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.

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.

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.

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.

Microevolution of the chromosomal region of acute disease antigen A (adaA) in the query (Q) fever agent Coxiella burnetii.

The acute disease antigen A (adaA) gene is believed to be associated with Coxiella burnetii strains causing acute Q fever. The detailed analysis of the adaA genomic region of 23 human- and 86 animal-derived C. burnetii isolates presented in this study reveals a much more polymorphic appearance and distribution of the adaA gene, resulting in a classification of C. burnetii strains of better differentiation than previously anticipated. Three different genomic variants of the adaA gene were identified which could be detected in isolates from acute and chronic patients, rendering the association of adaA positive strains with acute Q fever disease disputable. In addition, all adaA positive strains in humans and animals showed the occurrence of the QpH1 plasmid. All adaA positive isolates of acute human patients except one showed a distinct SNP variation at position 431, also predominant in sheep strains, which correlates well with the observation that sheep are a major source of human infection. Furthermore, the phylogenetic analysis of the adaA gene revealed three deletion events and supported the hypothesis that strain Dugway 5J108-111 might be the ancestor of all known C. burnetii strains. Based on our findings, we could confirm the QpDV group and we were able to define a new genotypic cluster. The adaA gene polymorphisms shown here improve molecular typing of Q fever, and give new insights into microevolutionary adaption processes in C. burnetii.

Presence of an emerging subclone of Francisella tularensis holarctica in Ixodes ricinus ticks from south-western Germany.

The zoonotic disease tularaemia is caused by the bacterial pathogen Francisella tularensis. Although the causative agent is known for 100 years, knowledge of its enzootic cycles is still rudimentary. Apart from tabanids and mosquitoes, hard ticks have been described as important vectors and potential reservoirs for F. tularensis. Available data on the incidence of human tularaemia indicate an increase in cases in the federal state of Baden-Wuerttemberg. To determine whether ticks are involved in the reported increase in F. tularensis infections in humans and wildlife in this south-western part of Germany, 916 Ixodes ricinus and 211 adult Dermacentor marginatus and D. reticulatus ticks were collected in two different locations. Screening for the presence of F. tularensis was performed by real-time PCR of the 16S rRNA gene. Of the 95 pools of I. ricinus ticks (representing 916 individual ticks), 8 tick pools (8.4%) were positive in this PCR. 30-bp deletion PCR confirmed that the F. tularensis subspecies holarctica was present. FtM24 VNTR analysis revealed that they belong to the emerging Franco-Iberian subclone group of F. tularensis holarctica. Of the 211 ticks of the genus Dermacentor, 35 randomly chosen DNAs were subjected to 16S rRNA gene screening PCR; 20 of these (57%) gave positive signals. For cluster analysis, the lpnA gene region of all Francisella-positive I. ricinus pools and 6 Dermacentor ticks with a positive reaction in the screening PCR was amplified and sequenced. In the resulting neighbour-joining tree, all Francisella-positive I. ricinus samples clustered with sequences of F. tularensis, whilst all Dermacentor tick samples clustered with FLE (Francisella-like endosymbiont) sequences. This study shows that I. ricinus ticks may serve as vectors and/or reservoirs of F. tularensis in Germany and supports the hypothesis that the state of Baden-Wuerttemberg represents an emerging endemic focus of tularaemia.

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.

'Real-time' PCR-based detection of Coxiella burnetii using conventional techniques.

The diagnosis of Q fever (Coxiella burnetii infection) relies primarily on the serological detection of specific antibodies. Recently, PCR-based methods have been introduced in diagnostic laboratories. Unfortunately, the fastest and most reliable 'real-time' detection method, which employs the 'online' detection of target nucleotide sequences while the amplification process is still in progress, requires expensive devices and consumables. In this study, we present a simple method that combines the simplicity of conventional PCR with new technical and methodical enhancements, resulting in a fast, specific and easy method for the molecular detection of C. burnetii. A collection of C. burnetii reference strains was tested with the modified conventional gel-based PCR approach applying a particluar PCR buffer (QIAGEN(®) Fast Cycling PCR kit) and using a closed ready-to-use gel-cassette-system (FlashGel(®)) for the visualization of specific PCR products. The modified conventional PCR method reached nearly the speed of the LightCycler(®) HybProbe real-time PCR assay (120 vs. 90 min) and showed equal sensitivity and specificity. The general cost per PCR run was 25% less than that for the LightCycler method. These improvements make this method suitable for small laboratories with limited resources and for deployable PCR diagnostics in field laboratories.

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.

Septic pneumonic tularaemia caused by Francisella tularensis subsp. holarctica biovar II.

This case of pneumonic tularaemia elucidates two aspects: it is believed to be the first documented case of bacteraemia caused by Francisella tularensis subsp. holarctica biovar II; furthermore, it illustrates the remission of septic pneumonic tularaemia without appropriate anti-infective therapy. A blood culture from a patient with community-acquired pneumonia was found to be positive for F. tularensis subsp. holarctica biovar II after 10 days of cultivation. Meanwhile, the patient had been treated with ceftriaxone, followed by sultamicillin and clindamycin. The patient continued suffering from fever of up to 40.7 degrees C and rising C-reactive protein (CRP) for 4 days before the fever and CRP declined. The isolated strain was later tested and found to be resistant to the antibiotics used. The present case underlines that F. tularensis subsp. holarctica infections may cause severe symptoms but mostly have a favourable outcome.

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.

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.

Description of Francisella hispaniensis sp. nov., isolated from human blood, reclassification of Francisella novicida (Larson et al. 1955) Olsufiev et al. 1959 as Francisella tularensis subsp. novicida comb. nov. and emended description of the genus Francisella.

Strain FhSp1T, isolated from human blood in Spain in 2003, was studied for its taxonomic allocation. By 16S rRNA and recA gene sequencing, the strain was shown to belong to the genus Francisella. In the 16S rRNA gene sequence, Francisella sp. FhSp1T shared similarity of more than 99% with strains of Francisella tularensis subspecies and Francisella novicida U112T, 98% with Francisella piscicida GM2212T and 98.4% with Francisella philomiragia ATCC 25015T. In the recA gene sequence, Francisella sp. FhSp1T exhibited 91.6-91.7% similarity to strains of F. tularensis subspecies, 91.2% to F. novicida U112T and 84% to F. philomiragia ATCC 25017. The genus affiliation was supported by a quinone system typical of Francisella (Q-8 as the major component), a complex polar lipid profile similar to that of F. tularensis with the major components diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unknown aminophospholipid (APL4) and a fatty acid profile consisting mainly of C10:0 (17.2%), C14:0 (11.2%), C16:0 (13.1%), C18:0 3-OH (14.2%) and C18:1omega9c (7.1%). DNA-DNA hybridization, which showed unambiguously that FhSp1T represents a novel species, and the results of biochemical tests allowed genotypic and phenotypic differentiation of the isolate from all hitherto-described Francisella species. A multiplex PCR developed in the course of this study discriminated FhSp1T from representatives of all other Francisella species and subspecies, clades A.I and A.II of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica biovar japonica and also between these representatives of the genus. Therefore, we propose the name Francisella hispaniensis sp. nov., with the type strain FhSp1T (=FnSp1T =FSC454T =F62T =DSM 22475T =CCUG 58020T). Furthermore, we formally propose the transfer of the species Francisella novicida to the species Francisella tularensis as Francisella tularensis subsp. novicida comb. nov. (type strain ATCC 15482T =CCUG 33449T =CIP 56.12T). We also present an emended description of the genus Francisella.

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.

Validation and comparison of an extrapolysaccharide (EPS)-based in-house ELISA and the PanBio melioidosis rapid cassette test-kits for serodiagnosis of melioidosis in a non-endemic area.

Detection of anti-Burkholderia pseudomallei antibodies in sera from melioidosis patients still represents a keystone in the confirmation of the clinical diagnosis, especially in non-endemic areas. An in-house assay was compared to lateral flow assays for the rapid detection of melioidosis-specific IgG or IgM. Employing 50 positive sera from patients and 200 negative sera from blood donors, sensitivity of the ELISA, the IgG and IgM assay were 84.0%, 90.0% and 84.0%, respectively. Specificity ranged from 98.0% (ELISA) to 99.5% (IgM assay). The application of the described diagnostic assays is a suitable method for the serodiagnosis of melioidosis in a non-endemic area.

[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.

Real-time PCR using hybridization probes for the rapid and specific identification of Francisella tularensis subspecies tularensis.

Tularemia is a plague-like infection caused by Francisella (F.) tularensis classified as a biological warfare agent. F. tularensis subsp. tularensis is the most virulent subspecies demanding rapid diagnosis. Typing systems for this fastidious bacterium to the subspecies level are laborious and time consuming. Therefore, the aim of this study was to develop a real-time PCR for the rapid and specific identification of F. tularensis subsp. tularensis. The specificity of the assay was determined using a comprehensive panel of Francisella strains, clinically relevant bacteria, and DNA preparations of potential hosts. F. tularensis subsp. tularensis was specifically detected but no other organisms. The range of linearity was determined to be 100 fg to 10 ng, the lower limit of detection was 25 fg of DNA (13 genome equivalents). An internal amplification control PCR system targeting lambda phage DNA was included. Neither the internal amplification control nor host DNA influenced the cycle threshold values obtained for F. tularensis subsp. tularensis. In conclusion, we have developed a highly sensitive and specific assay that can be integrated into real-time PCR-based identification procedures for biological agents. This is a major diagnostic improvement, as all other methods for the specific identification of F. tularensis subsp. tularensis are more time consuming.