Boydii Type Research Articles

DIARRHEA CAUSING ENTEROBACTERIACEAE AND THEIR ANTIBIOTIC RESISTANCE PATTERNS AMONG DIARRHEA PATIENTS FROM SANA’A CITY, YEMEN

Background and aims: The rise of antibiotic-resistant strains of the bacteria causing diarrhea remains a major worldwide health concern, especially in children under five. The purpose of this surveillance study was to ascertain the prevalence and antibiotic susceptibility profile of Enterobacteriaceae, which cause diarrhea. Materials and Methods: The National Center for Public Health Laboratories (NCPHL) in Sana'a city was the site of this cross-sectional investigation. Patient stool samples exhibiting diarrhea were gathered and cultivated using standard differential/selective medium. Standard biochemical assays and serological investigations were employed to identify the isolates. With the aid of Kirby-Bauer disk diffusion, the antibiogram was identified. Results: Enterobacteriaceae isolates were obtained from 972 stool samples collected; 15.3% (149/972) were diarrheagenic Enterobacteriaceae bacteria, including E. coli, Salmonella, and Shigella. There were 8.3% (81/972) Salmonella spp., 5.7% (56/972) Shigella spp., and 1.2% (12/972) E. coli. E. coli strains were grouped, with the most prominent being serogroups O153 (3, 2%) and O25 (2, 1.4%). Fifty-six (37.6%) Shigella species were identified as Shigella boydii type 2 and Shigella bodii type 17, which were the most common Shigella isolated (11, 8.1%, and 16, 10.7%, respectively). We detected high resistance of Salmonella isolates to tetracycline (29/81, 35.8%) but relatively low resistance to ciprofloxacin (7/81, 8.6%) and ceftriaxone (5/81, 6.3%). Conclusion: The many Enterobacteriaceae linked to instances of diarrhea in Sana'a, Yemen, were demonstrated in this study, along with the necessity of differential diagnosis for improved treatment results. Salmonella, Shigella, and Escherichia coli have all been linked to episodes of diarrhea in Sana'a city. To stop Yemen's antimicrobial resistance from spreading, regular antimicrobial susceptibility testing, efficient monitoring, and national surveillance of AMR bacteria should be put into place. Peer Review History: Received 3 February 2024; Revised 12 March 2024; Accepted 20 April; Available online 15 May 2024 Academic Editor: Dr. Gehan Fawzy Abdel Raoof Kandeel, Pharmacognosy Department, National Research Centre, Dokki, 12622, Giza, Egypt, gehankandeel9@yahoo.com Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 7.5/10 Reviewers: Dr. George Zhu, Tehran University of Medical Sciences, Tehran, Iran, sansan4240732@163.com Dr. Esther Marguerite Chase DJANGA, Faculty of Medicine and Biomedical Sciences. Department of Public Health. University of Yaoundé I, Cameroon. e.djanga@yahoo.com

Structural analysis of the O-antigen polysaccharide from Escherichia coli O188

The structure of the O-antigen from Escherichia coli reference strain O188 (E. coli O188:H10) has been investigated. The lipopolysaccharide shows a typical nonrandom modal chain-length distribution and the sugar and absolute configuration analysis revealed d-Man, d-Glc, d-GlcN and d-GlcA as major components. The structure of the O-specific polysaccharide was determined using one- and two-dimensional 1H and 13C NMR spectroscopy experiments, where inter-residue correlations were identified by 1H,13C-heteronuclear multiple-bond correlation and 1H,1H-NOESY experiments, which revealed that it consists of pentasaccharide repeating units with the following structure: ▪Biosynthetic aspects and NMR analysis are consistent with the presented structure as the biological repeating unit. The O-antigen of Shigella boydii type 16 differs only in that it carries O-acetyl groups to ~50% at O6 of the branch-point mannose residues. A molecular model of the E. coli O188 O-antigen containing 20 repeating units extends ~100 Å, which is similar to the height of the periplasmic portion of polysaccharide co-polymerase Wzz proteins that regulate the O-antigen chain length of lipopolysaccharides in the Wzx/Wzy biosynthetic pathway.

Prevalence of Shigella boydii in Bangladesh: Isolation and Characterization of a Rare Phage MK-13 That Can Robustly Identify Shigellosis Caused by Shigella boydii Type 1.

Shigellosis, caused by Shigella boydii type 1, is understudied and underreported. For 3 years, GEMS study identified 5.4% of all Shigella as S. boydii. We showed the prevalent serotypes of S. boydii in Bangladesh and phage-based diagnosis of S. boydii type 1, a rapid and low-cost approach. Previously typed 793 clinical S. boydii strains were used for serotype distribution. Twenty-eight environmental water samples were collected for isolation of Shigella phages. Forty-eight serotypes of Shigella and other enteric bacteria were used for testing the susceptibility to phage MK-13. Electron microscopy, restriction enzyme analysis, whole genome sequencing (WGS), and annotation were performed for extensive characterization. S. boydii type 1 is the second most prevalent serotype among 20 serotypes of S. boydii in Bangladesh. We isolated a novel phage, MK-13, which specifically lyses S. boydii type 1, but doesn’t lyse other 47 serotypes of Shigella or other enteric bacteria tested. The phage belongs to the Myoviridae family and distinct from other phages indicated by electron microscopy and restriction enzyme analysis, respectively. MK-13 genome consists of 158 kbp of circularly permuted double-stranded DNA with G + C content of 49.45%, and encodes 211 open reading frames including four tRNA-coding regions. The genome has 98% identity with previously reported phage, ΦSboM-AG3, reported to have a broader host range infecting most of the S. boydii and other species of Shigella tested. To our knowledge, MK-13 is the first phage reported to be used as a diagnostic marker to detect S. boydii type 1, especially in remote settings with limited laboratory infrastructure.

Straightforward sequential and one-pot synthesis of a pentasaccharide repeating unit corresponding to the cell wall O-antigen of Shigella boydii type 18

Synthesis of a pentasaccharide repeating unit corresponding to the cell of wall O-antigen Shigella boydii type 18 has been achieved by sequential as well as iterative glycosylations in one-pot. Use of p-methoxybenzyl group (PMB) as an in situ removable protecting group allowed obtaining the desired pentasaccharide derivative in a generalized glycosylation condition and in one-pot condition. Synthesis of a beta-L-rhamnosidic linkage present in the molecule has been successfully achieved using l-rhamnosyl thioglycoside donor having a picoloyl group at remote C-3 position influencing beta selectivity in the glycosylation. A combination of N-iodosuccinimide (NIS) and perchloric acid supported over silica (HClO4–SiO2) has been used as thiophilic glycosylation promoter in all glycosylation reactions. TEMPO mediated selective oxidation of the primary hydroxyl group has been carried out at the late stage of the synthetic strategy.

Antimicrobial Potential of Epiphytic Bacteria Associated With Seaweeds of Little Andaman, India.

Seaweeds of the intertidal regions are a rich source of surface associated bacteria and are potential source of antimicrobial molecules. In the present study, 77 epiphytic isolates from eight different algae collected from Little Andaman were enumerated. On testing for their antimicrobial activities against certain pathogens twelve isolates showed positive and six of them showed significant antimicrobial inhibition zone against Shigella boydii type 1, Shigella flexneri type 2a, Shigella dysenteriae type 5, Enterotoxigenic Escherichia coli O115, Enteropathogenic E. coli serotype O114, Vibrio cholera; O1 Ogawa, Aeromonas hydrophila, Klebsiella pneumoniae, Staphylococcus aureus. Based on the activity these six isolates (G1C, G2C, G3C, UK, UVAD, and Tor1) were identified by 16S rRNA gene sequence and were found to belong to the phyla Firmicutes and Proteobacteria. Purified antimicrobial compounds obtained from these isolates were identified by GC-MS. Furan derivatives were identified from G2C Pseudomonas stutzeri KJ849834, UVAD Alcanivorax dieselolei KJ849833, UK Vibrio sp. KJ849837, Tor1 Exiguobacterium profundum KJ849838. While 2-Pyrrolidinone, Phenol, 2, 4-bis (1, 1-dimethylethyl) were from G3C Vibrio owensii KJ849836 and (1-Allylcyclopropyl) methanol from the extracts of G1C Bacillus sp. KJ849835. The results of the present study shows that these six potent isolates isolated from the seaweeds are found to be a source of antimicrobial compounds.

Structure and genetics of the O-antigens of Escherichia coli O182–O187

O-polysaccharides (OPSs) were obtained by mild acid degradation of the lipopolysaccharides of Escherichia coli O182-O187, and their structures were established by sugar analysis, Smith degradation, and 1H and 13C NMR spectroscopy. In addition to the monosaccharides that occur often in E. coli OPSs (d-Glc, d-Gal, d-Man, d-GlcNAc, d-GalNAc, d-GlcA, l-Fuc, d-Rib), a number of less common components were identified as the OPS constituents, including 2-acetamido-2-deoxy-l-quinovose and 4-deoxy-4-[(S)-3-hydroxybutanoyl-l-alanyl]-d-quinovose (O186), 3-acetamido-3-deoxy-d-fucose (O187), 3-deoxy-3-[(R)-3-hydroxybutanoyl]-d-fucose (O184), and 2,3-diacetamido-2,3-dideoxy-l-rhamnose (O182). The OPS structures of E. coli O183 and O182 are identical to those of the OPS of Shigella boydii type 10 and the capsular polysaccharide of E. coli K48, respectively. The OPSs of E. coli O186 and O123 are closely related differing in the presence of a Glc residue in the former in place of a GlcNAc residue in the latter. The O-antigen gene clusters of the bacteria studied were analyzed and their contents were found to be consistent with the OPS structures. Predicted glycosyltransferases encoded in the gene clusters were tentatively assigned to glycosidic linkages based on similarities to sequences of other E. coli O-serogroups available from GenBank and taking into account the OPS structures established.

Structural Relationships Between Genetically Closely Related O-Antigens of Escherichia coli and Shigella spp.

Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using (1)H and (13)C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.

Structure and gene cluster of the O-antigen of Enterobacter cloacae G3421

The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G3421 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. In addition, partial solvolysis with anhydrous trifluoroacetic acid was applied, which cleaved selectively the α-l-rhamnopyranosidic linkages. The following structure of the branched hexasaccharide repeating unit was established. [Display omitted] The O-polysaccharide studied shares the β-l-Rhap-(1→4)-α-l-Rhap-(1→2)-α-l-Rhap trisaccharide fragment with the O-polysaccharide of Shigella boydii type 18. The O-antigen gene cluster of E. cloacae G3421 was sequenced. Functions of genes in the cluster, including those for glycosyltransferases, were tentatively assigned by a comparison with sequences in the available databases and found to be consistent with the O-polysaccharide structure.

Development of a cost-effective vaccine candidate with outer membrane vesicles of a tolA-disrupted Shigella boydii strain

Our previous studies on outer membrane vesicles based vaccine development against shigellosis, revealed the inability of Shigella to release significant amount of vesicles naturally, during growth. Disruption of tolA, one of the genes of the Tol–Pal system of Gram negative bacterial membrane, has increased the vesicle release rate of a Shigella boydii type 4 strain to approximately 60% higher. We also noticed the vesicles, released from tolA-disrupted strain captured more OmpA protein and lipopolysaccharide, compared to the vesicles released from its wild type prototype. Six to seven weeks old BALB/c mice, immunized with 25μg of three oral doses of the vesicles, released by tolA mutant, conferred 100% protection against lethal homologous challenge through nasal route, compared to only 60% protection after the same dose of wild type immunogen. Mice, immunized with the vesicles from tolA-mutant, manifested significant secretion of mucosal IgG and IgA. A sharp and significant response of pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) were also observed in the lung lavage of these groups of mice, within 6h post challenge; but at 24h, these inflammatory cytokines showed the sign of subsidence and the system was taken over by the release of anti-inflammatory cytokines (IL-4 and IL-10). Studies with naïve peritoneal macrophages, proved further, the potency of these vesicles to stimulate nitric oxide and TNF-α, IL-12p70, IL-6 and IL-10 productions in-vitro. The ability of these vesicles to trigger polarization of CD4+ T cells toward Th1 adaptive immune response, had also been observed along with the presence of anti-inflammatory cytokines in the system. Our study demonstrated, the vesicles from tolA-disrupted Shigella were able to suppress Shigella-mediated inflammation in the host and could balance between inflammation and anti-inflammation, promoting better survival and health of the infected mice. Outer membrane vesicles from tolA-mutant, could be a potential cost-effective vaccine candidate against shigellosis.

Structure and genetics of the O-antigen of Escherichia coli O169 related to the O-antigen of Shigella boydii type 6

The O-polysaccharide (O-antigen) of Escherichia coli O169 was studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the branched hexasaccharide repeating unit was established: [Display omitted] The O-polysaccharide of E. coli O169 differs from that of Shigella boydii type 6 only in the presence of a side-chain glucose residue. A comparison of the O-antigen biosynthesis gene clusters between the galF to gnd genes in the genomes of the two bacteria revealed their close relationship. The glycosyltransferase gene responsible for the formation of the β-d-Glcp-(1→6)-α-d-Galp linkage in the O-antigen was identified in the gene cluster.

Multi-serotype outer membrane vesicles of Shigellae confer passive protection to the neonatal mice against shigellosis

Recently, we have demonstrated, immunization of adult female mice with outer membrane vesicles (OMVs) of Shigella boydii type 4 protected their offspring passively from shigellosis. In our present study, we have advanced our research by formulating multi-serotype outer membrane vesicles (MOMVs), mixing the OMVs of Shigella dysenteriae 1 Δstx, Shigella flexneri 2a, 3a and 6, S. boydii type 4 and Shigella sonnei to achieve a broad spectrum protection against shigellosis. Adult mice were immunized orally with 50μg of MOMVs, four times at weekly intervals. Immunological parameters were observed at various time points, before, during and after immunization, in adult mice. Passive protection was examined in their offspring by measuring protective efficacy and studying intestinal colonization, after challenging with various Shigella strains. Immunized dams exhibited a consistent broad spectrum antibody response. 3–4 day-old offspring of immunized dams showed significant long term passive protection against wild type S. flexneri 2a, 3a, and 6, S. boydii type 2 and S. dysenteriae 1. Their stomach extracts, essentially containing mother's milk, have also exhibited significant levels of anti-MOMVs immunoglobulins. In conclusion, MOMVs formulation represents an easy, safe immunization strategy that was found suitable to provide complete passive protection to the neonatal mice against all four serogroups of Shigellae. It could be exploited for the development of a novel non-living vaccine against human shigellosis in near future.

Synthesis of a Common Tetrasaccharide Repeating Unit of the O-antigen of Enteroinvasive Escherichia coli O143 and Shigella boydii Type 8

Synthesis of a Common Tetrasaccharide Repeating Unit of the O-antigen of Enteroinvasive Escherichia coli O143 and Shigella boydii Type 8

Synthesis of a Common Tetrasaccharide Repeating Unit of the O-antigen of Enteroinvasive Escherichia coli O143 and Shigella boydii Type 81

A concise chemical synthesis of the common tetrasaccharide corresponding to the O-antigen of enteroinvasive Escherichia coli O143 and Shigella boydii type 8 has been achieved in excellent yield. Application of [2+2] block glycosylation and late stage oxidation of primary hydroxyl groups to the carboxylic groups are notable features in the synthetic strategy. Keywords: Escherichia coli, Shigella boydii, O-antigen, tetrasaccharide, dysentery

Outer membrane vesicles ofShigella boydiitype 4 induce passive immunity in neonatal mice

Like most other Gram-negative bacteria, Shigella releases outer membrane vesicles (OMVs) into the surrounding environment during growth. In this study, we have exploited OMVs of Shigella as a protective immunogen in a mice model against Shigellosis. Distinctive vesicle secretion was noticed from different Shigella strains. Among them, Shigella boydii type 4 (BCH612) was secreting relatively higher amounts. We immunized female adult mice orally with 32 μg of purified Shigella boydii type 4 (BCH612) OMVs four times at 1-week intervals. Antibodies against these vesicles were detected in immunized sera until 120 days, indicating a persistent immune response. To observe whether the passive immunity had been transferred to the neonates, the immunized female mice were mated and the offspring were challenged orally, with wild-type homologous and heterologous Shigella strains. All offspring of immunized mothers survived the challenge with homologous strain BCH612 and up to 81% protective efficacy was noted against heterologous strains Shigella dysenteriae 1, Shigella flexneri 2a, Shigella flexneri 3a, Shigella flexneri 6 and Shigella sonnei. Our results exhibited for the first time that oral immunization of adult female mice with purified OMVs of Shigella, without any adjuvant, conferred passive protection to their offspring against shigellosis. These findings will contribute to the future development of a potential non-living vaccine candidate against shigellosis.

Antimicrobial-resistant Shigella infections from Iran: an overlooked problem?

In this study, we wanted to assess the level of antimicrobial resistance, the presence of genes encoding resistance to cephalosporins and plasmid-mediated quinolone resistance (PMQR), and genetic relatedness among Shigella isolates obtained from Iranian patients. A total of 44 Shigella isolates were collected from Iranian patients admitted to Milad Hospital, Tehran, Iran, during 2008-10. Of these, 37 were serotyped and characterized by MIC determination. A subset of eight suspected extended-spectrum β-lactamase (ESBL) producers (six Shigella sonnei phase II and two Shigella flexneri type 1b) were examined for the presence of genes encoding cephalosporin resistance. The presence of PMQR was assessed in one S. flexneri isolate exhibiting low-level resistance to ciprofloxacin and susceptibility to nalidixic acid. PFGE was performed on 25 S. sonnei phase II isolates. Of the isolates, 25 (68%) were S. sonnei phase II, with 5 (14%) S. flexneri, 5 (14%) Shigella dysenteriae type 2, and 2 (5%) Shigella boydii type 2. Resistance to at least threeclasses of antimicrobials was detected in all species. The presence of bla(CTX-M-15) and the AmpC β-lactamase producer bla(CMY-2) was confirmed in five and one S. sonnei phase II isolates, respectively. One of the two S. flexneri type 1b that contained bla(CTX-M-15) also harboured a qnrS1 gene. PFGE identified sevenPFGE profiles; the main cluster included 15 of the strains, suggesting low genetic diversity between isolates or the presence of an endemic clone in Iran. This is the first known description of ESBL-producing and AmpC β-lactamase-producing Shigella and of PMQR Shigella in Iran. The emergence of CTX-15, CMY-2 and qnrS1 genes may compromise the treatment of shigellosis. Strategies to minimize the spread of ESBL-producing and AmpC-β-lactamase-producing Shigella should be implemented.

Convergent synthesis of the tetrasaccharide repeating unit of the O-antigen of Shigella boydii type 9

A convenient synthesis of the tetrasaccharide repeating unit of the O-antigen of Shigella boydii type 9 has been achieved in excellent yield using a [2 + 2] block glycosylation strategy. TEMPO-mediated selective oxidation of the primary alcohol of the tetrasaccharide derivative 8 to the carboxylic group followed by deprotection of the functional groups furnished target tetrasaccharide 1 as its 4-methoxyphenyl glycoside in high yield.

Emergence of a Novel Shiga Toxin-Producing Escherichia coli O Serogroup Cross-Reacting with Shigella boydii Type 10

This is the first report of the isolation of Shiga toxin-producing Escherichia coli (STEC) strains whose O antigens were genetically and serologically identical to those of Shigella boydii type 10, from human feces. The novel STEC O serogroup may be widespread in Japan and associated with diarrhea and hemorrhagic colitis.

Lipopolysaccharide core structures and their correlation with genetic groupings of Shigella strains. A novel core variant in Shigella boydii type 16

Bacteria Shigella, the cause of shigellosis, evolved from the intestinal bacteria Escherichia coli. Based on structurally diverse O-specific polysaccharide chains of the lipopolysaccharides (LPSs; O-antigens), three from four Shigella species are subdivided into multiple serotypes. The central oligosaccharide of the LPS called core is usually conserved within genus but five core types called R1-R4 and K-12 have been recognized in E. coli. Structural data on the Shigella core are limited to S. sonnei, S. flexneri and one S. dysenteriae strain, which all share E. coli core types. In this work, we elucidated the core structure in 14 reference strains of S. dysenteriae and S. boydii. Core oligosaccharides were obtained by mild acid hydrolysis of the LPSs and studied using sugar analysis, high-resolution mass spectrometry and two-dimensional NMR spectroscopy. The R1, R3 and R4 E. coli core types were identified in 8, 3 and 2 Shigella strains, respectively. A novel core variant found in S. boydii type 16 differs from the R3 core in the lack of GlcNAc and the presence of a D-glycero-D-manno-heptose disaccharide extension. In addition, the structure of an oligosaccharide consisting of the core and one O-antigen repeat was determined in S. dysenteriae type 8. A clear correlation of the core type was observed with genetic grouping of Shigella strains but not with their traditional division to four species. This finding supports a notion on the existing Shigella species as invalid taxa and a suggestion of multiple independent origins of Shigella from E. coli clones.

Convergent synthesis of the pentasaccharide repeating unit of the O-antigen of Shigella boydii type 14

An efficient synthesis of the pentasaccharide repeating unit of the O-antigen of Shigella boydii type 14 is reported. A one-pot, two step iterative glycosylation and [3+2] block synthetic strategy have been adopted for the construction of the pentasaccharide derivative 11, which was then transformed into target compound 1 after a series of functional group transformations. The primary hydroxyl group has been selectively oxidized to a carboxylic acid functionality without affecting the secondary hydroxyl groups. HClO 4–SiO 2 has been used as a solid acid substitute in all glycosylation and functional group transformations. The yields were excellent in all glycosylation steps.

Synthesis of tri- and pentasaccharide fragments corresponding to the O-antigen of Shigella boydii type 6

A convenient synthetic strategy for the synthesis of the acidic pentasaccharide repeating unit and its trisaccharide fragment corresponding to the O-antigen of Shigella boydii type 6 has been successfully developed. A stereoselective sequential glycosylation method has been exploited to obtain the target tri- and pentasaccharide derivatives. Most of the synthetic intermediates were solid and prepared in high yields from commercially available reducing sugars following a series of protection–deprotection reactions. A late-stage TEMPO mediated selective oxidation reaction finally resulted in the pentasaccharide containing a glucuronic acid unit. A 2-(4-methoxyphenoxy) ethyl group has been chosen as the anomeric protecting group to provide trisaccharide and pentasaccharide derivatives linked to an ethylene glycol linker.