Evaluation of Culture Media for the Isolation of Salmonellas from Sewage Sludge

Ten samples of sewage sludge were examined by various methods for the isolation of salmonellas using three types of enrichment broth: Muller-Kauffmann Tetrathionate Broth (MKTB), Selenite F Broth (SFB), and Brilliant Green Broth (BGB), two temperatures (37°C and 43°C) and three selective media: Deoxycholate Citrate Agar incubated aerobically (DCA), and anaerobically (DCA(N)), Brilliant Green Agar (BGA), and Bismuth Sulphite Agar (BSA). The results suggest that a combination of pre-enriched MKTB incubated at 37°C and plated on to BGA at 24 and 48 h was the best method, but when examining contaminated material such as sewage sludge, it appears unwise to rely on one single method.     

Comparison of methods for isolating Salmonella bacteria from faeces of naturally infected pigs

A series of experiments was conducted using faecal samples collected from commercial swine farms to evaluate the effects of variation in methods used for the detection of Salmonella bacteria. The primary objective of the studies was to compare the protocols routinely used in two laboratories in the USA. The studies included five experiments comparing the enrichment protocols used routinely in the respective laboratories (Method 1: 10 g faeces--buffered peptone water (BPW) pre-enrichment--selective enrichment in Rappaport/Vassiliadis (RV) broth; Method 2: approximately 1g faeces--primary enrichments in tetrathionate and Hajna GN broths--secondary enrichment in RV broth). The effects of enrichment temperatures (37 vs 42 degrees C) using RV broth (two experiments) and delayed secondary enrichment (four experiments) were also evaluated. Direct comparison of Method 1 and Method 2 indicated comparable results. However, when compared using faecal samples of equal weight, the Method 2 enrichment protocol was more sensitive for detecting Salmonella bacteria than the Method 1 protocol. Enrichment in RV at 42 degrees C was superior to 37 degrees C, particularly for samples that were pre-enriched in BPW. Delayed secondary enrichment increased detection of Salmonella bacteria in swine faeces. These results highlight the imperfect sensitivity of culture methods, and the need for researchers to consider the sensitivity of bacteriological methods in the design and interpretation of the results of epidemiologic studies based on faecal culture.     

Comparison of selective enrichment media for the detection of Salmonella in poultry faeces

AIMS: The aim of this study was to compare the results of semisolid media and Rappaport-Vassiliadis (RV) medium for the detection of Salmonella in faecal samples from broiler and layer flocks. METHODS AND RESULTS: Three different selective enrichment media were used: (a) RV medium; (b) diagnostic semisolid Salmonella medium (DIASALM) and (c) modified semisolid RV (MSRV) medium. The performance of DIASALM and MSRV was significantly better compared with RV. CONCLUSION: The results of this study indicate that approximately 95% of the samples containing Salmonella would be detected by a combination of a semisolid medium (MSRV or DIASALM) and RV. SIGNIFICANCE AND IMPACT OF THE STUDY: The International Standard method ISO 6579, including RV and selenite cystine broth as selective enrichment media, is most frequently used for the isolation of Salmonella from poultry faeces. This study reveals that there are more suitable combinations of selective enrichment media.     

Importance of pre-enrichment media for isolation of Salmonella spp. from swine and poultry

The performance of two new (1-day) culture methods, Salmonella Enrichment Broth (SEB) and Revive, and an alternative pre-enrichment broth, designated Universal pre-enrichment broth (UB), was compared to the internationally accepted buffered peptone water (BPW). The study was directed towards detection of Salmonella in 100 faecal samples from porcine and 100 neck-skin samples from poultry. The sensitivity (number of positive cases per method among all the positive cases) of the conventional pre-enrichment in BPW was found to be 0.77 for swine and 0.66 for poultry samples, while a combination of the BPW method with parallel pre-enrichment of the same sample in UB resulted in high sensitivity for swine (0.92) and poultry (0.95) samples. A 2-h pre-enrichment in the non-selective Revive, followed by overnight enrichment in selective broth, resulted in a low sensitivity, particularly for the neck-skin samples (0.16, P=0.001). The SEB method in the porcine samples resulted in a sensitivity (0.71) comparable to the standard method (P=0.31). In conclusion, additional pre-enrichment of samples in UB may substantially increase the culture sensitivity. During routine screening of large numbers of samples, it may be advantageous to use SEB rather than standard culturing.     

Thermophilic Campylobacter spp. in turkey samples: evaluation of two automated enzyme immunoassays and conventional microbiological techniques

AIMS: To determine the sensitivity and specificity of two automated enzyme immunoassays (EIA), EiaFoss and Minividas, and a conventional microbiological culture technique for detecting thermophilic Campylobacter spp. in turkey samples. METHODS AND RESULTS: A total of 286 samples (faecal, meat, neckskin and environmental samples) were collected over a period of 4 months at a turkey slaughterhouse and meat-cutting plant in Denmark. Faecal and environmental samples were tested by the conventional culture method and by the two EIAs, whereas meat and neckskin samples were tested by the two EIAs only. Two enrichment broths were used, Campylobacter Enrichment Broth (CEB) and Preston Broth (PB). Verification of positive test results was carried out by conventional culture on selective solid media. The specificities of all methods were high. The sensitivities of the EIAs were higher than that of the conventional culture technique but varied depending on the type of sample and enrichment broth. For neckskin samples, the Minividas had a significantly higher sensitivity than the EiaFoss and using PB instead of CEB as the enrichment broth significantly improved the sensitivity for both EIAs. CONCLUSIONS: Both EIAs provided more accurate results than the conventional culture technique. Furthermore, neckskin samples enriched in PB resulted in more positive test results and Campylobacter growth than samples enriched in CEB. SIGNIFICANCE AND IMPACT OF THE STUDY: The Eiafoss and Minividas proved to be reliable methods for detecting Campylobacter spp. in various samples. However, the results emphasize the need for the development of specific enrichment protocols for specific samples.     

Immunomagnetic separation and conventional culture procedure for detection of naturally occurring Salmonella in raw pork sausages and chicken meat

The aim of the study was to compare immunomagnetic separation (IMS) and conventional selective enrichment procedures using selenite cystine broth (SC) and Rappaport–Vassiliadis broth (RV) in 137 naturally contaminated food samples (69 raw pork sausages and 68 chicken meat). The utilization of SC or IMS appeared to be the most appropriate enrichment procedure: 15 out of 18 Salmonella -positive samples (83·3%) were detected by SC and 12 (66·7%) by IMS; RV yielded only seven positive isolations (38·9%). However, RV yielded the highest count of Salmonella colonies per plate and the lowest interference by competing organisms. IMS could become a reliable alternative to standard enrichment procedures and a combined IMS and selective enrichment broth could increase the chance of Salmonella recovery.     

Evaluation of new culture media for rapid detection and isolation of salmonellae in foods.

Conventional methods for Salmonella detection in foods can require up to 6 and at least 4 days. We have observed that the total analysis time can be reduced to 48 h by using Salmosyst broth as a liquid medium for both preenrichment and selective enrichment and Rambach agar (RA), a new selective plate medium. In samples of artificially contaminated ground beef Salmonella enteritidis was detected at a concentration of 0.4 CFU/g (10 CFU/25 g) by both a conventional method and the new method. Of 519 samples of foods for sale, 38 were Salmonella positive by both methods while 471 were negative. Nine samples which were negative by the conventional method were positive by the Salmosyst-RA method, while one sample positive by the first method was negative by the last. Therefore, the Salmosyst-RA method showed 97.9% sensitivity compared with the 81.2% sensitivity of the conventional method. The new method was also highly specific (98% specificity) in presumptive identification of Salmonella colonies. Furthermore, a 6-h preenrichment in Salmosyst broth has been proved sufficient for the repair of heat-injured Salmonella cells and for subsequent recovery by selective enrichment. In conclusion, the Salmosyst-RA method shows several advantages over both conventional and rapid noncultural methods: (i) only two media are required instead of the five media for conventional methods; (ii) in real time it is comparable to other rapid noncultural methods, which require 30 to 31 h; (iii) it is highly sensitive and specific; and (iv) it allows the isolation of Salmonella strains which can be characterized by appropriate phenotypic and genotypic typing methods for epidemiological investigations.     

Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples

We compared six procedures and investigated the optimal method for isolation of Campylobacter spp. from raw meat samples. Ninety-nine meat samples were enriched in Bolton broth and Preston broth, followed by plating on Skirrow, mCCDA, and blood agar (a membrane filter on its surface) media, respectively. Thirty-nine of 99 samples were positive and 71 Campylobacter were isolated by one or more methods. More than one species of Campylobacter were obtained in 8 (20.5 %) of 39 positive samples and two genotypes were yielded on the same medium (11 samples, 28.2 %) by pulsed-field gel electrophoresis (PFGE) genotyping. Enrichment by Preston broth was significantly better than by Bolton broth (P?<?0.05). Moreover, the latter failed to detect Campylobacter jejuni strains. Skirrow medium was significantly less efficient than mCCDA medium and membrane filtration method (P?<?0.05). Overall, the combination of PC (primary enrichment in Preston broth, followed by selective enrichment on mCCDA agar), PF (primary enrichment in Preston broth, followed by membrane filtration culture onto blood agar), and BF (primary enrichment in Bolton broth, followed by membrane filtration culture onto blood agar) methods provided the optimum isolation rate of Campylobacter spp.     

A note on the use of Rappaport-Vassiliadis medium (RIO/RV) for the isolation of salmonellas from sewage and sewage sludges

The use of a modified Rappaport broth for the selective enrichment of salmonellas in sewage sludge is described. Comparative trials were carried out using Muller Kauffman—Tetrathionate (MKT) broth and Rappaport-Vassiliadis (R10/RV) medium for selective enrichment. Results have indicated that R10/RV broth is more selective and is to be preferred for routine monitoring.     

A note on the use of Rappaport-Vassiliadis medium (R10/RV) for the isolation of salmonellas from sewage and sewage sludges

The use of a modified Rappaport broth for the selective enrichment of salmonellas in sewage sludge is described. Comparative trials were carried out using Muller Kauffman--Tetrathionate (MKT) broth and Rappaport-Vassiliadis (R10/RV) medium for selective enrichment. Results have indicated that R10/RV broth is more selective and is to be preferred for routine monitoring.     

Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively. CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42 degrees C) were higher even when MKT (43 degrees C) performance was improved by the use of brilliant green sul-phamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum. The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Modified enrichment broth for isolation of Yersinia enterocolitica from nonfood sources

Sorbitol-bile salts broth was modified by the addition of 0.5% peptone for the enrichment of Yersinia enterocolitica in nonfood samples. A quantitative comparison was made of the growth of Y. enterocolitica in sorbitol-bile salts broth and peptone-sorbitol-bile salts broth at 22 degrees C. Incubation for 48 h resulted in an average 7.79 log10 increase in a viable cell number for six serotypes with peptone-sorbitol-bile salts broth compared with an average 1.85 log10 increase for sorbitol-bile salts broth. Recovery from seeded seawater showed a greater than 100-fold increase in sensitivity with peptone-sorbitol-bile salts broth enrichment. Isolation of indigenous Y. enterocolitica from fresh and marine waters was also significantly enhanced.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42°C) were higher even when MKT (43°C) performance was improved by the use of brilliant green sulphamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Modified enrichment broth for isolation of Yersinia enterocolitica from nonfood sources.

Sorbitol-bile salts broth was modified by the addition of 0.5% peptone for the enrichment of Yersinia enterocolitica in nonfood samples. A quantitative comparison was made of the growth of Y. enterocolitica in sorbitol-bile salts broth and peptone-sorbitol-bile salts broth at 22 degrees C. Incubation for 48 h resulted in an average 7.79 log10 increase in a viable cell number for six serotypes with peptone-sorbitol-bile salts broth compared with an average 1.85 log10 increase for sorbitol-bile salts broth. Recovery from seeded seawater showed a greater than 100-fold increase in sensitivity with peptone-sorbitol-bile salts broth enrichment. Isolation of indigenous Y. enterocolitica from fresh and marine waters was also significantly enhanced.     

Methods for the isolation of water-borne Escherichia coli O157

AIMS: To develop improved methods for the detection of Escherichia coli O157 from water and sediments. METHODS AND RESULTS: The effects of different broth enrichment media (unsupplemented tryptic soya broth, tryptic soya broth with antibiotics, and gram-negative broth), incubation durations (5 and 24 hrs), incubation temperatures (37 and 44.5 degrees C) and the use of immunomagnetic separation (IMS) on the sensitivity of E. coli O157 detection were evaluated on artificially and naturally-contaminated water and sediment samples. The sensitivity of recovery of E. coli O157 from samples was dependent upon the media composition, temperature duration of incubation and the use of IMS. CONCLUSION: Use of high temperature (44.5 degrees C) incubation for 24 hrs in unsupplemented tryptic soya broth and the use of IMS improved the sensitivity of E. coli O157 culture from water and sediment samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods described can be used to increase the sensitivity of E. coli O157 detection from water and sediments.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

人体肠道细菌寡营养培养组条件的优化研究

【目的】探讨寡营养对人体肠道细菌培养组的条件。【方法】通过稀释富集培养基、固体平板和增菌肉汤培养基成分获得寡营养培养基。对健康人粪便样本分别用原液(0)、5、10、20、30和40倍稀释的富集培养基(添加羊血和瘤胃液的血培养瓶)连续增菌,在不同时间点(第0、3、6、9、15、27、30天)吸取增菌液,用YCFA (yeast casitone fatty acid)固体培养平板分离菌落;用YCFA增菌肉汤增菌后再次挑取单菌落,利用基质辅助激光解吸/电离飞行时间(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF)质谱和16S rRNA基因测序鉴定菌株。通过比较上述6种寡营养条件分离肠道菌群的效果,选取富集培养基原液、稀释10倍和30倍这3 种条件下分离效果较好的富集条件,与同样稀释倍数条件的固体平板和增菌肉汤分别组合成9种培养基条件,进一步优化肠道菌群的培养组条件。【结果】在6种寡营养富集培养基中,未稀释(原液)、10 倍和30倍稀释的富集培养基分离细菌的种类比其他...