Effects of 5-HT3 receptor antagonists on ethanol-induced hyperlocomotion in mice

DNA-based methodologies are considerably more powerful than other phenotype-based typing systems, providing a finer level of epidemiological discrimination, differentiating both closely and distantly related independent isolates that otherwise may appear as identical. In this study, plasmid analysis and pulsed-field gel electrophoresis were used to compare 28 isolates of Enterococci (respectively 13 strains of Enterococcus faecalis and 15 strains of Enterococcus faecium) with high-level resistance to aminoglycosides, isolated in Catania (Italy). Plasmid profile analysis resolved 20 different patterns among 24 plasmid harboring strains; many isolates showed one or two plasmids of the same size, but different plasmid content. Analysis of the PFGE-based RFLP patterns after SmaI digestion of genomic DNA resolved 26 different clones from 28 isolates: particularly, it resolved two different clones from three isolates showing identical plasmid profiles, and it identified as a single clone two isolates exhibiting different plasmid profiles. Thus, on the basis of our PFGE-based RFLP analysis data, we concluded that all the strains included in the study were genetically unrelated with two exceptions.     

Plasmid profiles of Klebsiella pneumoniae isolated from horses

Plasmid profiles of Klebsiella pneumoniae isolated from horses were examined. Thirty-nine strains of K. pneumoniae capsular type 1 (K1) isolated from cervical swabs of mares suffering from metritis, and from semen of stallions showed similar plasmid profile patterns, and all strains possessed a 125 megadaltons (Md) plasmid. There was no difference in plasmid profiles between the heavily-encapsulated and the less heavily-encapsulated strains of K. pneumoniae K1. Non-capsulated variants derived from the strains of K1 showed the same plasmid profile pattern as the parent strains. Plasmid profiles of K. pneumoniae other than K1 were various, and none of these strains possessed the 125 Md plasmid.     

Extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae from Zagreb, Croatia

Forty clinical isolates of Klebsiella pneumoniae, from various clinical specimens, with reduced susceptibility to ceftazidime, were tested for extended-spectrum beta-lactamase (ESBL) production. ESBL production was demonstrated by an 8-fold reduction in the minimum inhibitory concentration (MIC) of ceftazidime combined with clavulanate (2 mg/L) compared to ceftazidime alone in all strains. The aim of this investigation was the biochemical and molecular characterization of the ESBL produced by K. pneumoniae strains and their Escherichia coli transconjugants. Transfer of ceftazidime resistance was demonstrated in 23 of 40 strains. Thirteen strains produced an ESBL with the isoelectric point of 8.2 which was encoded by a self-transferable multiresistance plasmid of 150 kb. The substrate profile was similar to that of the SHV-5 isolated initially in Chile. Seven of these 12 strains had an additional TEM beta-lactamase. Six isolates and their transconjugants produced a plasmid-encoded ESBL with an isoelectric point close to 5.4. The remaining 21 strains produced an ESBL with an isoelectric point of 7.6 (thus probably SHV-2) which was encoded on a plasmid transferable to E. coli in 4 strains only. Four of those strains possessed an additional plasmid encoded TEM beta-lactamase with an isoelectric point close to 5.4. The transconjugants harbored a multiresistance plasmid of 150 kb. Thus SHV-2 and SHV-5 enzymes appear to have been the most common ESBLs in K. pneumoniae from Zagreb during 1994-1995.     

Shigellae isolated in 1997--plasmid profiles and antibiotic resistance

INTRODUCTION: Shigella spp is one of the most frequently isolated bacteria causing acute diarrhea with us. Genetics of pathogenicity of Shigella spp. includes chromosomal and plasmid genes. Most virulence factors are coded by invasion plasmid antigen genes residing on a 180-230 MDa plasmid. There is a big problem with multiple resistance of Shigella spp. strains, which is mostly plasmid-borne. Genetic analysis of bacterial cells, that is plasmid profile analysis, is important for investigation of sources and ways of spreading of the infection. All isolates originating from the same clone have identical plasmid profiles, i.e. number and size of plasmids. The aim of the investigation was: comparing the type of resistance to antimicrobical agents found in epidemic and nonepidemic. Shigella strains isolated in 1997, analyzing plasmid profiles of these isolates and confirming their epidemic connection. MATERIAL AND METHODS: Susceptibility to antibiotics was examined by a standard disc-diffusion method. Plasmid profiles of 40 strains (20 from the outbreak and 20 from sporadic cases) were tested using a method of alkaline lysis by Birnboim and Doly followed by electrophoresis in agarose gel. RESULTS: Shigella strains were resistant to antimicrobial agents which are most commonly used. Epidemic isolates shared the same resistance type, they were resistant to cephalexin, streptomycin and co-trimoxazole. The dominant type of resistance of nonepidemic strains was to ampicillin, streptomycin and co-trimoxazole. Strains isolated during the outbreak had identical plasmid profiles (2 plasmid bands of 55 and 1.5 MDa). Non-epidemic isolates had different plasmid profiles as well as type of resistance. CONCLUSION: Strains of Shigella spp. isolated during an outbreak had the same type of resistance and the same plasmid profiles, which indicated their origin from the same clone. The plasmid profile analysis is a reliable and precise method for determination of epidemic connection of Shigella isolates.     

Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in Taiwan

Thirty-one of 104 clinical isolates of Klebsiella pneumoniae collected over a period of 8 months were found to be putative extended-spectrum beta-lactamase (ESBL) producers. Isoelectric focusing and an iodine overlay agar method were used for preliminary identification of the ESBLs. They were further identified by DNA sequencing. Seventy-one percent of the isolates were found to produce SHV-5. The variation in the ESBL patterns of these isolates was slight, with only five patterns being identified. The strains were typed by pulsed-field gel electrophoresis (PFGE), and 16 different genotypes were identified. When the PFGE patterns were analyzed by the algorithmic clustering method called the unweighted-pair group method using arithmetic averages, five clusters were found. However, significant genetic variations were found among 11 isolates and between each cluster. A plasmid of 36 kb was found in all clinical isolates and in the transconjugants. Our results indicate that the increase in the number of ESBL-producing K. pneumoniae isolates in this hospital is due mainly to the dissemination of a resistance plasmid rather than to the clonal spread of a few epidemic strains.     

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One group (145 isolates) of Neisseria gonorrhoeae was collected from municipal clinics in Bloemfontein in 1994 and a second group (65 isolates) in 1995. Penicillin and tetracycline MICs were determined and plasmid analysis performed to monitor antimicrobial susceptibilities in conjunction with the occurrence of plasmids in these isolates. The prevalence of penicillin resistance caused by beta-lactamase plasmids remained constant at 9% during the study period. Three high-level tetracycline-resistant strains (MICs 16 mg/L), the first to be detected in South Africa, were isolated in 1994. Although there was a reduction in the percentage of isolates harbouring 24.5 MDa conjugative plasmids (from 79% in 1994 to 46% in 1995), this was partially counteracted by an increase in TetM-encoding conjugative plasmids (25.2 MDa) from 2% to 18.5%. The tetM genes of 13 isolates shown to exhibit high-level tetracycline resistance were characterized as the American type. The American-type tetracycline resistance plasmid was demonstrated in 11 isolates. Digestion with Bg/l showed that two isolates harboured tetM-encoding plasmids that differed from the American- and Dutch-type plasmids described previously: one isolate contained a plasmid that produced two fragments of different sizes from those of the American-type plasmid and the second isolate possessed an American/Dutch hybrid plasmid. Auxotyping/serotyping and random amplified polymorphic DNA analysis revealed a predominant tetracycline-resistant family (NR/IA-6, genomic group I) in Bloemfontein. As there is a high incidence of chlamydial infections in southern Africa requiring tetracycline therapy, selective pressures exist in the environment for the maintenance and rapid spread of high-level tetracycline-resistant N. gonorrhoeae. It is possible that tetM genes may have emanated from Botswana and/or Namibia to Bloemfontein. The establishment of high-level tetracycline-resistant N. gonorrhoeae in Bloemfontein was seen to be complex as a related group of strains was identified, plasmid dissemination was evident and two new TetM-encoding plasmids were demonstrated. The appearance of these TetM-encoding plasmids indicates either that the American- and Dutch-type plasmids are continuing to evolve or that tetM genes are being introduced into different families of 24.5 MDa conjugative plasmids.     

Resistance to beta-lactam antibiotics and aminoglycosides in gram negative bacteria. 1. Molecular and genetic characterization of R-factors (author's transl)

With frequent use of aminoglycoside antimicrobials and beta-lactam antibiotics in hospitals in the last few years, the number of bacterial strains resistant to these chemotherapeutics increased. Lately, strains of E. coli, Klebsiella, Enterobacter, Serratia, Proteus and Pseudomonas resistant to many antimicrobials (ampicillin, carbenicillin, cephalothin, chloramphenicol, gentamycin, tobramycin, sisomycin, neomycin, paromomycin, kanamycin, streptomycin, spectinomycin, tetracycline, sulphonamides) were isolated from patients of the university hospital in Zuerich. The resistant phenotype of two representative strains (Klebsiella pneumoniae 1 and Serratia marcescens 2) could be transferred by mixed cultivation to E. coli K-12. Multiple resistance of strain 1, and addition, could be transferred to Salmonella typhimurium, Serratia marcescens, Providencia, Proteus mirabilis and Klebsiella pneumoniae in varying frequencies. Transfer to Pseudomonas aeruginosa, however, could not be achieved. Spontaneous instability of resistance was observed in 0.15% of the cells of an overnight brothe culture and in 90% of the cells of a three months old culture. Conjugation, instability and the response to the sex phages MS-2 and If-1 suggested that resistance was mediated by a monomolecular R-factor, belonging to the fi+-type. This suggestion was confirmed by molecular characterization of the resistance plasmids. After transfer of the R-factors of K. pneumoniae 1 (R-FK 1) and Serratia marcescens 2 (R-FK2) into E. coli K-12, plasmid DNA was labelled with (methyl-3H) thymidine, and isolated by isopycnic centrifugation in cesiumchlorid-ethidium-bromide. Analysis of plasmid DNA then was carried out by sedimentation in a 5-20% neutral sucrose gradient together with reference plasmids of known molecular weights and sedimentation constants. The analysis revealed that R-FK1 had a molecular weight of 54 X 10(6) and R-FK2 of 50 X 10(6) daltons. The values were confirmed by contour length measurements of open circular forms with an electron microscope. A comparison of the sedimentation profile of labelled plasmid DNA from strain 1 and 14C-labelled DNA of E. coli K-12 (R-FK1) showed that the wild-type strain contained, besides the large resistance plasmid, at least two smaller "cryptic" plasmids. These smaller plasmid molecules were also found in antibiotic susceptible variants of strain 1, which did not contain the 54 X 10(6) dalton plasmid molecule, responsible for the resistant phenotype. The number of copies of R-FK1 in E. coli K-12 was determined to be 2, indicating stringent control of replication. It is discussed that the growing number of isolations of strains of Escherichia, Klebsiella, Serratia, Proteus, Providencia and Pseudomonas, exhibiting the same resistance phenotype, results from the spread of the R-factor described above among the hospital bacterial flora.     

beta-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa

Although resistance to the expanded-spectrum cephalosporins among members of the family Enterobacteriaceae lacking inducible beta-lactamases occurs virtually worldwide, little is known about this problem among isolates recovered in South Africa. Isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis resistant to expanded-spectrum cephalosporins recovered from patients in various parts of South Africa over a 3-month period were investigated for extended-spectrum beta-lactamase production. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Production of extended-spectrum beta-lactamases was evaluated by using the double-disk test, and the beta-lactamases were characterized by spectrophotometric hydrolysis assays and an isoelectric focusing overlay technique which simultaneously determined isoelectric points and general substrate or inhibitor characteristics. DNA amplification and sequencing were performed to confirm the identities of these enzymes. The P. mirabilis and E. coli isolates were found to produce TEM-26-type, SHV-2, and SHV-5 extended-spectrum beta-lactamases. An AmpC-related enzyme which had a pI of 8.0 and which conferred resistance to cefoxitin as well as the expanded-spectrum cephalosporins was found in a strain of K. pneumoniae. This is the first study which has identified organisms producing different extended-spectrum beta-lactamases from South Africa and the first report describing strains of P. mirabilis producing a TEM-26-type enzyme. The variety of extended-spectrum beta-lactamases found among members of the family Enterobacteriaceae isolated from major medical centers in South Africa is troubling and adds to the growing list of countries where these enzymes pose a serious problem for antimicrobial therapy.     

The plasmid profiles of fish pathogenic isolates of Aeromonas salmonicida, Vibrio anguillarum, and Vibrio ordalii from the Atlantic and Pacific coasts of Canada

The plasmid profiles of oxytetracycline- and streptomycin-resistant isolates of Aeromonas salmonicida, Vibrio anguillarum, and Vibrio ordalii were examined by agarose gel electrophoresis. Bacterial isolates were from disease outbreaks in fish on the Atlantic and Pacific coasts. Resistant isolates were examined when grown in the presence and absence of antibiotic. Alkaline lysis methods were used for plasmid isolation. Vibrio spp. were predominantly plasmidless, except for a 47-kilobase (kb) plasmid. Atlantic coast isolates of A. salmonicida possessed four or six plasmids, with four smaller plasmids ranging in size from 4.3 to 8.1 kb being consistently observed. The plasmid profiles of antibiotic-sensitive ATCC strains were identical. The plasmid profiles of the Pacific coast isolates of A. salmonicida varied slightly from those of the Atlantic coast isolates with six plasmids observed, ranging in size from 4.2 to 8.9 kb. Resistance to the antibiotics was not altered following plasmid curing experiments and resistance was not transferable to Escherichia coli. Thus, resistance to oxytetracycline and streptomycin did not appear to be plasmid mediated.     

Effect of US signal value on blocking of a CS-US association

To evaluate whether clinical Klebsiella isolates differ from nonclinical strains with respect to bacteriocin susceptibility patterns, a total of 452 Klebsiella pneumoniae and K. oxytoca strains isolated from different sources were examined. Bacteriocin typing was done by a modification of the scrape-and-point method, using a set of eight producer strains. 96% of the strains were typable. Forty-one different bacteriocin susceptibility patterns were observed. While two thirds of the K. oxytoca isolates belonged to only three different bacteriocin types, the K. pneumoniae strains showed a more heterogeneous distribution of patterns. No differences in pattern distribution were observed between isolates from clinical, fecal, or environmental sources. Certain bacteriocins showed a very broad spectrum of activity; e.g. 93% of all isolates were susceptible to bacteriocin type 3. The results suggest that nonclinical Klebsiella strains do not show other bacteriocin susceptibility types than clinical isolates do.     

Plasmid profiles and antimicrobial susceptibility of Campylobacter jejuni isolated from Israeli children with diarrhea

Thirty Campylobacter jejuni (C. jejuni) strains isolated from stools of Israeli children with enteritis were tested for sensitivity to eight antimicrobial agents (MIC) and the presence of plasmids. It was found that all the isolates were sensitive to ciprofloxacin, ofloxacin, furazolidone and erythromycin. Of the 30 strains tested, 21 (70%) were found to be tetracycline-resistant, a relatively high resistance rate as compared with data from other countries and previous reports from Israel. Plasmids were detected in 17 out of 30 C. jejuni isolates (55.6%). A total of nine different plasmid profiles could be distinguished; six profiles were represented by one strain each. Of the 21 tetracycline-resistant strains, plasmids were found in 17 isolates (80%) carrying from 1-2 to 5 plasmids of various sizes. No plasmids were found in tetracycline-sensitive strains, with the exception of one isolate which contained a 24.4 MDa plasmid and was co-trimoxazole-resistant. Our studies indicate a relatively high percentage of tetracycline-resistant C. jejuni isolates in the Tel Aviv area. In 80% of these strains, various plasmid profiles were detected.     

Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-D-treated field soils

Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis). A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species. Hybridization analysis revealed four groups. Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis. Group II, which exhibited homology only with the tfdA gene, was a small group and was probably a subset of group I. All group I and II strains had plasmids. Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains. One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area. The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe. The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group. The group III strains were identified as S. paucimobilis. The group IV strains, which hybridized to neither the tft prove nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses. Most of group IV strains could not be identified by the FAME analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimicrobial resistance and epidemiological study of Haemophilus influenzae strains isolated in Portugal. The Multicentre Study Group

In the course of a multicentric surveillance study, nine laboratories sent 375 isolates of Haemophilus influenzae to the Sector de Resistência aos Antibióticos (SRA) from the National Institute of Health in Lisbon, between 1 January and 31 December 1992. The majority of the H. influenzae isolates were from the respiratory tract (84.8%); only 5.1% were of invasive origin. Overall resistance for ampicillin was 11.7%, tetracycline 3.7%, and chloramphenicol 2.4%. All isolates tested were fully susceptible to cefotaxime, ceftriaxone, ciprofloxacin and rifampicin. Multiresistance was rare, occurring only in 2.4% of the isolates, although 50% of the ampicillin resistant strains had at least one additional resistance marker. Forty two isolates (11.2%) produced a TEM-1 type beta-lactamase, as shown by isoelectric focusing. beta-lactamase production was not detected in two of the ampicillin resistant strains. Fifteen of the 42 beta-lactamase producing strains (35.7%) contained detectable DNA plasmid: nine harboured large plasmids with an apparent molecular mass of 45 or 54 kb depending on their resistance phenotype and six harboured a small plasmid of 5 kb. In order to study transfer of resistance in both ampicillin and multiresistant strains conjugation experiments were performed for 14 isolates, seven of which harboured a large plasmid and seven had no detectable plasmid DNA. All 14 transferred their resistance phenotype but only a single large plasmid could be demonstrated in ten transconjugants. Restriction endonuclease analysis of plasmids from six representative transconjugants, isolated in different hospitals, revealed that there was no dissemination of a single R plasmid, which suggests an independent process of acquisition of resistance genes.     

Diversity among clinical isolates of Helicobacter pylori in Korea

Eleven strains of Helicobacter pylori were isolated from biopsy specimens obtained at the A-San Medical Center from December, 1995 to February, 1996. Every H. pylori positive patient was diagnosed to have chronic, erosive, or mild superficial gastritis. To determine the diversity in clinical isolates, the following were studied: total protein profile, plasmid profile, presence of cagA, and variation in DNA sequence. Protein profiles of nine isolates were similar to each other while two isolates had a 35 kDa protein which did not appear in others. The presence of cagA was detected with PCR in seven isolates (63%). Among eleven isolates, seven (63%) carried plasmids. Each isolate showed a big diversity with a PCR-based randomly amplified polymorphic DNA method. Even though all H. pylori isolates used in this study were isolated from gastritis patients at the same hospital, their molecular and biological characteristics were quite different from another showing a big diversity in H. pylori.     

Plasmids isolated from marine sediment microbial communities contain replication and incompatibility regions unrelated to those of known plasmid groups

Two hundred ninety-seven bacteria carrying plasmids that range in size from 5 to 250 kb were identified from more than 1,000 aerobic heterotrophic bacteria isolated from coastal California marine sediments. While some isolates contained numerous (three to five) small (5- to 10-kb) plasmids, the majority of the natural isolates typically contained one large (40- to 100-kb) plasmid. By the method of plasmid isolation used in this study, the frequency of plasmid incidence ranged from 24 to 28% depending on the samples examined. Diversity of the plasmids occurring in the marine sediment bacterial populations was examined at the molecular level by hybridization with 14 different DNA probes specific for the incompatibility and replication (inc/rep) regions of a number of well-characterized plasmid incompatibility groups (repB/O, FIA, FII, FIB, HI1, HI2, I1, L/M, X, N, P, Q, W, and U). Interestingly, we found no DNA homology between the plasmids isolated from the culturable bacterial population of marine sediments and the replicon probes specific for numerous incompatibility groups developed by Couturier et al. (M. F. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Our findings suggest that plasmids in marine sediment microbial communities contain novel, as-yet-uncharacterized, incompatibility and replication regions and that the present replicon typing system, based primarily on plasmids derived from clinical isolates, may not be representative of the plasmid diversity occurring in some marine environments. Since the vast majority of marine bacteria are not culturable under laboratory conditions, we also screened microbial community DNA for the presence of broad- and narrow-host-range plasmid replication sequences. Although the replication origin of the conjugally promiscuous broad-host-range plasmid RK2 (incP) was not detectable in any of the plasmid-containing culturable marine isolates, DNA extracted from the microbial community and amplified by PCR yielded a positive signal for RK2 oriV replication sequences. The strength of the signal suggests the presence of a low level of the incP replicon within the marine microbial community. In contrast, replication sequences specific for the narrow-host-range plasmid F were not detectable in DNA extracted from marine sediment microbial communities. With the possible exception of mercuric chloride, phenotypic analysis of the 297 plasmid-bearing isolates did not demonstrate a correlation between plasmid content and antibiotic or heavy metal resistance traits.     

Characterization of plasmids which mediate resistance to multiple antibiotics in gram-negative bacteria of nosocomial origin

BACKGROUND: The genetic and molecular mechanisms involved in antimicrobial resistance of 10 strains of gramnegative bacilli (1 Serratia marcescens; 2 Escherichia coli; 1 Proteus mirabilis; 4 Klebsiella pneumoniae; 1 Enterobacter cloacae y 1 Alcaligenes faecalis), isolated from adult patients with nosocomial pulmonary infection at the in-patient facilities of the University Hospital of Los Andes, Mérida, Venezuela, have been studied. METHODS: The antimicrobial susceptibility was determined by minimum inhibitory concentrations using the dilution method in agar. The study of extrachromosomal genes was carried out by conjugation, bacterial infection with the bacteriophage M13 and curing of plasmid by acridine orange. The plasmids were isolated by alkaline lysis and analysis of restriction endonuclease digestion was carried out separately using the enzymes EcoRI and HindIII. A DNA probe, derived from the region which encodes the TEM-1 beta-lactamase of the plasmid pBR322 was used for dot-blot hybridization tests. RESULTS: All of the gramnegative bacilli showed resistance to ampicillin, carbenicillin and cephalothin (> 128 micrograms/ml) and 3 strains also showed resistance to gentamicin (> 64 micrograms/ml). Genetic and molecular procedures showed the presence of conjugative plasmids of approximately 54 kb in all the 10 strains. The restriction patterns obtained by using EcoRI and HindIII indicated common DNA fragments in most of the plasmids studied. The dot-blot hybridization tests confirmed homology between the plasmids and the DNA probe used (TEM-1 beta-lactamase). CONCLUSIONS: In this study, the gramnegative bacteria of nosocomial origin harbored self-transferable plasmids of approximately 54 kb, which mediate resistance to gentamicin and encode a beta-lactamase of the TEM group.     

Virulence of Rhodococcus equi isolates from patients with and without AIDS

Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients. Thirty-nine isolates of R. equi from immunocompromised patients with and without AIDS were analyzed for the presence of virulence plasmid DNA, expression of 15- to 17-kDa antigens, and their pathogenicities in mice. Of the human isolates, eight contained an 85-kb virulence plasmid, expressed 15- to 17-kDa antigens, and were virulent in mice. Nineteen isolates carried cryptic plasmids of various sizes, and the remaining 12 isolates did not contain any plasmids. These 31 isolates did not express virulence-associated antigens and were not virulent in mice. The results suggested that opportunistic infections in immunocompromised patients could be caused by both virulent and avirulent R. equi strains and that the pathogenesis of R. equi infection in immunocompromised patients appears to be different from that which occurs in foals.     

Molecular markers for differentiation of multiresistant Klebsiella pneumoniae isolates in a pediatric hospital

OBJECTIVE: To study the spread of extended-spectrum beta-lactamase-producing, but aminoglycoside-susceptible, Klebsiella pneumoniae strains in our hospital over an 8-month period, by using two genotypic markers. DESIGN: Ribotyping (using two endonucleases) and randomly amplified polymorphic DNA analysis (RAPD; using two different 10-mer primers) were applied to the epidemiological typing of clinical K pneumoniae isolates from stools, ileal fluid, or urine of hospitalized children. SETTING AND PATIENTS: The surgical intensive-care ward (S1: 9 patients, 17 isolates), surgical unit (S2: 2 patients, 2 isolates), and gastroenterology ward (GE: 1 patient, 1 isolate) of the Robert Debré Hospital of Paris, France. RESULTS: Ribotyping of the 20 clinical isolates, the type strain of the species, and two epidemiologically unrelated isolates with EcoRI and HindIII revealed 6 and 5 different patterns, respectively. Six ribotypes were identified by using these two enzymes. RAPD generated 6 distinct patterns, in complete agreement with ribotyping. Our genotypic results showed that 11 patients from wards S1, S2, and GE harbored genotypically related strains, suggesting nosocomial transmission and cross-colonization between and within the three wards. CONCLUSIONS: Ribotyping and RAPD appear to be reliable methods for distinguishing K pneumoniae strains. The spread of one strain of K pneumoniae in different units of our hospital was demonstrated by both methods. However, RAPD has the advantage of simplicity and rapidity conferred by polymerase chain reaction.     

Outbreak of ceftazidime-resistant Klebsiella pneumoniae in a pediatric hospital in Warsaw, Poland: clonal spread of the TEM-47 extended-spectrum beta-lactamase (ESBL)-producing strain and transfer of a plasmid carrying the SHV-5-like ESBL-encoding gene

In 1996 a large, 300-bed pediatric hospital in Warsaw, Poland, started a program of monitoring infections caused by extended-spectrum beta-lactamase (ESBL)-producing microorganisms. Over the first 3-month period eight Klebsiella pneumoniae isolates were identified as being resistant to ceftazidime. Six of these were found to produce the TEM-47 ESBL, which we first described in a K. pneumoniae strain recovered a year before in a pediatric hospital in Lód?, Poland, which is 140 km from Warsaw. Typing results revealed a very close relatedness among all these isolates, which suggested that the clonal outbreak in Warsaw was caused by a strain possibly imported from Lód?. The remaining two isolates expressed the SHV-5-like ESBL, which resulted from the horizontal transfer of a plasmid carrying the blaSHV gene between nonrelated strains. The data presented here exemplify the complexity of the epidemiological situation concerning ESBL producers typical for large Polish hospitals, in which no ESBL-monitoring programs were in place prior to 1995.     

Genotypic characterization of Salmonella enteritidis phage types by plasmid analysis, ribotyping, and pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to resolve XbaI and SpeI macrorestriction fragments from 60 defined phage type (PT) reference strains of Salmonella enteritidis. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct XbaI pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb spv-associated fragment was observed for three PT reference strains, and the profile was designated PFP X1A. The XbaI macrorestriction profiles of a further four PT reference strains were altered by the presence of plasmid-associated bands. Twenty-six SpeI-generated PFPs (plus one subtype) were observed for the same strains. No SpeI fragment corresponding to the 38-MDa serovar-specific plasmid was detected. The distribution of XbaI and SpeI profiles did not always correspond, producing a total of 32 combined PFPs for the 60 PT reference strains. This compared with a total of 18 different plasmid profiles and three PvuII ribotypes generated by the same strains. The results of this study indicate that PFGE may offer an improved level of discrimination over other genotypic typing methods for the epidemiological typing of S. enteritidis.