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ABSTRACTIn Brazil, Salmonella entericaserovar Infantis resistant to various antimicrobials, including cephalosporins, has been identified as an etiological agent of severe gastroenteritis in hospitalized children since 1994. In this study, 35 serovar Infantis strains, isolated from children admitted to four different Rio de Janeiro, Brazil, hospitals between 1996 and 2001, were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing in order to determine their genetic relatedness and antimicrobial resistance profiles. Thirty-four serovar Infantis strains were resistant to at least two antibiotic classes, and all 35 strains were susceptible to fluoroquinolones, cephamycin, and carbapenem. Extended-spectrum beta-lactamase (ESBL) screening by double-disk diffusion indicated that 32 serovar Infantis strains (91.4%) produced beta-lactamases that were inhibited by clavulanic acid. Antimicrobial resistance gene profiles were determined by PCR for a subset of 11 multidrug-resistant serovar Infantis strains, and putative ESBLs were detected by isoelectric focusing. Ten serovar Infantis strains carried blaTEM, catI, ant(3")Iaand/or ant(3")Ib, sulIand/or sulII, and tet(D) genes as well as an integron-associated aac(6')-Iqcassette. Eight strains possessed at least four different beta-lactamases with pI profiles that confirmed the presence of both ESBLs and non-ESBLs. Our PFGE profiles indicated that 33 serovar Infantis strains isolated from Rio de Janeiro hospitals came from the same genetic lineage.

ABSTRACTThe etiological agent most commonly associated with bacillary dysentery is Shigella. As part of its mandate, the Bacteriology and Enteric Disease Program of Health Canada identifies and serotypes unusual isolates of Shigellareceived from provincial laboratories of public health. In this report, six unusual isolates from three provinces were analyzed biochemically and serologically using slide and tube agglutinations and molecularly using standard pulsed-filed gel electrophoresis (PFGE), PCR, and PCR-restriction fragment length polymorphism (RFLP) techniques. All six isolates were identical. PFGE analysis grouped these strains; biochemically, they were mannitol negative and consistent with the profile of Shigella. Serologically, these strains produced weak reactions in Shigella dysenteriaeserovars 4 and 16 and Escherichia coliO159 and O173 antisera. Molecular serotyping by PCR-RFLP of the rfbgene produced an S. dysenteriaeserovar 2/E. coliO112ac pattern. They were positive by PCR for ipaHand ialenteroinvasive genes but negative for all other genes tested. Antiserum was prepared from one of the isolates and tested against Shigellaand E. colireference strains as well as the other isolates. The antiserum reacted with the five remaining isolates and showed cross-reactivity with S. dysenteriaeserovars 1, 4, and 16; Shigella flexneritype 3; and E. coliO118, O159, O168, O172, and O173 antigens. Absorbing the sera with E. coliO159 and S. dysenteriaeserovar 4 antigen removed all cross-reactions and only slightly reduced the homologous titer. Based on biochemical, molecular, and complete serological analysis, we propose that these six isolates represent a new provisional serovar of S. dysenteriae, type strain BEDP 02-5104.

The etiological agent most commonly associated with bacillary dysentery is SHIGELLA: As part of its mandate, the Bacteriology and Enteric Disease Program of Health Canada identifies and serotypes unusual isolates of Shigella received from provincial laboratories of public health. In this report, six unusual isolates from three provinces were analyzed biochemically and serologically using slide and tube agglutinations and molecularly using standard pulsed-filed gel electrophoresis (PFGE), PCR, and PCR-restriction fragment length polymorphism (RFLP) techniques. All six isolates were identical. PFGE analysis grouped these strains; biochemically, they were mannitol negative and consistent with the profile of SHIGELLA: Serologically, these strains produced weak reactions in Shigella dysenteriae serovars 4 and 16 and Escherichia coli O159 and O173 antisera. Molecular serotyping by PCR-RFLP of the rfb gene produced an S. dysenteriae serovar 2/E. coli O112ac pattern. They were positive by PCR for ipaH and ial enteroinvasive genes but negative for all other genes tested. Antiserum was prepared from one of the isolates and tested against Shigella and E. coli reference strains as well as the other isolates. The antiserum reacted with the five remaining isolates and showed cross-reactivity with S. dysenteriae serovars 1, 4, and 16; Shigella flexneri type 3; and E. coli O118, O159, O168, O172, and O173 antigens. Absorbing the sera with E. coli O159 and S. dysenteriae serovar 4 antigen removed all cross-reactions and only slightly reduced the homologous titer. Based on biochemical, molecular, and complete serological analysis, we propose that these six isolates represent a new provisional serovar of S. dysenteriae, type strain BEDP 02-5104.

Specific primer pairs were selected for the PCR amplification of 14 tetracycline resistant genes commonly found in Gram positive and Gram negative organisms. Combinations of primer pairs were used in multiplex PCR reactions to detect specific groups of tetgenes as follows; Group I tet(B), tet(C), tet(D); Group IItet(A), tet(E), tet(G); Group III tet(K), tet(L), tet(M), tet(O), tet(S); Group IV tetA(P), tet(Q), tet(X). To test the multiplex PCR, Groups I and II were used on 25 clinical isolates of Salmonella entericaserovar Typhimurium DT104. Group III primers were used to investigate 19 clinical isolates of methicillin-resistant Staphylococcus aureus. Multiplex PCR should result in significant savings in terms of labour and cost in analysis of a large number of strains when compared with using an individual PCR for targeting each gene. It may also be a useful method to differentiate the types of tetracycline resistance when used as an additional marker for the purpose of outbreak investigation and surveillance.

Typical and atypical Campylobacter strains were isolated from the colons of cattle and swine by techniques that enabled the selective pressures of antibiotics to be avoided. Some cephalothin-susceptible strains and a strain with an indeterminate hippurate reaction were classified as Campylobacter coli by DNA homology testing. Tetracycline-resistant isolates were obtained from animals with no recorded exposure to antibiotics. A selection of 12 C. coli and 6 C. jejuni strains was used to determine the ability of fresh isolates to grow on a range of selective media. C. coli isolates were inhibited more than C. jejuni on selective media containing antibiotics. The least inhibitory media were Skirrow medium (M. B. Skirrow, Br. Med. J. 2:9-11, 1977) and the charcoal-based media developed by Hutchinson and Bolton (D. N. Hutchinson and F. J. Bolton, J. Clin. Pathol. 37:956-957, 1984) and Karmali et al. (M. A. Karmali, A. E. Simon, M. Roscoe, P. C. Fleming, S. S. Smith, and J. Lane, J. Clin. Microbiol. 23:456-459, 1986). The plasmid contents of the isolates did not appear to be related to their sensitivity to growth on selective (antibiotic-containing) media. The study indicates that selective media used to detect Campylobacter spp. could select against the isolation of C. coli.

Tetracycline-resistant strains of Campylobacter jejuni and Campylobacter coli from swine and cattle colons were isolated and characterized by hybridization with DNA probes. A probe consisting of the 1.8-kilobase (kb) HincII fragment from pUA466 was highly specific for the detection of tetracycline resistance (Tcr) in C. jejuni and C. coli. The 5-kb tetM DNA probe from Streptococcus agalactiae plasmid pJI3 which has homology with the 1.8-kb HincII fragment from pUA466 could also be used to detect Tcr Campylobacter strains. However, the tetM probe had a much lower sensitivity and required a lower stringency of hybridization. Therefore, the 1.8-kb HincII fragment appeared to be more appropriate for the classification of Tcr in Campylobacter spp. No homology was detected between the Tcr determinant from Campylobacter spp. and the tetL and tetN probes from Streptococcus spp. DNA homology was demonstrated between pUA649, a derivative of plasmid pUA466 which had lost most of the Tcr region, and Tcr plasmids from C. jejuni and C. coli isolated from animal and human sources. There was also homology between pUA649 and the chromosomes of C. jejuni and C. coli strains. In this study, all but one of the tetracycline-resistant C. coli and C. jejuni strains contained plasmids of approximately 50 kb which hybridized with the 1.8-kb HincII probe. In one C. coli strain (UA703), Tcr appeared to be chromosomally mediated.

The reaction of 15 aryl trifluoromethyl ketones with N-carbamoylmethyl-1,4-dihydronicotinamide has been studied in aqueous sulfolane buffer. The unsubstituted ketone and those containing electron-withdrawing groups in the ring have the following reaction characteristics: (a) a high yield of alcohol is obtained, (b) the observed reaction rate is independent of ring substituent; however, when corrections are made for degree of hydration of the ketones the rate is correlated with Hammett σ values with a rho of +1.98, (c) a secondary isotope effect of approximately 1.08 and primary isotope effects of 1.45–1.62 are observed at 43.4 °C for the reaction of the dihydronicotinamide containing one or two atoms of deuterium at C-4, (d) ΔH≠ = 15.2 kcal mol−1, and ΔS≠ = −27.0 cal deg−1 mol−1for the unsubstituted compound, uncorrected for ketone hydration; ΔS≠for reaction of the unhydrated ketone and dihydronicotinamide is estimated to be −45 to −50 cal deg−1 mol−1. The mechanism for the reduction is consistent with hydride transfer from reductant to oxidant, very possibly accompanied by blind-alley formation of an adduct between ketone hydrate and dihydronicotinamide. Ketones containing electron-donating groups in the ring react with the dihydronicotinamide in some undetermined way, giving little or no alcohol as product.

The rapid ID32A kit (bioMérieux Vitek, Inc., Hazelwood, Mo.) was evaluated for its ability to identify Peptostreptococcus species compared with conventional biochemical tests and gas-liquid chromatography (Virginia Polytechnic Institute), the current "gold standard" method. A total of 5 Peptostreptococcus American Type Culture Collection strains and 95 clinical isolates comprising Peptostreptococcus anaerobius, P. asaccharolyticus, P. magnus, P. micros, and P. prevotii isolates were included for analysis. Overall, the sensitivity and specificity of the rapid ID32A kit in the identification for five Peptostreptococcus species compared with the Virginia Polytechnic Institute method were 93 and 80%, respectively. All P. anaerobius (n = 20) and P. asaccharolyticus (n = 25) isolates were identified with 100% sensitivity and 100% specificity. For the identification of P. magnus (n = 24) and P. micros (n = 19), the rapid ID32A kit was 100% sensitive for both species; the specificity for P. magnus was 95.8% and that for P. micros was 57.9%. The sensitivity and specificity of the rapid ID32A kit for identification of P. prevotii (n = 12) were poor (41.7 and 8.3%, respectively). The rapid ID32A kit is a useful method for the rapid differentiation of P. anaerobius and P. asaccharolyticus from other Peptostreptococcus spp. Conventional methods should be used to identify to the species level isolates of P. magnus, P. micros, and P. prevotii.

With the aid of a range spectrograph the differential and integral muon intensities at sea level have been measured. The differential intensities at (0.85±0.03) GeV/c and (1.16±0.04) GeV/c are (4.09±0.21)·10−6cm−2s−1sr−1(MeV/c)−1and (3.29±0.19)·10−6cm−2s−1sr−1(MeV/c)−1respectively. These measurements confirm the recent results of Allkoferet al.at similar muon momenta, and suggest that the differential muon momentum spectrum below 5 GeV/c is slightly steeper than has hitherto been believed.

Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil for growth (PCU-) have homogeneous phenotypes; most are plasmid-free, belong to few serovars, and are significantly associated with intermediate levels of susceptibility to penicillin, tetracycline, erythromycin, and cefoxitin. Because of their lack of variation by these criteria, molecular typing methods, ribotyping (restriction fragment length polymorphism [RFLP] of rRNA genes), and multilocus enzyme electrophoresis were explored as tools for further distinguishing PCU- isolates. By ribotyping, selected PCU- isolates could be separated into four groups on the basis of the hybridization patterns (RFLPs) of SmaI- and AvaII-digested DNA with probes containing rRNA sequences. Most of the isolates (18 of 23 isolates) belonged to a single RFLP (group I). One isolate each was in groups II and IV, and three isolates were in group III. All isolates except one, isolate NS791, had similar multilocus enzyme electrophoresis patterns. Strain NS791 was unusual in that it contained a variant cryptic plasmid with an insert in the 0.46-kb MspI-HinfI fragment of the 4.2-kb plasmid, it was the only isolate belonging to RFLP group IV, and it differed in its multilocus enzyme electrophoresis pattern, having different mobilities for glyceraldehyde phosphate dehydrogenase, phosphoglucose isomerase, 6-phosphogluconate dehydrogenase, and glutamate dehydrogenase. Serovars of PCU- isolates appeared to be more indicative of strain divergence than RFLP or isoenzyme typing. Multilocus enzyme electrophoresis indicated that PCU- isolates are clonal.

The ability of Campylobacter coli and Campylobacter jejuni to grow in the presence of antibiotics used in selective growth media was compared. MIC data for C. coli indicated that some strains were more susceptible to the antibiotics than were the C. jejuni strains tested. A reduction of greater than 1 log cycle in the numbers of cells growing on plates containing antibiotics was considered to be a marked level of inhibition. Only one of nine of the antibiotic combinations studied did not markedly inhibit most of the C. coli strains tested. Although one C. coli strain was not inhibited by any of the antibiotic combinations, the other six strains were inhibited for up to 7 log cycles. The addition of blood or growth supplements reduced but did not eliminate the inhibitory effect. The inhibition of laboratory strains of C. coli on media developed for the isolation of Campylobacter spp. indicates that the incidence of C. coli may be underestimated.

Electron microscopic studies of Campylobacter revealed that different morphological forms predominate at different parts of a colony. At the periphery, cells were almost all spirals, while in the center of the colony cells were mainly coccus shaped. Unusual ring-shaped cells, "donuts", were observed in the raised, peripheral region of the colony. Donut or ring forms have not previously been reported for Campylobacter organisms. Our data indicate that young or actively growing cells are mainly spiral shaped. Older cells undergo a degenerative change to coccoid forms. The donut shape appears to be an intermediate stage between spirals and cocci. Comparisons of plate counts of actively growing and inactive cells confirmed that coccoid cells are probably nonviable.

Four strains of Campylobacter jejuni and four strains of Campylobacter coli were used to compare the quantitative growth of Campylobacter cells on blood agar base no. 2 (Oxoid), brucella agar (BBL Microbiology Systems and Difco Laboratories), campylobacter agar base (Difco), Columbia blood agar base (Difco and Oxoid), and Mueller-Hinton agar (Difco and Oxoid). Columbia blood agar base and blood agar base no. 2 were inhibitory to most of the strains tested, as evidenced by reduced (10- to 1,000-fold) colony counts compared with other basal media. One of the brucella agars was inhibitory to two of the C. coli strains. The inhibitory effect of these media could be eliminated by addition of FBP (0.05% each ferrous sulfate hydrate, sodium metabisulfite, and sodium pyruvate) or 7% defibrinated sheep blood. However, addition of FBP or blood to brucella agar, campylobacter agar base, or Mueller-Hinton agar did not significantly affect the count, indicating that supplements are not required in these media for growth of Campylobacter in pure culture.

Three oligonucleotide probes, complementary to tetM sequences, were labelled non-radiometrically using the DIG-oligonucleotide tailing kit and evaluated for their specificity for the detection of plasmid mediated tetracycline resistance in Neisseria gonorrhoeae. Only Probe 3, 5'-GCT CAA CAA TTC TGT TCC AGC-3', was specific for tetM. It hybridized with the tetM-containing 25·2-MDa plasmids from all of the 232 TRNG and the 130 PP/TRNG isolates used in the study. Its sensitivity, determined by dot-blot hybridization, was 0·1 pg of pJ13 plasmid DNA 10⁴ cells. It did not hypbridize with the DNA from non-PPNG, CMRNG and tetracycline susceptible isolates from seven other Neisseria species (N. meningitidis, N. subflava, N. cinerea, N. lactamica, N. sicca, N. mucosa, and N. flavescens), Moraxella spp. and Haemophilus influenzae. Probe 3 also hybridized to DNA of three tetracycline resistant P. magnus (OMIC = 16 µg ml^-1) isolates which presumptively carried the tetM determinant. Therefore, probe 3 can be used by reference laboratories as a confirmatory test for TRNG, as well isolates from the other genera containing the tetM determinant. Copyright 1994, 1999 Academic Press

The effects of δ9-tetrahydrocannabinol upon rat blood pressure were studied in control rats and in rats subjected to repeated immobilization stress over seven days. The immobilization stress was associated with a significant blood pressure increase in animals not receiving δ9-THC. δ9-THC resulted in a significant lowering of blood pressure in non-immobilized rats and blocked the appearance of hypertension in immobilized rats. These data support previous suggestions that THC compounds might be useful in an approach to the therapy of hypertension.

A combination of DNA macrorestriction analysis using pulsed-field gel electrophoresis and a serotyping method using three panels of monoclonal antibody was used to discriminate 43 epidemiologically unrelated Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil (PCU-) into 35 groups. This indicates that PCU- isolates of N. gonorrhoeae are not clonal.

The MICs for 101 isolates of Neisseria gonorrhoeae obtained by Etest (AB-Biodisk, Solna, Sweden) and the agar dilution method on GC medium base supplemented with 1% Kellog's defined supplement (GCMB) were compared. The overall percent agreement (within 1 log2 dilution) between methods was greater than 97.9. The Pearson's correlation coefficients for penicillin, tetracycline, erythromycin, and ceftriaxone for the two methods were 0.98, 0.97, 0.93, and 0.93 (P = 0.001), respectively, for comparisons on GCMB. The overall percent agreement was lower when hemoglobin-supplemented GCMB was used. Etest is an attractive alternative to the agar dilution method for gonococcal antimicrobial susceptibility testing and should be further analyzed in multicenter studies.

Nalidixic acid-resistant mutants of Campylobacter jejuni and C. coli as well as "C. laridis" strains showed cross-resistance to another DNA gyrase subunit A inhibitor, enoxacin (MIC, 32 micrograms/ml), whereas C. fetus subsp. fetus, C. fetus subsp. venerealis, and "C. hyointestinalis" strains were all susceptible to enoxacin (MIC, less than or equal to 2 micrograms/ml). All Campylobacter species were resistant to novobiocin (MIC, 32 to 512 micrograms/ml), but most strains were susceptible to the other DNA gyrase subunit B inhibitors coumermycin A1 and clorobiocin.