Draft genome sequence of a Shiga toxin-producing Escherichia coli strain from deer meat showing an IS-element integration in the B-subunit of the Shiga toxin Stx2b gene.

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens. Here we report sequence data of the STEC strain BfR-EC-18960, which has integrated IS elements in the B-subunit of the Shiga toxin Stx2b gene. The strain was isolated from deer meat at a local butchery in Germany in 2021.

Occurrence of selected bacterial pathogens in insect-based food products and in-depth characterisation of detected Bacillus cereus group isolates.

Insects are increasingly used as alternative protein sources and ingredients of foodstuffs produced in industrial scale. Previous studies on the microbial status of insect-based foods revealed that classical foodborne pathogens such as Salmonella spp., Campylobacter spp., Listeria monocytogenes or pathogenic Escherichia coli are rarely detected, whereas particularly spore-forming bacteria with pathogenic potential such as species of the Bacillus cereus group or Clostridium species may pose a food safety risk. However, detailed descriptions of the encountered pathogenic bacteria in insect foods are scarce. We investigated a variety of 73 food products with insect or other arthropod ingredients on the occurrence of potential bacterial pathogens. These included B. cereus (sensu lato (s.l.)), Clostridium perfringens and Clostridioides difficile as representatives of spore-formers and Salmonella spp. and Shiga toxin producing and enteropathogenic E. coli (STEC/EPEC) as representatives of non-spore-forming Enterobacteriaceae. Most of the investigated food products complied with food safety standards regarding the presence of pathogens considered. However, one cricket product contained two Salmonella enterica subspecies enterica serovars (S. Wandsworth and S. Stanley). B. cereus (s.l.) was found in 42 samples (58 %), of which six contained B. cereus (s.l.) at levels higher than 103 cfu/g. The highest B. cereus (s.l.) counts of 3.8 × 105 cfu/g were found in a product with boiled and dried scorpions. Clostridium perfringens was detected in twelve samples (16 %), whereas Clostridioides difficile and STEC/EPEC were not detected in any of the samples. Remarkably, five samples contained the B. cereus (s.l.) species B. cytotoxicus. Moreover, strikingly high numbers of B. cereus (s.l.) isolates carried the capsule syntheses genes capBCADE, which were presumably located on the B. cereus pBFI_2 plasmid. Whole genome sequencing-based phylogenetic analysis suggested a high relatedness for only very few of the B. cytotoxicus and cap-positive isolates, respectively.

Environmental Factors Associated With Soil Prevalence of the Melioidosis Pathogen Burkholderia pseudomallei: A Longitudinal Seasonal Study From South West India.

Melioidosis is a seasonal infectious disease in tropical and subtropical areas caused by the soil bacterium Burkholderia pseudomallei. In many parts of the world, including South West India, most cases of human infections are reported during times of heavy rainfall, but the underlying causes of this phenomenon are not fully understood. India is among the countries with the highest predicted melioidosis burden globally, but there is very little information on the environmental distribution of B. pseudomallei and its determining factors. The present study aimed (i) to investigate the prevalence of B. pseudomallei in soil in South West India, (ii) determine geochemical factors associated with B. pseudomallei presence and (iii) look for potential seasonal patterns of B. pseudomallei soil abundance. Environmental samplings were performed in two regions during the monsoon and post-monsoon season and summer from July 2016 to November 2018. We applied direct quantitative real time PCR (qPCR) together with culture protocols to overcome the insufficient sensitivity of solely culture-based B. pseudomallei detection from soil. A total of 1,704 soil samples from 20 different agricultural sites were screened for the presence of B. pseudomallei. Direct qPCR detected B. pseudomallei in all 20 sites and in 30.2% (517/1,704) of all soil samples, whereas only two samples from two sites were culture-positive. B. pseudomallei DNA-positive samples were negatively associated with the concentration of iron, manganese and nitrogen in a binomial logistic regression model. The highest number of B. pseudomallei-positive samples (42.6%, p < 0.0001) and the highest B. pseudomallei loads in positive samples [median 4.45 × 103 genome equivalents (GE)/g, p < 0.0001] were observed during the monsoon season and eventually declined to 18.9% and a median of 1.47 × 103 GE/g in summer. In conclusion, our study from South West India shows a wide environmental distribution of B. pseudomallei, but also considerable differences in the abundance between sites and within single sites. Our results support the hypothesis that nutrient-depleted habitats promote the presence of B. pseudomallei. Most importantly, the highest B. pseudomallei abundance in soil is seen during the rainy season, when melioidosis cases occur.

Complete Genome Sequence of a Salmonella enterica subsp. enterica Serovar Tennessee Strain from Tahini.

Salmonella sp. infections are associated with contaminated low-moisture foods (with high fat content) with increasing frequency. Here, we report the complete genome sequence of Salmonella enterica subsp. enterica serovar Tennessee, which was isolated from tahini (a paste made from ground sesame seeds) purchased at a local retailer in Berlin, Germany.

Chicken Skin Decontamination of Thermotolerant Campylobacter spp. and Hygiene Indicator Escherichia coli Assessed by Viability Real-Time PCR.

Thermotolerant Campylobacter spp. are fecal contaminants of chicken meat with serious implications for human health. E. coli is considered as hygiene indicator since, in contrast to Campylobacter. spp., the bacterium is generally present in the avian gut. Stress exposure may transiently cease bacterial division. Therefore, colony forming units (CFU) may underestimate the infection risk of pathogens. We developed a viability real-time PCR (v-qPCR) for the quantification of viable E. coli targeting the uidA gene, encoding ß-glucuronidase, which is usually detected for phenotypic species identification. The short- and long-term effects of decontaminating chicken skin on the survival of both C. jejuni and an ESBL-producing E. coli were evaluated by CFU and v-qPCR. The results showed that freezing and storage in cool conditions are potentially underestimated by CFU but not by v-qPCR. The effect of treatment with peroxyacetic acid on survival was consistently detected by CFU and v-qPCR. v-qPCR analysis detected bacterial survival upon the application of lactic acid, which awaits further analysis. Interestingly, both bacteria showed similar kinetics of inactivation upon the application of reduction strategies, suggesting that E. coli might be a complementary hygiene indicator. We conclude that v-qPCR can improve food safety under the consideration of some limitations.

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

Erratum to "Comparison of Oral Microbe Quantities from Tongue Samples and Subgingival Pockets".

[This corrects the article DOI: 10.1155/2018/2048390.].

Comparison of Oral Microbe Quantities from Tongue Samples and Subgingival Pockets.

OBJECTIVES: To improve understanding of periodontitis pathology, we need more profound knowledge of relative abundances of single prokaryotic species and colonization dynamics between habitats. Thus, we quantified oral microbes from two oral habitats to gain insights into colonization variability and correlation to the clinical periodontal status. METHODS: We analyzed tongue scrapings and subgingival pocket samples from 237 subjects (35-54 years) with at least 10 teeth and no recent periodontal treatment from the 11-year follow-up of the Study of Health in Pomerania. Relative abundances of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Streptococcus sanguinis, total bacteria, and Archaea were correlated to clinically assessed pocket depths (PD) and clinical attachment levels (CAL). RESULTS: Increased relative abundances of P. gingivalis, A. actinomycetemcomitans, and F. nucleatum were linked to increased levels of PD and CAL (i) on the subject level (mean PD, mean CAL) and (ii) in subgingival pockets. Relative abundances of Archaea from tongue samples correlated negatively with mean PD or mean CAL. Detection and quantity of bacterial species correlated weakly to moderately between the tongue and subgingival pocket, except for Archaea. CONCLUSIONS: Relative abundances of specific oral species correlated weakly to moderately between habitats. Single species, total bacteria, and Archaea were linked to clinically assessed severity of periodontitis in a habitat-dependent manner.

Caspase-6 mediates resistance against Burkholderia pseudomallei infection and influences the expression of detrimental cytokines.

Caspase-6 is a member of the executioner caspases and known to play a role in innate and adaptive immune processes. However, its role in infectious diseases has rarely been addressed yet. We here examined the impact of caspase-6 in an in vivo infection model using the Gram-negative rod Burkholderia pseudomallei, causing the infectious disease melioidosis that is endemic in tropical and subtropical areas around the world. Caspase-6-/- and C57BL/6 wild type mice were challenged with B. pseudomallei for comparing mortality, bacterial burden and inflammatory cytokine expression. Bone-marrow derived macrophages were used to analyse the bactericidal activity in absence of caspase-6. Caspase-6 deficiency was associated with higher mortality and bacterial burden in vivo after B. pseudomallei infection. The bactericidal activity of caspase-6-/- macrophages was impaired compared to wild type cells. Caspase-6-/- mice showed higher expression of the IL-1ß gene, known to be detrimental in murine melioidosis. Expression of the IL-10 gene was also increased in caspase-6-/- mice as early as 6 hours after infection. Treatment with exogenous IL-10 rendered mice more susceptible against B. pseudomallei challenge. Thus, caspase-6 seems to play a crucial role for determining resistance against the causative agent of melioidosis. To our knowledge this is the first report showing that caspase-6 is crucial for mediating resistance in an in vivo infection model. Caspase-6 influences the expression of detrimental cytokines and therefore seems to be important for achieving a well-balanced immune response that contributes for an efficient elimination of the pathogen.

Extended-Spectrum ß-Lactamase-Producing Klebsiella pneumoniae from Pharmaceutical Wastewaters in South-Western Nigeria.

Emergence and spread of Klebsiella pneumoniae isolates producing extended-spectrum ß-lactamases (ESBLs) present a major threat to public health. In this study, we characterized ß-lactam-resistant K. pneumoniae isolates from six wastewater samples obtained from two pharmaceutical industries located in Lagos and Ogun States, Nigeria. Bacteria were isolated by using MacConkey agar; species identification and antibacterial susceptibility testing were performed by Vitek 2. Etest was used for ESBL phenotype confirmation. The presence of ß-lactamase genes was investigated by PCR and sequencing. Bacterial strain typing was done by XbaI-macrorestriction and subsequent pulsed-field gel electrophoresis (PFGE) as well as multilocus sequence typing (MLST). Thirty-five bacterial species were isolated from the six samples; among them, we identified seven K. pneumoniae isolates with resistance to ß-lactams and co-resistance to fluoroquinolones, aminoglycosides, and folate pathway inhibitors. The ESBL phenotype was confirmed in six K. pneumoniae isolates that harbored ESBL genes blaCTX-M-15 (n = 5), blaSHV-2 (n = 1), and blaSHV-12 (n = 1). PFGE and MLST analysis revealed five clones belonging to four sequence types (ST11, ST15, ST37, ST101), and clone K. pneumoniae-ST101 was present in the wastewater samples from two different pharmaceutical industries. Additionally performed conjugation assays confirmed the location of ß-lactamase genes on conjugative plasmids. This is the first confirmation of K. pneumoniae isolates producing CTX-M-15-ESBL from pharmaceutical wastewaters in Nigeria. The co-resistance observed might be a reflection of the different drugs produced by these industries. Continuous surveillance of the environmental reservoirs of multidrug-resistant bacteria is necessary to prevent their further spread.

Multitarget Quantitative PCR Improves Detection and Predicts Cultivability of the Pathogen Burkholderia pseudomallei.

Burkholderia pseudomallei is present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification of B. pseudomallei in the environment are a prerequisite for assessing the risk of infection. We recently reported the direct detection of B. pseudomallei in soil samples using a quantitative PCR (qPCR) targeting a single type three secretion system 1 (TTSS1) gene. Here, we extend the qPCR-based analysis of B. pseudomallei in soil by validating novel qPCR gene targets selected from a comparative genomic analysis. Two hundred soil samples from two rice paddies in northeast Thailand were evaluated, of which 47% (94/200) were B. pseudomallei culture positive. The TTSS1 qPCR and two novel qPCR assays that targeted open reading frames (ORFs) BPSS0087 and BPSS0745 exhibited detection rates of 76.5% (153/200), 34.5% (69/200), and 74.5% (150/200), respectively. The combination of TTSS1 and BPSS0745 qPCR increased the detection rate to 90% (180/200). Combining the results of the three qPCR assays and the BPSS1187 nested PCR previously published, all 200 samples were positive by at least one PCR assay. Samples positive by either TTSS1 (n = 153) or BPSS0745 (n = 150) qPCR were more likely to be direct-culture positive, with odds ratios of 4.0 (95% confidence interval [CI], 1.7 to 9.5; P < 0.001) and 9.0 (95% CI, 3.1 to 26.4; P < 0.001), respectively. High B. pseudomallei genome equivalents correlated with high CFU counts by culture. In conclusion, multitarget qPCR improved the B. pseudomallei detection rate in soil samples and predicted culture positivity. This approach has the potential for use as a sensitive environmental screening method for B. pseudomalleiIMPORTANCE The worldwide environmental distribution of the soil bacterium Burkholderia pseudomallei remains to be determined. So far, most environmental studies have relied on culture-based approaches to detect this pathogen. Since current culture methods are laborious, are time consuming, and have limited sensitivity, culture-independent and more sensitive methods are needed. In this study, we show that a B. pseudomallei-specific qPCR approach can detect significantly higher numbers of B. pseudomallei-positive soil samples from areas where it is endemic compared with that from culture. The use of multiple independent B. pseudomallei-specific qPCR targets further increased the detection rate of B. pseudomallei compared with that from single targets. Samples with a high molecular B. pseudomallei load were more likely to be culture positive. We conclude that our quantitative multitarget approach might be useful in defining areas where there is a risk of B. pseudomallei infections in different parts of the world.

Quantitative molecular detection of putative periodontal pathogens in clinically healthy and periodontally diseased subjects.

Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that alterations in the proportion of these organisms relative to the total amount of oral microorganisms, namely their abundance, rather than their simple presence might be important in the transition from health to disease. Therefore, we developed a quantitative molecular method to determine the abundance of various oral microorganisms and the portion of bacterial and archaeal nucleic acid relative to the total nucleic acid extracted from individual samples. We applied quantitative real-time PCRs targeting single-copy genes of periodontal bacteria and 16S-rRNA genes of Bacteria and Archaea. Testing tongue scrapings of 88 matched pairs of periodontally diseased and healthy subjects revealed a significantly higher abundance of P. gingivalis and a higher total bacterial abundance in diseased subjects. In fully adjusted models the risk of being periodontally diseased was significantly higher in subjects with high P. gingivalis and total bacterial abundance. Interestingly, we found that moderate abundances of A. actinomycetemcomitans were associated with reduced risk for periodontal disease compared to subjects with low abundances, whereas for high abundances, this protective effect leveled off. Moderate archaeal abundances were health associated compared to subjects with low abundances. In conclusion, our methodological approach unraveled associations of the oral flora with periodontal disease, which would have gone undetected if only qualitative data had been determined.

BPSS1504, a cluster 1 type VI secretion gene, is involved in intracellular survival and virulence of Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide. B. pseudomallei harbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified a B. pseudomallei transposon mutant defective in the BPSS1504 gene that showed reduced plaque formation. The BPSS1504 locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis of B. pseudomallei in mammalian hosts. For further analysis, a B. pseudomallei BPSS1504 deletion (BpΔBPSS1504) mutant and complemented mutant strain were constructed. B. pseudomallei lacking the BPSS1504 gene was highly attenuated in BALB/c mice, whereas the in vivo virulence of the complemented mutant strain was fully restored to the wild-type level. The BpΔBPSS1504 mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lacking hcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS components vgrG, tssA, and hcp1, as well as the T6SS regulators virAG, bprC, and bsaN, was not dependent on BPSS1504 expression. However, secretion of Hcp1 was not detectable in the absence of BPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus.

Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR.

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.

Improved culture-based detection and quantification of Burkholderia pseudomallei from soil.

Environmental surveillance of the Gram-negative soil bacterium Burkholderia pseudomallei, the aetiological agent of melioidosis, is important in order to define human populations and livestock at risk of acquiring the infection. This study aimed to develop a more sensitive method for the detection of B. pseudomallei from soil samples in endemic areas compared with the currently used culture method based on soil dispersion in water. We report the development of a new protocol that involves soil dispersion in a polyethylene glycol (PEG) and sodium deoxycholate (DOC) solution to increase the yield of viable B. pseudomallei from soil samples. Comparative testing of soil samples from Northeast Thailand covering a wide range of B. pseudomallei concentrations demonstrated a significantly higher recovery (P<0.0001) of B. pseudomallei colony-forming units by the new method compared with the conventional method. The data indicate that using the detergents PEG and DOC not only results in a higher recovery of viable B. pseudomallei but also results in a shift in the bacterial species recovered from soil samples. Future studies on the geographical distribution and population structure of B. pseudomallei in soil are likely to benefit from the new protocol described here.

Testosterone-inducible regulator is a kinase that drives steroid sensing and metabolism in Comamonas testosteroni.

The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3alpha-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3alpha-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.

The ltp gene of temperate Streptococcus thermophilus phage TP-J34 confers superinfection exclusion to Streptococcus thermophilus and Lactococcus lactis.

The ltp gene, located within the lysogeny module of temperate Streptococcus thermophilus phage TP-J34, has been shown to be expressed in lysogenic strain S. thermophilus J34. It codes for a lipoprotein, as demonstrated by inhibition of cleavage of the signal sequence by globomycin. Exposure of Ltp on the surface of Lactococcus lactis protoplasts bearing a plasmid-encoded copy of ltp has been demonstrated by immunogold labeling and electron microscopy. Expression of ltp in prophage- and plasmid-cured S. thermophilus J34-6f interfered with TP-J34 infection. While plating efficiency was reduced by a factor of about 40 and lysis of strain J34-6f in liquid medium was delayed considerably, phage adsorption was not affected at all. Intracellular accumulation of phage DNA was shown to be inhibited by Ltp. This indicates interference of Ltp with infection at the stage of triggering DNA release and injection into the cell, indicating a role of Ltp in superinfection exclusion. Expression of ltp in L. lactis Bu2-60 showed that the same superinfection exclusion mechanism was strongly effective against phage P008, a member of the lactococcal 936 phage species: no plaque-formation was detectable with even 10(9) phage per ml applied, and lysis in liquid medium did not occur. In Lactococcus also, Ltp apparently inhibited phage DNA release and/or injection. Ltp appears to be a member of a family of small, secreted proteins with a 42 amino acids repeat structure encoded by genes of Gram-positive bacteria. Some of these homologous genes are part of the genomes of prophages.