKGROUND--Conservative treatment of malignant pericardial effusion by intrapericardial instillation of a sclerosing agent may be an alternative to surgery. METHODS--Twenty patients with malignant pericardial effusion were treated by ultrasound guided pericardiocentesis and the intrapericardial instillation of mitomycin C. RESULTS--Mitomycin C was effective in controlling the pericardial effusion in 70% of patients without causing side effects, except for pericardial constriction seven months later in one subject. CONCLUSIONS--Ultrasound guided intrapericardial instillation of mitomycin C is a suitable alternative in the management of malignant pericardial effusion.
PMCID: PMC474956  PMID: 8016797
8.  Diagnosis of pulmonary cryptococcosis by ultrasound guided percutaneous aspiration. 
Thorax  1993;48(1):75-78.
BACKGROUND: Ultrasound is useful for locating thoracic lesions and guiding biopsy procedures. The use of sonographic appearances and ultrasound guided needle aspiration has led to the diagnosis of pulmonary cryptococcosis at this hospital. METHODS: Six hundred and eight patients who had ultrasound guided lung aspirations were reviewed retrospectively and nine with documented pulmonary cryptococcosis were collected. All patients had nodules or infiltrates on the chest radiograph. The needle aspirates obtained under ultrasound guidance were stained by Riu's or Papanicolaou's method or with India ink, and six were sent for culture. Five patients also underwent bronchoscopy and biopsy. RESULTS: The nine patients had 18 pulmonary lesions, of which 15 were nodules and three infiltrates. Fifteen lesions were detectable by ultrasound, which showed the nodules to be hypoechoic with eccentrically located air echoes. In eight of the nine cases cryptococci were detected after the lung aspirates had been stained with Riu's or Papanicolaou stain or with India ink. In five of the six aspirates sent for fungal culture Cryptococcus neoformans was isolated. The diagnostic yield was higher than that of bronchoscopy. None developed post-aspiration pneumothorax or any evidence of late dissemination. CONCLUSIONS: Because they tend to be subpleural pulmonary cryptococcal lesions seem to be identifiable by ultrasound. Ultrasound guided lung aspiration is an effective, rapid, and safe method for diagnosis.
PMCID: PMC464253  PMID: 8434359
9.  Melioidosis: an emerging infection in Taiwan? 
Emerging Infectious Diseases  2001;7(3):428-433.
From January 1982 to May 2000, 17 infections caused by Burkholderia pseudomallei were diagnosed in 15 patients in Taiwan; almost all the infections were diagnosed from 1994 to May 2000. Of the 15 patients, 9 (60%) had underlying diseases, and 10 (67%) had bacteremic pneumonia. Thirteen (76%) episodes of infection were considered indigenous. Four patients died of melioidosis. Seventeen B. pseudomallei isolates, recovered from eight patients from November 1996 to May 2000, were analyzed to determine their in vitro susceptibilities to 14 antimicrobial agents, cellular fatty acid and biochemical reaction profiles, and randomly amplified polymorphic DNA patterns. Eight strains (highly related isolates) were identified. All isolates were arabinose non-assimilators and were susceptible to amoxicillin-clavulanate, piperacillin-tazobactam, imipenem, and meropenem. No spread of the strain was documented.
PMCID: PMC2631793  PMID: 11384520
11.  Ultrasound guided aspiration biopsy for pulmonary tuberculosis with unusual radiographic appearances. 
Thorax  1993;48(2):167-170.
BACKGROUND: Pulmonary tuberculosis can produce unusual radiographic appearances and negative results of sputum and bronchoscopic examinations are common. This study assessed the value of ultrasound guided aspiration biopsy in the diagnosis of pulmonary tuberculosis with unusual radiographic appearances. METHODS: Thirteen patients, ultimately diagnosed as having tuberculosis, underwent a chest ultrasonographic examination between June 1984 and August 1991. All had sputum available for examination and nine were also examined by bronchoscopy. Ten patients who had a negative sputum smear and negative bronchoscopic brushing smears underwent ultrasound guided aspiration or biopsy. Percutaneous aspiration was performed with a 22 gauge needle. If the smear did not reveal acid fast bacilli, a biopsy sample was taken with a 16 gauge Tru-cut needle to obtain a histological diagnosis. RESULTS: The ultrasonographic examination delineated the more complex nature of the lesions better than the chest radiograph. Ultrasound guided aspiration biopsy provided the diagnosis in nine of 10 patients, while the sputum smear and culture provided diagnosis in five of 13, and bronchoscopy in four of nine. In terms of rapid diagnosis, ultrasound guided aspiration biopsy gave the diagnosis in eight of 10 cases. No patient developed a major complication. CONCLUSION: Ultrasonography can direct the needle to the most suitable part of a lesion to obtain the relevant specimens. The diagnostic yield is high and the procedure is relatively safe. It is especially helpful in patients with negative results of sputum and bronchoscopic examinations.
PMCID: PMC464298  PMID: 8493633
12.  Use of a novel mobilizable vector to inactivate the scrA gene of Streptococcus sobrinus by allelic replacement. 
Journal of Bacteriology  1995;177(17):5028-5034.
The virulence factors of the cariogenic bacterium Streptococcus sobrinus have been difficult to assess because of a lack of tools for the genetic manipulation of this organism. The construction of an Escherichia coli-Streptococcus shuttle vector, pDL289, that can be mobilized into S. sobrinus by the conjugative plasmid pAM beta 1 was described in a previous report. The vector contains pVA380-1 for replication and mobilization in streptococci, the pSC101 replicon for maintenance in E. coli, a kanamycin resistance marker that functions in both hosts, and the multiple cloning site and lacZ from pGEM7Zf(-). pDL289 is stable with or without selection in several species of Streptococcus. In this study, a derivative with a deletion in the minus origin of the pVA380-1 component of pDL289 was constructed. This derivative, pDL289 delta 202, was less stable than pDL289 in Streptococcus gordonii Challis, Streptococcus mutans, and S. sobrinus. Both pDL289 and pDL289 delta 202 were mobilizable by pAM beta 1 into S. sobrinus, with frequencies of 3 x 10(-6) and 1 x 10(-7) transconjugants per recipient CFU, respectively. The cloned scrA gene of S. sobrinus 6715-10 coding for the EIISuc of the sucrose-specific phosphoenolpyruvate phosphotransferase system was interrupted by the insertion of a streptococcal spectinomycin resistance gene active in E. coli and streptococci. The interrupted scrA gene was subcloned into both pDL289 and pDL289 delta 202. Each recombinant plasmid was introduced into the DL1 strain of S. gordonii Challis, which was then used as a recipient for the conjugative transfer of pAM beta 1. The latter plasmid was used to mobilize each recombinant plasmid from S. gordonii Challis DL1 to S. sobrinus 6715-10RF. Subsequently, recombinants derived from a double-crossover event were isolated on the basis of resistance to spectinomycin and susceptibility to kanamycin. Recombinational events were confirmed by Southern hybridization, and the inactivation of the EII Suc in double crossovers was confirmed by phosphotransferase system assays. This is the first report of allelic replacement in S. sobrinus.
PMCID: PMC177280  PMID: 7665480
13.  Direct determination of cryptococcal antigen in transthoracic needle aspirate for diagnosis of pulmonary cryptococcosis. 
Journal of Clinical Microbiology  1995;33(6):1588-1591.
Pulmonary cryptococcosis causes significant morbidity and mortality in immunocompromised patients. Definitive diagnosis of pulmonary cryptococcosis is usually difficult. The use of direct determination of cryptococcal antigen in transthoracic needle aspirate to diagnose pulmonary cryptococcosis was investigated. Over a 2-year period, we studied a total of 41 patients with respiratory symptoms and pulmonary infiltrates of unknown etiology who were suspected of having pulmonary cryptococcosis. Twenty-two patients were immunocompetent patients and 19 patients were immunocompromised. A diagnosis of pulmonary cryptococcosis was based on cytological examination, culture for Cryptococcus neoformans, histopathologic examination, and clinical response to antifungal therapy. All patients underwent chest ultrasound and ultrasound-guided percutaneous transthoracic needle aspiration to obtain specimens for cryptococcal antigen determination. The presence of cryptococcal antigen was determined by the latex agglutination system (CALAS; Meridian Diagnostics, Cincinnati, Ohio). An antigen titer equal to or greater than 1:8 was considered positive. The specimens were also sent for cytological examination, fungal culture, and/or histopathologic examination. A final diagnosis of pulmonary cryptococcosis was made in eight patients. Direct determinations of cryptococcal antigen in lung aspirate were positive in all eight patients with pulmonary cryptococcosis (100% sensitivity, 97% specificity, a positive predictive value of 89%, and negative value of 100%), and there was only one false-positive in noncryptococcosis patients. The diagnostic accuracy was 97.5%. Serum cryptococcal antigen was positive in only three patients with pulmonary cryptococcosis (sensitivity, 37.5%). This study showed that direct measurement of cryptococcal antigen in lung aspirate can be a rapid and useful test for diagnosis of pulmonary cryptococcosis.
PMCID: PMC228221  PMID: 7650192
14.  Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids. 
Infection and Immunity  1991;59(12):4621-4627.
Actinobacillus actinomycetemcomitans, a periodontal pathogen, has been strongly implicated in human periodontal disease. Advances in the molecular analysis of A. actinomycetemcomitans virulence factors have been limited due to the unavailability of systems for genetic transfer, transposon mutagenesis, and gene complementation. Slow progress can be traced almost exclusively to the lack of gene vector systems and methods for the introduction of DNA into A. actinomycetemcomitans. An electrotransformation system that allowed at least five strains of A. actinomycetemcomitans to be transformed with stable shuttle plasmids which efficiently replicated in both Escherichia coli and A. actinomycetemcomitans was developed. One plasmid, a potential shuttle vector designated pDL282, is 5.7 kb in size, has several unique restriction enzyme sites, and codes for resistance to spectinomycin and ampicillin. E. coli and A. actinomycetemcomitans were transformed with equal efficiencies of approximately 10(5) transformants per micrograms of DNA. Similar transformation efficiencies were obtained whether the plasmid DNA was isolated from A. actinomycetemcomitans or E. coli. In addition, frozen competent cells of A. actinomycetemcomitans yielded comparable efficiencies of transformation. Restriction enzyme analysis of pDL282 isolated after transformation confirmed the presence of intact donor plasmids. A plasmid isolated from A. pleuropneumoniae was also capable of transforming some isolates of A. actinomycetemcomitans, although generally at a lower frequency. The availability of these shuttle plasmids and an efficient transformation procedure should significantly facilitate the molecular analysis of virulence factors of A. actinomycetemcomitans.
PMCID: PMC259087  PMID: 1937823
15.  Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis. 
Enterococcus faecalis LDR55, a human clinical isolate, is resistant to tetracycline (Tcr), erythromycin (Emr), and high levels (greater than 2,000 micrograms/ml) of spectinomycin (Spr) but not streptomycin. Filter matings between strain LDR55 and E. faecalis OG1-RF produced transconjugants with the following resistance phenotypes: Tcr Emr Spr, Tcr Emr, Tcr Spr, and Tcr only but never Emr or Spr only. The genetic determinant encoding resistance to spectinomycin was cloned in Streptococcus sanguis Challis from pDL55, a 26-kb plasmid harbored by a Tcr Spr transconjugant. Subcloning experiments yielded a 1.1-kb ClaI-NdeI fragment that encoded very high-level Spr in S. sanguis (10 mg/ml) and Escherichia coli (50 mg/ml). Cell extracts of cultures obtained from Spr strains expressed adenylating activity for spectinomycin but not for streptomycin, indicating that Spr was due to an AAD(9) activity. The nucleotide base sequence of the 1.1-kb ClaI-NdeI fragment contained a single 750-base open reading frame. The protein predicted from the open reading frame consisted of 250 amino acids and had a calculated size of approximately 28,000 daltons, similar to the size estimated from maxicell analysis (29,000 daltons). The deduced amino acid sequence of the streptococcal AAD(9) was compared with that of the AAD(9) encoded by staphylococcal transposon Tn554. The two proteins shared approximately 39% amino acid identity, which was expanded to 53% when conservative amino acid changes were included. When the streptococcal protein was compared with an AAD(3")(9) protein of E. coli, the degrees of identity were 27 and 47%, on the basis of actual amino acids and conservative replacements, respectively. The cloning and nucleotide base sequence analyses of the spectinomycin AAD(9) determinant from E. faecalis that results in high-level Spr when transferred to S. sanguis or E. coli are presented.
PMCID: PMC245272  PMID: 1659306
16.  Improved electroporation and cloning vector system for gram-positive bacteria. 
A protocol for transformation of intact Enterococcus faecalis cells by electroporation was developed through a systematic examination of the effects of changes in various parameters, including (i) growth conditions; (ii) composition of the electroporation solution; (iii) electroporation conditions, such as field strength and resistance; (iv) size, concentration, and purity of DNA used for transformation; and (v) conditions used to select for transformants. Key features of this protocol include the use of exponential-phase cells grown in inhibitory concentrations of glycine and the use of an acidic sucrose electroporation solution. Frequencies of greater than 2 x 10(5) transformants per microgram of plasmid DNA were obtained for E. faecalis cells, whereas various strains of streptococci and Bacillus anthracis were transformed at frequencies of 10(3) to 10(4) transformants per microgram of plasmid DNA with the same protocol. A novel Escherichia coli-Streptococcus and Enterococcus shuttle cloning vector, pDL276, was constructed for use in conjunction with the electroporation system. This vector features a multiple cloning site region flanked by E. coli transcription termination sequences, a relatively small size (less than 7 kb), and a kanamycin resistance determinant expressed in both gram-positive and gram-negative hosts. Various enterococcal and streptococcal DNA sequences were cloned in E. coli (including sequences that could not be cloned on other vectors) and were returned to the original host by electroporation. The vector and electroporation system was also used to clone directly into E. faecalis.
PMCID: PMC182867  PMID: 1905518
17.  Nucleotide sequence analysis of tetracycline resistance gene tetO from Streptococcus mutans DL5. 
Journal of Bacteriology  1988;170(8):3618-3626.
Streptococcus mutans DL5, isolated from the dental plaque of a pig, was resistant to high levels of streptomycin (Sm, 20 mg/ml), erythromycin (Em, 1 mg/ml), and tetracycline (Tc, greater than 100 micrograms/ml), but contained no detectable plasmid DNA. The Smr and Emr determinants were cloned from cellular DNA on the self-replicating 5-kilobase-pair (kbp) EcoRI fragment of pAM beta 1 and the 4.2-kbp cryptic plasmid pVA380-1, respectively, by transformation of Streptococcus sanguis Challis. Helper plasmid cloning, with a Challis host containing pVA380-1, was required to clone the Tcr determinant of strain DL5 on this vector. A single-colony isolate of the original Tcr clone contained a hybrid plasmid, pDL421, composed of 2.6 kbp of vector DNA and 11.4 kbp of S. mutans DNA. Plasmid pDL421 did not hybridize to plasmids containing the streptococcal Tcr determinants tetL, tetM, and tetN. A shortened derivative of this hybrid plasmid, pDL422, missing a 4.9-kbp HincII fragment from the S. mutans DNA but still encoding Tcr, was obtained by subcloning in S. sanguis Challis. The Tcr gene was located in a 1,917-base-pair open reading frame (ORF) corresponding to a 72-kilodalton protein. The ORF exhibited 99.4% sequence identity with the 1,917-base-pair tetO gene from a strain of Campylobacter coli (W. Sougakoff, B. Papadopoulou, P. Nordmann, and P. Courvalin, FEMS Microbiol. Lett. 44:153-160, 1987). A 1.67-kbp NdeI fragment, internal to the ORF from strain DL5, as well as pDL421 hybridized under stringent conditions to DNA from 10 of 10 Tcr strains of C. coli and Campylobacter jejuni from human and animal sources, but not to DNA from Tcs isolates of these two species.
PMCID: PMC211336  PMID: 2841293
18.  Molecular and genetic characterization of lactose-metabolic genes of Streptococcus cremoris. 
Journal of Bacteriology  1986;167(3):855-862.
Lac+ plasmid DNA from Streptococcus cremoris H2 was subcloned with an Escherichia coli vector on a 3.5-kilobase-pair PstI-AvaI fragment. Genetic analysis of the cloned DNA was possible because linear Lac+ DNA fragments were productive in the S. sanguis transformation system. Complementation of S. sanguis Lac-mutants showed that the 3.5-kilobase-pair fragment included the structural gene for 6-phospho-beta-D-galactosidase and either enzyme II-lac or factor III-lac of the lactose-specific phosphoenolpyruvate-dependent phosphotransferase system. Expression of the S. cremoris-like 40,000-dalton 6-phospho-beta-D-galactosidase in S. sanguis Lac+ transformants, rather than the 52,000-dalton wild-type S. sanguis enzyme, demonstrated the occurrence of gene replacement and not gene repair. The evidence supports chromosomal integration as the mechanism by which S. sanguis Lac- recipients are converted to a Lac+ phenotype after transformation with Lac+ DNA. Southern blot data suggest that the Lac+ DNA does not reside on a transposon, but that integration always occurs within a specific HincII fragment of the recipient chromosome. Hybridization experiments demonstrate homology between the S. cremoris Lac+ DNA and cellular DNA from Lac+ strains of Streptococcus lactis, S. mutans, S. faecalis, and S. sanguis.
PMCID: PMC215952  PMID: 3091581
19.  Broad geographical distribution of homologous erythromycin, kanamycin, and streptomycin resistance determinants among group D streptococci of human and animal origin. 
The Emr, Kmr, and Smr determinants of the Streptococcus faecalis R plasmid pJH1 were cloned in Streptococcus sanguis with a streptococcal plasmid vector, pVA380-1. Each cloned determinant was used as a probe in hybridization reactions with dot blots containing plasmid-enriched DNA from 91 group D streptococcal isolates resistant to erythromycin, kanamycin, and streptomycin; the isolates were obtained from animal and human sources in a variety of geographical locations. Nearly 70% of the strains contained DNA that hybridized to each of the three resistance determinants from pJH1. Five plasmids mediating resistance to erythromycin, kanamycin, and streptomycin were examined in more detail. These plasmids varied in size between 26 and 105 kilobase pairs (kbp) and exhibited very different EcoRI restriction patterns. However, each plasmid contained the resistance determinants on a single 13- to 20-kbp EcoRI fragment. Southern blot hybridizations and additional restriction endonuclease digests revealed extensive DNA sequence homology and virtually indistinguishable restriction endonuclease maps within a 9- to 11-kbp region of each plasmid which included the resistance determinants.
PMCID: PMC180439  PMID: 3010845
20.  Evidence for a disseminated erythromycin resistance determinant mediated by Tn917-like sequences among group D streptococci isolated from pigs, chickens, and humans. 
A total of 199 streptococci isolated from feces of healthy chickens, pigs, and beef cattle and 26 human clinical isolates were tested for resistance to kanamycin, streptomycin, tetracycline, erythromycin, and lincomycin. Of 66 isolates resistant to these antibiotics, 12 transferred one or more resistance traits by conjugation in broth. Erythromycin resistance (Emr) was transferred from 10 of the 12 successful donors. AvaI digests of plasmids isolated from Emr transconjugants derived from two human, two chicken, and one pig isolate contained three fragments similar in size to those produced from Tn3871, an Emr transposon. The three fragments from each of the five digests on Southern blots hybridized to radiolabeled Tn3871. Plasmid DNA from a transconjugant derived from a second pig isolate contained two of the three Tn3871-associated AvaI fragments. One of the AvaI fragments from each of the six plasmids hybridized with a radiolabeled probe containing a cloned AvaI fragment from Tn3871 that contained the Emr determinant. Transposition of the Emr trait was demonstrated for the plasmids derived from one human and one pig isolate. We concluded that extensive DNA homology existed between plasmids from streptococcal strains obtained from two human patients, two chickens, and two pigs and the Emr transposon Tn3871, which is very similar or identical to the well-characterized Emr transposon Tn917. The detection of Tn3871-like sequences in streptococcal isolates from Arkansas, Illinois, North Carolina, and Washington, D.C. indicates wide dissemination of Emr mediated by the same or closely related transposons.
PMCID: PMC180070  PMID: 2988428
21.  Physical and genetic analyses of streptococcal plasmid pAM beta 1 and cloning of its replication region. 
Journal of Bacteriology  1984;157(2):445-453.
Plasmid pAM beta 1, originally isolated from Streptococcus faecalis DS5, mediates resistance to the MLS (macrolide, lincosamide, and streptogramin B alpha) group of antibiotics. A restriction endonuclease map of the 26.5-kilobase (kb) pAM beta 1 molecule was constructed by using the enzymes AvaI, HpaII, EcoRI, PvuII, Kpn1, BstEII, HpaI, HhaI, and HindIII. A comparison of this map to those of four independently isolated deletion derivatives of pAM beta 1 located the MLS resistance determinant within a 2-kb DNA segment and at least one conjugative function within an 8-kb region. The 5.0-kb EcoRI-B fragment from pAM beta 1 was ligated onto the 4.0-kb Escherichia coli plasmid vector, pACKC1, and used to transform E. coli HB101. The 9.0-kb chimeric plasmid was then used to transform Streptococcus sanguis Challis with concurrent expression of the E. coli kanamycin resistance determinant. The 5.0-kb EcoRI-B fragment from pAM beta 1 was subsequently used as a vector to clone a streptomycin resistance determinant from a strain of Streptococcus mutans containing no detectable plasmid DNA. Subcloning experiments, using a HindIII partial digest of pAM beta 1 DNA, narrowed the replication region of this plasmid to a 2.95-kb fragment.
PMCID: PMC215268  PMID: 6319361
22.  Broad geographical distribution of a cytotoxin gene mediating beta-hemolysis and bacteriocin activity among Streptococcus faecalis strains. 
Infection and Immunity  1983;40(3):1015-1022.
Conjugative hemolysin-bacteriocin plasmids were isolated from Streptococcus faecalis var. zymogenes strains of diverse geographical origins. Cloned DNA fragments containing the hemolysin-bacteriocin gene(s) from one of these plasmids (pAD1) hybridized to two EcoRI fragments of identical size from each of the five plasmids examined. Results of hybridization experiments in which total plasmid DNA was used suggested that the plasmids shared extensive homology. Two of the plasmids, pAD1 from strain DS16 (Ann Arbor, Mich.) and pAM gamma 1 from strain DS5 (Miami, Fla.), were 100% homologous and had identical EcoRI restriction patterns (eight fragments each). There was no detectable homology between the plasmid-mediated hemolysin determinants of S. faecalis and DNA from other beta-hemolytic streptococci (Lancefield groups A, B, F, or H).
PMCID: PMC348152  PMID: 6303955
23.  Characterization of two tetracycline resistance determinants in Streptococcus faecalis JH1. 
Journal of Bacteriology  1982;150(2):835-843.
Streptococcus faecalis strain JH1 harbors two conjugative plasmids: pJH1, an R plasmid mediating resistance to kanamycin, streptomycin, gentamicin, erythromycin, and tetracycline, and pJH2, a hemolysin-bacteriocin plasmid. Studies of plasmid-cured derivatives of strain JH1 and of transconjugates obtained after mixed incubation of JH1 with the plasmid-free S. faecalis strain JH2-2 revealed the presence of two tetracycline resistance determinants in strain JH1. One determinant mediated constitutive resistance to 40 micrograms of tetracycline per ml and was associated with plasmid pJH1. The second determinant, either on the chromosome of strain JH1 or on an undetectable plasmid, was inducible by tetracycline and enabled the host strain, in the absence of pJH1, to grow in the presence of 80 micrograms of tetracycline per ml. One transconjugant, strain DL172, was resistant to 80 micrograms of tetracycline per ml, sensitive to kanamycin, streptomycin, and erythromycin, and hemolytic in the presence, but not in the absence, of tetracycline. A single plasmid, pDL172, from this strain consisted of plasmid pJH2 and a 17.8-kilobase segment of DNA homologous to total cell DNA from strain JH1 but did not contain plasmid pJH1. Whether the addition of heterologous DNA to plasmid pJH2 occurred by translocation of a 17.8-kilobase tetracycline resistance transposon or by classical recombination with pJH2 has not been determined.
PMCID: PMC216436  PMID: 6802800
24.  Tween 80 effect on glucosyltransferase synthesis by Streptococcus salivarius. 
Journal of Bacteriology  1978;133(1):231-239.
Streptococcus salivarius (ATCC 25975) produced very low or nondetectable amounts of the extracellular enzyme glucosyltransferase (GTase) when grown in a chemically defined medium. The addition of Tween 80 to this medium resulted in the production of markedly enhanced levels of the enzyme. Oleic acid, the methyl ester of oleic acid, and sucrose each could not substitute for Tween 80 in this regard. The surfactant had no direct activating effect on performed enzyme activity. Tween 80 also stimulated the production of GTase by concentrated cells suspended in defined medium during a time when no measurable growth occurred. Under these conditions, the stimulatory effect of Tween 80 was blocked by chloramphenicol. It was further found that the surfactant dramatically stimulated the differential rate of GTase synthesis. These and other data strongly suggest that Tween 80 stimulates the production of extracellular GTase by acting either directly or indirectly at the level of enzyme synthesis.
PMCID: PMC221999  PMID: 618839

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