Quantitative analyses of certain enteric bacteria and bacterial extracts: I. Standardization and sonic disruption of eight enteric bacterial species, each at five population levels

Standardized individual preparations of five population levels of eight enteric organisms [Escherichia coli (O4:H3), E. coli (O111:B4:H12), Salmonella enteritidis, S. paratyphi B, S. typhimurium, Shigella boydii, S. dysenteriae, and S. sonnei) were prepared. Dry weights, calculated mean cell weight, and nitrogen content of bacterial suspensions before, and of supernatant fluids after, ultrasonic disruption are tabulated. Percentages of disruption, estimated from nitrogen concentration ratios of the suspensions and supernatant fluids, are given. These data are presented as guidelines for the preparation of bacterial extracts prior to precipitin analyses.     

Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis

Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics—even below this breakpoint—is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA, which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M. tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M. tuberculosis survival during isoniazid treatment via reduced expression of katG, the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M. tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic.     

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80–100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.     

Shigella in Brazilian children with acute diarrhoea: prevalence,antimicrobial resistance and virulence genes

Diarrhoeal disease is still considered a major cause of morbidity and mortality among children. Among diarrhoeagenic agents, Shigella should be highlighted due to its prevalence and the severity of the associated disease. Here, we assessed Shigella prevalence, drug susceptibility and virulence factors. Faeces from 157 children with diarrhoea who sought treatment at the Children''s Hospital João Paulo II, a reference children´s hospital in Belo Horizonte, state of Minas Gerais, Brazil, were cultured and drug susceptibility of the Shigella isolates was determined by the disk diffusion technique. Shigella virulence markers were identified by polymerase chain reaction. The bacterium was recovered from 10.8% of the children (88.2% Shigella sonnei). The ipaH, iuc, sen and ial genes were detected in strains isolated from all shigellosis patients; set1A was only detected in Shigella flexneri. Additionally, patients were infected by Shigella strains of different ial, sat, sen and set1A genotypes. Compared to previous studies, we observed a marked shift in the distribution of species from S. flexneri to S. sonnei and high rates of trimethoprim/sulfamethoxazole resistance.     

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.     

Surface display of the HPV L1 capsid protein by the autotransporter Shigella IcsA

Autotransporters have become attractive tools for surface expression of foreign proteins in Gram-negative bacteria. In this study, the Shigella autotransporter IcsA, has been exploited to express the human papillomavirus (HPV) type 16 L1 capsid protein in Shigella sonnei and Escherichia coli. The L1 gene was fused in-frame to replace the coding sequence of the IcsA passenger domain that is responsible for actin-based motility. The resultant hybrid protein could be detected by an anti-L1 antibody on the surface of S. sonnei and E. coli. In E. coli, the protein was expressed on the entire surface of the bacterium. In contrast, the protein was detected mainly at one pole of the Shigella bacterium. However, the protein became evenly distributed on the surface of the Shigella bacterium when the icsP gene was removed. Our study demonstrated the possibility of exploiting autotransporters for surface expression of large, heterologous viral proteins, which may be a useful strategy for vaccine development.     

Modulation of Endotoxicity of Shigella Generalized Modules for Membrane Antigens (GMMA) by Genetic Lipid A Modifications: RELATIVE ACTIVATION OF TLR4 AND TLR2 PATHWAYS IN DIFFERENT MUTANTS*

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.     

Investigation of protective properties of Shigella sonnei carbohydrate biopolymers under experimental endotoxic shock models

We studied tolerogenic properties of new nonpyrogenic low-endotoxic complex preparation composed of Shigella sonnei carbohydrate biopolymers under D-galactosamine and direct experimental endotoxic shock models. Pretreatment with complex preparation (exopolysaccharide + lipopolysaccharide) S. sonnei protects mice (CBA × C57B1/6)F1 against lipopolysaccharide (E. coli O:55)-mediated death. We demonstrate correlation between increasing preimmunization dose of complex preparation with rate of survival and reduction of TNF-α serum level under a direct endotoxic shock model.     

Screening of probiotic lactobacilli for inhibition of Shigella sonnei and the macromolecules involved in inhibition

A total of 91 lactobacilli were screened for antimicrobial activity against Shigella sonnei. Agar-well assay showed that 16 lactobacilli displayed strong antibacterial activity against S. sonnei. The nature of these antimicrobial agents were investigated and shown to be dependent on their production of organic acids. Adhesion tests showed that 6 lactobacilli demonstrated good adherence to HT-29 cells, of these Lactobacillus johnsonii F0421 were selected for acid and bile salt tolerance properties. We further research on L. johnsonii F0421 inhibition of S. sonnei adhesion to HT-29 cells. The result showed that L. johnsonii F0421 exhibited significant inhibitory activity and excluded, competed and displaced adhered S. sonnei by 48%, 38% and 33%, respectively. In order to elucidate the inhibitory functions of macromolecules involved in L. johnsonii F0421, the cells were treated with 5 m LiCl, 0.05 m sodium metaperiodate and heating and assayed for inhibition activity. The results suggested a role of S-layer proteins on L. johnsonii F0421 cells in inhibition of the adhesion process, but carbohydrates do not seem to be involved. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant bands of approximately 40 kDa. In addition, 100 μg/well of S-layer proteins from L. johnsonii F0421 cells were effective in inhibiting adhesion of S. sonnei to HT-29 cells. These findings suggest that L. johnsonii F0421 possesses the capacity for inhibition of S. sonnei activity as well as probiotic properties, which could serve as a potential novel and effective probiotic strain for use in the food industry.     

Epidemiologic Study of Shigella sonnei from Sequential Outbreaks and Sporadic Cases Using Different Typing Techniques

We noted that eight outbreaks of Shigella sonnei from an unknown source occurred sequentially in Aichi Prefecture, Japan, between October 1992-June 1993. For comparative purposes we analyzed 53 outbreak-related isolates of Shigella sonnei using different subtyping methods and studied the epidemiology of the outbreaks. It appeared from our study that DNA-based techniques such as plasmid typing and pulsed-field gel electrophoresis (PFGE) were more useful tools for subtyping Shigella sonnei than colicin typing and the antimicrobial susceptibility test. Moreover, according to PFGE analysis, four genetically related isolates of Shigella sonnei were responsible for the eight sequential outbreaks. To further investigate the epidemiology of outbreaks, 58 sporadic isolates of Shigella sonnei from overseas travelers with shigellosis during the same period were also examined. We found that some sporadic isolates from travelers in Asia were genetically related to those of the outbreak-related isolates, indicating that genetically related isolates prevailed in Asia during this period, probably because of the extensive movement of people or food.     

Antimicrobial activity against Shigella sonnei and probiotic properties of wild lactobacilli from fermented food

Four lactobacilli strains (Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L, Lactobacillus casei Q8-L and L. rhamnosus GG (LGG), were systematically assessed for the production of antimicrobial substances active towards Shigella sonnei, Escherichia coli and Salmonella typhimurium. Agar-well assay showed that the four lactobacilli strains displayed strong antibacterial activity towards S. sonnei. The nature of antimicrobial substances was also investigated and shown to be dependent on the production of organic acids, in particular the lactic acid. Time-kill assay showed that the viability of the S. sonnei was decreased by 2.7–3.6 log CFU/ml after contact with CFCS (cell-free culture supernatants) of four lactobacilli for 2 h, which confirmed the result of the agar-well assay. Further analysis of the organic acid composition in the CFCS revealed that the content of lactic acid range from 227 to 293 mM. In addition, the aggregations properties, adherence properties and tolerance to simulated gastrointestinal conditions were also investigated in vitro tests. The result suggested that the M5-L, J10-L and Q8-L strains possess desirable antimicrobial activity towards S. sonnei and probiotic properties as LGG and could be potentially used as novel probiotic strains in the food industry.     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.     

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study

BackgroundAntimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei.ConclusionsThis study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.     

Rapid Exchange of Bound ADP on the Staphylococcus aureus Replication Initiation Protein DnaA

In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in Gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA+ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATP-DnaA to ADP-DnaA in S. aureus and thereby delaying the re-initiation of DNA replication.     

DiaA/HobA and DnaA: a pair of proteins co-evolved to cooperate during bacterial orisome assembly

Replication of the bacterial chromosome is initiated by binding the DnaA protein to oriC. Various factors control the ability of DnaA to bind and unwind DNA. Among them, Escherichia coli DiaA and Helicobacter pylori HobA have been characterized recently. They were found to interact with domain I of DnaA and stimulate DnaA binding to oriC. We examined HobA and DiaA functional homology and showed that, despite a high degree of structural similarity, they are not interchangeable because they are unable to interact with heterologous DnaA proteins. We revealed particular structural differences impeding formation of heterologous complexes and, consistently, we restored DiaA-enhanced oriC binding by the hybrid Ec^I-Hp^II-IVDnaA protein; i.e. H. pylori DnaA in which domain I was exchanged with that of E. coli. This proved that DiaA and HobA are functional homologs and upon binding to DnaA they exert a similar effect on orisome formation. Interestingly, we showed for the first time that the dynamics of DiaA- and HobA-stimulated orisome assembly are different. HobA enhances and accelerates HpDnaA binding to oriC, whereas DiaA increases but decelerates EcDnaA binding with oriC. We postulate that the different dynamics of orisome formation reflect the distinct strategies adopted by E. coli and H. pylori to regulate the frequency of the replication of their chromosomes. DiaA/HobA homolog have been identified in many proteobacteria and therefore might constitute a common, though species-specific, factor modulating bacterial orisome assembly.     

Rapid Diagnosis of Diarrhea Caused by Shigella sonnei Using Dipsticks; Comparison of Rectal Swabs,Direct Stool and Stool Culture

Background

We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs.

Methodology/Principal Findings

The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10⁶ CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%–98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%–88.6%) and 100 %, respectively.

Conclusion

This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.     

Characterization of a <Emphasis Type="Italic">Salmonella</Emphasis> Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria

In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.     

YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA

Control of DNA replication initiation is essential for cell growth. A unifying characteristic of DNA replication initiator proteins is their distinctive AAA+ nucleotide‐binding domains. The bacterial initiator DnaA assembles into a right‐handed helical oligomer built upon interactions between neighbouring AAA+ domains to form an active initiation complex. Recently we developed a unique cross‐linking assay that specifically detects ATP‐dependent DnaA helix assembly. Here we have utilized this assay to show that two DnaA regulatory proteins in Bacillus subtilis, YabA and DnaD, inhibit DnaA helix formation. These results, in combination with our previous finding that the regulatory factor Soj/ParA also targets DnaA filament formation, highlight the critical importance of regulating DnaA helix formation during the initiation reaction. Moreover, these observations lead us to suggest that DnaA oligomerization may be the main regulatory step of the initiator assembly pathway in B. subtilis, in contrast to the prevailing model of bacterial DNA replication based on Escherichia coli DnaA where ATP binding appears to be the targeted activity.     

Causative Species and Serotypes of Shigellosis in Mainland China: Systematic Review and Meta-Analysis

Background

Shigella, the causative agent of shigellosis, is a major global public health concern, particularly in developing countries with poor sanitation. A comprehensive and current understanding of the prevalent species and serotypes of shigellosis is essential for both disease prevention and vaccine development. However, no current data are available on the causative species/serotypes of shigellosis in mainland China during the past decade.

Methods and Findings

Relevant studies addressing the prevalent species of shigellosis in mainland China from January 2001 to December 2010 were identified from PubMed and the Chinese BioMedical Literature Database (in Chinese) until April 2012. A total of 131 eligible articles (136 studies) were included in this review. Meta-analyses showed that the prevalences of S. flexneri and S. sonnei were 76.2% (95% CI, 73.7%–78.5%) and 21.3% (95% CI, 19.0%–23.7%), respectively. Stratified analyses indicated a decrease in the prevalence of S. flexneri cases and an increase in the prevalence of S. sonnei cases concurrent with the rapid economic growth experienced by China in recent years. Moreover, significantly higher rates of S. sonnei were observed in the East, North and Northeast regions of China, as compared to the rest of the country. These phenomena imply the possible association between the prevalent species of Shigella and regional economic status; however, additional factors also exist and require further investigations. Moreover, the two major serotypes S. flexneri 2a and 4c accounted for 21.5% (95% CI, 16.7%–27.4%) and 12.9% (95% CI 9.8%–16.9%) of S. flexneri infections, respectively, in the past decade. However, these results were found to be frequently heterogeneous (p for Q tests <0.01).

Conclusions

This study provides an updated review of the causative agents of shigellosis in mainland China and focuses on the importance of strengthening prevention and research efforts on S. sonnei and the newly emerged S. flexneri serotype 4c.     

<Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> an environmental host for <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> and <Emphasis Type="Italic">Shigella sonnei</Emphasis>

The interaction between Shigella dysenteriae or Shigella sonnei and Acanthamoeba castellanii was studied by viable counts, gentamicin assay and electron microscopy. The result showed that Shigella dysenteriae or Shigella sonnei grew and survived in the presence of amoebae for more than 3 weeks. Gentamicin assay showed that the Shigella were viable inside the Acanthamoeba castellanii which was confirmed by electron microscopy that showed the Shigella localized in the cytoplasm of the Acanthamoeba castellanii. In conclusion, the relationship between Shigella dysenteriae and Shigella sonnei with Acanthamoeba castellanii is symbiotic, and accordingly free-living amoebae may serve as a transmission reservoir for Shigella in water.