Shigella Strains Research Articles

Tracing Back the Evolutionary Route of Enteroinvasive Escherichia coli (EIEC) and Shigella Through the Example of the Highly Pathogenic O96:H19 EIEC Clone.

Enteroinvasive Escherichia coli (EIEC) cause intestinal illness through the same pathogenic mechanism used by Shigella spp. The latter species can be typed through genomic and phenotypic methods used for E. coli and have been proposed for reclassification within E. coli species. Recently the first appearance of a highly pathogenic EIEC O96:H19 was described in Europe as the causative agent of two large outbreaks that occurred in Italy and in the United Kingdom. In contrast to Shigella spp and to the majority of EIEC strains, EIEC O96:H19 fermented lactose, lacked pathoadaptive mutations, and showed good fitness in extracellular environment, similarly to non-pathogenic E. coli, suggesting they have emerged following acquisition of the invasion plasmid by a non-pathogenic E. coli. Here we describe the whole genome comparison of two EIEC O96:H19 strains isolated from severe cases of diarrhea in Uruguay in 2014 with the sequences of EIEC O96:H19 available in the public domain. The phylogenetic comparison grouped all the O96:H19 strains in a single cluster, while reference EIEC strains branched into different clades with Shigella strains occupying apical positions. The comparison of the virulence plasmids showed the presence of a complete conjugation region in at least one O96:H19 EIEC. Reverse Transcriptase Real Time PCR experiments confirmed in this strain the expression of the pilin-encoding gene and conjugation experiments suggested its ability to mobilize an accessory plasmid in a recipient strain. Noteworthy, the tra region was comprised between two reversely oriented IS600 elements, which were also found as remnants in another EIEC O96:H19 plasmid lacking the tra locus. We hypothesize that an IS-mediated recombination mechanism may have caused the loss of the conjugation region commonly observed in EIEC and Shigella virulence plasmids. The results of this study support the hypothesis of EIEC originating from non-pathogenic E. coli through the acquisition of the virulence plasmid via conjugation. Remarkably, this study showed the ability of a circulating EIEC strain to mobilize plasmids through conjugation, suggesting a mechanism for the emergence of novel EIEC clones.

Metagenomic analysis identified microbiome alterations and pathological association between intestinal microbiota and polycystic ovary syndrome

To identify different microbial species in women with polycystic ovary syndrome (PCOS) and reveal a possible relationship between gut dysbiosis and pathological changes. Cross-sectional study. Academic institution. Reproductive-aged women with PCOS (n = 14) and controls (n = 14) from the Centre for Reproductive Medicine. Shotgun metagenomic sequencing on fecal samples from patients, and clinical parameters (including body mass index, endocrine hormone levels, and glycemia level) gathered for correlation analysis. Identification of different gut microbial strains and relativity between microbiota and clinical parameters. We found several microbial strains were statistically significantly more abundant in the PCOS group, including Parabacteroides merdae, Bacteroides fragilis, and strains of Escherichia and Shigella, whereas Faecalibacterium prausnitzii was enriched in the control group. Metagenomic species (MGS) analysis revealed that the microbes of the PCOS group were negatively correlated with those of the control group. Of note, we observed a positive correlation between MGS relevant to PCOS and endocrine disorders, including body mass index and elevated levels of serum testosterone, luteinizing hormone, and antimüllerian hormone. Functional alterations, reflected by Kyoto Encyclopedia of Genes and Genomes orthologues, could imply potential mechanisms of microbial involvement in the developmental progress of PCOS. Our findings suggest an intimate association and potential mechanisms linking microbial dysbiosis and the pathophysiologic changes of PCOS. We address the importance of monitoring and modulating microbial composition and functional shifts in future clinical practice.

Live attenuated Salmonella Typhimurium with monophosphoryl lipid A retains ability to induce T-cell and humoral immune responses against heterologous polysaccharide of Shigella flexneri 2a

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4+ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG+ B220low/int BM cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4+ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.

The characteristics and genome analysis of the novel Y. pestis phage JC221

The pathogen of plague is Yersinia pestis (Y. pestis), one of the deadliest pathogens in the world and belonging to the family Enterobacteriaceae. In this work, the biological characteristics and complete genome sequence analysis of a novel lytic Y. pestis-specific phage JC221 isolated from Yunnan Province, China, was studied. JC221 belongs to the Myoviridae family and has a regular icosahedral head and a long contractile tail. The double-stranded DNA genome of JC221 contains 174,931 bp, and the G + C content is 41.23 %. There are 274 predicted genes, of which only 103 hits of genes or gene products are found in database searches, and there are no known virulence-related or antibiotic resistance genes. The genome sequence of JC221 showed <80 % identity to other phages, and evolutionary analysis revealed that bacteriophage JC221 belongs to the Yersinia phage cluster. Furthermore, the bacteriophage could completely lyse most of the tested Y. pestis strains (12/13) at 28 °C and 37 °C, and some Shigella strains could be lysed at 37°C. Morphological and genomic analysis indicated that JC221 is a new Y. pestis phage and a new member of the Tequatrovirus phages. The novel Y. pestis phage JC221 has important reference value for the study of environmental microecology and epidemiology of plague foci.

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

BackgroundShigella is a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans. Shigella invades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strain S. flexneri serotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches.ResultsWe have sequenced, assembled, annotated and manually curated the full genome of S. flexneri 5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new hybrid strategy to prepare gapless, highly accurate genome sequences, which also cover AT-rich tracks or repetitive sequences that are transcribed. Furthermore, we have performed genome-wide analysis of transcriptional start sites (TSS) and determined the length of 5′ untranslated regions (5′-UTRs) at typical culture conditions for the inoculum of in vitro infection experiments. We identified 6723 primary TSS (pTSS) and 7328 secondary TSS (sTSS). The S. flexneri 5a M90T annotated genome sequence and the transcriptional start sites are integrated into RegulonDB (http://regulondb.ccg.unam.mx) and RSAT (http://embnet.ccg.unam.mx/rsat/) databases to use their analysis tools in the S. flexneri 5a M90T genome.ConclusionsWe provide the first complete genome for S. flexneri serotype 5a, specifically the laboratory reference strain M90T. Our work opens the possibility of employing S. flexneri M90T in high-quality systems biology studies such as transcriptomic and differential expression analyses or in genome evolution studies. Moreover, the catalogue of TSS that we report here can be used in molecular pathogenesis studies as a resource to know which genes are transcribed before infection of host cells. The genome sequence, together with the analysis of transcriptional start sites, is also a valuable tool for precise genetic manipulation of S. flexneri 5a M90T. Further, we present a new hybrid strategy to prepare gapless, highly accurate genome sequences. Unlike currently used hybrid strategies combining long- and short-read DNA sequencing technologies to maximize accuracy, our workflow using long-read DNA sequencing and short-read RNA sequencing provides the added value of using non-redundant technologies, which yield distinct, exploitable datasets.

OCCURRENCE OF SHIGELLA SPECIES IN RAW MILK AND KAREISH CHEESE WITH SPECIAL REFERENCE TO ITS VIRULENCE GENES

A total number of 100 random samples of raw milk and kareish cheese (50 for each) were collected from Assiut city farms and dairy shops. Shigella species were isolated, confirmed biochemically and by using Polymerase Chain Reaction (PCR). Also two virulence genes; invasive gene (invC) and plasmid- encoded virulence gene (ipaH) were identified using PCR technique. 10 isolated strains of Shigella isolates (37%) from dairy milk samples and 13 isolates (43%) from kareish cheese were identified as following: S. dysenteriae, S. flexneri, S. sonnei and S. boydii. The two virulence genes; (invC) and (ipaH) were detected in only six and four strains of the identified Shigella species, respectively. Four of them had both the virulence genes (isolated from milk and cheese samples). Even though conventional culture is considered the gold standard for Shigella detection and the PCR method is a useful tool which complements detection of foodborne pathogens such as Shigella. Chitosan was evaluated in this study as antibacterial substance on the identified Shigella strains by inoculating it in pasteurized milk, using 3 different concentrations: 0.25, 0.5 and 1% of chitosan. Chitosan reduced the inoculated Shigella strain mean counts with highly significant effect (P<0.01) at the 6th day reached to 2.10 ±0.17, 1.00 ±0.30 and th day we noticed that chitosan concentration of 0.5% only was highly significant (P<0.05). Generally, the 0.5% chitosan concentration showed the highly reduction effect on the count and survival of the Shigella strain involved.

Functional Annotation and Curation of Hypothetical Proteins Present in A Newly Emerged Serotype 1c of Shigella flexneri: Emphasis on Selecting Targets for Virulence and Vaccine Design Studies.

Shigella flexneri is the principal cause of bacillary dysentery, contributing significantly to the global burden of diarrheal disease. The appearance and increase in the multi-drug resistance among Shigella strains, necessitates further genetic studies and development of improved/new drugs against the pathogen. The presence of an abundance of hypothetical proteins in the genome and how little is known about them, make them interesting genetic targets. The present study aims to carry out characterization of the hypothetical proteins present in the genome of a newly emerged serotype of S. flexneri (strain Y394), toward their novel regulatory functions using various bioinformatics databases/tools. Analysis of the genome sequence rendered 4170 proteins, out of which 721 proteins were annotated as hypothetical proteins (HPs) with no known function. The amino acid sequences of these HPs were evaluated using a combination of latest bioinformatics tools based on homology search against functionally identified proteins. Functional domains were considered as the basis to infer the biological functions of HPs in this case and the annotation helped in assigning various classes to the proteins such as signal transducers, lipoproteins, enzymes, membrane proteins, transporters, virulence, and binding proteins. This study contributes to a better understanding of growth, survival, and disease mechanism at molecular level and provides potential new targets for designing drugs against Shigella infection.

FTIR-assisted MALDI-TOF MS for the identification and typing of bacteria

MALDI-TOF MS is well-recognized for microbial identification and widely used in research and clinical fields due to its specificity, speed of analysis, and low cost of consumables. However, the classification or identification accuracy is poor for E. coli and Shigella. In addition, FTIR is a promising tool for bacterial typing. In this study, 14 strains of E. coli and 9 strains of Shigella were typed by both MALDI-TOF MS and FTIR techniques. Alternatively, a data fusion strategy using these two approaches was attempted to achieve better typing accuracy. Hierarchical clustering analysis (HCA) revealed that the typing accuracies for selected E. coli and Shigella from blood agar were 65.2%, 78.3%, and 100% for MALDI-TOF MS, FTIR, and FTIR combined with MALDI-TOF MS, respectively. Based on these results, a library constructed of the MS-IR fusion data was employed to identify individual bacteria at the strain level, and the library-based typing accuracies for 207 test spectra of sample strains from three different culture media yielded 97.6% accuracy for distinguishing between E. coli and Shigella at genus level. For species level and strain level, it yielded 95.2% and 92.3% typing accuracy, respectively. These results indicate that FTIR is a better technique for typing selected E. coli and Shigella than MALDI-TOF MS, and the accuracy is increased by combining the data from MALDI-TOF MS and FTIR spectroscopy. FTIR could be used to supplement MALDI-TOF MS for the identification and typing of taxonomic microorganisms.

SRL pathogenicity island contributes to the metabolismof D-aspartate via an aspartate racemase in Shigella flexneri YSH6000.

In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence.

Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the Shigella protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall Shigella virulence. Here, we provide both in vitro and in vivo characterization of Spa47 oligomerization and activation in the presence and absence of engineered ATPase-inactive Spa47 mutants. The findings describe mechanistic details of Spa47-catalyzed ATP hydrolysis and uncover critical distinctions between oligomerization mechanisms capable of supporting ATP hydrolysis in vitro and those that support T3SS function in vivo. Concentration-dependent ATPase kinetics and experiments combining wild-type and engineered ATPase inactive Spa47 mutants found that monomeric Spa47 species isolated from recombinant preparations exhibit low-level ATPase activity by forming short-lived oligomers with active site contributions from at least two protomers. In contrast, isolated Spa47 oligomers exhibit enhanced ATP hydrolysis rates that likely result from multiple preformed active sites within the oligomeric complex, as is predicted to occur within the context of the type three secretion system injectisome. High-resolution fluorescence microscopy, T3SS activity, and virulence phenotype analyses of Shigella strains co-expressing wild-type Spa47 and the ATPase inactive Spa47 mutants demonstrate that the N-terminus of Spa47, not ATPase activity, is responsible for incorporation into the injectisome where the mutant strains exhibit a dominant negative effect on T3SS function and Shigella virulence. Together, the findings presented here help to close a significant gap in our understanding of how T3SS ATPases are activated and define restraints with respect to how ATP hydrolysis is ultimately coupled to T3SS function in vivo.

Антибиотикорезистентность распространенных штаммов сальмонелл и шигелл

Антибиотикорезистентность распространенных штаммов сальмонелл и шигелл

In silico functional and toxicity prediction of Shigella dysenteriae sd197 hypothetical proteins

Shigella dysenteriae is a gram-negative bacterium of the family Enterobacteriaceae, which causes Shigellosis, an acute form of gastroentritis in the children. The Sd197 strain of Shigella was fully sequenced, consisting of 1 circular chromosome comprising 4,369,232nt (4,664 genes). The objective of this research is to analyze the function forecast of earlier not allocated proteins in the genome of Shigella dysenteriae strain Sd197 using different bioinformatics instruments and databases. There were 828 hypothetical proteins available in Shigella dysenteriae strain Sd197 genome. These hypothetical proteins were evaluated to estimate their physicochemical characteristics using parameters such as the Theoretical PI, Extinction Coefficient, Aliphatic Index and Grand Hydropathicity Average (GRAVY) in the ProtParam server. It was further analyzed using bioinformatics tools for functional prediction, structural prediction, and virulent prediction. Of the 828 hypothetical proteins, 38 are enzymes, 11 have virulence factor, 85 are hydrolase proteins, 5 are isomerase, 4 are lyase, 8 are ligase proteins etc. This research will promote a stronger knowledge of drug resistance and the mechanism of pathogenesis in S.dysenteriae as well as the discovery of drug targets for disease therapy.

An In Silico Approach in Determining the Potency of Quercetin Against Infectious Diarrhoea Caused by Different Pathogenic Strains of Shigella

An In Silico Approach in Determining the Potency of Quercetin Against Infectious Diarrhoea Caused by Different Pathogenic Strains of Shigella

Colicin U from Shigella boydii Forms Voltage-Dependent Pores.

Colicin U is a protein produced by the bacterium Shigella boydii (serovars 1 and 8). It exerts antibacterial activity against strains of the enterobacterial genera Shigella and Escherichia Here, we report that colicin U forms voltage-dependent pores in planar lipid membranes; its single-pore conductance was found to be about 22 pS in 1 M KCl at pH 6 under 80 mV in asolectin bilayers. In agreement with the high degree of homology between their C-terminal domains, colicin U shares some pore characteristics with the related colicins A and B. Colicin U pores are strongly pH dependent, and as we deduced from the activity of colicin U in planar membranes at different protein concentrations, they have a monomeric pore structure. However, in contrast to related colicins, we observed a very low cationic selectivity of colicin U pores (1.5/1 of K+/Cl- at pH 6) along with their atypical voltage gating. Finally, using nonelectrolytes, we determined the inner diameter of the pores to be in the range of 0.7 to 1 nm, which is similar to colicin Ia, but with a considerably different inner profile.IMPORTANCE Currently, a dramatic increase in antibiotic resistance is driving researchers to find new antimicrobial agents. The large group of toxins called bacteriocins appears to be very promising from this point of view, especially because their narrow killing spectrum allows specific targeting against selected bacterial strains. Colicins are a subgroup of bacteriocins that act on Gram-negative bacteria. To date, some colicins are commercially used for the treatment of animals (1) and tested as a component of engineered species-specific antimicrobial peptides, which are studied for the potential treatment of humans (2). Here, we present a thorough single-molecule study of colicin U which leads to a better understanding of its mode of action. It extends the range of characterized colicins available for possible future medical applications.

International Spread of Multidrug-Resistant Campylobacter coli in Men Who Have Sex With Men in Washington State and Québec, 2015-2018.

Background Campylobacter species are among the most common causes of enteric bacterial infections worldwide. Men who have sex with men (MSM) are at increased risk for sexually transmitted enteric infections, including globally distributed strains of multidrug-resistant Shigella species.MethodsThis was a retrospective study of MSM-associated Campylobacter in Seattle, Washington and Montréal, Québec with phenotypic antimicrobial resistance profiles and whole genome sequencing (WGS).ResultsWe report the isolation of 2 clonal lineages of multidrug-resistant Campylobacter coli from MSM in Seattle and Montréal. WGS revealed nearly identical strains obtained from the 2 regions over a 4-year period. Comparison with the National Center for Biotechnology Information’s Pathogen Detection database revealed extensive Campylobacter species clusters carrying multiple drug resistance genes that segregated with these isolates. Examination of the genetic basis of antimicrobial resistance revealed multiple macrolide resistance determinants including a novel ribosomal RNA methyltransferase situated in a CRISPR (clustered regularly interspaced short palindromic repeats) array locus in a C. coli isolate.ConclusionsAs previously reported for Shigella, specific multidrug-resistant strains of Campylobacter are circulating by sexual transmission in MSM populations across diverse geographic locations, suggesting a need to incorporate sexual behavior in the investigation of clusters of foodborne pathogens revealed by WGS data.

Azithromycin non-susceptible Shigella circulating in Israel, 2014-2016.

Shigella species remains a major diarrhoeagenic agent, affecting mostly children, with global high incidence and high mortality rate specially in developing areas. Although azithromycin is recommended for treatment of shigellosis, there are currently no CLSI susceptibility breakpoints, accordingly no routine antimicrobial susceptibility test is performed in the clinical laboratory. The purpose of this study was to estimate the prevalence, resistance profile and molecular epidemiology of azithromycin non-susceptible Shigella strains in Israel during a three year period. Shigella isolates (n = 1,170) referred to the National Reference Center during 2014–2016, were included in this study. Serotyping was performed by slide agglutination. Resistance genes, mph(A) and erm(B), were identified by PCR and the phenotype profile was determined by broth microdilution (BMD). Genetic relatedness was assessed by wgMLST. Decreased susceptibility to azithromycin (DSA) phenotype and genotype were detected in various Shigella species and serotypes related to diverse genetic backgrounds and antimicrobial profiles: 6% (26/423) of Shigella flexneri and 2% (16/747) of Shigella sonnei displayed DSA (MIC16 mg/L). Correlation of this phenotype with the presence of mph(A) and erm(B) genes was confirmed. All DSA-strains displayed resistance to ≥3 different antimicrobial classes. Among DSA-strains, 14% were resistant to quinolones and 5% displayed resistance to ceftriaxone. Most of these strains (32/42) were isolated from children in the southern and central regions of Israel. Clonality and significant relatedness was confirmed by PFGE and wgMLST. The presence of macrolide resistance genes among the different species and lineages reflects the transmissible nature of these genes. The emergence of DSA-Shigella reinforces the necessity to establish clinical breakpoints that would warrant routine testing, reporting and surveillance for this drug of choice.

Resistance characteristics of CTX-M type Shigella flexneri in China.

The present study was to identify the drug resistance, resistance mechanism and the extended-spectrum β-lactamase (ESBLs) genotypes of Shigella flexneri (S. flexneri) in Jinan. Susceptibility tests were performed by MIC-determination. The genotypes of β-lactamase were identified using PCR and DNA sequencing. The resistance transfer ability of the ESBL-producing strains was examined by conjugation tests. A total of 105 S. flexneri isolates were collected, and 34 (32.4%) were ESBL-producing isolates. All ESBL-producing isolates were susceptible to cefoxitin and imipenem, and 35.3% isolates were resistant to ciprofloxacin. ESBL-producing isolates showed high level resistant to ampicillin (100%), cefotaxime (100%), tetracycline (100%), chloramphenicol (100%), trimethoprim/sulfamethoxazole (100%), ceftazidime (73.5%) and cefepime (73.5%). Three types of β-lactamase genes (blaTEM, blaOXA and blaCTX-M) were identified in all ESBL-producing isolates, and the genotypes were confirmed as blaTEM-1 (23/34), blaOXA-30 (34/34), blaCTX-M-14 (9/34) and blaCTX-M-15 (25/34) by sequencing. In conclusion, the Shigella strains isolated in Jinan are cross-resistant and multi-drug resistant. The main genotypes of ESBLs are CTX-M-14 and CTX-M-15.

Rapid culture-based identification of Shiga toxin-producing Escherichia coli and Shigella spp./Enteroinvasive E. coli using the eazyplex® EHEC complete assay

Shiga toxin-producing Escherichia coli (STEC) and Shigella spp./enteroinvasive E. coli (EIEC) are common diarrheagenic bacteria that cause sporadic diseases and outbreaks. Clinical manifestations vary from mild symptoms to severe complications. For microbiological diagnosis, culture confirmation of a positive stool screening PCR test is challenging because of time-consuming methods for isolation of strains, wide variety of STEC pathotypes, and increased emergence of non-classical strains with unusual serotypes. Therefore, molecular assays for the rapid identification of suspect colonies growing on selective media are very useful. In this study, the performance of the newly introduced eazyplex® EHEC assay based on loop-mediated isothermal amplification (LAMP) was evaluated using 18 representative STEC and Shigella strains and 31 isolates or positive-enrichment broths that were collected from clinical stool samples following screening by BD MAX™ EBP PCR. Results were compared to real-time PCR as a reference standard. Overall, sensitivities and specificities of the eazyplex® EHEC were as follows: 94.7% and 100% for Shiga toxin 1 (stx1), 100% and 100% for stx2, 93.3% and 97.1% for intimin (eae), 100% and 100% for enterohemolysin A (ehlyA), and 100% and 100% for invasion-associated plasmid antigen H (ipaH) as Shigella spp./EIEC target, respectively. Sample preparation for LAMP took only some minutes, and the time to result of the assay ranged from 8.5 to 13min. This study shows that eazyplex® EHEC is a very fast and easy to perform molecular assay that provides reliable results as a culture confirmation assay for the diagnosis of STEC and Shigella spp./EIEC infections.

Genomic and Proteomic Characterizations of Sfin-1, a Novel Lytic Phage Infecting Multidrug-Resistant Shigella spp. and Escherichia coli C.

Shigellosis is a public health threat in developed as well as developing countries like “India.” While antibiotic therapy is the mainstay of treatment for shigellosis, current emergence of multidrug-resistant strains of Shigella spp. has posed the problem more challenging. Lytic bacteriophages which destroy antibiotic resistant Shigella spp. have great potential in this context and hence their identification and detailed characterization is necessary. In this study we presented the isolation and a detailed characterization of a novel bacteriophage Sfin-1, which shows potent lytic activity against multidrug-resistant isolates of Shigella flexneri, Shigella dysenteriae, Shigella sonnei obtained from clinical specimens from shigellosis patients. It is also active against Escherichia coli C. The purified phage is lytic in nature, exhibited absorption within 5–10 min, a latent period of 5–20 min and burst size of ∼28 to ∼146 PFU/cell. The isolated phage shows stability in a broad pH range and survives an hour at 50°C. Genome sequencing and phylogenetic analyses showed that Sfin-1 is a novel bacteriophage, which is very closely related to T1-like phages (89.59% identity with Escherichia virus T1). In silico analysis indicates that Sfin-1 genome consists of double stranded linear DNA of 50,403 bp (GC content of 45.2%) encoding 82 potential coding sequences, several potential promoters and transcriptional terminators. Under electron microscopy, Sfin-1 shows morphology characteristics of the family Siphoviridae with an isometric head (61 nm) and a non-contractile tail (155 nm). This is most likely the first report of a lytic bacteriophage that is active against three of the most virulent multidrug-resistant Shigella species and therefore might have a potential role in phage therapy of patients infected with these organisms.

Kinetic modeling of selenium reduction by a defined co-culture in batch reactors

Abstract The kinetics of Se(VI) reduction by a defined co-culture of a Se(VI)-reducing strain Shigella fergusonii TB42616 (TB) and a Se(IV)-reducing strain Pantoea vagans EWB32213-2 (EWB) was investigated in batch reactors. Se(VI) reduction was a two-stage process from Se(VI) to Se(IV) and then from Se(IV) to Se(0). Biokinetic parameters were optimized with a Monod-type kinetic model using batch pure culture data through the Robust Global Optimization Algorithm embedded in a computer package including yield coefficients, decay coefficients, maximum specific growth rates, and half-velocity constants. Se(VI) reduction by the defined co-culture was then simulated and verified over a range of culture compositions and initial Se(VI) concentrations, respectively. An inter-species inhibition term was incorporated into the model to illustrate the competition for Se(IV) between these two species during Se(VI) reduction in the co-culture. The results indicated that the incubation time corresponding to the peak level of Se(IV) increased with initial Se(VI) concentrations and a significant increase of Se(IV) accumulation with higher initial Se(VI) concentration. However, Se(IV) accumulation can be reduced with increasing population ratio of EWB to TB. The relatively high correlation coefficients suggested that the model was robust and applicable in simulating Se(VI) reduction by the defined co-culture.