Clostridioides difficile in South American Camelids in Germany: First Insights into Molecular and Genetic Characteristics and Antimicrobial Resistance.

Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates.

MLVA-16 Genotyping of Brucella abortus and Brucella melitensis Isolates from Different Animal Species in Egypt: Geographical Relatedness and the Mediterranean Lineage.

Brucellosis is a common zoonotic disease in Egypt. However, there are limited data available on the genetic diversity of brucellae circulating in Egypt and other Mediterranean areas. One hundred and nine Brucella (B.) strains were isolated from different animal species in thirteen Egyptian governorates. Multi-locus variable number tandem repeats (VNTRs) analysis (MLVA-16) was employed to determine the geographical relatedness and the genetic diversity of a panel of selected Egyptian strains (n = 69), with strains originating from Italy (n = 49), Portugal (n = 52), Greece (n = 63), and Tunisia (n = 4). Egyptian B. melitensis strains clustered into two main clusters containing 21 genotypes. Egyptian B. abortus strains clustered into three main clusters containing nine genotypes. The genotypes were irregularly distributed over time and space in the study area. Egyptian strains of B. melitensis showed MLVA-16 patterns closer to that of Italian strains. Egyptian B. abortus strains isolated from cattle share the same genotype with strains from Portugal and similar to strains from Italy with low genetic diversity. Strains with similar MLVA patterns isolated from different governorates highlight the movement of the pathogen among governorates. Hence, it may also reflect the long endemicity of brucellosis in Egypt with earlier dispersal of types and great local genetic diversity. Open markets may contribute to cross-species transmission and dissemination of the new types nationwide. The presence of West Mediterranean lineages of B. melitensis and relatedness of B. abortus strains from the studied countries is a result of the socio-historical connections among the Mediterranean countries. Transnational eradication of brucellosis in the Mediterranean basin is highly demanded.

Draft Genome Sequence of an Acinetobacter baumannii Isolate Recovered from a Horse with Conjunctivitis in Germany.

Here, we announce the draft genome sequence of Acinetobacter baumannii strain 161514, which was recovered from a horse with conjunctivitis, and determine the genetic basis of its antimicrobial resistance phenotype. The genome has a size of 3,839,365 bp and a G+C content of 38.93% and is predicted to contain 3,529 coding sequences. The isolate belongs to sequence type 462 (ST462) according to the Oxford scheme (Abaumannii1) and to ST46 according to the Pasteur scheme (Abaumannii2).

Quantitative Proteome Profiling of Coxiella burnetii Reveals Major Metabolic and Stress Differences Under Axenic and Cell Culture Cultivation.

Coxiella burnetii is the causative agent of the zoonotic disease Q fever. To date, the lipopolysaccharide (LPS) is the only defined and characterized virulence determinant of C. burnetii. In this study, proteome profiles of C. burnetii Nine Mile phase I (RSA 493, NMI) and its isogenic Nine Mile phase II (RSA 439 NMII) isolate with a deep rough LPS were compared on L-929 mouse fibroblasts and in complex (ACCM-2), and defined (ACCM-D) media. Whole proteome extracts were analyzed using a label-free quantification approach. Between 659 and 1,046 C. burnetii proteins of the 2,132 annotated coding sequences (CDS) were identified in any particular experiment. Proteome profiles clustered according to the cultivation conditions used, indicating different regulation patterns. NMI proteome profiles compared to NMII in ACCM-D indicate transition from an exponential to a stationary phase. The levels of regulatory proteins such as RpoS, CsrA2, UspA1, and UspA2 were increased. Comparison of the oxidative stress response of NMI and NMII indicated that ACCM-2 represents a high oxidative stress environment. Expression of peroxidases, superoxide dismutases, as well as thioredoxins was increased for NMI. In contrast, in ACCM-D, only osmoregulation seems to be necessary. Proteome profiles of NMII do not differ and indicate that both axenic media represent similar oxidative stress environments. Deep rough LPS causes changes of the outer membrane stability and fluidity. This might be one reason for the observed differences. Proteins associated with the T4SS and Sec translocon as well as several effector proteins were detectable under all three conditions. Interestingly, none of these putatively secreted proteins are upregulated in ACCM-2 compared to ACCM-D, and L-929 mouse fibroblasts. Curiously, a higher similarity of proteomic patterns (overlapping up- and downregulated proteins) of ACCM-D and bacteria grown in cell culture was observed. Particularly, the proteins involved in a better adaptation or homeostasis in response to the harsh environment of the parasitophorous vacuole were demonstrated for NMI. This semi-quantitative proteomic analysis of C. burnetii compared axenically grown bacteria to those propagated in cell culture.

Multidrug-resistant Clostridium difficile ribotypes 078 and 014/5-FLI01 in piglets from Costa Rica.

Though an overlap of Clostridium difficile PCR ribotypes (RT) in humans and animals has been noted -particularly in piglets-information regarding C. difficile isolates from swine is scarce in Latin America. A characterization of 10 C. difficile isolates obtained from this origin in Costa Rica revealed the presence of the RT078 (n = 4) and RT014/5-FLI01 (n = 6) ribotypes. Unlike two previous reports from the region, all isolates were multidrug resistant (MDR). According to a minimum spanning tree (MST) analysis, our RT078 isolates formed a clonal complex with some German RT078 isolates and the already noted overlap of RT078 strains in humans and animals. This unanticipated high level of genetic relatedness confirms the transcontinental spread and geographically unlimited clustering of RT078.

Presence of Clostridium difficile in poultry and poultry meat in Egypt.

C. difficile has been recognized as a potential zoonotic agent encouraging investigations of C. difficile prevalence and ribotypes in animals. Here we report the prevalence and diversity of Egyptian C. difficile in I) samples from healthy poultry (n = 50), II) samples from diseased poultry (n = 54), and III) poultry meat (n = 150). Thirteen isolates were obtained from seven healthy and five diseased animals, but no C. difficile was cultured from poultry meat. The isolated C. difficile strains belonged to 3 different PCR-ribotypes (039/2, 205 and 001/FLI01). The detection of strains related to RT 001 known for its ability to cause disease in humans makes poultry a potential reservoir for pathogenic C. difficile.

Molecular typing of isolates obtained from aborted foetuses in Brucella-free Holstein dairy cattle herd after immunisation with Brucella abortus RB51 vaccine in Egypt.

Bovine brucellosis is endemic in Egypt in spite of application of surveillance and control measures. An increase of abortions was reported in a Holstein dairy cattle herd with 600 animals in Damietta governorate in Egypt after immunisation with Brucella (B.) abortus RB51 vaccine. Twenty one (10.6%) of 197 vaccinated cows aborted after 3 months. All aborted cows had been tested seronegative for brucellosis in the past 3 years. B. abortus was isolated from four foetuses. Conventional biochemical and bacteriological identification and polymerase chain reaction (PCR) confirmed two B. abortus biovar (bv.) 1 smooth and two B. abortus rough strains. None of the B. abortus isolates were identified as RB51. Genotyping analysis by multiple locus of variable number tandem repeats analysis based on 16 markers (MLVA-16) revealed two different profiles with low genetic diversity. B. abortus bv1 was introduced in the herd and caused abortions.

Clear distinction between Burkholderia mallei and Burkholderia pseudomallei using fluorescent motB primers.

BACKGROUND: A frame-shift mutation in the flagellum motor gene motB coding for the chemotaxis MotB protein of Burkholderia mallei has been utilized to design a conventional duplex PCR assay with fluorescent labelled primers. FINDINGS: Species specificity was tested with a panel of 13 Burkholderia type strains. A total of 41 B. mallei field strains, 36 B. pseudomallei field strains, and 1 B. thailandensis field strain from different geographic regions were tested and correctly identified. Testing of 55 non-Burkholderia bacterial species revealed 100% specificity of the assay. The minimum detection limit was 1 pg DNA or 160 GE for B. mallei and 130 GE for B. pseudomallei, respectively. CONCLUSIONS: This assay enables the clear distinction between B. mallei and B. pseudomallei/B. thailandensis.

Fate of Bacillus anthracis during production of laboratory-scale cream cheese and homemade-style yoghurt.

The viability of Bacillus anthracis during production and storage of cream cheese and yoghurt was evaluated. Experimental cheeses were manufactured from whole milk inoculated with a suspension of B. anthracis vegetative cells and spores at a final concentration of 10(4) cfu/ml. Lactic acid bacteria (LAB) and lab ferment were used to induce milk ripening and milk coagulation. The pH-value of the contaminated milk dropped below 4.5 within the first 6 h and the amount of LAB increased by approximately 2-logs. During cheese production and storage at 5-9 °C for 24 days no growth of B. anthracis was observed. The amount of vegetative cells and spores fluctuated by 1-log. Inoculation of whole milk with heat-treated spores at 10(4) cfu/ml resulted in a slight increase of vegetative cell counts during the first 6 h. This indicated that germination occurred, but replication of vegetative cells was still inhibited in the produced cheese. Incubation of cheeses at room temperature or heating after milk coagulation strongly reduced the amount of LAB but had no effect on the growth behaviour of B. anthracis. The vegetative cell and spore content remained steady at 10(4) cfu/100 mg. During yoghurt production the pH-value decreased within 5 h below 5 and growth of B. anthracis was inhibited throughout storage. A pH-value of 5 or less is likely a critical factor to control the growth of B. anthracis. However, spores remained viable in experimental cream cheeses and yoghurts and are a potential risk of infection.

DNA microarray-based detection of Coxiella burnetii, the causative agent of Q fever.

BACKGROUND: An easy-to-handle microarray assay based on the cost-effective ArrayTube™ platform has been designed for the rapid and unequivocal identification of Coxiella burnetii, the causative agent of Q fever. The gene targets include the chromosomally coded markers icd, omp/com1, and IS1111 as well as the plasmid coded markers cbbE and cbhE. RESULTS: A representative panel comprising 50 German C. burnetii isolates and 10 clinical samples was examined to validate the test. All tested isolates harboured plasmid QpH1 and were correctly identified, corresponding to 100% sensitivity. The assay's limit of detection was 100 genome equivalents (GE) for icd, omp/com1, cbbE and cbhE and 10 GE for IS1111. Assay specificity was 100% as determined by analysing a panel of 37 non-Coxiella strains. CONCLUSIONS: The present array is a rational assembly of established and evaluated targets for the rapid and unequivocal detection of C. burnetii. This array could be applied to the screening of vaginal swabs from small ruminants; screening of environmental samples e.g. on farms or screening of human samples.

Comparative evaluation of eleven commercial DNA extraction kits for real-time PCR detection of Bacillus anthracis spores in spiked dairy samples.

Spores of Bacillus anthracis are highly resistant and can survive conditions used for food preservation. Sample size and complexity represent the major hurdles for pathogen detection in food-related settings. Eleven commercial DNA extraction kits were evaluated for detection of B. anthracis spores by quantitative real-time PCR (qPCR) in dairy products. DNA was extracted from serial dilutions of B. anthracis spores in milk powder, cream cheese, whole milk and buttermilk. Three kits (QIAamp DNA mini kit, Invisorb Food kit I and II) were determined to produce the lowest limit of detections (LODs) with equally good performance. These kits employed lysozyme and proteinase K treatments or proteinase K in combination with cethyltrimethylamonium bromide-mediated (CTAB) precipitation of cell debris for cell disruption and DNA release. The LODs for these three kits were determined as 10(2) spores/ml of distilled water, 10(3)s pores/20 mg of powdered milk and 10(4) spores/100 mg of cream cheese, respectively. Performance testing of the QIAamp DNA mini kit demonstrated a good reproducibility and appropriate detection limits from 10(3)/ml for butter milk, 10(4)/ml for whole milk and 10(4)/100 mg for low fat cream cheese. However, DNA extraction efficiency was strongly inhibited by cream cheese with higher fat contents with an increased LOD of 10(6)/100 mg spores. This study demonstrated that qPCR detection depends directly on the appropriate DNA extraction method for an individual food matrix and bacterial agent.

Clostridium difficile genotypes in piglet populations in Germany.

Clostridium difficile was isolated from 147 of 201 (73%) rectal swabs of piglets from 15 farms of Lower Saxony and North Rhine-Westphalia. In 14 farms, 14 to 100% (mean, 78%) of the animals tested were culture positive. The rate of isolation was 68% postpartum, increased to 94% in animals 2 to 14 days of age, and declined to 0% for animals 49 days of age and older. There was no link between isolation and antibiotic treatment or diarrhea of piglets. Strains were assigned to 10 PCR ribotypes, and up to 4 PCR ribotypes were found to be present at the same time on a farm. The closely related PCR ribotypes 078 (55%) and 126 (20%) were most frequently recovered and were present in 13 of the 14 positive farms. The comparison of multilocus VNTR (variable number of tandem repeats) analysis (MLVA) data from this study and previously published data on human, porcine, and bovine PCR ribotype 078 isolates from 5 European countries revealed genetic differences between strains of different geographic origin and confirmed the relatedness of human and porcine C. difficile isolates. This study demonstrated that the human-pathogenic PCR ribotypes 078 and 126 are predominant in piglets in Germany. The results suggest that presence of C. difficile is correlated with animal age but not with antibiotic treatment or clinical disease. MLVA indicated that strains of the same geographical origin are often genetically related and corroborated the hypothesis of a close epidemiological connection between human and porcine C. difficile isolates.

Presence of Clostridium difficile PCR ribotype clusters related to 033, 078 and 045 in diarrhoeic calves in Germany.

This study provides data on the distribution and relationship of C. difficile PCR ribotypes in diarrhoeic calves in Germany. C. difficile was isolated from 176 of 999 (17.6 %) faecal samples or swabs of diarrhoeic calves from 603 farms collected between January 2010 and August 2012 by eight federal laboratories of six states. Strains were assigned to 17 PCR ribotypes. PCR ribotypes 033 (57 %), 078 (17 %) and 045/FLI01 (closest match to 045 in the WEBRIBO database; 9 %) were found the most frequently. Nine per cent of all culture-positive tested animals shed more than one multiple locus variable number tandem repeat analysis (MLVA) or PCR ribotype. Eight PCR ribotypes with related profiles (including 033, 078 and 045/FLI01) representing 92 % of all isolates were grouped into three clusters. Molecular relatedness was supported by the absence of the MLVA locus A6Cd only in clustered strains and identical toxin gene profiles for strains within each cluster. Previously reported mulitilocus sequence typing analysis for PCR ribotypes that were also recovered in this study found identical sequence types and a tcdC deletion (Δ39 bp) for 033, 045, 078 and 126 (ST-11), confirming this clustering. A different geographical occurrence of PCR ribotypes was shown for cluster 033 (found more frequently in southern Germany) and 045 (found more frequently in northern Germany). This study showed that clusters of C. difficile PCR ribotypes related to 033, 078 and 045 are predominant in diarrhoeic calves in Germany. The high number of strains belonging to PCR ribotype 078 demonstrated that diarrhoeic calves are also potential reservoirs for human pathogenic C. difficile strains.

German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity.

BACKGROUND: Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis. RESULTS: Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level. CONCLUSIONS: F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.

Isolation of Brucella melitensis biotype 3 from epidural empyema in a Bosnian immigrant in Germany.

Brucellosis is a regionally emerging infectious disease in Mediterranean countries with an increasing number of human cases and high morbidity rates. Here, we describe a case of severe B. melitensis biotype 3 infection in an immigrant who had contact with ruminants during a short-term stay in Bosnia before he returned to Germany. The patient developed thoracic spondylodiscitis accompanied by a large epidural empyema and neurological deficits. The isolated strain was characterized and compared to other strains from the Mediterranean region by multiple locus variable number of tandem repeat analysis, showing minor differences between emerging strains from neighbouring geographical areas.

Zoonotic agents in small ruminants kept on city farms in southern Germany.

Sheep and goats are popular examples of livestock kept on city farms. In these settings, close contacts between humans and animals frequently occur. Although it is widely accepted that small ruminants can carry numerous zoonotic agents, it is unknown which of these agents actually occur in sheep and goats on city farms in Germany. We sampled feces and nasal liquid of 48 animals (28 goats, 20 sheep) distributed in 7 city farms and on one activity playground in southern Germany. We found that 100% of the sampled sheep and 89.3% of the goats carried Shiga toxin-producing Escherichia coli (STEC). The presence of Staphylococcus spp. in 75% of both sheep and goats could be demonstrated. Campylobacter spp. were detected in 25% and 14.3% of the sheep and goats, respectively. Neither Salmonella spp. nor Coxiella burnetii was found. On the basis of these data, we propose a reasonable hygiene scheme to prevent transmission of zoonotic agents during city farm visits.

Prevalence of Coxiella burnetii in clinically healthy German sheep flocks.

BACKGROUND: Current epidemiological data on the situation of Coxiella (C.) burnetii infections in sheep are missing, making risk assessment and the implementation of counteractive measures difficult. Using the German state of Thuringia as a model example, the estimated sero-, and antigen prevalence of C. burnetii (10% and 25%, respectively) was assessed at flock level in 39/252 randomly selected clinically healthy sheep flocks with more than 100 ewes and unknown abortion rate. RESULTS: The CHECKIT™ Q-fever Test Kit identified 11 (28%) antibody positive herds, whereas real-time PCR revealed the presence of C. burnetii DNA in 2 (5%) of the flocks. Multiple-locus variable number of tandem repeats analysis of 9 isolates obtained from one flock revealed identical profiles. All isolates contained the plasmid QpH1. CONCLUSIONS: The results demonstrate that C. burnetii is present in clinically inconspicuous sheep flocks and sporadic flare-ups do occur as the notifications to the German animal disease reporting system show. Although C. burnetii infections are not a primary veterinary concern due to the lack of significant clinical impact on animal health (with the exception of goats), the eminent zoonotic risk for humans should not be underestimated. Therefore, strategies combining the interests of public and veterinary public health should include monitoring of flocks, the identification and culling of shedders as well as the administration of protective vaccines.

Cross-border molecular tracing of brucellosis in Europe.

To assess the general impact of endemic countries on the re-emergence of brucellosis in non-endemic regions of the European Union, the genetic fingerprints of Brucella melitensis strains imported to Germany were compared to ovine strains from Turkey in a molecular epidemiological study. Genotyping of 66 Brucella strains (based on Multiple Locus of Variable number of tandem repeats Analysis) isolated from German travellers and Turkish immigrants living in Germany revealed epidemiological concordance with 20 sheep isolates originating from Eastern Anatolia, Turkey. In summary, cross-border molecular tracing confirmed brucellosis being a zoonosis of concern for European public health.

Development of a diagnostic multiplex polymerase chain reaction microarray assay to detect and differentiate Brucella spp.

Brucellosis is a worldwide zoonosis leading to tremendous economic losses and severe human illness. Fast and reliable laboratory tests are needed to detect disease in both humans and animals and to monitor the production of safe food products and feed. For rapid identification of the genus Brucella and differentiation of its species, a multiplex polymerase chain reaction microarray assay based on 11 signature sequences and redundant oligonucleotide probes was developed. The gene targets included genus-specific sequences in bcsp31, perA, cgs, and omp2b, as well as chromosomal regions displaying species-specific hybridization patterns. Brucella reference strains and a representative panel of 102 field isolates were unambiguously identified by their hybridization patterns. The differentiation of species, however, was limited in members of the groups B. suis bv 3/4/B. canis and B. neotomae/B. microti. In summary, the newly developed Brucella ArrayTube® assay is an easy-to-handle molecular test for high-throughput and parallel analysis.

Diversity and coexistence of tick-borne pathogens in central Germany.

In total, 1000 Ixodes ricinus L. ticks were collected from a small recreational forest area in central Germany (Thuringia) and investigated for the presence of Borrelia spp., Babesia spp., Anaplasma spp., Rickettsia spp., Coxiella burnetii, and Francisella tularensis. Overall, 43.6% of the ticks were infected with at least one pathogen. In 8.4% of ticks double infections were detected, and 1.6% harbored more than two pathogens. In this study, we present data on the coexistence of established and emerging pathogens in questing nymphs and adult ticks in a recreational area in central Germany, indicating the need for further studies for a reliable risk assessment.