- common food- and waterborne enteric bacterium accounting for 60% of all bacterial disease outbreaks in the US
- even if disease is not directly caused by consumption of water, contaminated water can be considered an important source of transmission on food
- although only two species (Salmonella enterica and Salmonella bongori) have been identified, numerous subspecies and serotypes are known to exist and cause disease
- enhanced survival and increased resistance to disinfection by intracellular lifestyle in protozoa
- infectious dose estimated to be in the range between 104 to 107 cells
- well studied with several commercial detection kits available
, like Escherichia coli
, is an enteric pathogen belonging to the family Enterobacteriaceae ( FDA Bad Bug Book
enterica has been identified as the second most common cause of gastroenteritis in children under 5 years of age in the UK ( Crowley et al. 1997
). This organism is responsible for salmonellosis, the second most common cause of bacterial food poisoning reported to the Communicable Disease Surveillance Center (CDSC) ( Evans et al. 1998
) and accounts for 60% of all bacterial disease outbreaks in the US. It is estimated that globally over 4 million cases of non-typhoidal Salmonella
infections occur ( Feng P 1992
) of which in the United States of America, an estimated 1.4 million non-typhoidal Salmonella
infections occur, resulting in 168,000 visits to physicians, 15,000 hospitalizations and 580 deaths annually (WHO). Molecular methods have shown that the genus Salmonella
consists of only two species, Salmonella enterica
and Salmonella bongori
. Of these two species, S. bongori
only contributes 22 serovars. According to the official Kauffmann-White Scheme for naming bacteria ( Grimont and Weill 2007
), Salmonella enterica
is further divided into six subspecies and roughly 2,500 serovars that can be differentiated by biochemical and genetic tests. A characteristic feature of Salmonella
enterica is the broad host spectrum of this pathogen, comprising most animal species in addition to humans. Disease is caused by the penetration of the Salmonella
bacteria into the epithelium of the small intestine and subsequent enterotoxin production resulting in electrolytic imbalance. Although the primary cause of salmonellosis is consumption of Salmonella
-contaminated foods, Salmonella
may also be waterborne and outbreaks have been associated with the consumption of drinking water ( O'Reilly et al. 2007
). There is increasing evidence that this pathogen may be associated with biofilms on materials of different nature and under different growth conditions ( Solano et al. 2002
et al. 2004) and may be important in drinking water distribution systems ( Jones and Bradshaw 1996
). The transmission through water becomes evident considering that individuals infected with Salmonella
shed the organisms in their feces, which can enter domestic sewage and consequently contaminate drinking water sources. Even if disease is not directly caused by consumption of water, contaminated water can be considered an important source of transmission on food. The WHO's Global Foodborne Infections Network and specifically Salm-Serv has been established to reduce the global burden of foodborne and other enteric infections by enhancing laboratory-based surveillance and outbreak detection and response and to foster collaboration and communication between microbiologists and epidemiologists working in human health, veterinary and food sciences (http://www.who.int/gfn/supported/en/
has been isolated from both natural and treated waters. In the environment the concentration of Salmonella
can be naturally occurring at 50 organisms/L ( Lemarchand et al. 2004
) and as high 103
organisms/L as in raw wastewater in the US ( Bitton G 2005
) and even higher in developing nations ( September et al. 2007
). Since Salmonella
is frequently associate with fecal matter, numerous studies have been undertaken in wastewater environments. Removal of Salmonella
throughout a sewage treatment system is exemplified by the following numbers presented in a US study: Salmonella
concentrations have been shown to be in the region of 5x 103
- 8 x 104
CFU/L in raw sewage, 102
- 3 x 103
CFU/L after primary treatment only (primary sedimentation and disinfection), 3 - 103
CFU/L after secondary treatment (including tric kling filter or activated sludge), and 10-6
- 7 CFU/L after advanced secondary treatment (including coagulation, filtration and disinfection) ( Maier et al. 2000
). These figures however are merely representative and removal rates can vary greatly. In a study from Spain, raw water samples showed a high content of Salmonella
spp., with a mean MPN of 2667/L while treated water (after decanting and activated sludge) contained a MPN of 450/L, representing a reduction of only 83% ( Howard et al. 2004
Examples of studies on occurrence in water are summarized in the following:
- A recent outbreak in Alamosa, Colorado, left 111 people ill when Salmonella was found to be contaminating the untreated groundwater supplying the town's drinking water ( Berg R 2008). Alamosa's potable water comes from 5 deep groundwater wells and is not treated before being fed into homes. This is not unusual in smaller rural American towns where more than half of the drinking water comes from untreated groundwater. Although groundwater does interact with potentially- contaminated surface waters, it does not usually need treatment because there is a natural filtration process through the ground soils and clays (USGS). It was discovered that an in-ground water storage tank in poor condition may have been the point source where contamination may have entered the system, and PFGE analysis indicated that the serotype of Salmonella enterica responsible for the outbreak in the town was the same as that naturally occurring in the local bird and deer population.
- In a study conducted in California, effluents from 11 of 12 sewage treatment plants tested positive for Salmonella when samples were analyzed downstream of a chlorination/dechlorination site, before effluents merge with the receiving stream (outside the plant). Six hundred eighty-three Salmonella isolations were made from 26 of the 32 sampling sites. Eleven serogroups and 54 serotypes of Salmonella represented all the isolations. During the sampling period, people were observed swimming and fishing in the sewage treatment plant effluent within 30.5 m (100 ft) of the outfall. Subsequent to this study, an interesting theoretical exercise was carried out. The daily consumption of water per capita is estimated to be 400 liters resulting in the same volume of wastewater to be treated. A person with acute salmonellosis excretes Salmonella in quantities of 108- 1011 organisms per gram of feces. In a community with a population of 100,000 people consuming 400 liters of water per person per day, one might expect to find Salmonella concentrations of 1011 / 105 x 400 liters = 2.5 x 103 cells per liter of sewage ( Kinde et al. 1997).
- In 2004, an outbreak of gastroenteritis was investigated on South Bass Island, Ohio, an island of 900 residents that is visited by >500,000 persons each year. Between May and September 2004, 1,450 persons reported illness. Out of 70 stool specimens tested for bacterial pathogens, Salmonella enterica serotype Typhimurium was identified in only 1 person. The remaining cases were shown to be due to Campylobacter jejuni, norovirus and Giardia intestinalis. The environmental assessment demonstrated that contamination of the karst aquifer beneath the island had occurred from multiple land uses such as onsite septic systems, land application of septage, infiltration of land run-off, and, possibly, a direct hydraulic connection with Lake Erie ( O'Reilly et al. 2007). While this study did not show the dominance of Salmonella in the outbreak, it did serve to illustrate two important points about the pathogen's modus operandi: first, it is seldom the only pathogen in a waterborne outbreak, and second it is a pathogen capable of maintaining itself as a virulent agent even at low numbers in the environment.
- In a Japanese study, higher incidence of Salmonella spp. in river water than sea water suggested that salinity is a crucial factor in governing its distribution. The occurrence of Salmonella spp. in Fukuyama port marine samples might stem from an increased discharge of polluted waters from an adjacent land or coastal area ( Venkateswaran et al. 1989). In addition to simply the presence of the pathogen in water systems, regrowth and survival of attached Salmonella has been shown in rural communities' storage containers (polyethylene and galvanized steel) at low levels (< 1-15 CFU/cm2) ( Momba and Kaleni 2002), in domestic toilet bowls for up to 4 weeks after diarrhea had stopped ( Barker and Bloomfield 2000) and in UASB reactors in a wastewater treatment plant ( Keller et al. 2003).
- Salmonella Typhimurium has been linked to outbreaks of salmonellosis resulting from contact with water frogs including African Dwarf Frogs. Between 24 May and 30 December 2009, 85 infected individuals had been reported from 31 states in the USA. In addition, environmental samples taken from aquariums containing aquatic frogs in four homes of patients yielded isolates of Salmonella Typhimurium matching the outbreak strain. Preliminary traceback information indicated these frogs likely came from a single breeder in California ( CDC 2010a). At the same time, the CDC tracked a similar outbreak associated with ingestion of raw alfalfa sprouts. Between February and May 2009, 228 cases in 13 US states implicated the source as alfalfa sprouts produced at multiple facilities using seeds that likely originated from a common grower. The source of contamination was suggested to be rinse water ( CDC 2010b).
The ability of many human pathogens to survive in the environment and the identification of water sources as one of the preferred environments of these organisms has created an interest in the development of appropriate and effective disinfection strategies of environmental sources. A major frustration has been the observation that despite the development of many new technologies and disinfection strategies to which the target organisms are susceptible, still many more potential pathogens appear to be resistant. To learn whether cellulose might be responsible for chlorine resistance and therefore the survival of S. enterica
strains as biofilms in water supplies and food-processing chains, Solano et al. carried out survival experiments of the wild-type strain and cellulose-minus mutants ( Solano et al. 2002
). They used a concentration of NaOCl (30 ppm) that is 100- to 200-fold higher than the free chlorine concentrations typically obtained in municipal water supplies and is into the concentration range used as a sanitizer for food plants. After a 20 min exposure period, 75% of the wild-type cells survived NaOCl exposure. In contrast, only 0.3% of cellulose-deficient mutant cells survived under the same experimental conditions. Other important implications on the susceptibility to disinfection are brought by the facts that Salmonella
can associate with biofilms and be internalized by protozoa.
Selected studies on disinfection are summarized in the following:
- Oliver et al. showed that when sodium hypochlorite (free chlorine) solution was added to provide a final concentration of 1mg/L of free chlorine in the wastewater, culturability of control S. Typhimurium cells in the stationary phase remained at about 106 CFU/mL while cells exposed to this chlorination protocol typically declined to 106 total cells in all cases ( Oliver et al. 2005). On average, regardless of the physiological state of the cells, 0.39% of the treated cells responded to a viability assay (as opposed to culturability) after 60 min of chlorination, indicating a small portion of the cells were able to resist this treatment. While such a percentage is low, it equates to roughly 103-104 cells/mL.
- An often overlooked aspect of the susceptibility of pathogens to disinfection is their evolutionary ability to survive intracellularly within various protozoan species. To show this, King et al. 1988 performed disinfection experiments on some common waterborne pathogens including S. Typhimurium which were ingested by a variety of protozoans. As a baseline, they showed that the C*t99 values required to eliminate culturability of Salmonella when free-living are 0.4, 0.5 and 0.5 minutes with free chlorine residuals of 1.0, 0.5 and 0.25 mg/L respectively. However, when ingested by the protozoan Tetrahymena pyriformis, S. Typhimurium exhibited > 50-fold greater resistance to free chlorine, even at elevated levels. C*t99 values required for inactivation under these protected conditions were around 90, 90, 80 and 50 minutes at free chlorine residuals of 0.5, 1.0, 2.0 and 4.0 mg/L respectively.
- Berney et al. examined the efficacy of sunlight irradiation on the inactivation of some common waterborne pathogens including S. Typhimurium ( Berney et al. 2006). Resistance to sunlight at 37°C based on F99 values was in the following order: Salmonella Typhimurium > Escherichia coli > Shigella flexneri > Vibrio cholerae. While F90 values of S. Typhimurium and E. coli were similar, F99 values differed by 60% due to different inactivation curve shapes. These authors also point out that T90 values are not appropriate for the determination of irraditation efficacy because they do not take into account different irradiation intensities. They bemoan the fact that the display of T90 values has become very common in solar disinfection publications, making comparisons among different studies very difficult. Keller et al. showed that viable Salmonella in treated wastewater effluents could be considerably reduced after UV exposure, although the degree of inactivation depended on the turbidity of the effluent, with more turbid samples requiring greater doses ( Keller et al. 2003). They demonstrated that 2 log reductions in non-filtered effluent could be achieved at doses of about 30 mW sec/cm2 whereas only 20 mW sec/cm2 doses were required to achieve the same levels of inactivation in filtered effluent. These findings were corroborated in a 2005 study by Rodriguez-Romo and Yousef who demonstrated a 2 log reduction in viable Salmonella on the surfaces of egg shells at doses of about 24 mW sec/cm2 ( Rodriguez-Romo and Yousef 2005).
- The inactivation of Salmonella by ozone has been shown to be an effective means of controlling surface contamination on food surfaces such as egg shells, fruits and berries ( Rodriguez-Romo and Yousef 2005), and in liquids such as apple cider, orange juice and water ( Restaino et al. 1995; Lezcano et al. 1999; Williams et al. 2004). According to Restaino et al. 1995 more than 5 log units of S. Typhimurium cells per mL were killed instantaneously after exposure to ozonated water at a concentration of around 0.19 ppm. These authors point out however that the efficacy of ozonation is more dependant on the type of organic material present in the water rather than the amount of the organics as was previously thought. They also indicate that the currently held hypothesis that determining ozone concentrations for an all-or-none inactivation may be faulty, showing that death caused by ozone followed a biphasic pattern. The result of this finding suggests that the high dose-short time, or low dose-long time approach may need to be revisited. Lezcano et al. 1999 showed that an environmental isolate of S. Typhimuirium was more resistant in water to ozone than both ATCC strains and environmental isolates of E.coli and Shigella sonnei. They demonstrated t90 of 3.44, 3.59, 1.69 and 0.62 minutes and t100 (complete inactivation) of 13, 10, 5 and 3 minutes at 0.48, 0.58, 1.04 and 1.75 mg/L ozone respectively.
Although many would argue that the preferred environment for Salmonella
is the gut of animals, the entire lifecycle of this organism must be considered and there is clear evidence that much of the survival and persistence of this so-called pathogen is associated with environments outside of the host animal. Salmonella
discharged in the effluents from municipal WWTPs treating human sewage were reported to survive for an extended time in nutrient-rich river water ( AWWA 2006
). Several mechanisms of survival have been suggested, including the adoption of a viable-but-non culturable (VBNC) state, the integration of the pathogen into an existing biofilm ( Jones and Bradshaw 1996
; Barker and Bloomfield 2000
; Solano et al. 2002
; Stepanovic et al. 2003) and internalization of the pathogen into a variety of protozoan hosts. Although survival depends on a variety of factors, Salmonella
survival in water and its susceptibility to disinfection have been shown to be similar to those of coliform bacteria ( McFeters et al. 1974
; Mitchell and Starzyk 1975
; Health Canada 2006
). As both are of fecal origin, the absence of E.coli
should thus adequately indicate the absence of Salmonella
although exceptions are known ( Health Canada 2006
Several subspecies of Salmonella enterica, including enterica, salamae and arizonae, have been shown to enter a VBNC state after lengthy exposure to oligotrophic fresh and seawater under ambient temperature ( Roszak and Colwell 1987; Jiménez et al. 1989; Cho and Kim 1999). These and many other microbial pathogens for which this VBNC state has been reported have also been suggested to retain the capacity to cause disease and therefore be considered still active ( McDougald et al. 1998).
Evidence for a VBNC state of Salmonella can be found in the following cases:
- In a study on pathogens in bottled mineral water, Salmonella Typhimurium exhibited greatest survival (compared to Escherichiacoli, Staphylococcus aureus and Yersinia enterocolitica) under all storage conditions including light and dark incubation, different recovery media, presence or absence of autochthonous flora and different ages of water, based on culturability ( Ramalho et al. 2001).
- Domingo et al. showed that in 3 of 4 strains of Salmonella seeded into river water, the use of a variety of resuscitation techniques were useful for detecting viable pathogens for a lengthy period of time ( Domingo et al. 2000). Although the culturable counts of two bacterial strains in filtered water after 31 days represented approximately only 0.001% of the total Salmonella counts, direct viable counts and resuscitation studies with a dilution series suggested that the number of viable bacteria was at least four orders of magnitude higher.
Survival of Salmonella in protozoa is demonstrated in the following cases:
- Salmonella enterica serovar Typhimurium in water and sediments was tested using artificially contaminated aquaria. Water samples remained culture positive for Salmonella for up to 54 days. Sediment samples were culture positive up to 119 days ( Moore et al. 2003). Larval chironomids (midges) raised in contaminated sediments became culture positive, and the bacteria were carried over to adults after emergence. Uptake of Salmonellae by chironomid larvae and adults suggests that they are possible vectors in both aquatic and terrestrial environments.
- The ability of Salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. ( Tezcan-Merdol et al. 2004). The survival of Salmonella enterica serovar Typhimurium within Acanthamoeba castellanii during chlorination was also reported, suggesting a protective intracellular habitat for the bacteria ( King et al. 1988). Results show that A. rhysodes was able to ingest Salmonellae and that subsequent events included intracellular bacterial replication. The study also detected a bacterium-mediated cytotoxicity that appeared to be dependent on previously identified virulence genes, implying that genetic determinants of Salmonellae used for invasion and intracellular proliferation in mammals could also be operative in the environment.
Since there is such genetic diversity within the Salmonella
genus, it is almost impossible to determine a blanket value for the infectious dose. Based on studies including a variety of species and conditions, a suggested range of infectious dose would be 104
cells ( Bitton G 2005
). In one study, the oral infectious dose of Salmonella enterica
serovar Typhi was estimated to be around 104
cells ( Health Canada 2006
). However, the infectious dose can be as low as 15 - 20 cells ( FDA Bad Bug Book
). The infectious dose can also be expressed relative to the probability of infecting specific tissues: The probability of infection of tissue (or, perhaps more appropriately for this outcome variable, the probability of invasion) assessed by the analysis of spleens was estimated to be r = 1.2x10-3
/CFU of the inoculum. This is equivalent to an ID50
of 580 CFU ( Takumi et al. 2002
In a study on the development of a biosensor for Salmonella, Pathirana et al. 2000 indicated that their biosensor was capable of detecting below 105 cells which is around the suggested the infectious dose.
Johnson in "OSHA Infectious Dose White Paper" summarises the challenges in establishing blanket values for infectious doses and specifically highlights the difficulty in the case of Salmonella ( Johnson B 2003)
Most diagnosticians would be familiar with the standard detection techniques for Salmonella
based on traditional biochemical and culture-based techniques. These include ONE Broth-Salmonella
Base and Brilliance Salmonella
Agar (Oxoid). While mostly effective and relatively simple, these methods are often costly in terms of time (often more than 36 hours) and labour and highly reliant on the ability of the organisms to be successfully cultured. Recognizing the possibility of the VBNC state in Salmonella
as well as the increasing need for rapid diagnostic techniques to keep pace with the increased pace of product supply and demand, numerous other rapid detection methodologies are being investigated.
A wide spectrum of immunological and nucleic acid based methods is available as Salmonella serves as a model pathogen in many studies and while enrichment techniques are still occasionally used prior to PCR for sensitive detection of Salmonella, these procedures typically take hours and such practices are incompatible with the goal of rapid detection. In order to improve the detection limit, automated concentration mechanisms are under development for the purpose of increasing the cell densities of liquid test samples ( Eyigor et al. 2002; Bhagwat AA 2004). Currently, there are a small number of PCR kits commercially available for the detection of Salmonellae. These systems include BAX(r) System PCR Assay for Salmonella (DuPont Qualicon, Wilmington, DE.), TaqMan (Perkin Elmer, Applied Biosystems, Foster City, CA), and Probelia (Sanofi Diagnostics Pasteur, Marnes La Coquette, France) (Lin et al. 2004). However, among these systems, none has been approved as an AOAC official method. Only the BAX system for Salmonella detection in food samples has received Performance Tested Method status from the AOAC Research Institute ( Bhagwat AA 2004). Sequences of the PCR primers used in these systems have not been reported, and these systems are still too expensive to be used as prescreening systems.
As early as 1992, Rhan et al. utilized sequences from the invA gene for PCR-based detection of Salmonella in environmental samples and subsequent research has primarily focused on this gene (Rhan et al. 1992; Csordas et al. 2004). The inv group of genes has been shown to be responsible for entry of the pathogen into host epithelial cells ( Galan and Curtiss 1989). Furthermore, this group of genes has also been shown to have widespread presence through the Salmonella species ( Galan and Curtiss 1991). The sensitivity (limit of detection) of PCR with invA has shown an extremely wide range depending on reaction conditions: between 300 cells to 2.6 x 104 CFU/mL ( Galan and Curtiss 1991; Rahn et al. 1992; Fey et al. 2004). A non-selective pre-enrichment step would obviously further increase the limit of detection and Moganedi et al. 2007 have shown this limit to be 26 CFU/mL. Only two out of 14 strains tested showed no amplification.
When samples were taken from natural sources and spiked with different number of cells of Salmonella, the detection limit was as high as 1,000 cells per ml using a novel set of primers amplifying the enterotoxin (stn) gene. To overcome this problem, a two-step nested PCR was applied. The nested PCR increased the sensitivity of the assay tremendously, being able to detect a single cell per ml of natural water samples with total viable counts of background microflora ranging from 1 x 103 CFU/mL to 5 x 103 CFU/mL. The study indicates that Salmonella enterotoxin gene is highly conserved and the protocol devised in this study can be used as rapid and reliable method for detection of Salmonella spp. in water samples ( Riyaz-Ul-Hassan et al. 2004).
A comparison of two commercially available kits utilizing MB-PCR (iQ-Check, Bio-Rad Laboratories) and conventional Association of Official Analytical Chemists (AOAC)-approved PCR (BAX, Dupont Qualicon) was performed on artificially inoculated produce. As few as 4 CFU/25 g of produce were detected after 16 h of enrichment in buffered peptone broth ( Liming and Bhagwat 2004).
Selected non-cultivation based methods tested on water samples are listed in the following:
- Multiplex PCR, specifically targeting flostR, intI1 and invA genes, has also been studied in the detection of Salmonella and has shown as much success as conventional single-target PCR ( Ebner and Mathew 2001).
- qPCR: More recently, qPCR has been used to quantify the presence of Salmonella in environmental samples. In one example, qPCR was used on municipal wastewater based on a primer/probe set and showed a detection limit of 100 fg genomic DNA (=22 gene copies based on an Aeromonas standard curve) ( Shannon et al. 2007).
- qPCR with IAC: Real time PCR is a useful tool for the quantification of a specific target gene. Unfortunately, amplification efficiencies can be different from sample to sample due to the effects caused by inhibition of amplification, human failures or preparation errors. The application of internal amplification controls targeted toward so-called housekeeping genes can provide an effective means of standardizing results. In one study using primer/probe qPCR targeting himA ( Chen et al. 2000), invA ( Hoorfar et al. 2000) or spaQ genes( Kurowski et al. 2002) and an IAC targeting a GFP-coding sequence, a quantifiable detection limit of 200 CFU/reaction was achieved, and for qualitative detection 2 CFU/reaction ( Klerks et al. 2004).
- RT-qPCR: In an alternative attempt at standardizing qPCR results, Fey et al. made use of SYBR Green (QIAGEN) RT-qPCR for the amplification of a specific 16S rRNA and the invA RNA as control. In this study, detection limits of 2 and 20 copies/reaction of DNA and RNA respectively based on invA were determined ( Fey et al. 2004).
- Diagnostic microarray technology is an exciting advance in the field of molecular biology. In one study, an oligonucleotide microarray was designed based on 16S rRNA and cpn60 genes of several pathogens. Also included on this array were wecE-specific probes for the further discrimination within the Enterobacteriaceae family. Using this PCR microarray technique detection sensitivity was shown to be around 104 Salmonella genome copies ( Maynard et al. 2005). Bayardelle et al. 2002 further showed that PCR using primers for the wec gene was highly efficient in the detection of the most frequent Enterobacteriaceae pathogens.
- Molecular typing: The amplification of the 16S-23S ribosomal spacer regions and the size polymorphism of the resulting products have proven useful in the species-level identification of a broad range of bacteria. These sequences are generally found in multiple copies in most bacterial genomes. When multiple amplification products, which contain homologous sequences flanking heterologous intervening sequences, are generated, these products can cross-hybridize to form heteroduplex DNA structures. Homoduplex double-stranded DNA structures and heteroduplex structures have already been successfully used to identify Salmonella serotypes isolated from clinical samples. Thus, the comparison of pattern groups of ribosomal spacer-heteroduplex polymorphism (RS-HP) types may provide a rapid and convenient alternative method for identifying Salmonella serotypes isolated from aquatic environmental samples. While more a qualitative than a quantitative technique, and when used in combination with selective enrichment, RS-HP serves as an efficient tool to discriminate species at both serotype and interserotype levels ( Baudart et al. 2000).
- Several immunoassay-based systems and devices are commercially available for detection of a variety of food-borne pathogens including Salmonella ( Baylis et al. 2000; Paula et al. 2002; Fratamico PM 2003), however few of these immunological methods have been shown to be amiable to use in the water industry. In one study the objective was to develop a rapid (8 h) most probable number (MPN)-enzyme-linked immunosorbant assay (ELISA) for the detection and enumeration of Salmonella Typhimurium in poultry wastewater and compare it to the traditional MPN method. The traditional MPN method used a 48-h enrichment period followed by an analysis, while the MPN-ELISA used a 5-h enrichment period followed by a 3-h ELISA analysis. No differences (P < 0.05) were found between the traditional MPN and the MPN-ELISA, indicating promise of the faster detection technique (Goodridge et al. 2003).
A technology employed to increase sensitivity of DNA-based molecular assays and reduce the need for enrichment has been the use of FTA filters. FTA filters were used directly as templates to demonstrate their sensitivity and applicability in PCR-based detection assays. With pure cultures, the sensitivities of detection by FTA filter-based PCR were 30 to 50 CFU for the gram-negative enterics. The amount of extraneous material centrifuged concurrently with bacterial cells was very significant, and this reduced the sensitivity of the PCR assay. Whether this was due to material that blocked binding of the bacteria to the filter, thus preventing lysis, or due to introduction of PCR inhibitors into the reaction mixture is not clear. Additional preparation prior to application of the material to the FTA filters may be required to overcome diminished sensitivity ( Lampel et al. 2000).
The following pictures are part of the Public Health Image Library (PHIL) from the Centers for Disease Control and Prevention (CDC). The photos are in the public domain and thus free of copyright restrictions. We would like to express our appreciation for providing these images.
Color-enhanced scanning electron micrograph showing Salmonella typhimurium (red) invading cultured human cells.
Credit: Rocky Mountain Laboratories, NIAID, NIH
Under a very high magnification of 12000X, this colorized scanning electron micrograph (SEM) revealed the presence of a large grouping of Gram-negative Salmonella typhimurium bacteria that had been isolated from a pure culture.
Photo ID: 10983
Content provider(s): Centers for Disease Control and Prevention/ Bette Jensen
Photo Credit: Janice Haney Carr
Under a high magnification of 12000X, this colorized scanning electron micrograph (SEM) revealed the presence of numbers of clustered Gram-negative Salmonella typhimurium bacteria, which had been grown in a pure culture.
Photo ID: 10971
Content provider(s): Centers for Disease Control and Prevention/ Bette Jensen
Photo Credit: Janice Haney Carr, Centers for Disease Control and Prevention
After having just handled a turtle, this young child was appropriately washing his hands, which potentially could have been contaminated with Salmonella bacteria that may have been carried by the pet turtle in this classroom. The sale of turtles less than 4 inches in length has been banned in the United States since 1975. The ban by the U.S. Food and Drug Administration (FDA) has prevented an estimated 100,000 cases of salmonellosis annually in children.
Photo ID: 8277
Content provider(s): Centers for Disease Control and Prevention/ James Gathany
Credit: James Gathany