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PubMed1997

Influence of genes encoding proton-translocating enzymes on suppression of Salmonella typhimurium growth and colonization.

Twenty-four-hour-old, aerobically grown, Luria-Bertani broth cultures of Salmonella typhimurium F98 suppressed the growth of a spectinomycin-resistant (Spcr) derivative of the same strain inoculated at 10(3) CFU ml(-1). This growth suppression is genus specific and RpoS independent, and it is not solely a result of nutrient depletion (P. A. Barrow, M. A. Lovell, and L. Zhang-Barber, J. Bacteriol. 178:3072-3076, 1996). Mutations in three genes are shown here to significantly reduce growth suppression under these conditions. The mutations were located in the nuo, cyd, and unc operons, which code for the NADH dehydrogenase I, cytochrome d oxidase, and F0F1 proton-translocating ATPase complexes, respectively. When cultures were grown under strictly anaerobic conditions, only the unc mutant did not suppress growth. Prior colonization of the alimentary tract of newly hatched chickens with the S. typhimurium F98 wild type or nuo or cyd mutants suppressed colonization by an S. typhimurium F98 Spcr derivative inoculated 24 h later. In contrast, the S. typhimurium unc mutant did not suppress colonization. The nuo and unc mutants showed poorer growth on certain carbon sources. The data support the hypothesis that growth suppression operates because of the absence of a utilizable carbon source or electron acceptor.

Anaerobiosis↗
PubMed1997

Construction and characterization of a mutant of alkaliphilic Bacillus firmus OF4 with a disrupted cta operon and purification of a novel cytochrome bd.

The caa3-type terminal oxidase of Bacillus firmus OF4 has been proposed to play an important role in the growth and bioenergetics of this alkaliphile (A. A. Guffanti and T. A. Krulwich, J. Biol. Chem. 267:9580-9588, 1992). A mutant strain was generated in which the cta operon encoding the oxidase was disrupted by insertion of a spectinomycin resistance cassette. The mutant was unable to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Absorption spectra of membranes confirmed the loss of the enzyme and indicated the presence of a cytochrome bd-type terminal oxidase. The mutant could grow on glucose but was unable to grow on malate or other nonfermentative carbon sources, despite the presence of the cytochrome bd. The cytochrome bd was purified from the mutant. The enzyme consisted of two subunits and, with menadiol as substrate, consumed oxygen with a specific activity of 12 micromol of O2 x min(-1) x mg(-1). In contrast to both cytochromes bd of Escherichia coli, the enzyme did not utilize TMPD as an electron source. A number of additional features, including subunit size and spectral properties, distinguish this cytochrome bd from its counterparts in E. coli and Azotobacter vinelandii.

Alkalies↗
PubMed1999

Pkg2, a novel transmembrane protein Ser/Thr kinase of Streptomyces granaticolor.

A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.

Amino Acid Sequence↗
PubMed2000

Role of the dpr product in oxygen tolerance in Streptococcus mutans.

We have previously identified and characterized the alkyl hydroperoxide reductase of Streptococcus mutans, which consists of two components, Nox-1 and AhpC. Deletion of both nox-1 and ahpC had no effect on the sensitivity of S. mutans to cumene hydroperoxide or H(2)O(2), implying that the existence of another antioxidant system(s) independent of the Nox-1-AhpC system compensates for the deficiency. Here, a new antioxidant gene (dpr for Dps-like peroxide resistance gene) was isolated from the S. mutans chromosome by its ability to complement an ahpCF deletion mutant of Escherichia coli with a tert-butyl hydroperoxide-hypersensitive phenotype. The dpr gene complemented the defect in peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. Under aerobic conditions, the dpr disruption mutant carrying a spectinomycin resistance gene (dpr::Spc(r) mutant) grew as well as wild-type S. mutans in liquid medium. However, the dpr::Spc(r) mutant could not form colonies on an agar plate under air. In addition, neither the dpr::Spc(r) ahpC::Em(r)::nox-1 triple mutant nor the dpr::Spc(r) sod::Em(r) double mutant was able to grow aerobically in liquid medium. The 20-kDa dpr gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of S. mutans cells to air. We propose a mechanism by which Dpr confers aerotolerance on S. mutans.

Aerobiosis↗
PubMed2001

Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality.

The discovery of toxin-antitoxin gene pairs (also called addiction modules) on extrachromosomal elements of Escherichia coli, and particularly the discovery of homologous modules on the bacterial chromosome, suggest that a potential for programmed cell death may be inherent in bacterial cultures. We have reported on the E. coli mazEF system, a regulatable addiction module located on the bacterial chromosome. MazF is a stable toxin and MazE is a labile antitoxin. Here we show that cell death mediated by the E. coli mazEF module can be triggered by several antibiotics (rifampicin, chloramphenicol, and spectinomycin) that are general inhibitors of transcription and/or translation. These antibiotics inhibit the continuous expression of the labile antitoxin MazE, and as a result, the stable toxin MazF causes cell death. Our results have implications for the possible mode(s) of action of this group of antibiotics.

Anti-Bacterial Agents↗
PubMed2003

Accumulation of S-adenosyl-L-methionine enhances production of actinorhodin but inhibits sporulation in Streptomyces lividans TK23.

S-Adenosyl-L-methionine synthetase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. Despite extensive research with many organisms, its role in Streptomyces sp. remains unclear. In the present study, the putative SAM-s gene was isolated from a spectinomycin producer, Streptomyces spectabilis. The purified protein from the transformed Escherichia coli with the isolated gene synthesized SAM from L-methionine and ATP in vitro, strongly indicating that the isolated gene indeed encoded the SAM-s protein. The overexpression of the SAM-s gene in Streptomyces lividans TK23 inhibited sporulation and aerial mycelium formation but enhanced the production of actinorhodin in both agar plates and liquid media. Surprisingly, the overexpressed SAM was proven by Northern analysis to increase the production of actinorhodin through the induction of actII-ORF4, a transcription activator of actinorhodin biosynthetic gene clusters. In addition, we found that a certain level of intracellular SAM is critical for the induction of antibiotic biosynthetic genes, since the control strain harboring only the plasmid DNA did not show any induction of actII-ORF4 until it reached a certain level of SAM in the cell. From these results, we concluded that the SAM plays important roles as an intracellular factor in both cellular differentiation and antibiotic production in Streptomyces sp.

Amino Acid Sequence↗
PubMed1969

Phenotypic suppression in Escherichia coli by chloramphenicol and other reversible inhibitors of the ribosome.

Antibiotics that interfere reversibly with various aspects of ribosomal function (chloramphenicol, tetracycline, erythromycin, and spectinomycin) are shown to antagonize, at barely inhibitory concentrations, the inhibitory effect of low concentrations of streptomycin (SM) on the growth of Escherichia coli. Paradoxically, these compounds can also replace SM in supporting the growth of conditionally SM-dependent mutants. Chloramphenicol produced about as much phenotypic suppression as SM in SM-sensitive strains, but less than that attainable with high concentrations of SM in resistant strains. The antagonism to SM inhibition and the phenotypic suppression appear to be specific for those growth inhibitors that act on the ribosome. Since inhibitors of the 50S subunit of the ribosome (chloramphenicol, erythromycin) are as active as inhibitors of the 30S subunit, it is suggested that phenotypic suppression by borderline concentrations of ribosome inhibitors does not necessarily depend on an alteration of the recognition region of the ribosome. Alternatively, partial inhibition of the ribosomes might change the environment in a way that would influence the frequency of misreading. Phenotypic suppression by a low concentration of SM as well as by chloramphenicol was found to depend on the presence of a trace of the required growth factor.

Bacterial Proteins↗
PubMed2006

Clonal relationship among Vibrio cholerae O1 El Tor strains causing the largest cholera epidemic in Kenya in the late 1990s.

Eighty Vibrio cholerae O1 strains selected to represent the 1998-to-1999 history of the largest cholera epidemic in Kenya were characterized by ribotyping, antimicrobial susceptibility, and random amplified polymorphic DNA patterns. Except for 19 strains from 4 local outbreaks in North Eastern Province along the Somalia border, the other 61 strains from 25 outbreaks occurring in districts scattered around the country were all ribotype B27 and resistant to chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim. The 61 strains showed similar and specific amplified DNA patterns. These findings indicate that the predominant strains that caused the Kenyan epidemic had a clonal origin and suggest that ribotype B27 strains, which first appeared in West Africa in 1994, have had a rapid spread to eastern Africa.

Anti-Bacterial Agents↗
PubMed1982

Isolation of Treponema hyodysenteriae from wild rodents.

Rodents from swine-producing farms were examined for the presence of Treponema hyodysenteriae. Wild mice (n = 257) and rats (n = 41) were trapped on eight farms. Ceca were removed aseptically, and the contents and mucosal scrapings were cultured on selective medium (blood agar containing 400 micrograms of spectinomycin per ml). T. hyodysenteriae was detected in the cecal scrapings of four mice from three different farms where swine dysentery had occurred. Gross lesions were detected in the ceca in two of the four mice. In addition, Treponema innocens was detected in the cecal scrapings of 12 mice and 13 rats. Three of the four T. hyodysenteriae isolates were pathogenic when inoculated intragastrically into swine. The results of this investigation suggest that wild rodents may be carriers of T. hyodysenteriae.

Animal Population Groups↗
PubMed1985

Antimicrobial susceptibility and characterization of outer membrane proteins of Haemophilus ducreyi isolated in Thailand.

One hundred strains of Haemophilus ducreyi isolated in Thailand from patients with chancroid were tested by the agar dilution method against 10 antimicrobial agents and typed by outer membrane protein pattern by using sodium dodecyl sulfate gel electrophoresis. All strains produced beta-lactamase and were resistant to tetracycline, kanamycin, and sulfonamides. Most had a decreased susceptibility to trimethoprim (MIC for 50% of the strains [MIC50], 0.5 micrograms/ml) and chloramphenicol (MIC50, 8 micrograms/ml). Strains were susceptible to ciprofloxacin (MIC90, 0.001 micrograms/ml), ceftriaxone (MIC50, 0.0015 micrograms/ml), erythromycin (MIC50, 0.015 micrograms/ml), rosoxacin (MIC50, 0.03 micrograms/ml), and spectinomycin (MIC50, 8 micrograms/ml). The degree of antimicrobial resistance found in Thailand is higher than that reported for H. ducreyi isolated in other regions. Five different outer membrane protein patterns were found by analyzing proteins in the range of 29 to 61 kilodaltons, but 98% of the Thai strains fell into three patterns which did not differ greatly. Outer membrane protein patterns of Thai strains were also seen in strains from other geographic areas. A new outer membrane protein type was found among nine strains isolated in Singapore.

Bacterial Outer Membrane Proteins↗
PubMed1986

Microbiological and biochemical characterization of spirochetes isolated from the feces of homosexual males.

Spirochetes were isolated from the feces of 11 homosexual males who had diarrhea. The anaerobic organisms were isolated from a selective medium that consisted of Trypticase soy agar supplemented with either 5% horse or human blood, 400 micrograms of spectinomycin per ml, and 5 micrograms of polymyxin B per ml. Nonselective media that permitted good growth of these fastidious organisms were developed, and selected biochemical tests were performed. The tests included carbohydrate utilization, detection of certain enzymes, and determination of volatile fatty-acid end products of metabolism. Two growth patterns were noted on solid media, a haze of growth and production of small colonies. Based on the results of biochemical tests, patterns of preformed enzymes, and volatile fatty-acid production, we believe that the 11 isolates represent a heterogeneous group of spirochetes. The data suggest that the human colon may harbor unique strains of cultivable spirochetes; additional study of the taxonomy of the organisms and assessment of their virulence for humans are needed.

Culture Media↗
PubMed1992

Evaluation of the standardized disk diffusion and agar dilution antibiotic susceptibility test methods by using strains of Neisseria gonorrhoeae from the United States and Southeast Asia.

Presently, most Neisseria gonorrhoeae testing is based on beta-lactamase tests and agar dilution with common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) recently described a disk diffusion test that produced results similar to the reference agar dilution method for the antibiotic susceptibility of N. gonorrhoeae. We obtained 71 gonococcal isolates from active-duty males aboard a United States Navy vessel while deployed in the Western Pacific during 1989. In addition, 47 isolates of N. gonorrhoeae were obtained from sexually transmitted disease clinics within the branch clinic operations of the Naval Hospital, San Diego (SD), and tested. Antibiotic susceptibility tests by using the NCCLS agar dilution and disk diffusion techniques were compared. Among the Southeast Asia (SEA) isolates, 47% were beta-lactamase producers compared with 10.5% of the SD isolates. The mean MICs (SEA/SD) in micrograms per milliliter for both groups were as follows: penicillin, 88/15; tetracycline, 2.2/0.95; erythromycin, 1.2/0.49; ceftriaxone, 0.016/0.012; cefotaxime, 0.034/0.03; cefuroxime, 0.44/0.17; cefoxitin, 1.3/0.97; spectinomycin, 150/131; ciprofloxacin, 0.07/0.034; norfloxacin, 0.77/0.29; lomefloxacin, 0.15/0.0.056; and ofloxacin, 0.07/0.036. The established NCCLS interpretive criteria for both susceptibility methods appear applicable to domestic gonococcal strains. However, modifications may be necessary for the more antimicrobial agent-resistant SEA isolates on the basis of the clinical success and cure rates following the indicated single-dose regimens for the geographic region.

Asia, Southeastern↗
PubMed1993

Incidence of Neisseria gonorrhoeae isolates negative by Syva direct fluorescent-antibody test but positive by Gen-Probe accuprobe test in a sexually transmitted disease clinic population.

To determine the accuracy of the Syva (Palo Alto, Calif.) direct fluorescent-antibody (DFA) test in comparison with the Gen-Probe (San Diego, Calif.) Accuprobe culture confirmation test, we tested 395 isolates of Neisseria gonorrhoeae from cultures obtained from patients attending a sexually transmitted disease clinic from 1 July 1991 through 30 June 1992. All isolates were tested for DFA reactivity with a polyclonal reagent (Difco Laboratories, Detroit, Mich.) and a monoclonal reagent (Syva, Inc., direct specimen test) and for specific molecular probe reactivity by the Gen-Probe Accuprobe culture confirmation test for N. gonorrhoeae. The 395 isolates gave positive results for the Gen-Probe culture confirmation test and the Difco polyclonal direct specimen test. However, 18 (4.6%) of the isolates were negative for N. gonorrhoeae by the Syva DFA test. With the exception of six beta-lactamase-positive isolates, all isolates that were negative by Syva DFA were sensitive to penicillin, tetracycline, spectinomycin, and ceftriaxone by disk-diffusion susceptibility testing. Auxotyping and serotyping studies indicated that strains negative by Syva DFA consisted of several variants. The frequency of N. gonorrhoeae isolates showing negative results by Syva DFA in this patient population ranged from 0 to 11.5%/month. Laboratories using only the Syva DFA test for confirmation of N. gonorrhoeae may incur a significant risk of misidentification.

Adolescent↗
PubMed1994

A 4-year survey of antimicrobial susceptibility trends for isolates from cattle with bovine respiratory disease in North America.

The antimicrobial susceptibility trends of bovine respiratory disease (BRD) pathogens isolated from 1988 to 1992 were determined. A total of 880 isolates representing Pasteurella haemolytica, Pasteurella multocida, and Haemophilus somnus were used in the study. Overall, resistance to ampicillin, tetracycline, erythromycin, and sulfamethazine was frequently encountered among strains of P. haemolytica and P. multocida. Ceftiofur, an extended-spectrum cephalosporin originally marketed in 1988 for the treatment of BRD, was very active against the BRD pathogens tested; the MIC of ceftiofur for 90% of isolates tested was < or = 0.06 microgram/ml. Resistance to spectinomycin varied on the basis of the breakpoint used. Substantial variation in the year-to-year susceptibility of BRD pathogens to tilmicosin, a new macrolide antimicrobial agent, was observed. The proportion of susceptible P. haemolytica isolates ranged from 84.7% in the second year to 7.1% in the third year and 78.2% in the fourth year. Similar fluctuations were observed with strains of P. multocida.

Animals↗
PubMed1999

Cholera in Vietnam: changes in genotypes and emergence of class I integrons containing aminoglycoside resistance gene cassettes in vibrio cholerae O1 strains isolated from 1979 to 1996.

The number of cholera cases and the mortality rates reported from different regions of Vietnam varied considerably in the period from 1979 to 1996, with between 2,500 and 6,000 cases reported annually from 1992 to 1995. Annual mortality rates ranged from 2.0 to 9.6% from 1979 to 1983 to less than 1.8% after 1983. Major cholera outbreaks were reported from the High Plateau region for the first time in 1994 and 1995; this is an area with limited access to health services and safe drinking-water supplies. All cases were associated with Vibrio cholerae O1. Using ribotyping, cholera toxin (CT) genotyping, and characterization of antibiotic susceptibility patterns and antibiotic resistance genes by PCR, we show that strains isolated after 1990 were clearly different from strains isolated before 1991. In contrast to strains isolated before 1991, 94% of 104 strains isolated after 1990 showed an identical ribotype R1, were resistant to sulfamethoxazole and streptomycin, and showed a different CT genotype. Furthermore, PCR analysis revealed that sulfamethoxazole-resistant strains harbored class I integrons containing a gene cassette ant(3")-1a encoding resistance to streptomycin and spectinomycin. This is, to our knowledge, the first report of class I integrons in V. cholerae. The development of cholera and the changes in the phenotypic and genotypic properties of V. cholerae O1 shown in the present study highlight the importance of monitoring V. cholerae O1 in Vietnam as in other parts of the world. In particular, the emergence of the new ribotype R1 strain containing class I integrons should be further studied.

Aminoglycosides↗
PubMed1999

Antimicrobial susceptibilities and plasmid contents of Neisseria gonorrhoeae isolates from commercial sex workers in Dhaka, Bangladesh: emergence of high-level resistance to ciprofloxacin.

Commercial sex workers (CSWs) serve as the most important reservoir of sexually transmitted diseases (STD), including gonorrhea. Periodic monitoring of the antimicrobial susceptibility profile of Neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. A study concerning the prevalence of gonococcal infection among CSWs was conducted in Bangladesh. The isolates were examined with regards to their antimicrobial susceptibility to, and the MICs of, penicillin, tetracycline, ciprofloxacin, cefuroxime, ceftriaxone, and spectinomycin by disk diffusion and agar dilution methods. The total plasmid profile of the isolates was also analyzed. Of the 224 CSWs, 94 (42%) were culture positive for N. gonorrhoeae. There was a good correlation between the results of the disk diffusion and agar dilution methods. Some 66% of the isolates were resistant to penicillin, and 34% were moderately susceptible to penicillin. Among the resistant isolates, 23.4% were penicillinase-producing N. gonorrhoeae (PPNG). 60.6% of the isolates were resistant and 38.3% were moderately susceptible to tetracycline, 17.5% were tetracycline-resistant N. gonorrhoeae, 11.7% were resistant and 26.6% had reduced susceptibility to ciprofloxacin, 2.1% were resistant and 11.7% had reduced susceptibility to cefuroxime, and 1% were resistant to ceftriaxone. All PPNG isolates contained a 3.2-MDa African type of plasmid, and a 24.2-MDa conjugative plasmid was present in 34.1% of the isolates. Since quinolones such as ciprofloxacin are recommended as the first line of therapy for gonorrhea, the emergence of significant resistance to ciprofloxacin will limit the usefulness of this drug for treatment of gonorrhea in Bangladesh.

Anti-Infective Agents↗
PubMed1999

Detection of an archaic clone of Staphylococcus aureus with low-level resistance to methicillin in a pediatric hospital in Portugal and in international samples: relics of a formerly widely disseminated strain?

Close to half of the 878 methicillin-resistant Staphylococcus aureus (MRSA) strains recovered between 1992 and 1997 from the pediatric hospital in Lisbon were bacteria in which antibiotic resistance was limited to beta-lactam antibiotics. The other half were multidrug resistant. The coexistence of MRSA with such unequal antibiotic resistance profiles prompted us to use molecular typing techniques for the characterization of the MRSA strains. Fifty-three strains chosen randomly were typed by a combination of genotypic methods. Over 90% of the MRSA strains belonged to two clones: the most frequent one, designated the "pediatric clone," was reminiscent of historically "early" MRSA: most isolates of this clone were only resistant to beta-lactam antimicrobials and remained susceptible to macrolides, quinolones, clindamycin, spectinomycin, and tetracycline. They showed heterogeneous and low-level resistance to methicillin (MIC, 1.5 to 6 microg/ml), carried the ClaI-mecA polymorph II, were free of the transposon Tn554, and showed macrorestriction pattern D (clonal type II::NH::D). The second major clone was the internationally spread and multiresistant "Iberian" MRSA with homogeneous and high-level resistance to methicillin (MIC, >200 microg/ml) and clonal type I::E::A. Surprisingly, the multidrug-resistant and highly epidemic Iberian MRSA did not replace the much less resistant pediatric clone during the 6 years of surveillance. The pediatric clone was also identified among contemporary MRSA isolates from Poland, Argentina, The United States, and Colombia, and the overwhelming majority of these were also associated with pediatric settings. We propose that the pediatric MRSA strain represents a formerly widely spread archaic clone which survived in some epidemiological settings with relatively limited antimicrobial pressure.

Anti-Bacterial Agents↗
PubMed2000

Comparison of Pasteurella spp. simultaneously isolated from nasal and transtracheal swabs from cattle with clinical signs of bovine respiratory disease.

Twenty-four matched pairs of isolates of Pasteurella haemolytica and three matched pairs of isolates of Pasteurella multocida were isolated by using a nasal swab and a transtracheal swab from individual calves with clinical signs of bovine respiratory disease. The identity of each matched pair was confirmed biochemically and serologically. The similarity of the isolates obtained from a nasal swab and from a transtracheal swab was compared by using ribotyping and antibiotic susceptibility analyses. Although the calves were sampled only once with a nasal and a transtracheal swab, when both samples were bacteriologically positive the nasal swab identified the same bacterial species as the transtracheal swab 96% of the time. The nasal swab isolate was genetically identical to the transtracheal isolate in 70% of the matched pairs. Six different ribotypes were observed for the P. haemolytica isolates, while only one ribotype was observed for the limited number of P. multocida isolates. Of the six P. haemolytica ribotypes, two ribotypes predominated. All the paired isolates displayed similar susceptibility to ceftiofur, erythromycin, tilmicosin, trimethoprim-sulfamethoxazole, and florfenicol, with some minor variations for ampicillin and spectinomycin. These results suggest that a nasal swab culture can be predictive of the bacterial pathogen within the lung when the isolates are from an acutely ill animal and can be used to determine antibiotic susceptibility.

Animals↗