Evaluation of the BBL Minitek System for the Identification of Enterobacteriaceae

Thirty-one different substrate disks were tested in parallel with comparable, prepared media (BBL) against a minimum of 300 cultures of Enterobacteriaceae. An overall correlation of 98% was observed with all the disks tested. In addition, the system was used to identify 461 fresh isolates of Enterobacteriaceae in parallel with conventional media using the schema used at the Veterans Administration Hospital, Baltimore. An overall correlation of 97% was observed. Minitek is a time and space saving system. It is accurate and easily adapted to the clinical laboratory. A wide variety of substrates are available, allowing most laboratories to use their own schema. The long shelf life of most disks is a definite advantage.     

Rapid Confirmation of Suspect Salmonella Colonies by Use of the Minitek System in Conjunction with Serological Tests

Colonies of 40 members of the Enterobacteriaceae family (26 Salmonella serotypes and 14 other organisms) were picked from selective agar plates and inoculated into Minitek inoculum broth (BBL) and then onto Minitek discs of dextrose, lactose, sucrose, mannitol, maltose, dulcitol, lysine and H₂S. After incubation for 6 h, the inoculum broth was tested with salmonella Poly O and after 24 h with salmonella Poly H antisera. The results of the biochemical tests were read after 24 h incubation. With this procedure, all the salmonella cultures used in this study were confirmed as salmonella and differentiated from all the other organisms, which were rejected. This procedure provides an alternative to the time consuming conventional procedures for the biochemical and serological confirmation of suspect salmonella colonies on selective agar plates.     

Biochemical characteristics and identification of Enterobacteriaceae isolated from meats.

The isolation and identification of 2,220 Enterobacteriaceae from meats indicated that Escherichia coli biotype I, Enterobacter agglomerans, and Serratia liquefaciens were the principal types to be differentiated in meats. Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter hafniae were also commonly identified. Identification of isolates by the Encise II (Roche Diagnostics Inc., Nutley, N.J.) and Minitek (BBL Microbiology Systems, Cockeysville, Md.) coding systems gave similar results with only 255 (11.5%) discrepancies in identity, but both systems required large numbers of supplementary tests for identification of the isolates. Not only the distribution of Enterobacteriaceae types isolated from meats but also some of the biochemical reactions of the isolates differed from those of clinical isolates. The Minitek technique is recommended because of its versatility. However, with the addition of cellobiose and salicin disks and the inclusion of methyl red to the Minitek test and the use of the Voges-Proskauer test and gas production in EC medium at elevated temperature as standard tests, the identification of these Enterobacteriaceae from meats would be greatly facilitated. The inclusion of the motility test, for example, using nitrate motility agar, would also be of value to Enterobacteriaceae identification.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Comparison of miniaturized multitest systems with conventional methodology for identification of Enterobacteriaceae from foods.

Four miniaturized multiple test systems were compared with tube methodology used to identify Enterobacteriaceae encountered in foods. Identification aids supplied with each system were used to assign names to isolates at the species level. For the 129 strains tested, the Minitek system demonstrated a 96.9 percent agreement with reactions in tubed media. The Inolex, Analytab, and PathoTec test systems exhibited 94.3, 93.8, and 92.7 percent agreement, respectively. Analytab identified 96.1 percent of the isolates to the species level, whereas the Minitek, PathoTec, and Inolex systems were able to identify 78.3, 32.6, and 27.1 percent, respectively. The results indicate that the Analytab and Minitek systems are acceptable substitutes for the tube methodology routinely employed in identifying enterics from foods. Although the PathoTec system might be used to screen isolates for their identity, neither the presently available PathoTec nor the Inolex systems should be substituted for current methodology when definitive identification of foodborne organisms is required.     

Comparison of miniaturized multitest systems with conventional methodology for identification of Enterobacteriaceae from foods.

Four miniaturized multiple test systems were compared with tube methodology used to identify Enterobacteriaceae encountered in foods. Identification aids supplied with each system were used to assign names to isolates at the species level. For the 129 strains tested, the Minitek system demonstrated a 96.9 percent agreement with reactions in tubed media. The Inolex, Analytab, and PathoTec test systems exhibited 94.3, 93.8, and 92.7 percent agreement, respectively. Analytab identified 96.1 percent of the isolates to the species level, whereas the Minitek, PathoTec, and Inolex systems were able to identify 78.3, 32.6, and 27.1 percent, respectively. The results indicate that the Analytab and Minitek systems are acceptable substitutes for the tube methodology routinely employed in identifying enterics from foods. Although the PathoTec system might be used to screen isolates for their identity, neither the presently available PathoTec nor the Inolex systems should be substituted for current methodology when definitive identification of foodborne organisms is required.     

Evaluation of the Minitek system for characterization of Bacillus species

Minitek (BBL Microbiology Systems, Cockeysville, Md.) substrate disks were evaluated as alternatives to conventional tests for the characterization of Bacillus species. Results were compared for 10 reference isolates and 87 isolates from food sources. The overall agreement of results between the Minitek and conventional tests was 92% for reference strains and 86% for food isolates.     

Evaluation of the Minitek system for characterization of Bacillus species.

Minitek (BBL Microbiology Systems, Cockeysville, Md.) substrate disks were evaluated as alternatives to conventional tests for the characterization of Bacillus species. Results were compared for 10 reference isolates and 87 isolates from food sources. The overall agreement of results between the Minitek and conventional tests was 92% for reference strains and 86% for food isolates.     

Using SIB and API kits to identify and biotype enterobacteria of different origin

A collection of 26 Enterobacteriaceae reference strains provided by Reference Centres in Moscow (USSR) and Copenhagen (Denmark) as well as a collection of 660 freshly isolated cultures of Gram-negative bacteria of different origin were investigated using SIB indicator systems manufactured at the Gorky Institute of Epidemiology and Microbiology (USSR) and API-20E, Rapid-20E and API-10S kits (API, France) with the aim of species determination. In analyzing freshly isolated cultures, API-20E, API-10S and SIB-B kits proved to be of approximately equal efficiency, whereas the Rapid-20E system enabled species identification in no more than 78% of the tested cultures. In a model biotyping of 284 E. coli cultures of different origin, SIB-B and API-20E kits in combination with the Analytical Profile Index enabled sufficiently rapid and standard identification of Enterobacteriaceae biovars.     

API System: a Multitube Micromethod for Identification of Enterobacteriaceae

The API system for identification of Enterobacteriaceae was evaluated with 366 cultures. Overall accuracy of identification was 96.4%; of the 13 cultures misidentified, 7 were atypical strains.     

r/b Expanders: Their Use in Identifying Routinely and Unusually Reacting Members of Enterobacteriaceae

This cooperative study between a large clinical laboratory and a reference laboratory evaluated the performance of the expanded r/b system for identifying Enterobacteriaceae. The 2,200 cultures isolated in the normal hospital routine presented no problem of identification to the r/b system. About 250 "atypical" cultures which were exchanged between the collaborating laboratories were also identified accurately. The expanded r/b system was found to perform as well as most biochemical-physiological identification systems, and when used appropriately was highly satisfactory as a system for identification of Enterobacteriaceae.     

Pathogenicity islands in opportunistic Enterobacteria

The presence of fragments of genomes hlyA, hlyB, papAH, papC, sfaG, sfaA and kps MT, associated with the pathogenicity islands of Escherichia coli, in clinical strains of other genera of the family Enterobacteriaceae, has been experimentally evaluated with the use of PCR. The presence of DNA fragments specific to the known genes of the pathogenicity clusters of E. coli in representatives of the genera Citrobacter, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Serratia and Yersinia of rarely occurring groups has been established. In Enterobacteriaceae cultures isolated from the intestine amplicons homologous to hlyB were detected significantly less frequently than among strains of nonintestinal origin. In Enterobacteriaceae strains isolated in respiratory pathology amplicons of the pili gene (sfaG) were detected significantly more frequently than in collection cultures. The total evaluation of the detection rate of the genes of pathogenicity islands among Enterobacteriaceae clinical strains under study in comparison with E. coli showed that they occurred significantly less frequently. Klebsiella spp. were found to differ most essentially from E. coli as regards the occurrence of fragments of the genes of pathogenicity islands. The conclusion was made on the high probability of genetic exchange in DNA fragments between different species of bacteria with corresponding changes in their pathogenicity.     

Comparison of the Effects of Liquid Medium Repair and the Incorporation of Catalase in MacConkey Type Media on the Recovery of Enterobacteriaceae Sublethally Stressed by Freezing

Nine pure cultures of species of Enterobacteriaceae were stressed by rapid freezing in tryptone soya broth (TSB) to — 22°C and subsequent storage at that temperature for 7 d. About one to two log cycles kill and at least one additional log cycle sublethal impairment was achieved. Numbers of colonies of these cultures in poured plates of violet red bile glucose (VRBG) agar, with 67 u/ml of catalase added at 47°C, were only slightly higher than those in plain VRBG, both incubated overnight at 30°C. Two hours incubation of TSB suspensions at 17–25° C resulted in almost complete restoration of the ability of cells to develop colonies in VRBG, without, however, leading to any significant multiplication.
Similar experiments with 32 samples of frozen minced meat, 27 samples of frozen surface water, 18 of frozen chicken liver and 14 of fresh sausage substantiated the results obtained in the studies on pure cultures.
In the experiments with the nine pure cultures the influence of the nutrient composition of the solid enumeration media: 'minimal' agar, TSB agar (TSBA) and Mueller-Hinton agar with Polyvitex nutrient supplement (MHA), on the recovery of Enterobacteriaceae stressed by freezing was also studied. Colony numbers in TSBA and MHA were virtually identical. The glucose mineral salts medium led to lower recovery, indicating that so-called 'minimal medium recovery' of stressed bacterial populations is not a common phenomenon.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37 degrees and 42.5 degrees C (thermotrophs) and 30 degrees C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37 degrees and 42.5 degrees C. At 42.5 degrees C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37 degrees C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42.5 degrees C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Evaluation of the Redesigned Enterotube—a System for the Identification of Enterobacteriaceae

The redesigned Enterotube has been evaluated with 414 unknown Enterobacteriaceae cultures from the stock culture collection of the Center for Disease Control. When the Enterotube was used as recommended by the manufacturer, an average of 96.4% of these cultures were correctly identified. Only two groups (Salmonella and Edwardsiella) were identified with less than 90% accuracy (89.2 and 87.5%, respectively). The Enterotube now provides a convenient, rapid, and accurate test system for the identification of typically reacting enteric bacteria.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37° and 42°5°C (thermotrophs) and 30°C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37° and 42°5°C. At 42°5°C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37°C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42°5°C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Comparison of frequency and susceptibility to antimicrobials of bacterial strains of the family Enterobacteriaceae isolated from patients hospitalised in intensive care units

The aim of the study was estimation of frequency and susceptibility to antimicrobial agents of gram-negative rods isolated from clinical specimens obtained from patients requiring intensive care, with emphasis on profile of the unit. The analysis comprised strains of gram-negative rods isolated from patients of two intensive care units (ICUs) of a tertiary care hospital (1200 beds). Identification of cultured isolates was done using automated VITEK and API systems (bioMerieux, France). Susceptibility to antimicrobial agents was tested by a disk-diffusion method according to the NCCLS recommendations. In total the analysis comprised 722 strains of gram-negative rods. In blood cultures predominated strains of Enterobacter spp. (42.5%) and Klebsiella pneumoniae (37.5%). In cultures of clinical specimens other than blood 41.6% comprised strains of Klebsiella pneumoniae, 14.8% Escherichia coli and 14.4% Proteus mirabilis. Frequency of multi-drug resistant strains of bacteria of the family Enterobacteriaceae was much higher among blood isolates in comparison to strains cultured from other clinical specimens. There was a relatively high percentage of strains of Enterobacteriaceae susceptible to piperacillin and tazobactam (69.0%) and ceftazidime (54.6%). Conclusions: 1. All strains were susceptible to carbapenems. 2. There was a relatively high percentage of strains of gram-negative rods susceptible to piperacillin/tazobactam and ceftazidime. 3. Bacteria isolated from blood cultures were characterised by a much higher percentage of resistant strains in comparison to other specimens. 4. Longer stay in ICU promoted selection of strains resistant to antimicrobials.     

Sensitivity to antibiotics and antibacterial preparations of conditionally pathogenic bacteria isolated from patients with gastrointestinal tract dysfunctions]

Sensitivity of 690 cultures of the conditionally pathogenic microbes of Enterobacteriaceae and Pseudomonadaceae to 17 drugs was studied with the agar diffusion method. It was found that 98.6 per cent of the cultures had multiple resistance to 2--10 drugs. Most of the cultures were resistant to erythromycin, carbenicillin and ampicillin. Different species of the conditionally pathogenic microorganisms were resistant to different numbers of the drugs. Thus, Ps. aeruginosa cultures were resistant to 6--10 drugs, the cultures of Citrobacter were resistant to 3--8 drugs and the cultures of Kl. pneumonia were resistant to 2--5 drugs. Levomycetin, tetracycline, streptomycin and biseptol proved to be the most active antibacterial drugs.     

Use of pyrrolidonyl peptidase to distinguish Citrobacter from Salmonella

In the routine testing of foods for Salmonella, Citrobacter and other members of the Enterobacteriaceae often produce colonies which are almost indistinguishable from Salmonella on commonly used selective agars. Biochemical confirmation of such colonies can be expensive and time-consuming. It has been suggested that the enzyme pyrrolidonyl peptidase (PYRase) could be used as a rapid test to distinguish Citrobacter colonies (PYRase-positive) from Salmonella (PYRase-negative). Pure cultures of Salmonella, Citrobacter and other Enterobacteriaceae were tested for PYRase activity; all strains of Salmonella tested were PYRase-negative, and all Citrobacter tested were PYRase-positive. Inoculated and naturally contaminated food samples were tested for the presence of Salmonella by a standard cultural method. A PYR test was used to test Salmonella-like colonies isolated on selective agar and potentially, eliminate PYR-positive isolates from further biochemical testing. The test was able to screen out 6% of colonies selected from samples inoculated with Salmonella, and 43% of colonies selected from uninoculated samples.