A chromogenic plating medium for the isolation and identification of Enterobacter sakazakii from foods, food ingredients, and environmental sources

A chromogenic agar, R&F Enterobacter sakazakii chromogenic plating medium (ESPM), was developed for isolating presumptive colonies of E. sakazakii from foods and environmental sources. ESPM contains two chromogenic substrates (5-bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside and 5-bromo-4-chloro-3-indoxyl-beta-D-cellobioside), three sugars (sorbitol, D-arabitol, and adonitol), a pH indicator, and inhibitors (bile salts, vancomycin, and cefsulodin), which all contribute to its selectivity and differential properties. On ESPM, 79 pure culture strains of E. sakazakii (10 clinical isolates and others from food and environmental sources) yielded blue-black (three strains were blue-gray) raised colonies, 1 to 2 mm in diameter with and without halos after 24 h at 35 degrees C. Other enteric organisms plus Pseudomonas aeruginosa yielded white, yellow, green, or clear colonies with and without clear halos. Of these genera, only Shigella sonnei and one Pantoea strain produced blue-black to blue-gray colonies. ESPM was used to isolate E. sakazakii from a variety of foods: corn, wheat, and rice flours; powdered infant formula; dairy products (dried milk, whey, and caseinates); cereals; and environmental sources. Most false-positive results on ESPM were eliminated by observing acid production on either sucrose or melibiose after 6 h at 35 degrees C on a R&F E. sakazakii screening medium (ESSM) biplate. In an analysis of 240 samples, the number of samples positive for E. sakazakii by the ESPM-ESSM method and the U.S. Food and Drug Administration protocols (violet red bile glucose agar and tryptic soy agar) were 27 and 16, respectively, with sensitivity and specificity values of 100.0 and 96.9% versus 59.3 and 43.7%, respectively. These data support the fact that E. sakazakii confirmation should be based on more than one confirmation system. Both the API 20E and Biolog Microlog3 4.20 systems should be used for confirmation of E. sakazakii isolates.     

Comparison of three chromogenic media and evaluation of two molecular-based identification systems for the detection of Enterobacter sakazakii from environmental samples from infant formulae factories

Enterobacter sakazakii is an occasional contaminant of powdered infant formula that can cause rare but severe foodborne infections in infants. To determine optimal methods for the detection and identification of E. sakazakii, 38 naturally contaminated samples from infant formulae factories were analyzed by two PCR-based methods and by a method (TS 22964/RM 210) developed by the International Organization for Standardization and the International Dairy Federation (ISO-IDF) using three different commercial chromogenic agars. The ISO-IDF method includes two enrichment steps, plating of the second enrichment broth on E. sakazakii isolation agar (a chromogenic selective agar), picking of five typical colonies for transfer onto tryptone soy agar, and subsequent confirmation of yellow-pigmented colonies by biochemical characterization. Twenty-two of the 38 samples were positive by the culture method. E. sakazakii isolation agar (ESIA; AES Laboratoires), COMPASS agar (Biokar Diagnostics), and Druggan-Forsythe-Iversen agar (Oxoid) compared favorably with violet red bile glucose agar (VRBG, a selective medium for Enterobacteriaceae), with positive predictive values of 86.96, 88, and 74.07%, respectively, in contrast to 47.83% for VRBG. One additional positive sample was detected using the nonpatented real-time PCR method evaluated, and those results were in 97.3% concordance with the ISO-IDF results. Some discrepancies between the results of the DuPont Qualicon BAX system and those of the ISO-IDF method could be explained by heterogeneity of contamination and sampling. Thus, both PCR-based systems were suitable for detecting and specifically identifying E. sakazakii within 1 to 2 days, and COMPASS agar and ESIA could be used interchangeably as a first-step medium to isolate presumptive E. sakazakii colonies.     

A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter sakazakii cells from infant formula

This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan-Forsythe-Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1x10(7)CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 degrees C for 10min), a freeze-thaw cycle (-20 degrees C for 24h, thawed at room temperature, frozen again at -20 degrees C, and thawed), acidic pH (pH 3.56 for 15min), alkaline pH (pH 11.04 for 15min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1x10(6)CFU/g, held at 21 degrees C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.     

A selective differential medium for Enterobacter sakazakii, a preliminary study

Enterobacter sakazakii can cause fatal invasive infection of neonates associated with the presence of this organism in powdered infant milk formula. A new chromogenic medium (Druggan-Forsythe-Iversen agar, DFI) is described for the selective detection of this emergent pathogen. The medium is based on the alpha-glucosidase reaction which is detected using 5-bromo-4-chloro-3-indolyl-alpha,D-glucopyranoside (XalphaGlc). Ent. sakazakii hydrolyses this substrate to an indigo pigment, producing blue-green colonies on this medium. DFI was compared with the current method of detection on violet red bile glucose agar (VRBGA) followed by pigment production on tryptone soy agar (TSA) after 48-72 h at 25 degrees C and subsequent biochemical profile determination using Biomerieux API20E. Ninety-five clinical and food strains of Ent. sakazakii were detected on the DFI chromogenic medium 2 days sooner than the alternative method. The characteristics of 148 strains representing 17 genera of non-Ent. sakazakii Enterobacteriaceae were compared using the two methods. Only 16/18 Escherichia vulneris strains, 2/3 strains of Pantoea spp. and 1/8 Citrobacter koseri strains gave false positive results on DFI agar. Eight alpha-glucosidase positive strains were identified as Pantoea using their API20E biochemical profile, but had higher percentage identification as Ent. sakazakii using ID32E. Therefore the DFI medium enables the detection of Ent. sakazakii within mixed cultures of Enterobacteriaceae, whereas the organism could be missed when using VRBGA since the latter is a general Enterobacteriaceae selective medium. In addition, the common use of API20E to check yellow pigmented colonies on TSA may lead to false negative results and consequently the acceptance of a batch of infant formula milk (IFM) that contains Ent. sakazakii.     

Isolation of Yersinia enterocolitica from foods.

Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24 degrees C; plating on SSDC agar (2 days at 30 degrees C). (2) Pre-enrichment in TSB for 1 day at 24 degrees C; enrichment in BOS for 5 days at 24 degrees C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCl for 5 s); plating on CIN agar (2 days at 24 degrees C).     

A new protocol for the detection of Enterobacter sakazakii applied to environmental samples

Enterobacter sakazakii is a motile, peritrichous, gram-negative rod that was previously known as a yellow pigmented Enterobacter cloacae. It is documented as a rare cause of outbreaks and sporadic cases of life-threatening neonatal meningitis, necrotizing enterocolitis, and sepsis. E. sakazakii has been isolated from milk powder-based formulas, and there is thus a need to investigate whether and where E. sakazakii occurs in these manufacturing environments. For this purpose, a simple detection method was developed based on two features of E. sakazakii: its yellow pigmented colonies when grown on tryptone soy agar and its constitutive alpha-glucosidase, which is detected in a 4-h colorimetric assay. Using this screening method, E. sakazakii strains were isolated from three individual factories from 18 of 152 environmental samples, such as scrapings from dust, vacuum cleaner bags, and spilled product near equipment. The method is useful for routine screening of environmental samples for the presence of E. sakazakii.     

Survival of Enterobacter sakazakii in powdered infant formula as affected by composition, water activity, and temperature

A study was done to determine survival characteristics of Enterobacter sakazakii in four milk-based and two soybean-based powdered infant formulas. A 10-strain mixture of E. sakazakii was inoculated into the six infant formulas at water activity (aw) 0.25 to 0.30, 0.31 to 0.33, and 0.43 to 0.50 to give low (0.80 log CFU/g) and high (4.66 to 4.86 log CFU/g) populations. At an initial population of 0.80 log CFU/g, E. sakazakii was detected by enrichment in six of six, four of six, and one of six formulas stored for 12 months at 4, 21, and 30 degrees C, respectively. In four of six formulas at aw values of 0.25 to 0.30, initially high populations decreased significantly (P < or = 0.05), although by less than 1 log CFU/g, within 6 months at 4 degrees C. Populations decreased significantly in all formulas in the aw range of 0.25 to 0.50 during storage for 1 month at 21 or 30 degrees C and again between 1 and 6 months in most formulas. Significant reductions occurred between 6 and 12 months in some formulas. At all storage temperatures, reductions in populations tended to be greater in formulas at aw 0.43 to 0.50 than in formulas at aw 0.25 to 0.30. The rate of inactivation of E. sakazakii in formulas was not markedly influenced by formula composition. Cells from mucoid and nonmucoid colonies formed by two strains on violet red bile glucose agar supplemented with pyruvate were inoculated into a milk-based powdered infant formula and a soybean-based powdered infant formula having a high aw range of 0.43 to 0.86 and stored at 4, 21, and 30 degrees C for up to 36 weeks. With few exceptions, populations of both strains decreased significantly in both formulas within 2 weeks at all temperatures; rates of death increased with increased aw and storage temperature. The presence of mucoidal extracellular materials on the surface of E. sakazakii cells was not associated with protection against death. This study shows that the retention of viability of E. sakazakii in powdered infant formula is affected by a, and temperature. Increases in both parameters cause an increase in the rate of death.     

Comparison of sampling methods for the detection of Salmonella on whole broiler carcasses purchased from retail outlets

An experiment was conducted to compare the effectiveness levels of two methods in recovering Salmonella from the same carcass. One hundred fresh whole broiler chickens were purchased from retail outlets over a 5-week period (20 carcasses per week). After carcasses had been aseptically removed from the packages and giblets had been removed, the carcasses were placed in sterile bags containing 400 ml of 1% buffered peptone water, the bags were shaken for 60 s, and a 30-ml aliquot was removed and incubated for 24 h at 37 degrees C (aliquot sample). Then, an additional 130 ml of 1% buffered peptone water was immediately added to the bag with the carcass (bringing the volume to 500 ml), the bag was reshaken, and the carcass and rinse were incubated for 24 h at 37 degrees C (whole-carcass enrichment sample). Following incubation, 0.5-ml samples for the two methods were placed into 10 ml of Rappaport-Vassiliadis broth and into 10 ml of tetrathionate (Hajna) broth and incubated at 42 degrees C for 24 h. Each broth was then streaked onto BG Sulfa agar and modified lysine iron agar and incubated for 24 h at 35 degrees C. Suspected Salmonella colonies were inoculated onto triple sugar iron and lysine iron agar slants and incubated at 35 degrees C for 24 h. Presumptive positive results were confirmed by Poly O and Poly H agglutination tests. Over the 5-week period, 13% of the aliquot samples tested positive for Salmonella, compared with 38% of the whole-carcass enrichment samples from the same carcasses. Recovery rates ranged from 0 of 20 samples to 4 of 20 samples for aliquot method and from 4 of 20 samples to 10 of 20 samples for the whole-carcass enrichment method over the 5-week period. These results indicate that when small numbers of Salmonella are expected, the sampling method has a major influence on the identification of Salmonella-positive carcasses.     

Growth and survival of Enterobacter sakazakii in human breast milk with and without fortifiers as compared to powdered infant formula

Enterobacter sakazakii infections often involve debilitated neonates consuming contaminated reconstituted powdered infant formula. There is the possibility that expressed human breast milk can become contaminated with E. sakazakii in the hospital or home setting and through the use of contaminated breast milk fortifiers. In addition, although breast milk has been shown to have some antimicrobial effects, this has not been extensively researched in regards to E. sakazakii. Thus, we examined the survival and growth of 9 strains of E. sakazakii in breast milk, human breast milk with fortifiers and powdered infant formula at 10, 23 and 37 degrees C. The average generation times for clinical, food and environmental isolates in breast milk were 0.94+/-0.04, 0.75+/-0.04 and 0.84+/-0.04 h at 23 degrees C; and 0.51+/-0.03, 0.33+/-0.03 and 0.42+/-0.03 h at 37 degrees C, respectively. E. sakazakii was able to survive up to 12 days in breast milk with fortifiers at 10 degrees C. However, its average generation times among replicates and isolate sources ranged from 11.97+/-3.82 to 27.08+/-4.54 h in breast milk at 10 degrees C. Interestingly, average generation times in breast milk with fortifiers at 23 degrees C (0.83+/-0.05, 0.93+/-0.06 and 0.96+/-0.06 h) and 37 degrees C (0.41+/-0.04, 0.51+/-0.05 and 0.54+/-0.05 h) were longer than in powdered infant formula and breast milk at the same temperatures, indicating that human breast milk fortifiers may have an inhibitory effect on the growth of E. sakazakii. However, the intrinsically ascribed antimicrobial properties of breast milk do not appear to inhibit the growth of this foodborne pathogen in-vitro.     

Characterization of a collection of Enterobacter sakazakii isolates from environmental and food sources

Enterobacter sakazakii has emerged as a rare cause of neonatal meningitis, septicemia and enterocolitis. Contaminated infant milk formula (IMF) has been identified as one infection route. A small number of clinical outbreaks have been epidemiologically linked to IMF contaminated post-pasteurization during manufacture and/or mishandled when reconstituted. Currently no agreed standardized typing protocol has been developed to trace E. sakazakii. The objectives of this study were to apply biochemical and genetic methods to characterize 51 environmental and food E. sakazakii isolates and 6 E. sakazakii type strains. Isolates were presumptively identified using biochemical profiles based on API 20E and ID32E methods and by culture on differential selective Druggan Forsythe Iversen (DFI) agar. Identification was subsequently confirmed by real time polymerase chain reaction (PCR). All but one of the isolates was identified as E. sakazakii by biochemical profiling. One isolate was identified as Escherichia vulneris by ID 32E and as Pantoea agglomerans by API 20E. All isolates produced green/blue colonies on DFI medium characteristic of this organism. Real time PCR could differentiate between E. sakazakii, Enterobacter spp. and other Enterobacteriacae. Analysis of RAPD banding patterns revealed 3 major clusters of E. sakazakii. There was a large degree of diversity noted amongst the remaining isolates. Our findings indicate that RAPD may be applied as a useful and reliable tool for direct comparison of E. sakazakii isolates providing traceability through the infant formula food chain.     

Detection, occurrence, and characterization of Escherichia coli O157:H7 from raw ewe's milk in Spain

A study was carried out in the Castilla y León region of Spain to investigate the presence of Escherichia coli O157:H7 in raw ewe's milk samples collected from several cheese factories during 1 year. All specimens were examined for E. coli O157:H7 by selective enrichment at 41.5 +/- 1.0 degrees C, after both 6 and 22 h of incubation, and then immunomagnetically separated and plated on cefixime-potassium tellurite-sorbitol MacConkey agar. No growth was obtained in the enrichment broth after a 6-h incubation. Presumptive colonies obtained after 22 h of incubation were screened by a multiplex PCR assay for the presence of rfbO157 and fliCH7 genes. Of all the ewe's milk samples studied, three were positive for E. coli O157:H7. The E. coli O157:H7 strains that were positive for the rfbO157 and fliCH7 genes were then analyzed by multiplex PCR for the presence of virulence genes (stx1, stx2, ehxA, and eaeA). All E. coli O157:H7 isolates were Shiga toxigenic and harbored additional genes related to virulence (ehxA and eaeA). The predominant Stx toxin type was stx2. These results demonstrate that raw ewe's milk used in cheesemaking may be sporadically contaminated with E. coli O157:H7 strains that are potentially pathogenic for humans.     

克罗诺杆菌属(阪崎肠杆菌)的分离与鉴定能力验证结果与分析

目的 通过参加“奶粉中克罗诺杆菌属(阪崎肠杆菌)的检测能力验证计划”,验证实验室对克罗诺杆菌属(阪崎肠杆菌)的检测能力,提高实验室检测技能。方法 依据GB 4789.40-2016 《食品安全国家标准 食品微生物学检验 克罗诺杆菌属(阪崎肠杆菌)检验》中的第一法,对能力验证样品进行检验,对分离出的可疑菌落进行生化鉴定,同时用BIOLOG鉴定系统对可疑菌落进行鉴定。 结果 编号G785和编号V995的两个能力验证样品皆检出克罗诺杆菌属(阪崎肠杆菌) 结论 本次能力验证获得满意结果,证明本实验室具备克罗诺杆菌属(阪崎肠杆菌)的检测能力。在本次能力验证实验过程中,在以国标法检测的同时,辅以BIOLOG进行鉴定,与传统生化鉴定结果互为印证,提高了检测结果的准确性,对以后的检测工作具有一定的参考价值。     

Bacteriology of unshelled frozen blue swimming crab (Portunus pelagicus)

In this study, 45 (10 whole specimens and 35 frozen claws) frozen samples of Portunus pelagicus imported into Sicily (Italy) from the west coast of Africa were examined to assess their bacteriological characteristics and suitability for consumption. Bacteriological examination was performed on two subsamples for each whole crab. The first was the body and claw muscle; the second was a pool of viscera and gills. In the case of frozen claws, each muscle claw was a sample. An aerobic plate count at 30 degrees C (mesophilic aerobic plate count [MAPC]) and 18 degrees C (psychrotrophic aerobic plate count [PAPC]) for 3 days, sulfite-reducing anaerobes, total coliforms, Escherichia coli, enterococci, and Aeromonas spp. were enumerated. Detection of halophilic Vibrio spp. was also performed using salt polymixin broth as an enrichment medium and thiosulfate citrate bile salts sucrose agar as a selective medium; a further morphological and biochemical identification of suspected colonies was performed. The bacterial load of muscle and viscera and gills was low. The MAPC ranged from 0.78 to 3.26 log CFU/g, and the PAPC ranged from 0.48 to 2.41 log CFU/g. Vibrio spp., Aeromonas spp., sulfite-reducing anaerobes, and E. coli were never isolated from muscles or viscera and gills. In contrast to the findings of others, this study showed good bacteriological quality of crabs imported into Sicily from the west coast of Africa. This study also demonstrated the positive influence of the characteristics of environment of origin and postharvest handling hygiene; these parameters could be useful in the context of the application of the hazard analysis critical control point system to this production.     

Evaluation of different methods for the detection and identification of Enterobacter sakazakii isolated from South African infant formula milks and the processing environment

Enterobactersakazakii is an emerging pathogen associated with life-threatening neonatal infections resulting from the consumption of contaminated powdered infant formula milk (IFM). Recent taxonomic analyses have determined that E. sakazakii comprises a number of genomospecies, and it has been proposed that E. sakazakii be reclassified as a novel genus, "Cronobacter". Accurate methods are required for the rapid detection and identification of this group of micro-organisms, since even low cell numbers have been reported to cause disease. The aim of this study was to evaluate various E. sakazakii detection methods in order to ascertain the most suitable method for detection and identification of these pathogenic agents. Samples from IFM and the environment were evaluated for the presence of E. sakazakii using the isolation steps (pre-enrichment, enrichment and selection) described in the Food and Drug Administration (FDA) method for E. sakazakii detection. Sixty-four isolates (50 from IFM and 14 from the environment) were selected from tryptone soy agar (TSA), regardless of colony appearance, and these isolates were identified by 16S ribosomal DNA (rDNA) sequencing. Thereafter, different culture-dependent and culture-independent methods were evaluated to accurately detect and identify the E. sakazakii isolates. These methods included the assessment of yellow pigment production on TSA, typical colonies on chromogenic Druggan-Forsythe-Iversen (DFI) and Chromocult(R) Enterobacter sakazakii (CES) media and polymerase chain reaction (PCR) using six different species-specific primer pairs described in the literature. Identification of E. sakazakii using yellow pigment production was demonstrated to have a low sensitivity, specificity and accuracy (87%, 71% and 74%, respectively), which lowers the suitability of the FDA method. Chromogenic DFI and CES media were sensitive, specific and accurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. The specificity of the PCR amplifications ranged from 8% to 92%, emphasising the need for rigorous primer testing against closely related species. Of the primer pairs evaluated, Esakf/Esakr were the most suitable for E. sakazakii detection and identification. The detection limit of Esakf/Esakr was found to be 10(4) CFU/ml. This study demonstrated that no single method was capable of unambiguously confirming the presence and identity of E. sakazakii isolates, that each method had inherent advantages and disadvantages, and that in most cases several methods were required for accurate detection and identification. Further, it was demonstrated that the current FDA method for E. sakazakii detection should be revised in the light of the availability of more sensitive, specific and accurate detection methods.     

IMPROVED MEDIUM FOR DETECTION OF KLEBSIELLA IN POWDERED MILK

The selectivities to pathogenic Klebsiella strains of different isolation media were compared by known standard strains. The modified MacConkey-inositol-carbenicillin (MCIC) medium (Named MCIAC, MacConkey-inositol-adonitol-carbenicillin) supplemented with adonitol gave no false-negative colonies, and exhibited higher selectivity. MCIC and Simmons citrate agar with inositol (SCAI) media gave two false-negative colonies, respectively. These three media all gave two false-positive colonies, respectively. Salmonella Shigella medium gave four false-negative colonies and five false-positive colonies. Violet red bile glucose agar medium gave the most false-positive colonies, although it gave no false-negative colonies. One hundred samples of powdered milk were examined by MCIAC, MCIC and SCAI plates. The typical positive colonies were further identified using Vitek GNI Auto Microbic system and API 20E system. The results showed that the specificity of the MCIAC medium was higher than MCIC and SCAI media.

PRACTICAL APPLICATIONS

MacConkey-inositol-carbenicillin (MCIC) is the most commonly used selective medium for the detection of Klebsiella . But some inositol-nonfermenting Klebsiella strains would be missed when selected by this medium. We improved the MCIC medium by supplementing with 1% adonitol. The new modified medium (MacConkey-inositol-adonitol-carbenicillin, MCIAC) had advantages over other selective Klebsiella media in having a higher selectivity and an incubation time of only 16–24 h. MCIAC could be routinely used for pathogenic Klebsiella selection of powdered milk and other food samples.     

Evaluation of immunomagnetic separation and plating media for recovery of Salmonella from meat

Immunomagnetic separation (IMS) was compared with selective enrichment in selenite cystine (SC) broth for isolation of Salmonella from 86 artificially contaminated ground beef samples. Both Rambach agar (RA) and Hektoen enteric (HE) agar were used as selective plating media. The highest count of Salmonella colonies per plate was obtained after enrichment in SC broth and plating on RA (mean value: 111.1+/-58.1 CFU per plate); the lowest count was obtained after IMS and plating on HE agar (mean value: 65.4+/-36.6 CFU per plate). Salmonella in preenrichment was concentrated 1.7-fold by IMS and represented 31% of the microbial population captured by the beads, but nonspecific binding was high. As a result of the large numbers of competing bacteria, isolations on both RA and HE agar following IMS were quite difficult (mean value for Salmonella colonies: 79.9+/-42.7 CFU per plate; mean value for non-Salmonella colonies: 144.1+/-75.7 CFU per plate; ratio of Salmonella to non-Salmonella colonies: 0.8). This study indicates that SC broth is superior to IMS in the isolation of Salmonella from raw ground meat.     

Inhibition of growth of Enterobacter sakazakii in reconstituted infant formula by the lactoperoxidase system

Neonatal bacteremia and meningitis caused by the opportunistic pathogen Enterobacter sakazakii have been associated with the consumption of reconstituted powdered infant formula. Lactoperoxidase (LPO), present in mammalian milk, is known to inhibit the growth of enteric pathogens. We undertook a study to determine if the lactoperoxidase system (LPOS) will inhibit the growth of E. sakazakii in a milk-based powdered infant formula reconstituted with water. Initially at 0.04 CFU/ml, E. sakazakii grew to 2.40 to 2.74 log CFU/ml in reconstituted infant formula held at 30 or 37 degrees C for 8 h and to 0.6 log CFU/ ml in formula held for 12 h at 21 degrees C. The pathogen was not detected (less than 1 CFU/227 ml) by enrichment of formula treated with 10 to 30 microg/ml LPO and stored for 24 h at 37 degrees C or 30 microg/ml LPO and stored for 24 h at 30 degrees C. Populations of E. sakazakii, initially at 4.40 log CFU/ml of reconstituted infant formula containing 5 microg/ml LPO, did not increase significantly (P > 0.05) for up to 12 h at 21 and 30 degrees C. Populations either decreased significantly or were unchanged in formula supplemented with 10 microg/ml LPO and stored at 21, 30, or 37 degrees C for up to 24, 8, and 8 h, respectively. Results indicate that LPOS can be used to control the growth of E. sakazakii in reconstituted infant formula, thereby potentially reducing the risk of neonatal infections resulting from consumption of formula that may be contaminated with the pathogen.     

Isolation of Salmonella typhimurium from outbreak-associated cake mix

During May and June of 2005, 26 persons in several states were infected by a single strain (isolates indistinguishable by pulsed-field gel electrophoresis) of Salmonella enterica serotype Typhimurium after eating cake batter ice cream. The cake mix used to prepare the cake batter in the ice cream was implicated by epidemiologic investigation as the source of Salmonella contamination. Initial tests did not detect Salmonella in cake mix collected during the outbreak investigation. The objective of this study was to evaluate different procedures to isolate Salmonella from the implicated cake mix, cake, and ice cream. All outbreak-associated food samples (14 samples) were collected during the outbreak investigation by health departments of several of the states involved. Different combinations of Salmonella isolation procedures, including sample size, preenrichment broth, enrichment broth, enrichment temperature, and isolation medium, were used. Salmonella Typhimurium was isolated from two cake mix samples; the food isolates were indistinguishable from the outbreak pattern by pulsed-field gel electrophoresis subtyping. Universal preenrichment broth was substantially better than was lactose broth for preenrichment, and tetrathionate broth was better than was Rappaport-Vassiliadis broth for isolating Salmonella from the two positive cake mix samples. Although more typical Salmonella colonies were observed on plates from enrichment cultures grown at 35 degrees C, more confirmed Salmonella isolates were obtained from plates of enrichment cultures grown at 42 degrees C. Brilliant green agar, xylose lysine tergitol 4 agar, xylose lysine desoxycholate agar, Hektoen enteric agar, and bismuth sulfite agar plates were equally effective in isolating Salmonella from cake mix. The best combination of preenrichment-enrichment conditions for isolating the outbreak strain of Salmonella was preenrichment of cake mix samples in universal preenrichment broth at 35 degrees C for 24 h, followed by enrichment in tetrathionate broth at 42 degrees C for 24 h.     

Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay

Enterobacter sakazakii is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5' nuclease assay (TaqMan). A set of primers and probe was designed using the E. sakazakii partial macromolecular synthesis operon: the rpsU gene 3' end and the primase (dnaG) gene 5' end. The specificity of the assay was evaluated using 68 Enterobacter and 55 non-Enterobacter strains. The newly developed assay enables us to detect 100 CFU/ ml in pure culture and in reconstituted infant formula in 50 cycles of PCR without enrichment. The assay was specific enough to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to 5 days and eliminate the need for plating samples on selective or diagnostic agars and for biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.     

副溶血性弧菌与弧菌科易混淆菌的鉴别方法

目的 快速鉴别副溶血性弧菌检测工作中常见的几种易混淆菌。方法 首先筛选出7株在副溶血性弧菌检测工作中的易混淆菌株, 分别通过选择性平板、初步生化筛选和干制生化鉴定试剂盒的方法, 对包括副溶血性弧菌标准菌株在内的8株菌进行鉴别分析。结果 各菌株培养24 h后, 只有创伤弧菌能在改良纤维二糖多粘菌素B多粘菌素E琼脂平板上生长; 硫代硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基对弧菌科菌株鉴别性较强; 结合硫代硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基以及弧显平板上的菌落大小和颜色特征, 可对以上8株菌进行鉴别, 利用各菌株在副溶血性弧菌的典型生化反应上的不同点, 可进一步进行验证。结论 掌握各检测阶段菌株的不同特征, 通过常规检测方法实现快速鉴别, 节省检测时间, 提高检测效率, 及时发现其它可疑致病菌。