Whole genome sequence of a non-toxigenic Corynebacterium diphtheriae strain from a hospital in southeastern China

Background Sporadic cases of infection with non-toxigenic Corynebacterium diphtheriae (C. diphtheriae) isolates have been reported in regions covered by the Diphtheria-Tetanus-Pertussis vaccine, but no information describing the whole genome of non-toxigenic strains collected in China is available. Therefore, in this work, the complete genome of a non-toxigenic strain of C. diphtheriae from a hospital located in southeastern China was performed. Results This non-toxigenic isolate belonged to the belfanti biotype and possessed a unique ST (assigned as ST799 in pubMLST). ErmX was present in the genome sequence and this isolate owned the resistance to erythromycin and clindamycin. Genes coding for virulence factors involved in adherence, iron-uptake and regulation of diphtheria toxin were also found. Two genes were involved in the interaction between pathogen and host. The phylogenetic analysis revealed that this newly isolated strain was similar to the strain NCTC10838, CMCNS703 and CHUV2995. Conclusion Non-toxigenic C. diphtheriae strain contained virulence factors, thus it is able to cause an infectious disease, aspect that could be clarified by performing the whole genome sequencing analysis.

C. difficile-infection and microbyota in newborns: resolved and outstanding issues

The article presents an analytical review of the works of domestic and foreign authors on the problem of studying the peculiarities of the formation of the intestinal microbiota and risk factors for the development of microdysbiosis in newborns with the activation of opportunistic bacteria, including C. difficile. Special attention is paid to the effect of antibiotics on the state of the intestinal microbiota and the activation of toxigenic strains of C. difficile. Divergent views on the role of toxigenic strains of C. difficile in the development of infectious pathology of newborns are presented.

Volatile Organic Compounds Emitted by Aspergillus flavus Strains Producing or Not Aflatoxin B1

Aspergillus flavus is a phytopathogenic fungus able to produce aflatoxin B1 (AFB1), a carcinogenic mycotoxin that can contaminate several crops and food commodities. In A. flavus, two different kinds of strains can co-exist: toxigenic and non-toxigenic strains. Microbial-derived volatile organic compounds (mVOCs) emitted by toxigenic and non-toxigenic strains of A. flavus were analyzed by solid phase microextraction (SPME) coupled with gas chromatography–mass spectrometry (GC-MS) in a time-lapse experiment after inoculation. Among the 84 mVOCs emitted, 44 were previously listed in the scientific literature as specific to A. flavus, namely alcohols (2-methylbutan-1-ol, 3-methylbutan-1-ol, 2-methylpropan-1-ol), aldehydes (2-methylbutanal, 3-methylbutanal), hydrocarbons (toluene, styrene), furans (2,5-dimethylfuran), esters (ethyl 2-methylpropanoate, ethyl 2-methylbutyrate), and terpenes (epizonaren, trans-caryophyllene, valencene, α-copaene, β-himachalene, γ-cadinene, γ-muurolene, δ-cadinene). For the first time, other identified volatile compounds such as α-cadinol, cis-muurola-3,5-diene, α-isocomene, and β-selinene were identified as new mVOCs specific to the toxigenic A. flavus strain. Partial Least Square Analysis (PLSDA) showed a distinct pattern between mVOCs emitted by toxigenic and non-toxigenic A. flavus strains, mostly linked to the diversity of terpenes emitted by the toxigenic strains. In addition, the comparison between mVOCs of the toxigenic strain and its non-AFB1-producing mutant, coupled with a semi-quantification of the mVOCs, revealed a relationship between emitted terpenes (β-chamigrene, α-corocalene) and AFB1 production. This study provides evidence for the first time of mVOCs being linked to the toxigenic character of A. flavus strains, as well as terpenes being able to be correlated to the production of AFB1 due to the study of the mutant. This study could lead to the development of new techniques for the early detection and identification of toxigenic fungi.

Aspergillus Goes Viral: Ecological Insights from the Geographical Distribution of the Mycovirome within an Aspergillus flavus Population and Its Possible Correlation with Aflatoxin Biosynthesis

Microbial multi-level interactions are essential to control the success of spreading and survival of most microbes in natural environments. Phytopathogenic mycotoxigenic fungal species, such as Aspergillus flavus, represent an important issue in food safety. Usually, non-toxigenic strains are exploited for biocontrol strategies to mitigate infections by toxigenic strains. To comprehend all the biological variables involved in the aflatoxin biosynthesis, and to possibly evaluate the interplay between A. flavus toxigenic and non-toxigenic strains during intraspecific biocompetition, the “virological” perspective should be considered. For these reasons, investigations on mycoviruses associated to A. flavus populations inhabiting specific agroecosystems are highly desirable. Here, we provide the first accurate characterization of the novel mycovirome identified within an A. flavus wild population colonizing the maize fields of northern Italy: a selection of A. flavus strains was biologically characterized and subjected to RNAseq analysis, revealing new mycoviruses and a peculiar geographic pattern distribution in addition to a 20% rate of infection. More interestingly, a negative correlation between viral infection and aflatoxin production was found. Results significantly expanded the limited existent data about mycoviruses in wild A. flavus, opening new and intriguing hypotheses about the ecological significance of mycoviruses.

Genomic stability among O3:K6 V. parahaemolyticus pandemic strains isolated between 1996 to 2012 in American countries

Background The V. parahaemolyticus pandemic clone, results in the development of gastrointestinal illness in humans. Toxigenic strains of this species are frequently isolated from aquatic habitats and organisms such as mollusks and crustaceans. Reports on the isolation of the pandemic clone started in 1996, when a new O3:K6 clone was identified in Asia, that rapidly spread worldwide, becoming the predominant clone isolated from clinical cases. In this study whole genome sequencing was accomplished with an Illumina MiniSeq platform, upon six novel V. parahaemolyticus strains, that have been isolated in Mexico since 1998 and three representative genomes of strains that were isolated from reported outbreaks in other American countries, and were deposited in the GenBank. These nine genomes were compared against the reference sequence of the O3:K6 pandemic strain (RIMD 2210633), which was isolated in 1996, to determine sequence differences within American isolates and between years of isolation. Results The results indicated that strains that were isolated at different times and from different countries, were highly genetically similar, among them as well as to the reference strain RIMD 2210633, indicating a high level of genetic stability among the strains from American countries between 1996 to 2012, without significant genetic changes relative to the reference strain RIMD 2210633, which was isolated in 1996 and was considered to be representative of a novel O3:K6 pandemic strain. Conclusions The genomes of V. parahaemolyticus strains isolated from clinical and environmental sources in Mexico and other American countries, presented common characteristics that have been reported for RIMD 2210633 O3:K6 pandemic strain. The major variations that were registered in this study corresponded to genes non associated to virulence factors, which could be the result of adaptations to different environmental conditions. Nevertheless, results do not show a clear pattern with the year or locality where the strains were isolated, which is an indication of a genomic stability of the studied strains.

Resistance Profile and Molecular Characterization of Toxigenic Vibrio cholerae Strains Responsible for the 2016 to 2018 Epidemic in Republic of Benin (West Africa)

Aims: This study aims to characterize of toxigenic Vibrio cholerae strains for improved cholera surveillance in Benin. Methodology: 304 diarrheal stool samples were collected from people with watery diarrhea of unknown etiology and vomiting during epidemics from 2016 to 2018 in Benin. Toxigenic Vibrio cholerae strains were isolated and then biochemical tests, serogrouping and serotyping were performed. Antibiotic susceptibility tests were performed using the disc diffusion method and E-tests. Multiplex and real-time PCR were used to identify and detect virulence genes (CtxA, OmpW and TcpA). Results: The results showed a 21.71% prevalence of toxigenic Vibrio cholera in Benin. All strains were Vibrio cholerae O1 serotype Inaba (100%) and showed a high sensitivity to doxycycline (96.97 %), chloramphenicol (95.45 %) and ciprofloxacin (90.91 %). However, antibiotic resistance was observed, especially for erythromycin (74.24 %), Trimethoprim-sulfamethoxazole (71.21 %) and ampicillin (43.94 %). The CtxA and TcpA virulence genes were respectively detected in 100% and 96.97% of the toxigenic strains of V. cholerae isolated. While the OmpW gene was identified on all the toxigenic strains of Vibrio cholerae isolated. Conclusion: Vibrio cholerae strains isolated from patients suspected of cholera were highly virulent and resistant to antimicrobials.

Dried fruits marketed in Russian: toxigenic mold contamination

Introduction. Dried fruits are a valuable source of dietary fibre, many vitamins and minerals in the population’s diet. However, the high content of readily available carbohydrates makes this type of product vulnerable to mould contamination. The greatest danger among which are toxigenic species. But there is practically no scientific information about the contamination presented on the Russian market dried fruits with moulds producing mycotoxins. That does not allow judging about this aspect of food safety. Materials and methods. Contamination with moulds and bacteria of 57 samples of dried fruits of 7 species popular in Russia was studied using cultural methods of analysis. Monospore isolates of moulds were isolated from dried fruits; in vitro mycotoxins production studied; by UHPLC-MS / MS analyzed mycotoxins in the multidetection mode. Results and discussion. In general, the microbial contamination of dried fruits was low: 87.7% of the samples met the established microbiological standards, in most cases, moulds caused it. At the same time, the highest frequency and levels of contamination were found in dates. Aspergillus sp. dominated in the micoflora of all types of dried fruits. Among the isolated 33 strains of moulds, 45.5% turned out to be toxigenic and, in vitro, were capable of biosynthesis of significant amounts of several types of mycotoxins, including emergent mycotoxins. Fumonisin- and ochratoxin-producing activities have been found in Aspergillus strains of the Nigri section. In model experiments, the accumulation of mycotoxins in individual strains exceeded the level normalized in grain products, including (in μg/kg): for aflatoxins B1 - more than 32000 and B2 - 3230; fumonisin B2 - more than 3100; ochratoxin A up to 4.3; for emergent accumulation reached: sterigmatocystin up to 6218220 and citreoviridine - 153. Conclusion. Moulds are the main type of microflora that contaminates dried fruits. The ability of mould isolates from dried fruits to form mycotoxins has been established, among which highly toxigenic strains have been identified. This indicates the presence of a potential risk of contamination of this type of food with unregulated mycotoxins and a possible increase in their content in the diets of consumers. The results obtained substantiate the need for extensive monitoring of mycotoxin producers in dried fruits. This is important for predicting the risk of toxin formation and identifying the relationship of specific mycotoxins with certain types of dried fruits. The presence of toxigenic activity of moulds isolated from dried fruits has been shown in Russia for the first time.

Retail chicken meats as potential sources of Clostridioides difficile in Al-Jouf, Saudi Arabia

Introduction: Presence of Clostridioides difficile in stool of food birds and animals is a risk for contamination of their meats to become potential sources of human infection. The main virulence factors of C. difficile are its resistance to antibiotics, production of toxins and spores. As far as I know, this is the first study to evaluate C. difficile prevalence in chicken meats, its toxigenic activities and antibiotics sensitivity patterns in Al-Jouf, Saudi Arabia. Methodology: Totally, 250 raw chicken meat samples were examined. Standard microbiological and biochemical procedures were used for C. difficile isolation and identification. The suspected colonies were tested by L-proline and C. difficile test kits then confirmed by Vitek 2 compact system. Xpect C. difficile toxin A/B test was used to detect A/B toxins production. Antibiotics susceptibility patterns were detected by Epsilon tests. Results: C. difficile was isolated from 11/250 (4.40%) chicken meat samples; 5/65 (7.69%) legs, 3/65 (4.61%) thighs, 2/60 (3.33%) wings and 1/60 (1.67%) breasts (p = 0.4). All isolates were non-toxigenic. Although all isolates were vancomycin sensitive, some isolates were intermediate/resistant to metronidazole, tetracycline, clindamycin or moxifloxacin antibiotics with variable degrees. Conclusions: C. difficile might contaminate retail chicken meats. Although low level of contamination by non-toxigenic strains was detected, chicken meats should be investigated as C. difficile infection sources for humans especially elders, immune-compromised and long terms wide spectrum antibiotics-used persons. Decreased sensitivity of C. difficile to antibiotics is emerging.

Molecular surveillance of shiga toxigenic Escherichia coli in selected beef abattoirs in Osun State Nigeria

Shiga toxigenic strains of E. coli (STEC) known to be etiological agents for diarrhea were screened for their incidence/occurrence in selected abattoirs sources in Osogbo metropolis of Osun State, Nigeria using a randomized block design. Samples were plated directly on selective and differential media and E. coli isolates. Multiplex PCR analysis was used to screen for the presence of specific virulence factors. These were confirmed serologically as non-O157 STEC using latex agglutination serotyping kit. Sequence analysis of PCR products was performed on a representative isolate showing the highest combination of virulence genes using the 16S gene for identification purposes only. Results showed that the average cfu/cm2 was significantly lower in the samples collected at Sekona-2 slaughter slab compared with those collected at Al-maleek batch abattoir and Sekona-1 slaughter slab in ascending order at P = 0.03. Moreover, the average cfu/cm2E. coli in samples collected from butchering knife was significantly lower when compared with that of the workers’ hand (P = 0.047) and slaughtering floor (P = 0.047) but not with the slaughter table (P = 0.98) and effluent water from the abattoir house (P = 0.39). These data suggest that the abattoir type may not be as important in the prevalence and spread of STEC as the hygiene practices of the workers. Sequence analysis of a representative isolate showed 100% coverage and 96.46% percentage identity with Escherichia coli O113:H21 (GenBank Accession number: CP031892.1) strain from Canada. This sequence was subsequently submitted to GenBank with accession number MW463885. From evolutionary analyses, the strain from Nigeria, sequenced in this study, is evolutionarily distant when compared with the publicly available sequences from Nigeria. Although no case of E. coli O157 was found within the study area, percent occurrence of non-O157 STEC as high as 46.3% at some of the sampled sites is worrisome and requires regulatory interventions in ensuring hygienic practices at the abattoirs within the study area.