Detection of virulence genes in Malaysian Shigellaspecies by multiplex PCR assay

Members of the genus Shigella, namely S. flexneri, S. dysenteriae, S. sonnei and S. boydii have caused and continue to be responsible for mortality and/or morbidity in high risk populations such as children under five years of age, senior citizens, toddlers in day-care centres, patients in custodial institutions, homosexual men and, war- and famine-engulfed people. Yearly episodes of shigellosis globally have been estimated to be 164.7 million and of these, 163.2 million were in developing countries and the remaining in industrialized nations. The mortality rate was approximately 0.7% [1]. A recent study by Lee & Puthucheary [2] on bacterial enteropathogens in childhood diarrhoea in a Malaysian urban hospital showed that Shigella spp. was the third most common bacteria isolated. S. flexneri and S. dysenteriae type 1 infections are usually characterized by frequent passage of small amounts of stool and mucus or blood. At times, watery stool followed by typical dysenteric stool maybe present with S. dysenteriae type 1 infection. S. sonnei and S. boydii infections are less severe with watery faeces but little mucus or blood.

Shigellosis is usually a self-limiting infection, however when it subsides, the intestinal ulcers heal with scar tissue formation. Uncomplicated recovery is usual and the organisms rarely cause other types of infections. Adversely, in 3 to 50% of cases, depending on the virulence of the strain, the nutritional and immune status of the host, the initial infection maybe followed by neurological complications or kidney failure. Serious complications do occur at greatest frequencies in malnourished infants, toddlers, older adults and immunocompromised individuals [3, 4].

Virulence genes responsible for the pathogenesis of shigellosis may be located in the chromosome or on the inv plasmid borne by the organism. They are often multifactorial and coordinately regulated, and the genes tend to be clustered in the genome. Previously reported PCR-based detection methods concentrated mainly on the ipaH gene alone [5, 6] or on ipaH and ial genes in two separate PCR assays [7, 8]. As ial is found on the large inv plasmid which is prone to loss or deletions, this gene-based detection may give false negative results. ipaH, on the other hand, is present on both the Shigella chromosome and on a large plasmid and hence, it is a more stable gene to detect. However, the sole presence of ipaH is not an absolute indicator of virulence as loss or deletion of the plasmid renders the bacterium noninvasive and therefore, avirulent. set1A and set1B are chromosomal genes encoding Shigella enterotoxin 1 (ShET1), which cause the watery phase of diarrhoea in shigellosis [9, 10]. ial and ipaH are responsible for directing epithelial cell penetration by the bacterium and for the modification of host response to infection, respectively [11‐13].

Here, we describe the application of a multiplex PCR (mPCR) design for simultaneous detection of four virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. and to determine the prevalence of these virulence genes in a random selection of Malaysian Shigella strains.

Numerous studies had been performed to detect virulence genes in Shigella by monoplex PCRs [8, 17, 18]. Studies involving the combination of chromosomal- and plasmid-encoded virulence genes in a single assay for Shigella detection, on the other hand, are scarce. Although the optimization of mPCR is more tedious and difficult to achieve than monoplexes, the ease of screening a large number of specimens, once the system is optimized, far outweighs the initial problems. The present mPCR system encompasses the presence of virulence genes found in the Shigella chromosome and on the large inv plasmid. Hence, it can determine if the pathogenesis of a particular strain is attributable to its chromosome or the plasmid, or if the strain is still invasive or otherwise, in a single reaction.

Initially, the monoplex PCRs were carried out following reaction conditions as proposed by a previous report [17]. However, we could not reproduce their results and hence had to modify the PCR conditions. Our failure to reproduce identical results despite using similar reagent concentrations and amplification conditions maybe attributed to the different makes of PCR reagents and primers used. Broude et al. [19] had compared amplification efficiencies of two commercial Taq DNA polymerases and found that they displayed different specificity in PCR.

Preferential amplification of one target sequence over another is a known phenomenon in mPCRs and it is usually overcome by increasing the amount of primers for the weaker amplification simultaneously with a decrease of primer concentrations for the stronger amplification. Buffer concentration may also affect mPCR amplifications despite it being seldom considered during monoplex optimization works [20]. Upon adjustment of primers and buffer concentrations, specific and consistent amplification of all the genes in the multiplex combination was achieved.

Although other studies have demonstrated the presence of ial and ipaH in strains of enteroinvasive Escherichia coli (EIEC) [11, 13, 21], we had not applied the mPCR assay to EIEC strains. It is unfortunate that these strains were not available for our study as EIEC gives rise to similar illness as Shigellosis.

Our study supported the observations of Noriega et al. [9] and Vargas et al. [17] in local Shigella strains. Their studies showed that both set1A and set1B were present exclusively in S. flexneri 2a. The complete correlation between the presence of both set1A and set1B showed that both genes are indeed found in tandem in the Shigella genome. In this study, almost all the Shigella strains positive for the presence of set1A and set1B (41/45 strains) belonged to S. flexneri 2a, thus confirming previous works that both genes are highly conserved in this particular serotype [14].

Both the prevalence of ial and ipaH were independent of the four different species of Shigella tested. Though both ial and ipaH are responsible for invasion-related processes and are found on the inv plasmid, the ial gene cluster resides near a region of the plasmid, which is a hot spot for spontaneous deletions [22]. This probably explains the lower prevalence of ial (45/110 strains) than ipaH (110/110 strains) in the Malaysian Shigella strains. Since invasiveness is a prerequisite for virulence in shigellae and since most of these virulence genes are located on the large plasmid, these strains would have possessed the plasmid when first isolated from patients. Due to storage/subculturing, the plasmid might have been lost together with the virulence-associated genes. By virtue of multiple copies being present on both the chromosome and the inv plasmid [23], ipaH seemed to be less compromised by plasmid loss and/or deletions. As the sole presence of ipaH is not indicative of the invasive phenotype, our mPCR design, which incorporated three other virulence genes, could determine the invasiveness of Shigella strains in epidemiological studies.

Dilution of the faecal sample with BHI was performed to lower the levels of PCR inhibitors such as bilirubin, bile salts and heme in the faeces [16]. An additional step of preincubating the spiked faecal samples also helped to eliminate the natural inhibitors [24]. The short 4-h enrichment step would increase the total number of target sequences caused by more bacterial growth and the overall detection sensitivity of the assay. Although PCR cannot differentiate between dead and viable bacteria, enrichment helped to dilute the concentrations of dead bacteria, thus reducing the probability of detecting them by the subsequent mPCR assay. The sensitivity level achieved in the study was found to be comparable to other studies. Houng et al. [25] detected up to 7.4 × 10⁴ cfu shigellae ml^-1 by amplifying the IS 630 sequences in shigella spp.. Yavzori et al. [24] reported a detection level of 10⁴ cfu shigellae per gram of faeces with the use of virF primers. Although it has been reported that ingestion as low as 100 shigellae resulted in clinical disease [26], the highest percentage of volunteers having diarrhoea were administered doses of at least 10⁴ viable organisms. Thus, the average detection limit of mPCR described in this study (5.0 × 10⁴ cfu/ml) is within the common infectious dose for shigellae.

Results from the preliminary clinical screening were promising. Nevertheless, the consideration of other diarrhoeal pathogens being present in the clinical samples cannot be negated. More patient samples are warranted to thoroughly vet the robustness and applicability of the developed mPCR in clinical environments.

One limitation of the present mPCR system is its inability to differentiate Shigella spp., unlike the multiplex reactions based on specific rfc genes developed by Houng et al. [25]. For future research, either set1A or set1B may be omitted from the multiplex system as both genes are shown to exist tandemly. rfc primers of different Shigella origins maybe incorporated to enable the discrimination of Shigella spp. as well as the identification of virulent strains in one assay.

We conclude that the mPCR system is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. The described assay is reproducible, sensitive, can be easily performed and is able to give a presumptive identification of the causal pathogen, which could be confirmed by culture techniques using selective media. An added advantage would be that EIEC, which gives a similar illness, might also be detected by this method, as EIEC also harbours ial and ipaH genes.