Molecular analysis of Coxiella burnetii in Germany reveals evolution of unique clonal clusters.

The causative agent of Q fever, Coxiella burnetii, is a query agent occurring naturally all over the world. We studied 104 German Coxiella burnetii strains/DNA samples obtained between 1969 and 2011 using a 14 microsatellite marker Multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA) technique. We were able to divide our collection into 32 different genotypes clustered into four major groups (A-D). Two of these (A and C) formed predominant clonal complexes that covered 97% of all studied samples. Group C consisted exclusively of cattle-associated isolates/DNA specimens, while group A comprised all other affected species including all sheep-derived strains/DNA samples. Within this second cluster, two major genotypes (A1, A2) were identified. Genotype A2 occurred in strains isolated from ewes in northern and central Germany, whereas genotype A1 was found in most areas of Germany. MLVA analysis of C. burnetii strains from neighbouring countries revealed a close relationship to German strains. We thus hypothesize that there is a western and central European cluster of C. burnetii. We identified predominant genotypes related to relevant host species and geographic regions which is in line with findings of the Dutch Q fever outbreak (2007-2010). Furthermore three of our analyzed German strains are closely related to the Dutch outbreak clone. These findings support the theory of predominant genotypes in the context of regional outbreaks. Our results show that a combination of 8 MLVA markers provides the highest discriminatory power for attributing C. burnetii isolates to genotypes. For future epidemiological studies we propose the use of three MLVA markers for easy and rapid classification of C. burnetii into 4 main clusters.

Treatment of tularemia in patient with chronic graft-versus-host disease.

We describe a case of human tularemia caused by Francisella tularensis subsp. holarctica in a stem cell transplant recipient with chronic graft-versus-host disease who was receiving levofloxacin prophylaxis. The infection was characterized by pneumonia with septic complications. The patient was successfully treated with doxycycline.

Phylogeography of Francisella tularensis subsp. holarctica, Europe.

Francisella tularensis subsp. holarctica isolates from Austria, Germany, Hungary, Italy, and Romania were placed into an existing phylogeographic framework. Isolates from Italy were assigned to phylogenetic group B.FTNF002-00; the other isolates, to group B.13. Most F. tularensis subsp. holarctica isolates from Europe belong to these 2 geographically segregated groups.

Standardized broth microdilution antimicrobial susceptibility testing of Francisella tularensis subsp. holarctica strains from Europe and rare Francisella species.

OBJECTIVES: Tularaemia is a widespread zoonosis in Europe caused by Francisella tularensis subsp. holarctica. Because of a lack of standardized CLSI-approved antibiotic susceptibility data from European Francisella strains, the antibiotic susceptibilities of a selection of F. tularensis subsp. holarctica isolates originating from Germany, Austria, France, Spain and other European countries were determined. Rarely isolated species and subspecies of Francisella such as Francisella philomiragia, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica as well as the type strain of Francisella hispaniensis were included in this study. METHODS: MIC data were obtained using cation-adjusted Mueller-Hinton broth with a 2% growth supplement. The broth microdilution testing system comprised 14 antibiotics, including gentamicin, streptomycin, ciprofloxacin and tetracycline. RESULTS: All of the 91 strains tested were susceptible to aminoglycosides, quinolones, tetracycline and chloramphenicol. The antimicrobial susceptibility of rare Francisellae was similar to the antibiotic profile of F. tularensis subsp. holarctica strains. For erythromycin, we detected two geographically distinct groups of F. tularensis subsp. holarctica isolates in western Europe. One group was resistant and the other one was susceptible. Both groups overlapped in a small region in Germany. CONCLUSIONS: Being performed in accordance with CLSI criteria, this study provides reliable data on antibiotic susceptibility patterns of European Francisella isolates. The standardized methodology of this study can be used for testing of suspicious colonies from clinical specimens for therapeutic guidance. Based on the results, aminoglycosides or quinolones are recommended as first-choice antibiotics for the therapy of F. hispaniensis, F. philomiragia or F. tularensis subsp. novicida infections in immunocompromised patients.

Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis.

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

Extensive host sharing of central European Tula virus.

To examine the host association of Tula virus (TULV), a hantavirus present in large parts of Europe, we investigated a total of 791 rodents representing 469 Microtus arvalis and 322 Microtus agrestis animals from northeast, northwest, and southeast Germany, including geographical regions with sympatric occurrence of both vole species, for the presence of TULV infections. Based on serological investigation, reverse transcriptase PCR, and subsequent sequence analysis of partial small (S) and medium (M) segments, we herein show that TULV is carried not only by its commonly known host M. arvalis but also frequently by M. agrestis in different regions of Germany for a prolonged time period. At one trapping site, TULV was exclusively detected in M. agrestis, suggesting an isolated transmission cycle in this rodent reservoir separate from spillover infections of TULV-carrying M. arvalis. Phylogenetic analysis of the S and M segment sequences demonstrated geographical clustering of the TULV sequences irrespective of the host, M. arvalis or M. agrestis. The novel TULV lineages from northeast, northwest, and southeast Germany described here are clearly separated from each other and from other German, European, or Asian lineages, suggesting their stable geographical localization and fast sequence evolution. In conclusion, these results demonstrate that TULV represents a promiscuous hantavirus with a large panel of susceptible hosts. In addition, this may suggest an alternative evolution mode, other than a strict coevolution, for this virus in its Microtus hosts, which should be proven in further large-scale investigations on sympatric Microtus hosts.

Molecular Characterization of German Acinetobacter baumannii Isolates and Multilocus Sequence Typing (MLST) Analysis Based on WGS Reveals Novel STs.

Acinetobacter baumannii (A. baumannii) is a major cause of severe nosocomial infections worldwide. The emergence of infections associated with A. baumannii poses a significant health risk in Germany. A. baumannii is part of the ACB complex and is difficult to distinguish from other species phenotypically, necessitating its reliable identification. The current study analyzed 89 A. baumannii strains from human and non-human origins by matrix-assisted laser desorption/ionization (MALDI-TOF) and PCR detection of intrinsic blaOXA-51-like carbapenemase, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and ISAba 1 genes. Whole-genome sequencing (WGS) was applied for species confirmation and strain type determination. Combining the molecular detection of the intrinsic blaOXA-51-like carbapenemase gene together with MALDI-TOF with a score value of >2.300 proved to be a suitable tool for A. baumannii identification. WGS data for all of the sequenced strains confirmed the identity of all A. baumannii strains. The Pasteur scheme successfully assigned 79.7% of the strains into distinct STs, while the Oxford scheme succeeded in allocating only 42.7% of isolates. Multilocus sequence typing (MLST) analysis based on the Pasteur scheme identified 16 STs. ST/241 was the most prevalent in samples from non-human origin, whereas ST/2 was predominant in human samples. Furthermore, eight isolates of non-human origin were allocated to seven new STs (ST/1410, ST/1414, ST/1416, ST/1417, ST/1418, ST/1419, and ST/1421). Ten isolates from non-human origin could not be typed since new alleles were observed in the loci Pas_cpn60, Pas_rpoB, and Pas_gltA. MLST analysis based on the Pasteur scheme was more appropriate than the Oxford scheme for the current group of A. baumannii.

Investigating an airborne tularemia outbreak, Germany.

In November 2005, an outbreak of tularemia occurred among 39 participants in a hare hunt in Hesse, Germany. Previously reported tularemia outbreaks in Germany dated back to the 1950s. We conducted a retrospective cohort study among participants and investigated the environment to identify risk factors for infection. Ten participants had serologic evidence of acute Francisella tularensis infection; 1 other participant died before laboratory confirmation was obtained. Presence within 5 meters of the place where disemboweled hares were rinsed with a water hose was the risk factor most strongly associated with infection (risk ratio 22.1; 95% confidence interval 13.2-154.3). Swabs taken at the game chamber and water samples were PCR negative for F. tularensis. Eleven of 14 hare parts showed low-level concentrations of F. tularensis, compatible with cross-contamination. More than half of case-patients may have acquired infection through inhalation of aerosolized droplets containing F. tularensis generated during rinsing of infected hares.

Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA.

BACKGROUND: Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories. RESULTS: The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes.Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples. CONCLUSION: We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples.

Phenotypic and WGS-derived antimicrobial resistance profiles of clinical and non-clinical Acinetobacter baumannii isolates from Germany and Vietnam.

OBJECTIVES: This study aimed to combine in vitro phenotyping analysis and whole-genome-sequencing (WGS) to characterise the phenotype and genetic determinants associated with intrinsic resistance in 100 clinical and non-clinical Acinetobacter baumannii strains originating from Germany and Vietnam. Moreover, it aimed to assess whether powdered milk as a food source functions as a potential reservoir of antibiotic resistance and possesses similar antimicrobial resistance (AMR) genes as in clinical strains isolated from Germany. METHODS: Antimicrobial susceptibility testing was performed using the broth microdilution method and the minimum inhibitory concentration (MIC) was determined for 18 antibiotics. The WGS data from all isolates were mapped to intrinsic genes known to be associated with phenotypic AMR. RESULTS: The highest resistance frequency was observed for chloramphenicol (100%), followed by fosfomycin (96%) and cefotaxime (95%). The lowest resistant rates were observed for colistin (3%), trimethoprim/sulfamethoxazole (17%), tigecycline (19%), and amikacin (19%). Thirty-five percent of tested strains displayed resistance to at least one of the carbapenems. Resistance to fluoroquinolones, aminoglycosides, tigecycline, penicillins, trimethoprim/sulfamethoxazole, and fourth-generation cephalosporins was determined only in human strains. About one-quarter of isolates (24%) was multidrug-resistant (MDR) and all were of human origin. Among them, 16 isolates were extensively drug resistant (XDR) and 10 from those 16 isolates showed resistance to all tested antibiotics except colistin. In silico detection of intrinsic AMR genes revealed the presence of 36 ß-lactamases and 24 non-ß-lactamase resistance genes. Two colistin-resistant and 10 ertapenem-resistant strains were isolated from powdered milk produced in Germany. Thirty-eight AMR genes associated with resistance to antibiotics were found in isolates recovered from milk powder. Several resistance mechanisms towards many classes of antibiotics existed in A. baumannii including ß-lactamases, multidrug efflux pumps and aminoglycoside-modifying enzymes. CONCLUSION: The use of WGS for routine public health surveillance is a reliable method for the rapid detection of emerging AMR in A. baumannii isolates. Milk powder poses a risk to contain MDR Acinetobacter strains or resistance genes in Germany.

Ulceroglandular tularemia in a toddler in Germany after a mosquito bite.

INTRODUCTION: Although tularemia is a long-known disease, its significance had diminished over the last decades in Middle Europe. However, over the past years, there is new evidence suggesting that tularemia has re-emerged in Germany. In 2007, the highest number of human cases for almost 50 years has been notified. Beside typical vectors, new ways of transmission seem to gain significance. So far, mosquito bite-transmitted tularemia has only been known from Scandinavia but not from Middle Europe. CASE REPORT: We report the first case of a 1-year-old toddler from Southwestern Germany with mosquito bite-associated ulceroglandular tularaemia. The new and interesting features of this case are the young age of the patient and the unusual transmission route. The available data suggesting changes in the epidemiology for tularemia in Germany are reviewed. This is an interesting case of infantile tularemia with a very unusual transmission route, highlighting ongoing changes in the epidemiology of tularemia in Germany.

Re-emergence of tularemia in Germany: presence of Francisella tularensis in different rodent species in endemic areas.

BACKGROUND: Tularemia re-emerged in Germany starting in 2004 (with 39 human cases from 2004 to 2007) after over 40 years of only sporadic human infections. The reasons for this rise in case numbers are unknown as is the possible reservoir of the etiologic agent Francisella (F.) tularensis. No systematic study on the reservoir situation of F. tularensis has been published for Germany so far. METHODS: We investigated three areas six to ten months after the initial tularemia outbreaks for the presence of F. tularensis among small mammals, ticks/fleas and water. The investigations consisted of animal live-trapping, serologic testing, screening by real-time-PCR and cultivation. RESULTS: A total of 386 small mammals were trapped. F. tularensis was detected in five different rodent species with carrier rates of 2.04, 6.94 and 10.87% per trapping area. None of the ticks or fleas (n = 432) tested positive for F. tularensis. We were able to demonstrate F. tularensis-specific DNA in one of 28 water samples taken in one of the outbreak areas. CONCLUSION: The findings of our study stress the need for long-term surveillance of natural foci in order to get a better understanding of the reasons for the temporal and spatial patterns of tularemia in Germany.

Optimized application of surface-enhanced laser desorption/ionization time-of-flight MS to differentiate Francisella tularensis at the level of subspecies and individual strains.

Francisella tularensis, the causative agent of tularaemia, is a potential agent of bioterrorism. The phenotypic discrimination of the closely related F. tularensis subspecies and individual strains with traditional methods is difficult and time consuming, often producing ambiguous results. Surface-enhanced laser desorption/ionization time-of-flight MS (SELDI-TOF MS) was used in this study to discriminate the different species and subspecies of the genus Francisella. We tested 18 Francisella strains including at least one representative of each species/subspecies on four different types of chromatographic chip surfaces. Multivariate analysis (hierarchical clustering and principal component analysis) allowed grouping of the strains according to their designated subspecies. Furthermore, single strains within F. tularensis subspecies could be discriminated.

An immunochromatographic test for the diagnosis of ricin inhalational poisoning.

INTRODUCTION: Aerosolized ricin is a feared bioweapon for which no diagnostic protocol is currently available. METHODS: We obtained antibodies to develop an immunochromatographic test (ICT) that would be part of such a protocol. RESULTS: Our ICT, that can be read with the naked eye, has a sensitivity of 1 ng ricin/mL buffer, without enhancement, making it the most sensitive rapid test available for this toxin as far as we know. Its dynamic range extends to 10 microg/mL, with a plateau from 10 to at least 250 microg/mL (no "hook effect"), and it has limited cross-reactivity with other lectins. DISCUSSION: Calculated from experimental and animal data, sampling with nasal swabs and testing with the devised ICT should give more than a thousand times the sensitivity required to diagnose ricin inhalational poisoning. Such a margin is particularly valuable in the absence of clinical data. CONCLUSION: This cost-effective and easy to use diagnostic tool could assist in the diagnosis of inhalational exposure from ricin aerosols.

[Mission oriented diagnostic real-time PCR]. / Einsatzrelevanz der PCR als diagnostisches Verfahren.

In out of area military missions soldiers are potentially exposed to bacteria that are endemic in tropical areas and can be used as biological agents. It can be difficult to culture these bacteria due to sample contamination, low number of bacteria or pretreatment with antibiotics. Commercial biochemical identification systems are not optimized for these agents which can result in misidentification. Immunological assays are often not commercially available or not specific. Real-time PCR assays are very specific and sensitive and can shorten the time required to establish a diagnosis markedly. Therefore, real-time PCRs for the identification of Bacillus anthracis, Brucella spp., Burkholderia mallei und Burkholderia pseudomallei, Francisella tularensis und Yersinia pestis have been developed. PCR results can be false negative due to inadequate clinical samples, low number of bacteria in samples, DNA degradation, inhibitory substances and inappropriate DNA preparation. Hence, it is crucial to cultivate the organisms as a prerequisite for adequate antibiotic therapy and typing of the agent. In a bioterrorist scenario samples have to be treated according to rules applied in forensic medicine and documentation has to be flawless.

Antimicrobial susceptibilities of Austrian Francisella tularensis holarctica biovar II strains.

The antibiotic susceptibilities of 50 Francisella tularensis subsp. holarctica biovar II strains isolated from hares and human patients from the eastern part of Austria were examined. Minimum inhibitory concentrations (MICs) of 24 antimicrobial agents were determined using Eteststrade mark on cysteine heart agar plates supplemented with 10% sheep blood. All isolates were sensitive to tetracyclines, aminoglycosides, quinolones, chloramphenicol and rifampicin. Resistance was observed in all isolates against macrolides, penicillins and aztreonam. Bacteria were resistant to cephalosporins and carbapenems, except for 8% of strains investigated that were susceptible or intermediately susceptible. Our in vitro susceptibility data can be applied for the detection and comparison of resistance development and to provide in vitro data for the guidance of therapy.

Rapid high resolution genotyping of Francisella tularensis by whole genome sequence comparison of annotated genes ("MLST+").

The zoonotic disease tularemia is caused by the bacterium Francisella tularensis. This pathogen is considered as a category A select agent with potential to be misused in bioterrorism. Molecular typing based on DNA-sequence like canSNP-typing or MLVA has become the accepted standard for this organism. Due to the organism's highly clonal nature, the current typing methods have reached their limit of discrimination for classifying closely related subpopulations within the subspecies F. tularensis ssp. holarctica. We introduce a new gene-by-gene approach, MLST+, based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains and apply this approach to investigate an epidemic of lethal tularemia among non-human primates in two animal facilities in Germany. Due to the high resolution of MLST+ we are able to demonstrate that three independent clones of this highly infectious pathogen were responsible for these spatially and temporally restricted outbreaks.

Oxidative stress in phagocytes--"the enemy within".

Phagocytes represent a powerful defense system against invading microorganisms that threaten the life or functional integrity of the host. The capacity to generate and release substantial amounts of reactive oxygen species is a unique property of activated polymorphonuclear and mononuclear phagocytes. The crucial role of these molecules in killing microorganisms and their consecutive contribution to tissue damage during injury and inflammation is widely known. Although much research has been done to explore the molecular events involved in the interaction of oxygen intermediates with microbes or host tissue, surprisingly little attention has been paid to the effect of reactive metabolites on the phagocyte itself. This fact is especially surprising, since it is apparent that the activated phagocyte is directly exposed to its own toxic metabolites. The potential damage occurring during excessive radical formation might notably alter the vital functions of these primarily immunocompetent cells. Moreover, the critical role of oxygen radicals in apoptosis of leukocytes has been recently revealed. Apoptosis is now supposed to represent a key mechanism in neutrophil deactivation and resolution of inflammation. Therefore, this review will focus on the delicate balance between released oxidants and antioxidative protection within the phagocytes themselves. General and phagocyte-specific antioxidative mechanisms, which have co-evolved with the radical generating machinery of phagocytes, are discussed, since the outcome of local inflammation can directly depend on this antioxidative capacity and might range from adequate elimination of the pathogen with minimal acute tissue damage to progression towards a systemic inflammatory response syndrome.

Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague.

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera (n=11) and buboes (n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.