Evaluation of Two Commercially Available Media for Detection of Bacteremia

Analysis of the results of 13,162 blood cultures during a 9-month interval has shown that Pseudomonas aeruginosa statistically was recovered more frequently from Trypticase soy broth (TSB) than from Thioglycollate-135C and that contaminants, including Staphylococcus epidermidis and aerobic and anaerobic Corynebacterium species, were isolated with statistically greater frequency from Thioglycollate-135C than from TSB. No other statistically significant differences were found.     

Comparison of Two Commercially Available Media for Detection of Bacteremia

An analysis of 3,795 positive blood cultures obtained from 1,718 patients in a 2.5-year evaluation of Tryptic Soy Broth (TSB) and Thiol Broth is reported. Isolation rates of Actinobacillus and Pseudomonas were significantly greater in TSB, whereas isolation rates of Streptococcus and Corynebacterium (aerobic and anaerobic) were significantly greater in Thiol. Otherwise, the two media were similar. Disregarding contaminants, anaerobic bacteria represented 11% of positive cultures and 20% of patients with bacteremia. Eleven per cent of the patients had polymicrobial bacteremia.     

Comparison of three enrichment media for the isolation of Campylobacter spp. from foods

AIM: This study compared the performance of three Campylobacter enrichment broths: Bolton broth (BB), Campylobacter Enrichment broth (CEB) and Preston broth (PB). METHODS AND RESULTS: Pure cultures of target and competitor organisms, and naturally-contaminated food samples, were used to establish the performance of these media. In pure culture the PB supported the growth of the greatest number of strains of Campylobacter spp. but failed to inhibit some competitor organisms. The CEB showed the opposite result, inhibiting all 15 competitor organisms used but failing to support the growth of five Campylobacter strains. By comparison, BB showed the best compromise between inhibition of competitors and growth of Campylobacter. CONCLUSIONS: Plates inoculated with BB and CEB food enrichments resulted in more Campylobacter growth than those inoculated with PB, which supported significantly less typical growth (P < or = 0.001). The most common competitor organism isolated from PB was Escherichia coli, and Pseudomonas spp. were frequently isolated from BB and CEB. Both BB and CEB were better than PB for the isolation of Campylobacter from naturally-contaminated foods, although BB yielded more confirmed Campylobacter growth than CEB. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in performance of media used to isolate Campylobacter spp. from foods.     

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Inhibitory activity of 2-nitropropanol against select food-borne pathogens in vitro

AIMS: To test the inhibitory activity of 2-nitro-1-propanol (2NPOH) against Salmonella Typhimurium, Escherichia coli O157:H7 and Enterococcus faecalis. METHODS AND RESULTS: Specific growth rates (h(-1)) of S. Typhimurium, E. coli O157:H7 and Ent. faecalis were determined during culture in tryptic soya broth (TSB) supplemented with 0-10 mm 2NPOH. Growth rates were inhibited by 2NPOH, with nearly complete inhibition observed with 10 mm. Studies with S. Typhimurium revealed that its survivability during culture in TSB containing 5 or 10 mm 2NPOH was lower (P < 0.05) under aerobic than anaerobic conditions. The survivability of Salmonella during anaerobic culture in TSB containing 2.5 mm 2NPOH was less at pH 5.6 than at pH 7.0 and 8.0. No Salmonella survived anaerobic incubation in TSB supplemented with 10 mm 2NPOH regardless of pH. When incubated in suspensions of freshly collected populations of ruminal and faecal bacteria, Salmonella concentrations were lower (P < 0.05) in suspensions containing 10 mm 2NPOH than in suspensions containing no 2NPOH. CONCLUSIONS: 2NPOH inhibited S. Typhimurium, E. coli O157:H7 and Ent. faecalis. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that 2NPOH may be a useful antimicrobial supplement to reduce carriage of certain food-borne pathogens in food animals.     

Culturing enterohemorrhagic Escherichia coli in the presence and absence of glucose as a simple means of evaluating the acid tolerance of stationary-phase cells.

Prior growth of seven enterohemorrhagic and one nonenterohemorrhagic strains of Escherichia coli in tryptic soy broth with (TSB+G) and without (TSB-G) 1% glucose was evaluated for its effect on acid tolerance. The final pHs of 18-h TSB+G and TSB-G cultures were 4.6 to 5.2 and 6.9 to 7.0, respectively. Cells were then transferred to brain heart infusion broth adjusted to pH 2.5 or 3.0 with HCl, incubated at 37 degrees C for up to 7 h, and assayed periodically for viable populations with brain heart infusion and MacConkey agars. All enterohemorrhagic strains were acid resistant (< 0.5 log decline after 7 h) when initially cultured in TSB+G, but substantial differences in acid tolerance were observed among strains cultured in TSB-G (log declines ranged from < 0.3 to > 3.8). The results indicated that prior growth in a medium with and without a fermentable carbohydrate is a convenient way to studying the induction of acid tolerance, that acid inactivation is preceded by a period of acid injury, and that pH-independent and pH-dependent stationary-phase acid tolerance phenotypes may exist among strains of enterohemorrhagic E. coli.     

Antibacterial Effect of Bovine Lactoferrin Against Udder Pathogens

The antibacterial effect of lactoferrin (Lf) was tested on isolates of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and coagulase-negative staphylococci (CNS) as well as on Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae), originally isolated from bovine mastitis. Concentrations of Lf used were 0.67 mg/ml, 1.67 mg/ml, and 2.67 mg/ml. Growth of udder pathogens was monitored by turbidometry either in broth culture or in whey prepared from normal milk. We focused on 3 different growth variables: lag time, slope, and maximum absorbance of bacterial growth curves. Growth inhibition was seen in the broth but hardly at all in whey. The isolates of E. coli and CNS did not grow sufficiently well in whey to draw any conclusions. The most effective inhibitory activity of Lf was seen against E. coli and P. aeruginosa. All 5 E. coli isolates had similar growth patterns. Inhibition of growth by Lf was concentration-dependent. The concentration of 0.67 mg/ml in broth and whey was generally too low for a significant inhibitory effect.     

Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner’s 2nd Agar or Broth (R2A or R2AB), Brain–Heart Infusion Broth (BHIB), Mueller–Hinton Broth (MHB), and Ashdown’s (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.     

Prevalence of Escherichia coli O157 in Cattle Feeds in Midwestern Feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

Evaluation of the Sterifil Lysis-Filtration Blood Culture System

This paper describes the comparison of the Sterifil lysis-filtration (SLF) blood culture procedure with a standard Trypticase soy broth (TSB) technique. The lysing solutions employed in the SLF system, Triton X-100 (alkyl phenoxy polyethoxy ethanol) and sodium carbonate, were deleterious to most bacteria commonly encountered in bacteremia except staphylococci and enterococci. Candida was not adversely affected. There was a positive correlation between the tolerance of the microbial isolants to the lysing solutions and their recovery by the SLF technique. A total of 3,554 cultures were run in parallel and 398 isolants were obtained. Of 201 gram-positive isolants, 135 were recovered by both techniques, 43 were detected by the TSB technique only, and 23 were recovered only with the SLF method. In sharp contrast, of 168 gram-negative isolants, 28 were recovered in common, 130 were isolated only by TSB, and 10 were recovered only with the SLF method. The SLF method detected all cases of candidemia detected by the TSB method plus an additional 12 for a total of 29 cases. The SLF method, as currently described, is generally too toxic to bacteria for routine use in a clinical laboratory.     

Role of glucose in enhancing the temperature-dependent growth inhibition of Escherichia coli O157:H7 ATCC 43895 by a Pseudomonas sp

Growth of Escherichia coli O157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25 degrees C in both pure and mixed (1:1) cultures with a gluconate-producing Pseudomonas sp. found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition. The number of colonies of the Pseudomonas strain exceeded 9 log CFU/ml under all conditions tested. The pathogen grew better as the temperature increased from 10 to 15 and 25 degrees C and grew better in pure culture than in mixed cultures. Pseudomonas sp. inhibited E. coli O157:H7 in cocultures with glucose at 10 degrees C, while at 15 degrees C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period. Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15 degrees C and, irrespective of glucose, at 25 degrees C. These results indicate that glucose enhances the growth inhibition of E. coli O157:H7 by some Pseudomonas spp., potentially due to its rapid uptake and conversion to gluconate, at low (< or = 15 degrees C) temperatures.     

Addition of a surfactant to tryptic soy broth allows growth of a lactic acid bacteria food antimicrobial, Escherichia coli O157:H7, and Salmonella enterica

Aims: This study aimed to determine the survival and growth of Escherichia coli O157:H7 and Salmonella enterica subsp. enterica in a medium supporting the growth of a Lactic Acid Bacteria (LAB) food antimicrobial culture. Methods and Results: Foodborne pathogens and LAB were cultured individually in tryptic soy broth (TSB), tryptic soy broth supplemented with one g l^?1 Tween 80^® (TSB‐T80), and de Man, Rogosa and Sharpe (MRS) broth. Growth of E. coli O157:H7 and Salmonella was similar in TSB and TSB‐T80 but was significantly less in MRS. Conversely, LAB growth was similar in MRS and TSB‐T80 but was significantly less in TSB. Conclusions: Supplementation of TSB with Tween 80^® allows growth of LAB to levels similar to that observed with MRS but does not inhibit the growth of E. coli O157:H7 and Salmonella. We present the formulation of a medium useful in studies useful for evaluating competitive inhibition of foodborne pathogens by LAB in vitro. Significance and Impact of the Study: This study reports the utility of TSB‐T80 for the completion of in vitro competitive inhibition assays incorporating a Lactic Acid Bacteria food safety culture.     

Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples

Three different enrichment broths and two selective growth media were compared for isolating thermophilic campylobacters by combined membrane filtration and enrichment techniques from surface waters of different physical, chemical and bacteriological characteristics. Fifty-two strains of campylobacters were isolated from total of 1668 cultures. The various broth/medium combinations did not affect the dominance of C. jejuni over C. coli (total 49 C. jejuni and three C. coli). The most efficient combinations of enrichment broth and growth media were either Oosterom broth/blood-free charcoal-cefoperazone-deoxycholate agar (CCDA) medium or blood-free charcoal-cefoperazone-deoxycholate (CCD) broth/CCDA medium. Modified Preston broth (sheep blood instead of horse blood) with either of the growth media gave significantly lower yields although it suppressed efficiently the growth of contaminants. Skirrow medium had lower selectivity than CCDA medium and gave slightly lower isolation rate. Enrichment time (24 or 48 h) did not affect the isolation frequency of campylobacters but longer enrichment time increased the growth of contaminants. Prefiltration through membranes of pore sizes 5.0 and 1.2 microns decreased the growth of contaminants. However, these membranes retain campylobacters and must be cultured to avoid underestimation. From more polluted waters campylobacters were isolated most frequently with CCD broth and CCDA medium.     

Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli O111 and O26

Polymyxin-based enzyme-linked immunosorbent assay (polymyxin-ELISA) systems were developed for the detection of Escherichia coli O111 and O26 in ground beef after enrichment. Polymyxin immobilized in the wells of a microtiter plate served as a high affinity adsorbent for lipopolysaccharide (LPS) antigens, which were detected immunoenzymatically using commercially available anti-E. coli O111 or anti-E. coli O26 antisera. The polymyxin-ELISA sensitively detected E. coli strains bearing the O111 and O26 LPS antigens, discriminating between these target strains and a panel of various non-target Gram negative and Gram positive bacteria. The detection of E. coli O111 and O26 strains inoculated into ground beef was achieved after enrichment in either modified trypticase soy broth (TSB) with novobiocin, or the serotype-specific medium TSB supplemented with cefixime and vancomycin (E. coli O111), and the same medium containing potassium tellurite (E. coli O26). The polymyxin-ELISA shows promise as a rapid, simple and inexpensive screening tool for E. coli O111 and O26 in enrichment cultures of ground beef.     

Tinctorial Properties of Spherical Bodies in Broth Cultures of Nocardia asteroides GUH-2

In yeast extract-supplemented brain heart infusion (BHI) broth cultures of Nocardia asteroides GUH-2, many spherical bodies (SBs) were frequently seen nearby filamentous cells. They showed no Gram-positivity when Gram stain was applied. When acridine orange stain was applied, many of them showed different green fluorescence from bright orange fluorescence of the filamentous nocardiae under ultraviolet light. Their acid-fastness appeared to depend on the presence of paraffin. Using the polymerase chain reaction (PCR) method, 16S rRNA genes were detected in SB-containing broth cultures inoculated with culture filtrates from broth cultures of the strain and identical to that of N. asteroides. These results suggest that SBs are cell wall-defective (CWD) forms which result from the spontaneous mutation of N. asteroides GUH-2.     

THE HEAT RESISTANCE OF ESCHERICHIA COLI CELLS FROM CULTURES OF DIFFERENT AGES

SUMMARY: From the mortality curves of Escherichia coli cells heated at 55° in Ringer's solution both Decimal Reduction Times (DRTs) and 99–9% mortality times were obtained. In terms of these measures of heat resistance, cells harvested from broth cultures 0–8 hr old were more susceptible than those from more mature cultures. The time of commencement and the approximate duration of the logarithmic phase of growth of the organism in broth were determined from growth curves, and it was observed that the heat resistance was minimal during that phase.
Death rates were not always uniform for the whole of a given population. Particularly among young cultures, a period of rapid death was frequently followed by the slower death of a relatively small number of survivors. In one instance only was an initial period of slow death followed by one of more rapid death.     

Effect of Available Water on Thermal Resistance of Three Nonsporeforming Species of Bacteria

Cells of Escherichia coli, Pseudomonas fluorescens, and Staphylococcus aureus, previously grown in Trypticase Soy Broth (TSB) at a high level of available moisture (a(w) 0.994) and at low levels produced by addition of NaCl or glucose, were heated in neutral phosphate buffer, and in this buffer adjusted to low levels of available moisture by means of NaCl or glucose. Glucose in the heating medium was more protective than NaCl for E. coli and P. fluorescens, but hastened the thermal destruction of S. aureus. Added protection was given P. fluorescens during heating in glucose-buffer solution at a(w) 0.97 by previous growth in TSB adjusted to that a(w) value with glucose. Added protection was given E. coli during heating in NaCl-buffer solution at a(w) 0.98 by previous growth in TSB adjusted to that value with NaCl. With S. aureus, however, previous growth in TSB plus NaCl or glucose had little effect on heat resistance, but the solute in the heating medium had great influence, in that NaCl was very protective and glucose destructive. Opportunity may have been given during tempering of the cell suspension at 30 C in the heating medium prior to heating for the NaCl and glucose to diffuse into the staphylococcal cells.     

Growth of starved Escherichia coli O157 cells in selective and non-selective media.

Escherichia coli O157 strains starved in sterile deionized water (SDW) and filter-sterilized natural river water (SRW) were investigated with specific reference to their culturability in selective and non-selective media. Growth of the strains starved in both SDW and SRW were markedly suppressed with time in selective liquid media such as modified trypticase soy broth supplemented with novobiocin (mTSB+n) and modified E. coli broth supplemented with novobiocin (mEC+n). This suppression was more pronounced when incubated at 42 C than at 37 C, especially with mEC+n. By contrast, such growth suppression was seldom observed when cultured at 37 C in non-selective liquid media such as trypticase soy broth (TSB) and buffered peptone water. In mEC+n at 42 C, the non-starved cells from overnight cultures with an initial density of less than 10(3) colony-forming units (CFU)/ml grew to the density of over 10(7) CFU/ml after 24 hr incubation, whereas those starved for 6 weeks in SRW were only to maintain their initial density or died off after 24 hr incubation under the same culturing conditions. These results indicated that the isolation of starved cells of E. coli O157 from water samples would be most difficult with selective enrichment or direct plating on the selective plate media. It is thus highly recommended that a "resuscitation" of the cells with non-selective enrichment should be performed as a routine practice for maximum recovery of E. coli O157 from water systems.     

Mineral and carbon usage of two synthetic pyrethroid degrading bacterial isolates

AIMS: To investigate the biodegrading ability and cometabolism of synthetic pyrethroid (SP) utilizing bacteria in cultures with various minerals and carbon sources. METHODS AND RESULTS: Previously isolated SP-degrading Pseudomonas sp. and Serratia sp. were used in cultures containing either flumethrin SP or cypermethrin SP formulations. The culture media consisted of either (i) water only, (ii) water and sucrose, (iii) mineral broth or (iv) mineral broth and sucrose. The growth of both organisms was greatest in the mineral broth and sucrose medium, but the growth-limiting factor for Pseudomonas sp. strain Circle was the mineral content whereas for Serratia sp. strain White it was the carbon substrate. CONCLUSION: The greatest extent of degradation of both SP-based compounds occurred with Pseudomonas sp. strain Circle but was dependant on the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation could lead to the development of a relatively inexpensive medium supplement to enhance the microbial biodegradation of undesirable compounds, either in situ or ex situ. In this particular case, for the biodegradation of SPs used in sheep dip.