A range of molecular biological tools based on characterization of the genotype of the organism by analysis of plasmid and chromosomal DNA have now been developed either to supplement the more traditional phenotypic methods of typing (serotyping, phage typing, biotyping) or, in some cases, as methods of discrimination in their own right (see Threlfall, 2005b; Cook et al., 2006).
Plasmid Typing
Many strains of salmonellae carry plasmids differing in both molecular mass and number. Plasmid typing based on the numbers and molecular mass of plasmids after extraction of partially purified plasmid DNA has been used for differentiation within serovars. Plasmid typing is therefore restricted to serovars possessing plasmids and is of limited use in those serovars in which the majority of isolates contain only one plasmid, or are plasmid-free. The sensitivity of the plasmid profile typing may be increased by cleaving plasmid DNA with a limited number of restriction endonucleases and the resultant plasmid ‘fingerprint’ may be used to discriminate between plasmids of similar molecular mass. More recently the characterization of plasmids by identification of specific replicon areas has added a new dimension to plasmid typing. This method, developed by Caratollli and colleagues (2005), has considerable potential not only for the identification of plasmid incompatibility groups but also for investigating the spread of such plasmids, and the resistance genes encoded thereon, through the food chain.
Identification Of Chromosomal Heterogeneity
Molecular typing methods based on the characterization of plasmid DNA include plasmid profile typing, plasmid fingerprinting, and the identification of plasmidmediated virulence genes. Chromosomally based methods have sought to identify small regions of heterogeneity within the bacterial chromosome. Of the latter the most commonly used have been ribotyping, insertion sequence (IS) 200 typing, and pulsed field gel electrophoresis (PFGE). The latter method permits analysis of the whole bacterial genome on a single gel and is currently regarded as the gold standard for the molecular subtyping of salmonella. This method is used as the basic method of subtyping of salmonella in the United States, and for subdivision within PTs in those countries which use phage typing as the primary method for the discrimination of epidemiologically important serovars. The method has become standardized and networks have been developed – PulseNet in the United States (Swaminathan et al., 2001) and SalmGene in Europe (Fisher and Threlfall, 2005) – to provide common, harmonized molecular typing methods, and to facilitate the rapid electronic transfer of the images captured by them in a digitized format. More recently the SalmGene database of PFGE types has been expanded to form the basis of PulseNet Europe, which is fully compatible with PulseNet USA and other PulseNet networks, thereby providing an encompassing network for the molecular subtyping of salmonella worldwide.
With the development of the polymerase chain reaction (PCR), methods based on amplification of specific DNA sequences to produce characteristic groups of fragments dependent on the origin of the template DNA have been developed and used, with some success. Such methods include random amplified polymorphic DNA typing (RAPD), enterobacterial repetitive intergenic consensus typing (ERIC-PCR), repetitive extragenic palindromic element typing (REP-PCR), amplified fragment length polymorphism fingerprinting (AFLP), and variable number of tandem repeats (VNTR) fingerprinting. VNTR fingerprinting is based on the presence and subsequent identification of units of repeated DNA elements in the genome. Such elements, known as VNTRs, range from about 10 to 100 base pairs (bps). VNTR fingerprinting has been applied to the subtyping of S. Typhi and S. Typhimurium. A major drawback of the method is that for meaningful results VNTRs for typing should be based on the published genome sequence of a serovar. As only a limited number of serovar sequences have been published, the applicability of this method is somewhat limited.
Antimicrobial Drug Resistance
Resistance to key antimicrobials is particularly important in the treatment of infections caused by S. Typhi and S. Paratyphi A. The increasing occurrence of multiple resistance in serovars other than Typhi has also had a profound effect in the treatment of salmonella septicemia in infants and young children in developing countries, where multiple resistant strains have been implicated in numerous outbreaks in the community and in hospital pediatric units for the past 30 years.
Salmonella Typhi And Paratyphi A
Salmonella Typhi
An appropriate antibiotic is essential for the treatment of patients with typhoid fever and should commence as soon as clinical diagnosis is made. Since the emergence of plasmid-mediated chloramphenicol resistance in the typhoid bacillus in the early 1970s, the efficacy of chloramphenicol as a first-line drug has been increasingly undermined by outbreaks caused by strains with resistance to this antimicrobial in countries as far apart as Mexico and India. A feature of chloramphenicol-resistant strains from such outbreaks was that although the strains belonged to different Vi PTs, resistance to chloramphenicol – often in combination with resistance to streptomycin, sulfonamides, and tetracyclines (R-type CSSuT) – was encoded by a plasmid of the H1 incompatibility group (now termed HI1). Since 1989 there have been many outbreaks caused by Typhi strains resistant to chloramphenicol, ampicillin, and trimethoprim, and additional resistances to streptomycin, sulfonamides, and tetracyclines (R-type ACSSuTTm), particularly in the Indian subcontinent (see Threlfall 2005a, b; Cooke et al., 2006). The emergence of strains with resistance to trimethoprim and ampicillin, in addition to chloramphenicol, has caused many treatment problems.
Without exception, in all outbreaks studied thus far involving MR Typhi, the complete spectrum of multiple resistance has been encoded by plasmids of the HI1 incompatibility (inc) group and it has been suggested, incorrectly, that this plasmid group is specific for the typhoid bacillus. Evolutionary diversity within the HI1 group has recently been observed in plasmids from MR strains of S. Typhi from Vietnam over a 10-year period during the 1990s.
Since 1989, following the emergence of strains with resistance to chloramphenicol, ampicillin and trimethoprim, ciprofloxacin (CpL) has become the first-line drug in both developing and developed countries. Regrettably, strains of S. Typhi with decreased susceptibility to CpL (minimal inhibitory concentration (MIC): 0.25–1.0 mg/L) have been increasingly reported. In such strains CpL is chromosomally encoded. Such strains have caused substantive outbreaks in several developing countries, notably Tajikistan and Vietnam, and have also caused treatment problems in developed countries. Azithromycin, a macrolide antibiotic, has also been evaluated for the treatment of infections caused by MR typhoid, with encouraging results.
Salmonella Paratyphi A
Infections caused by S. Paratyphi A may also require antimicrobial intervention. S. Paratyphi A with decreased susceptibility to CpL has been reported in India since the late 1990s. An increase in strains of S. Paratyphi A with decreased susceptibility to CpL from patients from 10 European countries between 1999 and 2001 has also been observed and in the UK in 2005 over 80% of isolates of S. Paratyphi A exhibited decreased susceptibility to CpL.
Other Salmonella Serovars
Developed countries
In developed countries salmonella infections are primarily zoonotic in origin. When resistance is present, it has often been acquired prior to transmission of the organism through the food chain to humans. The most important serovars in the UK and Europe are Enteritidis and Typhimurium and, in the United States, Typhimurium, Enteritidis, and more recently, Newport. For all these serovars the main method of spread is through the food chain. In most cases the clinical presentation is that of mild to moderate enteritis. The disease is usually self-limiting and antimicrobial therapy is seldom required.
Since 1991 there has been an epidemic in cattle and humans in England and Wales of MR strains of S. Typhimurium DT 104 of R-type ACSSuT (see the previous section ‘Developed countries’ under ‘Nontyphoidal salmonellas’).
In MR DT 104 of R-type ACSSuT, resistances are contained in a 16-kilobase (kb) region of the 43-kb SGI-1, made up of integrons containing (respectively) the ASu (blaCARB-2 and sul1) and SSp (aadA2) genes (Sp, spectinomycin), with intervening plasmid-derived genes coding for resistance to chloramphenicol/florphenicol (florR) and tetracyclines (tetG). Although chromosomally encoded in recent years, SGI-1 has been identified in several different salmonella serovars, including S. Agona, S. Albany, and S. Paratyphi B variant Java, which is indicative of phage-mediated transfer of resistance, or transfer by an as yet unidentified method. Such strains have caused infections in humans and cattle and there is speculation of a connection with ornamental fish originating in the Far East.
In the United States, multiple resistance has been reported in serovars Saintpaul, Heidelberg, and Newport, in addition to S. Typhimurium DT 104. More recently, MDR S. Newport with plasmid-encoded resistance to ceftriaxone has caused numerous infections in both cattle and humans in North America (Threlfall, 2005a). This organism commonly shows resistance to ACSSuT, with additional resistance to third-generation cephalosporins mediated by the CMY-2 beta-lactamase gene. Similarly there have been increasing reports of resistance to extended-spectrum beta-lactamases in salmonella from humans and food animals in numerous countries worldwide. For example CTX-M-9, -15, and -17 to -18 enzymes have recently been reported in six different serovars isolated from humans in the UK, and CTX-M-like enzymes have been reported in S. Virchow in Spain and in S. Anatum in Taiwan. In Taiwan a particularly alarming development has been the emergence of a highly virulent strain of S. Cholerae-suis with high-level resistance to Cp and with plasmid-mediated resistance to ceftriaxone. As far as is known, the strain has not spread and no further infections with this highly drug-resistant organism have been detected.
Developing Countries
An additional feature of strains on nontyphoidal salmonellas in developing countries has been the possession of plasmid-mediated MDR, often with resistance to seven or more antimicrobials, mediated by a plasmid of the FI incompatibility group. In addition to coding for multiple resistance, this plasmid also codes for production of the hydroxamate siderophore aerobactin, a known virulence factor for some enteric and urinary tract pathogens. These plasmids, first identified in a strain of S. Typhimurium DT 208 that caused numerous epidemics in many Middle Eastern countries in the 1970s, have subsequently been identified in a strain of S. Wien responsible for a massive epidemic that began in Algiers in 1969 but spread rapidly thereafter through pediatric and nursery populations in many countries throughout North Africa, Western Europe, the Middle East, and eventually the Indian subcontinent over the next 10 years. A retrospective molecular study of this group of plasmids has demonstrated that the plasmids have evolved through sequential acquisition of integrons carrying different arrays of antibiotic resistance genes. Although not clinically proven, the epidemiological evidence strongly suggests that possession of this class of plasmid has contributed to the virulence and epidemicity of such strains.
Virulence Aspects Of Salmonella
Salmonella virulence is a highly complex phenomenon, and has been the subject of many investigations. Some serovars such as Typhi, Pullorum, Gallinarum, Dublin, Cholerae-suis, and Enteritidis are highly host-specific, with their reservoirs of infection being anthropoids (Typhi), poultry (Pullorum, Gallinarum, Enteritidis), swine (Cholerae-suis), and cattle (Dublin). Some of these serovars – for instance, Typhi, Gallinarum, and Pullorum – cause disease for the most part only in their natural reservoir. Others, such as Cholerae-suis and Dublin, cause disease in their food animal reservoir but when infections occur outside of their normal reservoir (e.g., in humans), the symptoms can be very severe, often resulting in septicemia with subsequent mortality. Other host-adapted serovars (e.g., Enteritidis) cause little overt disease in their natural reservoir but when transmitted to humans can be a major cause of salmonellosis. Still other serovars (e.g., Typhimurium) have a wide host range and cause disease both in their food animal reservoir (e.g., cattle) and in humans.
Salmonella Pathogenicity Islands
Many of the major components required by S. enterica to cause infections are chromosomally encoded. The regions responsible for the virulence functions are termed salmonella pathogenicity islands (SPI); 14 such islands have now been identified, termed SPI-1–SPI-14. The size, distribution, and virulence functions of these SPIs have been extensively reviewed by Morgan (2006). It is noteworthy that not all serovars possess all the islands, and that differential pathogenicity in different hosts may be related to the presence or absence of such islands.
Salmonella Enterica Virulence Plasmids
In addition to possessing pathogenicity islands certain serovars of subspecies I harbor serovar-specific plasmids ranging from 40–90 kb, which poses a gene cluster promoting virulence in mice. This gene cluster, termed the salmonella plasmid virulence (spv) cluster has been identified in the epidemiologically important serovars Enteritidis, Typhimurium, Dublin, and Gallinarum. The biology of the spv cluster (Tezcan-Merdol et al., 2006) indicates involvement in serum resistance and invasion, but not in the initial phase of the disease in human salmonellosis.
Control
Control of salmonella disease can be exerted at three levels – the individual, the community (the herd), and the environment. Such control may be exerted by vaccination, by eradication and/or withdrawal of an infected product, and by general hygiene and cooking practices. Factors exacerbating the emergence and spread of particular strains, such as the indiscriminate use of antibiotics, may also be important, and the control of antibiotics, particularly in animal husbandry, has been highlighted as an important factor in combating the emergence of strains with resistance to key antibiotics.
Vaccination
Individuals may be vaccinated and, for the control of typhoid, a range of vaccines are available for Salmonella Typhi. The oral, live attenuated Ty21a vaccine (marketed as Vivotif) remains in use in many countries, particularly in the Indian subcontinent. Possibly the most current is the polysaccharide capsular Vi vaccine (Typhim), but assessment of the relative efficacy of the different vaccines is difficult. Several field trials are currently in progress, both in developing and developed countries, with potentially promising results reported for a Vi-conjugate vaccine.
In the control of S. Enteritidis in poultry in the UK, the development and use of vaccines for breeders and layers has been extremely effective, and the use of such vaccines is currently being assessed for use in other countries, particularly within the EU.
Eradication And Withdrawal
In many outbreaks control has been exerted either by eradication of the reservoir of infection (e.g., infected poultry flocks) or withdrawal of the contaminated food products. There are many examples of the latter method of control, one of the most recent being the withdrawal of contaminated confectionary products worldwide following contamination with S. Montevideo. In such instances the existence of an international rapid response network has proved invaluable (see Fisher and Threlfall, 2005 for other examples of product withdrawal at an international level).