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A comparative study of the BBL crystal enteric/nonfermenter identification system and the biomerieux API20E and API20NE identification systems after overnight incubation.

The BBL Crystal Enteric/Nonfermenter (Crystal, Becton Dickinson Microbiology Systems) is a new multi-test identification system for Gram negative rods requiring no oil overlay or addition of reagents. One hundred and three selected Gram negative rod isolates from routine clinical specimens were tested in parallel using the appropriate API20E or API20NE (BioMerieux) reading after overnight incubation. The isolates included in the study, and the number tested, were as follows: Acinetobacter sp, 8; Aeromonas hydrophila, 1; Citrobacter diversus, 1; Citrobacter freundii, 1; Escherichia coli, 13; Enterobacter aerogenes, 10; Enterobacter agglomerans, 2; Enterobacter cloacae, 6; Klebsiella oxytoca, 6; Klebsiella pneumoniae, 12; Morganella morganii, 2; Proteus mirabilis, 6; Pseudomonas aeruginosa, 21; Salmonella sp, 2; Salmonella typhi, 1; Serratia marcescens, 1; Shigella sonnei, 2; Shigella sp, 2; Vibrio metschnikovii, 1; Xanthomonas maltophilia, 3; and, Yersinia enterocolitica, 2. The API20E and API20NE systems combined identified 74.8% (77/103) and the Crystal 97.1% (100/103) of isolates. Twenty six isolates required repeat and/or additional tests for correct identification with the API systems compared to 3 with the Crystal. Crystal is more convenient than API for routine clinical use because it requires fewer repeat and/or extra tests, and is easier and less time consuming to use.

Bacterial Typing Techniques↗

Peritoneal infection in acute intermittent peritoneal dialysis.

A prospective study was done to evaluate the incidence and microbiological trend of peritoneal infection in patients undergoing acute intermittent peritoneal dialysis (PD). Complete sterile procedure was ensured and at the completion of the procedure PD fluid was sent for bacteriological culture, sensitivity, and total and differential cell count. During the period September 2000 to February 2001 a total of 100 patients were evaluated. Male female ratio was 72:28. Mean age was 43.17 +/- 17.2 years. In 26 patients cyclers were used. Bacterial culture was positive in total of 30 cases (30%). Gram positive, Gram negative and mixed infection was found in 10%, 15%, and 5% respectively. Number of exchanges (31.61 +/- 7.7 vs. 31.3 +/- 6, p = 0.8) were similar and number of repositioning was significantly more in the infected group (23.3% vs. 11.4%, p < 0.01). Total cell count was significantly higher in infected group (274.3 +/- 502 vs. 31.25 +/- 79.34, p < 0.01). Among Gram +ve organisms Staphylococcus was found in 7, Enterococcus faecalis in 4 and Coryne bacterium sps. in 2 cases. Among Gram -ve organisms, E. coli was found in 4, Enterobacter in 3, Klebsiella 1, Pseudomonas 1, Acinetobacter arinatus 5, Acinetobacter baumani 3, and Citrobacter freundii 3. Mixed flora comprised of Enterococcus faecalis 3, Enterobacter 1, Staphlococcus 1, E. coli 3, Citrobacter 1, Acinobacter baumani 1. Although with the cyclers using collapsible bags, staphylococcus was not isolated, the total incidence of infection (11/26 cases) was not decreased with the use of cyclers. We conclude that in acute intermittent peritoneal dialysis the incidence of bacterial infection is 30% with preponderance of Gram -ve over Gram +ve organisms and organism of fecal origin being commoner than those of skin origin. Use of cycler-assisted over manual PD do not improve the incidence of infection. Repositioning of the stiff catheter significantly increases the incidence of infection.

Adult↗

Comparison of two methods for same-day identification of Enterobacteriaceae.

Two commercial methods, API 20E (as modified for same-day enterobacterial identification) and Micro-ID, were evaluated for ability to provide useful same-day information of 368 clinically isolated Enterobacteriaceae. Organisms included Escherichia coli (54), Shigella (7), Edwardsiella tarda (1), Salmonella enteritidis (10), Citrobacter (30), Klebsiella (55), Enterobacter (68), Hafnia alvei (2), Serratia (33), Proteus (64), Morganella morganii (24), Providencia (18), and Yersinia enterocolitica (2). Methods were those of manufacturers without supplemental tests. API at five hours identified 78.5% of strains to species, 9.5% to genus only, 10.1% as part of a spectrum of identifications (SI), and 1.9% incorrect. Micro-ID at four hours yielded 90.0% correct identification to species and 3.3% to genus only, 4.0% SI, and 2.7% incorrect. API identification of many Serratia, Citrobacter, Providencia strains was to genus only; most incorrect results occurred in Serratia marcescens. Micro-ID identified most organisms to species; incorrect identifications were mainly S. marcescens and Klebsiella pneumoniae. Both systems provided excellent identification of E. coli. Both methods sacrifice a degree of accuracy that varies with the species tested, as compared to overnight systems, but both provide rapid information of potential clinical value.

Culture Media↗

Sulbactam/ampicillin vs. chloramphenicol/ampicillin for the treatment of meningitis in infants and children.

Eighty-one patients ages one month to 14 years with meningitis were randomized to receive either sulbactam (50 mg/kg per day) and ampicillin (400 mg/kg per day; 41 patients) or chloramphenicol and ampicillin (40 patients). The groups were comparable in terms of sex and degree of illness; however, more patients treated with chloramphenicol/ampicillin than patients treated with sulbactam/ampicillin were younger than 12 months of age (78% vs. 56%). Pathogens were isolated from the cerebrospinal fluid (CSF) of 65 (80%) of the 81 patients. In the sulbactam/ampicillin group, there were 18 Haemophilus influenzae isolates (one resistant to ampicillin), five Streptococcus pneumoniae, five Neisseria meningitidis, one Klebsiella pneumoniae, one Pseudomonas aeruginosa, and one Listeria. In the chloramphenicol/ampicillin group, there were 19 H. influenzae isolates, 10 S. pneumoniae, three N. meningitidis, one Haemophilus parainfluenzae, and one Citrobacter. Of 63 patients with assessable CSF pathogens, one (3%) of 29 treated with sulbactam/ampicillin died (S. pneumoniae) and six (18%) of 34 treated with chloramphenicol/ampicillin died (two, H. influenzae; three, S. pneumoniae; and one, Citrobacter). Twelve percent in the sulbactam/ampicillin group and 18% in the chloramphenicol/ampicillin group had neurologic sequelae. No clinically significant reactions or toxicities were noted. Sulbactam/ampicillin was as effective as chloramphenicol/ampicillin in the treatment of meningitis.

Adolescent↗

A virulence plasmid in Escherichia coli enterotoxigenic for humans: intergenetic transfer and expression.

We studied the colonization-factor antigen I (CFA/I) fimbriae- and heat-stable enterotoxin (ST)-coding plasmid of enterotoxigenic Escherichia coli (strain H10407, serotype O78:H11) pathogenic for humans. With use of conjugal-transfer system of E. coli H10407 and transposon-labeling techniques, the virulence plasmid was shown to be transferable to many species of the family Enterobacteriaceae, including the enteropathogens, Shigella and Salmonella species, and the opportunistic pathogens, Klebsiella, Enterobacter, Citrobacter, Edwardsiella, Serratia, and Proteus species. The virulence plasmid-carrying transconjugants produced both CFA/I fimbriae and ST (exotoxin). Moreover, most of the transconjugants stably inherited the virulence plasmid, although plasmid stability was greatly dependent on culture temperature. Finally, administration of the virulence plasmid-carrying living bacterial cells of Klebsiella, Enterobacter, and Citrobacter species and E. coli K12 resulted in fluid accumulation in both infant-mouse and rabbit ileal-loop tests.

Animals↗

Mezlocillin, ceftizoxime, and amikacin alone and in combination against six Enterobacteriaceae in a neutropenic site in rabbits.

Closed-space, locally neutropaenic infection sites were simulated in rabbits by implanting subcutaneous semipermeable chambers inoculated with 5 X 10(4) cfu/ml of Escherichia coli (one strain), Citrobacter diversus (one strain), Klebsiella pneumoniae (two strains) or Serratia marcescens (two strains). Four hours after inoculation, treatment was begun with amikacin (15 mg/kg), mezlocillin (100 mg/kg), or ceftizoxime (50 mg/kg) alone or in two-drug combinations. Antibiotics were given intramuscularly every 6 h for 16 doses. Chambers were sampled for bacterial counts at the start of therapy, and 20, 44, and 92 h thereafter. Blood and chamber antibiotic levels were determined during the treatment course. In-vivo results were compared to in-vitro tests of inhibition, killing and synergism. Ceftizoxime alone was successful in vivo (greater than 6 log reduction in count) against the three strains of klebsiella and citrobacter, mezlocillin against one serratia strain, and amikacin alone against none of the strains. The best in-vitro correlation was with the minimum inhibitory concentration (MIC) at a high (10(8] inoculum for the beta-lactams and with the anaerobic MIC for amikacin. Among combinations, amikacin plus mezlocillin alone was successful against the three strains of klebsiella and serratia, but neither amikacin nor mezlocillin added to ceftizoxime were more successful than ceftizoxime alone. In-vitro chequerboard synergism was not predictive of in-vivo success. Mezlocillin alone was inactivated in vivo by all the strains except Ser. marcescens, and the E. coli strain inactivated both mezlocillin and ceftizoxime alone and in combination.

Agranulocytosis↗

Gram-negative bacilli resistant to third-generation cephalosporins: beta-lactamase characterization and susceptibility to Sch 34343.

We studied 192 recent clinical isolates, comprising six species of Gram-negative bacilli resistant either to cefotaxime or latamoxef (Moxalactam), from several hospitals. All isolates were resistant to several other third-generation cephalosporins or a monobactam. Two to five types of chromosomal beta-lactamases, as defined by isoelectric focusing, were readily identified in each species. Isolates of Citrobacter, Enterobacter and Serratia produced higher levels of chromosomal beta-lactamase than corresponding cefotaxime-susceptible strains. In addition, 20 of 57 produced one or two plasmid-determined beta-lactamases, TEM-1, OXA-2, or a novel enzyme, OHIO-1. The penem and carbapenem antibiotics, Sch 34343 and imipenem, were more active than cefotaxime, ceftazidime, ceftriaxone, latamoxef and aztreonam against isolates of Acinetobacter, Citrobacter, Ent. aerogenes, Ent. cloacae and Morganella, whereas imipenem, ceftazidime, and aztreonam were more active against Serratia isolates. The addition of plasmid-determined beta-lactamase increased resistance to piperacillin, cefoperazone and cefamandole but not to cefotaxime, ceftazidime, ceftriaxone, latamoxef, aztreonam, Sch 34343, or imipenem. Of 24 strains susceptible to aminoglycosides, none produced a plasmid-determined beta-lactamase, whereas 20 were found among the 33 strains resistant to aminoglycosides. Resistance of clinical isolates to newer beta-lactams appears to be due primarily to a high level of chromosomal cephalosporinase present without inducing agents. The plasmid-determined beta-lactamases, TEM-1 and OHIO-1, contributed little to resistance to most of the newer beta-lactams but were strongly associated with aminoglycoside resistance in these selected isolates. The greater in-vitro efficacy of the penem and carbapenem antibiotics, Sch 34343 and imipenem, against most of these isolates makes them promising candidates as first line agents against these pathogens.

Acinetobacter↗

In-vitro activity and beta-lactamase stability and inhibitory properties of a new penem antibiotic, Sch 34343.

Sch 34343 is a new penem antibiotic. Its in-vitro activity was determined against Gram-positive and -negative aerobic and anaerobic isolates. Sch 34343 inhibited 90% of strains of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp, Citrobacter spp. and Shigella spp. at less than 1 mg/1. Ninety per cent of strains of Enterobacter, Morganella and Providencia spp. were inhibited by less than 4 mg/1. Sch 34343 did not inhibit Pseudomonas spp., and it had slightly less activity against Enterobacteriaceae than did cefotaxime or latamoxef (moxalactam), but it inhibited organisms resistant to cefoxitin, cefoperazone and piperacillin. Sch 34343 inhibited methicillin-susceptible Staphylococcus aureus at less than or equal to 0.25 mg/1, and it inhibited haemolytic streptococci at less than or equal to 0.06 mg/1. Sch 34343 inhibited Bacteroides fragilis at 0.06 mg/1, including some cefoxitin-resistant isolates. Sch 34343 was not hydrolysed by plasmid- or chromosomally-mediated beta-lactamases, was an excellent inhibitor of Richmond-Sykes type Ia beta-lactamases, and also inhibited, although less effectively, the common plasmid beta-lactamases. It induced beta-lactamases, but inhibited Enterobacter and Citrobacter spp. in which it induced beta-lactamase activity.

Anti-Bacterial Agents↗

In-vitro activity of ofloxacin, a quinolone carboxylic acid compared to other quinolones and other antimicrobial agents.

Ofloxacin is a new quinolone carboxylic acid compound. Its activity against 900 bacterial isolates was determined. It inhibited 90% of Escherichia coli, Klebsiella sp., Aeromonas hydrophila, Salmonella spp., Shigella spp., Citrobacter spp., Enterobacter spp., Morganella morganii, Proteus mirabilis, Yersinia enterocolitica at less than or equal to 0.8 mg/l. Branhamella catarrhalis, Haemophilus sp., Neisseria sp. were inhibited by less than or equal to 0.1 mg/l. Pseudomonas aeruginosa and other Pseudomonas species were inhibited by less than or equal to 6.3 mg/l. Although most staphylococcal species, including methicillin-resistant staphylococci were inhibited by 3.1 mg/l, many streptococcal species had higher MIC values, and most Bacteroides species were inhibited at less than or equal to 6.3 mg/l. The overall activity of ofloxacin was similar to enoxacin and norfloxacin. Ofloxacin inhibited organisms resistant to nalidixic acid, ampicillin, cephalexin, piperacillin, and gentamicin including Enterobacter spp., Citrobacter freundii and Serratia marcescens resistant to cefotaxime. The activity of ofloxacin was lower at an acid pH and in urine, but serum had no effect on MICs or MBCs. Increase in ofloxacin MICs for various bacteria could be achieved by repeated subculture in the presence of ofloxacin.

Animals↗

Antibacterial properties of imipenem with special reference to the activity against methicillin-resistant staphylococci, cefotaxime-resistant Enterobacteriaceae and Pseudomonas aeruginosa.

Imipenem was examined with standardized agar dilution procedures against a wide range of bacteria. Geometric mean MICs against the genera Escherichia, Klebsiella, Enterobacter, Citrobacter and Serratia were 0.1-0.4 mg/l, and Proteus and Providencia spp. were inhibited by 0.25-4 mg/l. Acinetobacter calcoaceticus var. anitratum strains were inhibited by concentrations ranging from 0.12-0.5 mg/l. Methicillin-susceptible staphylococci were highly susceptible to the drug (MICs: less than or equal to 0.03 mg/l) and enterococci were inhibited by 0.25-16 mg/l. Most of the multi-resistant JK corynebacteria were resistant to imipenem. Imipenem was more active than any other beta-lactam against methicillin-resistant staphylococci; this was also demonstrated in a population analysis. Imipenem-resistant minorities in populations, however, were also observed. Cefotaxime-resistant and -intermediate Enterobacter and Citrobacter strains were inhibited by concentrations of 0.5 mg/l or less. No third-generation cephalosporin nor any other beta-lactam showed similarly high activity against these groups of organisms. Among 20 ceftazidime-resistant and 20 ceftazidime-susceptible isolates of Pseudomonas aeruginosa, no strain was resistant and only five ceftazidime-resistant strains were intermediately susceptible (MIC, 8 mg/l) to imipenem.

Anti-Bacterial Agents↗

In-vitro interactions of FCE 22101 with aminoglycosides against gram-negative rods.

Agar dilution MICs of FCE 22101 were measured for 894 consecutively-isolated Gram-negative rods from clinical specimens (413 Escherichia coli, 104 Klebsiella spp., 54 Enterobacter spp., 19 Citrobacter spp., 131 Proteus spp., 43 Acinetobacter spp., 9 Serratia spp., 3 Providencia spp., 4 Yersinia spp., 3 Hafnia spp. and 111 Pseudomonas spp.). Excluding Pseudomonas spp., 98% of these isolates were susceptible to 8 mg/l FCE 22101. Resistance (MIC greater than 8 mg/l) varied from 11% in Serratia spp. to 0% in Citrobacter spp. Interactions between FCE 22101 and gentamicin, tobramycin and amikacin were tested by the chequerboard method for 150 of the isolates. The geometric mean sigma FICs were 0.80 for FCE + gentamicin, 0.78 for FCE + tobramycin and 0.79 for FCE + amikacin. Synergy, defined at FIC index (sigma FIC) less than 0.5, for at least one combination, was found in only 16/150 (11%) of the isolates. Most other isolates showed an additive response (sigma FIC = greater than 0.5-1). No antagonism was detected. No significant difference was observed between the various species tested, except that sigma FIC values were lower (geometric mean sigma FIC = 0.61-0.68) for non-fermentative species than for fermentative species (geometric mean sigma FIC = 0.83-0.86). Likewise, geometric sigma FIC values were lower (0.67-0.69) for isolates resistant to FCE and/or aminoglycosides than for those susceptible to both components (sigma FIC 0.85-0.88).

Amikacin↗

Characterization of ceftriaxone-resistant Enterobacteriaceae: a multicentre study in 26 French hospitals. Vigil'Roc Study Group.

During a multicentre study performed in 26 French hospitals, 287 (3.2%) of 9038 Enterobacteriaceae isolated, mainly Enterobacter spp., Serratia spp., Citrobacter spp. and Klebsiella spp. were classified as ceftriaxone resistant on the basis of an MIC > 4 mg/L or the presence of an extended-spectrum beta-lactamase. Extended-spectrum beta-lactamase was present mainly in Klebsiella pneumoniae (65 strains, 10.2%) and very rarely in Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, Citrobacter spp. and Enterobacter spp. The extended-spectrum beta-lactamases conferred low-level resistance to ceftriaxone in nearly 60% of the strains harbouring them, emphasizing the need for routine testing for the presence of these enzymes. Among transconjugants three types of extended-spectrum beta-lactamase were identified. Those resembling TEM-3 were the most common, but TEM-21, and SHV-4 were also found. Clavulanate and to a lesser extent sulbactam inhibited all the extended-spectrum beta-lactamases encountered in this study.

Ceftriaxone↗

In-vitro microbiological assessment of a new penem, Men 10700.

The in-vitro antibacterial activity of Men 10700, a novel penem, has been compared with that of ritipenem, ciprofloxacin, amikacin, cefotaxime and co-amoxiclav against 539 strains taken from 17 genera. Men 10700 was most active against staphylococci and streptococci (MIC90 < 0.5 mg/L), slightly less active against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Citrobacter spp., Moraxella catarrhalis and peptostreptococci (MIC90 0.5-2 mg/L), moderately active against Enterococcus faecalis, members of the tribe Proteae, Serratia marcescens, Acinetobacter spp., clostridia and Bacteroides spp. (MIC90 4-16 mg/L), and inactive against Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Enterococcus faecium. Its antibacterial spectrum is thus slightly less broad than that of imipenem, but it compares favourably with an oral third-generation cephalosporin. Men 10700 was more active than ritipenem against many species, especially Enterobacter spp. and Citrobacter spp.

Anti-Bacterial Agents↗

Susceptibility to levofloxacin of clinical isolates of bacteria from intensive care and haematology/oncology patients in Switzerland: a multicentre study.

The objective of this study was to examine the susceptibility of clinical isolates to levofloxacin, a fluoroquinolone with extended activity against Gram-positive bacteria, and other antibiotics in 12 Swiss clinical microbiology laboratories using the NCCLS disc diffusion technique. Isolates were prospectively collected from intensive care units (ICUs (59%), oncology wards (7%) and other units with haematology/oncology patients (34%) from June 1995 to March 1996. The levofloxacin breakpoints used were as recommended by the manufacturer. A total of 310 Gram-positive and 580 Gram-negative isolates from the respiratory tract (36%), skin/wounds (12%), blood (16%), urine (17%) and other sources (19%) were tested. The percentage of isolates susceptible to levofloxacin was 100% for Enterococcus spp. (38 strains), Streptococcus agalactiae (13), Streptococcus pneumoniae (65), Acinetobacter spp. (11), Citrobacter diversus (6), Citrobacter freundii (17), Klebsiella oxytoca (39), Morganella morganii (16), Proteus mirabilis (20), Proteus vulgaris (23), Serratia spp. (19), Stenotrophomonas maltophilia (10) and Haemophilus influenzae (41). The percentage of isolates susceptible to levofloxacin for Staphylococcus aureus (95 strains, including 2% MRSA) was 94%, coagulase-negative staphylococci (85) 65%, Enterobacter spp. (75) 99%, Escherichia coli (111) 97%, Klebsiella pneumoniae (45) 98% and Pseudomonas aeruginosa (124) 87%. In conclusion, levofloxacin is a new fluoroquinolone to which the most common clinical isolates in Switzerland are susceptible. The susceptibility of Enterococcus spp. and S. pneumoniae to levofloxacin was particularly remarkable. This compound appears to be a promising therapeutic alternative for the treatment of Gram-positive infections.

Anti-Infective Agents↗

Molecular insights into carbapenemase-producing Enterobacterales from Senegal.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are an urgent global health threat, especially in resource-limited countries. Here we determined the prevalence and the molecular characteristics of CPE isolated from infections in Dakar, Senegal. METHOD: From January 2019 to December 2020, Enterobacterales with reduced susceptibility to ertapenem (diameter < 25&#x2005;mm) from infections were collected at the Pasteur Institute of Dakar. Carbapenemases were detected using biochemical and immunochromatographical assays. WGS was used to determine resistome, MLST, plasmids, virulence genes and genetic relatedness. RESULTS: Of the 1045 Enterobacterales collected during the study period, 86 had a diameter around ertapenem of <25&#x2005;mm (8%) and 19 were confirmed as CPE (2%). These included Escherichia coli (n&#x200a;=&#x200a;6) [ST410 (n&#x200a;=&#x200a;3), ST405 (n&#x200a;=&#x200a;2) and ST2083], Enterobacter spp. (n&#x200a;=&#x200a;6) [ST231 (n&#x200a;=&#x200a;3), ST245, ST760 and ST960] and Klebsiella spp. (n&#x200a;=&#x200a;5) (ST22, ST25, ST231, ST1535, ST4843), Citrobacter freundii ST22 (n&#x200a;=&#x200a;1) and Citrobacter koseri with unknown ST (n&#x200a;=&#x200a;1). blaOXA-48 (n&#x200a;=&#x200a;7; 35%), blaOXA-181 (n&#x200a;=&#x200a;7; 35%) and blaNDM-5 (n&#x200a;=&#x200a;6; 30%) genes were identified. C. freundii ST22 harboured blaNDM-5, blaOXA-48 and blaCTX-M-15 genes. Some E. coli isolates belonging to the high-risk clone ST410 were closely related (<20 SNPs) to isolates recovered in France from patients returning from Senegal, suggesting transnational spread. In addition, 5/6 carbapenemase-producing E. coli isolates possessed a four amino acid insertion in PBP3, conferring reduced susceptibility to aztreonam/avibactam and cefiderocol. CONCLUSIONS: This study highlights the spread of NDM-5 and OXA-181 in Senegal, and reports the first co-occurrence of NDM-5 and OXA-48 in sub-Saharan Africa. The spread of CPE, especially in high-risk clones, underscores the urgent need for continued surveillance and targeted interventions.

Senegal↗

The phylogenetic position of Serratia, Buttiauxella and some other genera of the family Enterobacteriaceae.

The phylogenetic relationships of the type strains of 38 species from 15 genera of the family Enterobacteriaceae were investigated by comparative 16S rDNA analysis. Several sequences of strains from the genera Citrobacter, Erwinia, Pantoea, Proteus, Rahnella and Serratia, analysed in this study, have been analysed previously. However, as the sequences of this study differ slightly from the published ones, they were included in the analysis. Of the 23 enterobacterial genera included in an overview dendrogram of relatedness, members of the genera Xenorhabdus, Photorhabdus, Proteus and Plesiomonas were used as a root. The other genera formed two groups which could be separated, although not exclusively, by signature nucleotides at positions 590-649 and 600-638. Group A contains species of Brenneria, Buttiauxella, Citrobacter, Escherichia, Erwinia, Klebsiella, Pantoea, Pectobacterium and Salmonella. All seven type strains of Buttiauxella share 16S rDNA similarities greater than 99%. Group B embraces two phylogenetically separate Serratia clusters, a lineage containing Yersinia species, Rahnella aquatica, Ewingella americana, and also the highly related pair Hafnia alvei and Obesumbacterium proteus.

DNA, Ribosomal↗

Use of two 16S DNA targeted oligonucleotides as PCR primers for the specific detection of Salmonella in foods.

A 16S DNA targeted polymerase chain reaction (PCR) method specific for the detection of Salmonella isolates with various serotypes was developed. The primers used for such a PCR method were 16SF1 and 16SIII. 16SF1 is the reverse and complementary strand of 16SI which has been shown to be able to hybridize with Salmonella and Citrobacter spp. 16III on the other hand, is able to hybridize with Klebsiella and Serratia spp. in addition to Salmonella. Although 16SF1 and 16SIII were not specific to Salmonella only, when they were used as PCR primers, only the Salmonella isolates could be specifically detected. The interference from Citrobacter, Klebsiella and Serratia spp. could be prevented. None of the other non-Salmonella isolates including strains of the family of Enterobacteriaceae closely related to Salmonella would generate the false-positive reaction. When this PCR system was used for the detection of Salmonella cells artificially contaminated in food samples, results obtained were satisfactory. A detection limit of N x 10(0) cells per assay could be obtained.

Base Sequence↗

Cloning and characterization of the eae gene from a dog attaching and effacing Escherichia coli strain 4221.

We have cloned and determined the nucleotide sequence of the eae gene from a dog attaching and effacing (A/E) Escherichia coli (DEPEC) strain 4221. When comparing the predicted amino acid sequence of the eaeDEPEC to that of the Eae proteins from enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli O157:H7 (EHEC), Citrobacter freundii biotype 4280, and a swine A/E E. coli strain O45 (PEPEC), the overall sequence identity was 84, 81, 83 and 83%, respectively, with the greatest divergence at the C-terminal end, the putative receptor-binding portion. Interestingly, the DEPEC Eae shares the greatest identity at the C-terminal region with the Citrobacter freundii Eae protein. We have constructed and purified a maltose-binding fusion protein (MBP) containing the product of the entire eae gene of the DEPEC strain 4221. Binding of MBP-EaeDEPEC fusion protein to HEp-2 cells was demonstrated by immunofluorescence microscopy. In addition, the Eae protein of DEPEC (4221) demonstrated a strong serological relationship with that of EPEC (E2348/69) as observed using a polyclonal antiserum against MBP-EaeDEPEC fusion protein.

Adhesins, Bacterial↗