An outbreak of Shigella boydii serotype 20 in January 2015 amongst United Kingdom healthcare workers involved in the Ebola response in Sierra Leone.

In January 2015, Public Health England and the United Kingdom (UK) Ministry of Defence investigated cases of diarrhoea and fever in military personnel recently returned to the UK after supporting the response to the Ebola epidemic in Sierra Leone. Tests for Ebola virus infection were negative. PCR tests detected the ipaH gene in 10/12 faecal specimens, and Shigella boydii serotype 20 was isolated from 7 patients. A case control study was undertaken and analysed using multivariable logistic regression. Consumption of a coronation chicken lunch at the transit camp in Sierra Leone (SL) 24-48 h prior to departure for the UK was significantly associated with disease [adjusted odds ratio (OR) 28.15, 95 % CI: 1.87-422.65]. In the context of heightened concern during the Ebola epidemic, this outbreak highlights the importance of rapid and effective microbiological and epidemiological investigations to identify the aetiological agent in patients presenting with fever and diarrhoea.

Simultaneous quantification of Escherichia coli O157:H7 and Shigella boydii using a visual-antibody-macroarray.

Being high throughput, rapid, automated, economical, convenient to operate and highly sensitive, protein arrays have been widely used in the analysis of tumor markers and veterinary drug residues. Pathogenic microbes also can be detected qualitatively by DNA array or protein array; however, their high throughput detection and quantification remains a big obstacle. To evaluate the potentiality of protein arrays for multiple quantitative detection of microorganisms with naked eye examination without the help of any equipment, here we developed a visual-antibody-macroarray (VAMA) aiming at rapid and simultaneous quantification of Escherichia coli O157:H7 and Shigella boydii. The results show that this VAMA is highly specific and is able to distinguish mixed Escherichia coli O157:H7 and Shigella boydii synchronously. The detection limits are equivalent to 3.4 × 10(5) CFU mL(-1) and 3.2 × 10(5) CFU mL(-1), respectively, which conform to the results of plate counting and ELISA. Importantly, the examination can be solely performed with the naked eye. Therefore, we provide an easy, reliable and rapid method for quantitative analysis of microorganisms.

Molecular characterization of serologically atypical provisional serovars of Shigella isolates from Kolkata, India.

During 2000-2004, 13 Shigella strains that were untypable by commercially available antisera were isolated from children <5 years of age with acute diarrhoea in Kolkata. These strains were subsequently identified as Shigella dysenteriae provisional serovar 204/96 (n = 3), Shigella dysenteriae provisional serovar E23507 (n = 1), Shigella dysenteriae provisional serovar I9809-73 (n = 1), Shigella dysenteriae provisional serovar 93-119 (n = 1), Shigella flexneri provisional serovar 88-893 (n = 6) and Shigella boydii provisional serovar E16553 (n = 1). In this study, characterization of those provisional serovars of Shigella was performed with respect to their antimicrobial resistance, plasmids, virulence genes and PFGE profiles. The drug resistant strains (n = 10) of Shigella identified in this study possessed various antibiotic resistance genetic markers like catA (for chloramphenicol resistance); tetA and tetB (for tetracycline resistance); dfrA1 and sul2 (for co-trimoxazole resistance); aadA1, strA and strB (for streptomycin resistance) and blaOXA-1 (for ampicillin resistance). Class 1 and/or class 2 integrons were present in eight resistant strains. Three study strains were pan-susceptible. A single mutation in the gyrA gene (serine to leucine at codon 83) was present in four quinolone resistant strains. The virulence gene ipaH (invasion plasmid antigen H) was uniformly present in all strains in this study, but the stx (Shiga toxin) and set1 (Shigella enterotoxin 1) genes were absent. Other virulence genes like ial (invasion associated locus) and sen (Shigella enterotoxin 2) were occasionally present. A large plasmid of 212 kb and of incompatibility type IncFIIA was present in the majority of the strains (n = 10) and diversity was noticed in the smaller plasmid profiles of these strains even within the same provisional serovars. PFGE profile analysis showed the presence of multiple unrelated clones among the isolates of provisional Shigella serovars. To the best of our knowledge, this is the first report on the phenotypic and molecular characterization of provisional serovars of Shigella isolates from Kolkata, India.

In vitro development and analysis of Escherichia coli and Shigella boydii azithromycin-resistant mutants.

The aim of this study was to develop and analyze in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella boydii. Three clinical isolates of E. coli and one S. boydii isolated from feces samples collected from children under 5 years of age with diarrhea in Lima, Peru were inoculated onto Mueller-Hinton plates containing increasing serial dilutions of AZM ranging from their specific minimal inhibitory concentration (2 or 4 mg/l) to 64 mg/l. From these plates, 16 AZM-resistant mutants were selected to determine the stability of the resistance and the presence of cross resistance with other antibiotics. The role of Phe-Arg-ß-Naphthylamide (PAßN)-inhibitible efflux pumps as well as the presence of mutations in the rplV, rplD, and rrlH (23S rRNA) genes and alterations in the outer membrane profiles were determined in these 16 mutants. The rate of mutation ranged from < 2.70×10(-10) to 2.17×10(-7) for E. coli and from < 9.58×10(-10) to 1.05×10(-8) for S. boydii. E. coli mutants showed an increase in the AZM-MIC up to sixfold with one strain achieving a MIC >256 mg/l. In contrast, S. boydii only presented increases of up to twofold in MIC levels. All the strains obtained, but one showed stable AZM resistance. In the presence of PAßN, the AZM MICs decreased to parental levels in Shigella mutants, while no MIC returned to parental levels among the E. coli mutants. No cross resistance to other classes of antibiotics was found. These results show the relevance of PAßN-inhibitible efflux pumps in the basal levels and development of AZM resistance. Further studies to characterize the remaining unidentified mechanisms of AZM resistance are needed.

Genetic diversity and antibiotic resistance in Shigella dysenteriae and Shigella boydii strains isolated from children aged <5 years in Egypt.

Diversity within Shigella dysenteriae (n=40) and Shigella boydii (n=30) isolates from children living in Egypt aged <5 years was investigated. Shigella-associated diarrhoea occurred mainly in summer months and in children aged <3 years, it commonly presented with vomiting and fever. Serotypes 7 (30%), 2 (28%), and 3 (23%) accounted for most of S. dysenteriae isolates; 50% of S. boydii isolates were serotype 2. S. dysenteriae and S. boydii isolates were often resistant to ampicillin, chloramphenicol and tetracycline (42%, 17%, respectively), although resistance varied among serotypes. Pulsed-field gel electrophoresis separated the isolates into distinct clusters correlating with species and serotype. Genetic differences in trimethoprim/sulfamethoxazole and ß-lactam-encoding resistance genes were also evident. S. dysenteriae and S. boydii are genetically diverse pathogens in Egypt; the high level of multidrug resistance associated with both pathogens and resistance to the most available inexpensive antibiotics underlines the importance of continuing surveillance.

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.

Arginine-dependent acid-resistance pathway in Shigella boydii.

Ability to survive the low pH of the human stomach is considered be an important virulent determinant. It was suggested that the unique acid tolerance of Shigella boydii 18 CDPH, the strain implicated in a 1998 outbreak, may have played an important role in surviving the acidic food (bean salad). The strain was capable of inducing arginine-dependent acid-resistance (ADAR) pathway. This pathway was assumed to be absent in Shigella sp. Here, we have examined occurrence and efficacy of ADAR pathway in 21 S. boydii strains obtained from the American Type Culture Collection (ATCC) along with strains of S. flexneri (n = 7), S. sonnei (n = 4), and S. dysenteriae (n = 2). The eight S. boydii strains were able to induce ADAR to survive the acid challenge at pH 2.0; additional 8 strains could tolerate acid challenge at pH 2.5 but not at pH 2.0. The remaining five S. boydii strains were not able to induce ADAR pathway and could not survive acid challenge even at pH 2.5. ADAR pathway also appears to be present in all four Shigella sp. Shigella ADAR pathway was induced when cells were grown under partial oxygen pressure while its expression in E. coli required mere fermentative growth on glucose.

Analysis of a temporal cluster of Shigella boydii isolates in Mpumalanga, South Africa, November to December 2007.

BACKGROUND: Shigellosis is a global human health problem. The disease is most prevalent in developing countries with poor access to safe potable water and sanitation. Shigella boydii is of particular epidemiological importance in developing nations such as African and Asian countries. In the present study, we report on the analysis of a temporal cluster of 29 S. boydii serotype 2 strains, isolated in the Mpumalanga Province of South Africa (SA) over the period of November to December 2007. METHODOLOGY: Bacteria were identified as S. boydii using standard microbiological identification techniques and serotyped using commercially available antisera. Susceptibility testing to antimicrobial agents was determined by the Etest. Genotypic relatedness of strains was investigated by pulsed-field gel electrophoresis (PFGE) analysis of digested genomic DNA. RESULTS: The cluster of 29 isolates revealed comparable antimicrobial susceptibility profiles, while dendrogram analysis of PFGE patterns showed that the cluster of isolates grouped together and could clearly be differentiated from a random selection of unrelated S. boydii serotype 2 strains. Our data has strongly suggested that this cluster of isolates may share a common ancestry. However, this cannot be substantiated by epidemiological data because a detailed epidemiological investigation was not conducted. CONCLUSIONS: We have documented the first cluster of S. boydii infection in SA. Due to the lack of adequate epidemiological investigation, we cannot emphatically state that an outbreak had occurred. However, we do hypothesis that this was an outbreak for which a waterborne source cannot be excluded. This study has highlighted the urgent need for timely and appropriate systems of epidemiological investigation of all suspected outbreaks of disease in developing countries.

Antimicrobial resistance in Shigella species isolated in Dakar, Senegal (2004-2006).

From May 2004 to October 2006, a prospective study was carried out in Dakar, Senegal, to update information about the antimicrobial susceptibility of Shigella spp. isolated from stool specimens. Among the 165 non-duplicate strains collected, 81 (49%) were identified as Shigella flexneri, 75 (45%) as Shigella sonnei, 5 (3%) as Shigella boydii, and 4 (2%) as Shigella dysenteriae. Disk diffusion testing revealed that the majority of isolates were resistant to sulphonamides, trimethoprim-sulfamethoxazole, streptomycin, and tetracycline (respective overall resistance rates: 90, 90, 96, and 94%). More than half of the S. flexneri isolates were resistant to amoxicillin, amoxicillin-clavulanic acid, and chloramphenicol (respective resistance rates: 59, 58, and 52%), and almost all of the S. sonnei isolates were susceptible to these antimicrobials (respective resistance rates: 4, 1, and 4%). Only one isolate (belonging to the species S. sonnei) was resistant to nalidixic acid and displayed reduced susceptibility to ciprofloxacin.

Traveler's myopericarditis.

Colitis caused by Shigella is an uncommon etiology of infectious diarrhea in developed countries, usually presenting as traveler's diarrhea. Aside from clinical intestinal manifestations, shigellosis can present with a wide variety of extra-intestinal symptoms. We present the case of a 38-year-old man with diarrhea, fever, and chest pain that started after a holiday in Cape Verde (Africa). Blood samples revealed an increase in cardiac enzymes. An electrocardiogram revealed a widespread elevation of the ST segment. Echocardiography showed a swift pericardial effusion, confirming the diagnosis of acute myopericarditis. Shigella boydii was identified in stool cultures. The patient was treated with ciprofloxacin and acetylsalicylic acid, resulting in improvement in clinical and laboratory findings.

Escherichia coli, Shigella and Salmonella species in acute diarrhoea in Hamedan, Islamic Republic of Iran.

This study investigated the frequency of Escherichia col, Shigella and Salmonella species in stool specimens from patients with diarrhoea presenting to health centres in Hamedan province, Islamic Republic of Iran. From 144 samples, Shigella strains were isolated in 17 cases (11.8%): 10 Sh. flexneri, 3 Sh. sonnei, 2 Sh. boydii and 2 untyped strains. No Salmonella strains were isolated. Using molecular diagnostic methods, diarrheogenic E. coli were detected in 37 cases (25.7%), the majority were enterotoxigenic (ETEC) (22 cases) and Shiga toxin-producing (STEC) strains (15 cases). In 14 cases (9.7%) there was co-infection.

Xylose utilization and short-chain fatty acid production by selected components of the intestinal microflora of a rodent pollinator (Aethomys namaquensis).

Namaqua rock mice (Aethomys namaquensis) consume nectar xylose when visiting Protea flowers. Whole-animal metabolism studies suggest that the gastrointestinal microflora plays an important role in xylose metabolism in A. namaquensis. We collected caecal contents under anaerobic conditions, cultured caecal microflora both aerobically and anaerobically, and assessed caecal microbial xylose utilization using a (14)C-xylose incubation assay. All four mice sampled hosted culturable caecal micro-organisms that tested positive for xylose utilization. These were classified by 16S rRNA based taxonomy as: Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Shigella boydii, Arthrobacter sp. and members of the fungal genera Aspergillus and Penicillium. Cultures of these isolates were then analyzed by gas chromatography to determine the types and quantities of short-chain fatty acids produced by xylose fermentation. These results are discussed in the context of other studies of gut microflora in vertebrates.

Immunocapture UPPCR combined with DGGE for rapid detection of Shigella species.

AIMS: To develop an immunocapture universal primer PCR (iUPPCR) combined with denaturing gradient gel electrophoresis (DGGE) and evaluate it as a method permitting rapid detection of Shigella species and their serotypes. METHODS AND RESULTS: This method amplifying the conserved regions of bacterial 16S rRNA genes of different species or serotypes of Shigella dysentery bacilli captured and enriched by polyvalent antibodies can detect and distinguish causative pathogens rapidly. Four serotypes from three Shigella species including Shigella dysenteriae serotype 1, Shigella boydii serotype 1, Shigella flexneri serotypes 1a and 3a were examined. CONCLUSION: Our approach could be adopted for not only axenic bacterial population but also mixed communities and achieve rapid detection of various bacteria from the same genus or species in one sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The iUPPCR-DGGE method was shown to be more convenient than serotype-specific-antibody-based method of iUPPCR for Shigella species detection and it could be also applied to the quick detection for other kinds of pathogens with many serotypes.

[Dysentery in Poland in 2003]. / Czerwonka bakteryjna w 2003 roku.

The notified number of dysentery cases is the lowest in this century and even every year decreasing. Only 75 cases were notified in the year 2003 (incidence rate 0.20/100 000 population) while 220 cases were registered in 2002 (incidence 0.58/100 000 population). No one death case was notified. The last three death cases were notified in 1999. Only one outbreak of dysentery (due to S. flexneri 2a) was registered involving 23 patients/111 residents of Social Home for Mentally Disabled Men. Source of infection was probably one of residents who were infected during an outbreak of dysentery in the same institution four years earlier in 1999. The outbreak changed the overall etiology of dysentery cases in 2003: 52% was due to S. sonnei, but 46% was due to S. flexneri and 2% to S. dysenteriae 2, S. boydii were not found among persons examined bacteriologically by laboratory service of Epidemiological and Sanitary Service. The external quality control of procedures for selective investigation of Shigella infections in stool probe was done in 37 laboratories of Sanitary Epidemiological Stations with the use of control strain S. boydii 6. It was shown that in nearly all laboratories the strain was unable to grow on media SS and Hektoen after enrichment in the phosphate selenine--medium (SF) used by them. In the period of low frequency of Shigella infections the external control of the quality of bacteriological media and laboratory procedures is needed and should be done regularly.

Identification and characterization of Shigella boydii 20 serovar nov., a new and emerging Shigella serotype.

Analysis of 163 putative Shigella isolates from Canada and the USA showed biochemical reactions consistent with Shigella species, although none of the isolates reacted with antiserum raised against any of the well-established or provisional Shigella serotypes. All these isolates, provisionally designated serotype SH108, were positive for the ipaH gene and the invasion-associated locus. All fermented mannitol, were serologically indistinguishable from each other and showed no reaction in antisera prepared against Escherichia coli serotypes O1 to O181. PCR-RFLP analysis of the genes involved in O-antigen synthesis revealed a common pattern among these isolates that was distinct from recognized Shigella serotypes and E. coli. Between 1999 and 2003, isolates from across Canada were submitted to the National Laboratory for Enteric Pathogens for antibiotic susceptibility testing, phage typing and PFGE. These assays revealed heterogeneity among the members of this serotype. Antimicrobial susceptibility testing with seven antibiotics identified six profiles, with 90 % (45/50) of the isolates resistant to four or more antibiotics and 72 % (36/50) resistant to five or more. All isolates were typable using a panel of 16 phages, with 11 different phage types (PTs) represented. The most common PTs found were PT 3 (64 %), PT 6 (10 %) and PT 16 (6 %). Analysis of XbaI-restricted genomic DNA revealed 16 highly related patterns that were not readily distinguishable from those obtained for some other Shigella serotypes. The World Health Organization Collaborating Center for Shigella has added serotype SH108 to the Shigella scheme as S. boydii serotype 20 (serovar nov.). Strain SH108 (isolate 99-4528) is the reference strain for this serotype.

Survival of Shigella boydii 18 in bean salad.

A strain of Shigella boydii 18 involved in a bean salad outbreak and S. boydii 18 ATCC 35966 were used to inoculate bean salad. Bean salad samples were stored at 4 or 23 degrees C. At 4 degrees C, the S. boydii survived for the duration of the shelf life of the salad but did not grow. At 23 degrees C, both strains increased by two orders of magnitude by day 2 and decreased rapidly thereafter. This demonstrates the importance of proper storage in preventing the outgrowth of foodborne pathogens.

Comparative analysis of Shigella boydii 18 foodborne outbreak isolate and related enteric bacteria: role of rpoS and adiA in acid stress response.

Shigella boydii CDPH (Chicago Department of Public Health) serotype 18 was implicated in an outbreak of foodborne illness in 1998. The suspected food vehicles were parsley and cilantro imported from Mexico used to prepare bean salad. Previous studies revealed that S. boydii CDPH serotype 18 can survive in bean salad, which contains organic acids and whose pH decreases over time. Acid challenge assays in acidified tryptic soy broth at pH 4.5, acidified Luria-Bertani broth at pH 4.5, and acidified M9 minimal salts medium at pH 2.5 containing amino acids, arginine, or glutamic acid were performed using S. boydii CDPH, S. boydii ATCC 35966, S. flexneri 3136, Escherichia coli O157:H7 dd8872, and E. coli O157:H7 dd642 to compare differences in acid tolerance. Differences in survival of exponential-phase cells were detected in acidified tryptic soy broth and Luria-Bertani broth at pH 4.5. In acidified minimal medium containing arginine, S. boydii strains were able to survive at pH 2.5. The arginine decarboxylase gene (adiA) present in S. boydii is involved in survival at extremely low pH. The discovery of adiA expression in S. boydii serotype 18 by use of an acidified minimal medium challenge and arginine decarboxylase biochemical assay is significant because arginine decarboxylase activity was thought to be unique to E. coli. Sequencing of the rpoS gene from the S. boydii outbreak strain indicates that it is 99% conserved compared with the E. coli K-12 rpoS gene and plays a vital role in survival under acidic conditions.

Comparison of chromogenic Shigella spp. plating medium with standard media for the recovery of Shigella boydii and Shigella sonnei from tomato surfaces.

Isolation of Shigella spp. from food is difficult because of a lack of appropriate selective media and the presence of low numbers of shigellae relative to competitive microorganisms. Chromogenic Shigella spp. plating medium (CSPM) was evaluated for use with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) enrichment procedure for isolation of artificially contaminated Shigella boydii UI02 and Shigella sonnei UI05 from tomato surfaces. Tomatoes were inoculated with various concentrations of S. boydii UI02 or S. sonnei UI05 and rinsed using a shake-rub-shake procedure. Tomato rinses were enriched overnight according to the BAM procedure and streaked for isolation on CSPM, Salmonella-Shigella agar (SSA), and MacConkey agar (MAC). To access the isolation of S. boydii UI02 and S. sonnei UI05 without competition from natural tomato microflora, experiments were repeated using rifampin-adapted inocula and enrichments supplemented with 50 microg/ml rifampin. Isolation of S. boydii UI02 and S. sonnei UI05 with or without natural tomato microflora was not significantly different (P > 0.05) on CSPM, MAC, or SSA. Colony color enhancements created by CSPM may ease differentiation of Shigella colonies from those of closely related competitors.

Comparison of conventional culture methods and FTA filtration-nested PCR for the detection of Shigella boydii and Shigella sonnei on tomato surfaces.

Detection of Shigella boydii UI02 and Shigella sonnei UI05 artificially inoculated onto tomatoes was evaluated using enrichment protocols of the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) and the American Public Health Association's Compendium of Methods for the Microbiological Examination of Food (CMMEF), enrichment in Enterobacteriaceae enrichment (EE) broth supplemented with 1.0 microg/ml novobiocin and incubated at 42 degrees C, and FTA filtration-nested PCR. To assess the effect of natural tomato microflora on recovery, conventional culture enrichments were repeated using rifampin-adapted inocula and enrichment medium supplemented with 50 microg/ml rifampin. The lowest detection levels for S. boydii UI02 were > 5.3 x 10(5) (BAM, CMMEF, and EE broth) and 6.2 CFU per tomato (FTA filtration-nested PCR). For S. sonnei UI05, the lowest detection levels were 1.9 x 10(1) (BAM), 1.5 x 10(3) (CMMEF), 1.1 x 10(1) (EE broth), and 7.4 CFU per tomato (FTA filtration-nested PCR). Natural tomato microflora had a large impact on recovery of S. sonnei UI05 and completely inhibited recovery of S. boydii UI02. EE broth was inhibitory to S. boydii UI02. FTA filtration-nested PCR provided superior detection (P < 0.05) compared with the conventional culture enrichment protocols.

Spectroscopic quantification of bacteria using artificial neural networks.

Fourier transform-infrared spectroscopy, in conjunction with artificial neural networks, has been used for identification and classification of selected foodborne pathogens. Five bacterial species (Enterococcus faecium, Salmonella Enteritidis, Bacillus cereus, Yersinia enterocolitica, Shigella boydii) and five Escherichia coli strains (O103, O55, O121, O30, O26) suspended in phosphate-buffered saline were enumerated to provide seven different concentrations ranging from 10(9) to 10(3) CFU/ ml. The trained artificial neural networks were then validated with an independent subset of samples and compared with the traditional plate count method. It was found that the concentration-based classification of the species was 100% correct and the strain-based classification was 90 to 100% accurate.