Combined TCBS and CHROMagar Analyses Allow for Basic Identification of Vibrio vulnificus within a 48 h Incubation Period in the Coastal Baltic Sea.

With rising infection rates in recent years, Vibrio vulnificus poses an increasing threat to public safety in the coastal brackish Baltic Sea. It is therefore important to monitor this organism and assess the V. vulnificus infection risk on a more regular basis. However, as the coastline of the Baltic Sea is 8000 km long and shared by nine nations, a convenient, fast, inexpensive, yet efficient V. vulnificus identification method is essential. We evaluated the effectiveness of a two-step agar-based approach consisting of successive Vibrio isolation and cultivation on thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar™ Vibrio for V. vulnificus in comparison with V. cholerae, V. parahaemolyticus, and V. alginolyticus. Our study contains isolates from water and sediment across a broad expanse of the Baltic Sea including 13 locations and two different summers, the time of year during which Vibrio infections are usually much more frequent. Confirmation of isolate species identity was carried out using molecular analyses. The two-step agar plating method performed well across different locations and timeframes in correctly identifying V. vulnificus by more than 80%, but the sensitivity in other Vibrio species varied. Thus, our approach yielded promising results as a potential tool for early V. vulnificus detection across a broad timeframe and transect of the Baltic Sea and potentially other brackish environments.

Virulence and resistance patterns of Vibrio cholerae non-O1/non-O139 acquired in Germany and other European countries.

Global warming has caused an increase in the emergence of Vibrio species in marine and estuarine environments as well as fresh water bodies. Over the past decades, antimicrobial resistance (AMR) has evolved among Vibrio species toward various antibiotics commonly used for the treatment of Vibrio infections. In this study, we assessed virulence and resistance patterns of Vibrio cholerae non-O1/non-O139 strains derived from Germany and other European countries. A total of 63 clinical and 24 environmental Vibrio cholerae non-O1/non-O139 strains, collected between 2011 and 2021, were analyzed. In silico antibiotic resistances were compared with resistance phenotypes according to EUCAST breakpoints. Additionally, genetic relatedness between isolates was assessed by two cgMLST schemes (SeqSphere +, pubMLST). Both cgMLST schemes yielded similar results, indicating high genetic diversity among V. cholerae non-O1/non-O139 isolates. Some isolates were found to be genetically closely related (allelic distance < 20), which suggests an epidemiological link. Thirty-seven virulence genes (VGs) were identified among 87 V. cholerae non-O1/non-O139 isolates, which resulted in 38 virulence profiles (VPs). VPs were similar between clinical and environmental isolates, with the exception of one clinical isolate that displayed a higher abundance of VGs. Also, a cluster of 11 environmental isolates was identified to have the lowest number of VGs. Among all strains, the predominant virulence factors were quorum sensing protein (luxS), repeats-in-toxins (rtxC/rtxD), hemolysin (hlyA) and different type VI secretion systems (T6SS) genes. The genotypic profiles revealed antibiotic resistance genes (ARGs) associated with resistance to beta-lactams, quinolones, macrolides, tetracycline, antifolate, aminoglycosides, fosfomycin, phenicols and sulfonamide. Carbapenemase gene VCC-1 was detected in 10 meropenem-resistant V. cholerae non-O1/non-O139 isolates derived from surface water in Germany. The proportion of resistance among V. cholerae non-O1/non-O139 species isolates against first line treatment (3rd generation cephalosporin, tetracycline and fluoroquinolone) was low. Empirical treatment would likely have been effective for all of the clinical V. cholerae non-O1/non-O139 isolates examined. Nevertheless, carbapenem-resistant isolates have been present in fresh water in Germany and might represent a reservoir for ARGs. Monitoring antimicrobial resistance is crucial for public health authorities to minimize the risks for the human population.

Analysis of Sporulation in Bacillus cereus Biovar anthracis Which Contains an Insertion in the Gene for the Sporulation Factor σK.

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction-modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed.

Impact of climate change on waterborne infections and intoxications.

Progressive climate change holds the potential for increasing human health risks from waterborne infections and intoxications, e. g. through an increase in pathogen concentrations in water bodies, through the establishment of new pathogens or through possible changes in pathogen properties. This paper presents some examples of potential impacts of climate change in Germany. Non-cholera Vibrio occur naturally in seawater, but can proliferate significantly in shallow water at elevated temperatures. In the case of Legionella, climate change could lead to temporary or longer-term increased incidences of legionellosis due to the combination of warm and wet weather. Higher temperatures in piped cold water or lower temperatures in piped hot water may also create conditions conducive to higher Legionella concentrations. In nutrient-rich water bodies, increased concentrations of toxigenic cyanobacteria may occur as temperatures rise. Heavy rainfall following storms or prolonged periods of heat and drought can lead to increased levels of human pathogenic viruses being washed into water bodies. Rising temperatures also pose a potential threat to human health through pathogens causing mycoses and facultatively pathogenic micro-organisms: increased infection rates with non-tuberculous mycobacteria or fungi have been documented after extreme weather events.

Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples.

Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays amplifying a total of 28 bacteria, microsporidal and parasitic nucleic acid sequence targets in human stool samples in a multicentric approach. In the assessments with strongly positive samples, sensitivity values ranging between 13% and 100% were recorded for bacteria, between 0% and 100% for protozoa and between 7% and 100% for helminths and microsporidia; for the weakly positive samples, the recorded sensitivity values for bacteria ranged from 0% to 100%; for protozoa, from 0% to 40%; and for helminths and microsporidia, from 0% to 53%. For bacteria, the recorded specificity was in the range between 87% and 100%, while a specificity of 100% was recorded for all assessed PCRs targeting parasites and microsporidia. The intra- and inter-assay variations were generally low. Specifically for some helminth species, the sensitivity could be drastically increased by applying manual nucleic acid extraction instead of the manufacturer-recommended automatic procedure, while such effects were less obvious for the bacteria and protozoa. In summary, the testing with the chosen positive control samples showed varying degrees of discordance between the evaluated Allplex assays and the applied in-house reference assays associated with higher cycle threshold values in the Allplex assays, suggesting that samples with very low pathogen densities might be missed. As the targeted species can occur as harmless colonizers in the gut of individuals in high-endemicity settings as well, future studies should aim at assessing the clinical relevance of the latter hint.

Heatwave-associated Vibrio infections in Germany, 2018 and 2019.

BackgroundVibrio spp. are aquatic bacteria that prefer warm seawater with moderate salinity. In humans, they can cause gastroenteritis, wound infections, and ear infections. During the summers of 2018 and 2019, unprecedented high sea surface temperatures were recorded in the German Baltic Sea.AimWe aimed to describe the clinical course and microbiological characteristics of Vibrio infections in Germany in 2018 and 2019.MethodsWe performed an observational retrospective multi-centre cohort study of patients diagnosed with domestically-acquired Vibrio infections in Germany in 2018 and 2019. Demographic, clinical, and microbiological data were assessed, and isolates were subjected to whole genome sequencing and antimicrobial susceptibility testing.ResultsOf the 63 patients with Vibrio infections, most contracted the virus between June and September, primarily in the Baltic Sea: 44 (70%) were male and the median age was 65 years (range: 2-93 years). Thirty-eight patients presented with wound infections, 16 with ear infections, six with gastroenteritis, two with pneumonia (after seawater aspiration) and one with primary septicaemia. The majority of infections were attributed to V. cholerae (non-O1/non-O139) (n = 30; 48%) or V. vulnificus (n = 22; 38%). Phylogenetic analyses of 12 available isolates showed clusters of three identical strains of V. vulnificus, which caused wound infections, suggesting that some clonal lines can spread across the Baltic Sea.ConclusionsDuring the summers of 2018 and 2019, severe heatwaves facilitated increased numbers of Vibrio infections in Germany. Since climate change is likely to favour the proliferation of these bacteria, a further increase in Vibrio-associated diseases is expected.

[Non-cholera Vibrio species - currently still rare but growing danger of infection in the North Sea and the Baltic Sea]. / Nicht-Cholera-Vibrionen ­ derzeit noch seltene, aber wachsende Infektionsgefahr in Nord- und Ostsee.

BACKGROUND: The abundance of non-cholera Vibrio spp. in the aquatic environment shows a positive correlation with water temperatures. Therefore, climate change has an important impact on the epidemiology of human infections with these pathogens. In recent years large outbreaks have been repeatedly observed during the summer months in temperate climate zones. OBJECTIVE: To inform medical professionals about the potentially life-threatening diseases caused by non-cholera Vibrio spp. MATERIAL AND METHODS: Review of the current literature on infections with non-cholera Vibrio spp. in general and on the epidemiological situation in Germany in particular. RESULTS: Non-cholera Vibrio spp. predominantly cause wound and ear infections after contact with contaminated seawater and gastroenteritis after consumption of undercooked seafood. As there have not been mandatory notification systems for these pathogens in Germany up to March 2020, a high number of unreported cases must be assumed. Immunosuppressed and chronically ill patients have a much higher risk for severe courses of diseases. If an infection with non-cholera Vibrio spp. is suspected anti-infective treatment should be promptly initiated and surgical cleansing is often necessary for wound and soft tissue infections. CONCLUSION: Due to the ongoing global warming an increased incidence of human infections with non-cholera Vibrio spp. must be expected in the future. Medical professionals should be aware of these bacterial pathogens and the potentially life-threatening infections in order to enable timely diagnostics and treatment.

Serological evidence for human exposure to Bacillus cereus biovar anthracis in the villages around Taï National Park, Côte d'Ivoire.

Bacillus cereus biovar anthracis (Bcbva) is an untypical anthrax-causing pathogen responsible for high wildlife mortality in Taï National Park (TNP), Côte d'Ivoire. However, nothing is known about its effect on the rural population living in the region bordering TNP. Contact to bushmeat is a known risk factor for exposure to a variety of zoonotic pathogens, but no human infections with Bcbva were noted so far. Therefore, we performed a retrospective seroprevalence analysis with sera from 1,386 study volunteers. We used assays which detect antibodies against the protective antigen PA, which is synthesized by both Bcbva and classic B. anthracis, and against the recently described antigen pXO2-60, a 35-kDa protein only produced by Bcbva. We found a high seroprevalence (22.37%) of antibodies against PA, and approximately half of those sera (10.46%) were also positive for the Bcbva-specific antigen pXO2-60. All sera negative for PA were also negative for antibodies against pXO2-60, confirming specificity and suitability of the PA/pXO2-60 combined serological assay. The fact that a large fraction of sera was positive for PA but negative for pXO2-60 can most likely be explained by lower immunogenicity of pXO2-60, but exposure to classic B. anthracis cannot be excluded. As only Bcbva has been detected in the TNP area so far, exposure to Bcbva can be suspected from the presence of antibodies against PA alone. In a questionnaire, most study participants reported contact to bushmeat and livestock carcasses. Unfortunately, risk factor analysis indicated that neither animal contacts, sex, age, nor country of origin were significant predictors of Bcbva seroprevalence. Nevertheless, our study added to an assessment of the distribution of Bcbva and its impact on the human population, and our data can serve to raise awareness of anthrax in the affected regions.

Low antibody prevalence against Bacillus cereus biovar anthracis in Taï National Park, Côte d'Ivoire, indicates high rate of lethal infections in wildlife.

Bacillus cereus biovar anthracis (Bcbva) is a member of the B. cereus group which carries both B. anthracis virulence plasmids, causes anthrax-like disease in various wildlife species and was described in several sub-Saharan African rainforests. Long-term monitoring of carcasses in Taï National Park, Côte d'Ivoire, revealed continuous wildlife mortality due to Bcbva in a broad range of mammalian species. While non-lethal anthrax infections in wildlife have been described for B. anthracis, nothing is known about the odds of survival following an anthrax infection caused by Bcbva. To address this gap, we present the results of a serological study of anthrax in five wildlife species known to succumb to Bcbva in this ecosystem. Specific antibodies were only detected in two out of 15 wild red colobus monkeys (Procolobus badius) and one out of 10 black-and-white colobus monkeys (Colobus polykomos), but in none of 16 sooty mangabeys (Cercocebus atys), 9 chimpanzees (Pan troglodytes verus) and 9 Maxwell's duikers (Cephalophus maxwellii). The combination of high mortality and low antibody detection rates indicates high virulence of this disease across these different mammalian species.

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest.

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation.

Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa-Chromosomal Monophyly and Broad Geographic Distribution.

Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats) in West and Central Africa (Côte d'Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo). The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans.

Characterization of Vibrio cholerae Strains Isolated from the Nigerian Cholera Outbreak in 2010.

We examined clinical samples from Nigerian patients with acute watery diarrhea for Vibrio cholerae during the 2010 cholera outbreak. A total of 109 suspected isolates were characterized, but only 57 V. cholerae strains could be confirmed using multiplex real-time PCR as well as rpoB sequencing and typed as V. cholerae O:1 Ogawa biotype El Tor. This finding highlighted the need for accurate diagnosis of cholera in epidemic countries to implement life-saving interventions.

Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis.

Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

Serological evidence of asymptomatic infections during Escherichia coli O104:H4 outbreak in Germany in 2011.

INTRODUCTION: The largest known outbreak caused by a rare hybrid strain of Shiga toxin-producing E.coli (STEC) and enteroaggregative E. coli (EAEC) (E.coli O104:H4) of serotype O104:H4 occurred in Germany in 2011. Fenugreek sprouts acted as a transmission vehicle and were widely consumed in the outbreak area at the time of the epidemic. In total 3,842 people developed a clinical illness caused by this strain; however the rates of asymptomatic infections remain unclear. We aimed to develop a serological assay for detection of E.coli O104 LPS specific antibodies and to establish the post-outbreak levels of seropositivity among people with documented exposure to contaminated sprouts. RESULTS AND DISCUSSION: Developed serological assays (ELISA with 84% sensitivity, 63% specificity and Western Blot with 100% sensitivity, 82.5% specificity) identified 33% (16/49) level of asymptomatic infection. Relatively small sample size and a significant time- lapse between the onset of symptoms and serum samples collection (appr. 8 weeks) might explain the assay variability. No association was found between clinical or demographic characteristics and assay positivity. Larger studies are needed to understand the complexity of human immune response and factors influencing development of clinical symptoms. Development of intra-outbreak research plans will substantially aid the conduct of more thorough scientific investigation during an outbreak period.

The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids.

Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as "B. cereus variety (var.) anthracis".

Shedding and transmission of novel influenza virus A/H1N1 infection in households--Germany, 2009.

Essential epidemiologic and virologic parameters must be measured to provide evidence for policy/public health recommendations and mathematical modeling concerning novel influenza A/H1N1 virus (NIV) infections. Therefore, from April through August of 2009, the authors collected nasopharyngeal specimens and information on antiviral medication and symptoms from households with NIV infection on a daily basis in Germany. Specimens were analyzed quantitatively by using reverse transcriptase-polymerase chain reaction. In 36 households with 83 household contacts, 15 household contacts became laboratory-confirmed secondary cases of NIV. Among 47 contacts without antiviral prophylaxis, 12 became cases (secondary attack rate of 26%), and 1 (8%) of these was asymptomatic. The mean and median serial interval were 2.6 and 3 days, respectively (range: 1-3 days). On average, the authors detected viral RNA copies for 6.6 illness days (treated in time = 5.7 days, not treated in time = 7.1 days; P = 0.06), but they estimated that most patients cease to excrete viable virus by the fifth illness day. Shedding profiles were consistent with the number and severity of symptoms. Compared with other nasopharyngeal specimen types, nasal wash was the most sensitive. These results support the notion that epidemiologic and virologic characteristics of NIV are in many aspects similar to those of seasonal influenza.