Antimicrobial Susceptibility from a One Health Perspective Regarding Porcine Escherichia coli from Bavaria, Germany.

Antimicrobial resistance is one of the most crucial One Health topics worldwide. Consequently, various national and international surveillance programs collect data and report trends regularly. Ceftiofur, colistin and enrofloxacin belong to the most important and critical class of anti-infective medications in both human and veterinary medicine. In the present study, antimicrobial resistance was analyzed using the epidemiological cut-off (ECOFF) value on 6569 Escherichia coli isolated from pigs in Bavaria, Germany, during five years, from 2016 to 2020. The statistically relevant results regarding antimicrobial resistance revealed a decrease for colistin, an increase for enrofloxacin, and a constant level for ceftiofur. In Germany, the usage of all three antimicrobial substances in livestock has fallen by 43.6% for polypeptides, 59.0% for fluoroquinolones and 57.8% for the 3rd + 4th generation cephalosporines during this time. Despite the decline in antimicrobial usage, a reduction regarding antimicrobial resistance was solely observed for colistin. This finding illustrates that in addition to the restriction of pharmaceutical consumption, further measures should be considered. Improved biosecurity concepts, a reduction in crowding, and controlled animal movements on farms may play a key role in finally containing the resistance mechanisms of bacteria in farm animals.

Seroprevalence of Brucella Infection in Wild Boars (Sus scrofa) of Bavaria, Germany, 2019 to 2021 and Associated Genome Analysis of Five B. suis Biovar 2 Isolates.

Brucella species are highly pathogenic zoonotic agents and are found in vertebrates all over the world. To date, Germany is officially declared free from brucellosis and continuous surveillance is currently limited to farm ruminants. However, porcine brucellosis, mostly caused by B. suis biovar 2, is still found in wild boars and hares. In the present study, a three-year monitoring program was conducted focusing on the wild boar population in the state of Bavaria. Serologic screening of 11,956 animals and a direct pathogen detection approach, including a subset of 681 tissue samples, was carried out. The serologic incidence was 17.9%, which is in approximate accordance with previously published results from various European countries. Applying comparative whole genome analysis, five isolated B. suis biovar 2 strains from Bavaria could be assigned to three known European genetic lineages. One isolate was closely related to another strain recovered in Germany in 2006. Concluding, porcine brucellosis is endemic in Bavaria and the wild boar population represents a reservoir for genetically distinct B. suis biovar 2 strains. However, the transmission risk of swine brucellosis to humans and farm animals is still regarded as minor due to low zoonotic potential, awareness, and biosafety measures.

Whole-Genome Investigation of Salmonella Dublin Considering Mountain Pastures as Reservoirs in Southern Bavaria, Germany.

Worldwide, Salmonella Dublin (S. Dublin) is responsible for clinical disease in cattle and also in humans. In Southern Bavaria, Germany, the serovar was identified as a causative agent for 54 animal disease outbreaks in herds between 2017 and 2021. Most of these emerged from cattle herds (n = 50). Two occurred in pig farms and two in bovine herds other than cattle. Genomic analysis of 88 S. Dublin strains isolated during these animal disease outbreaks revealed 7 clusters with 3 different MLST-based sequence types and 16 subordinate cgMLST-based complex types. Antimicrobial susceptibility investigation revealed one resistant and three intermediate strains. Furthermore, only a few genes coding for bacterial virulence were found among the isolates. Genome analysis enables pathogen identification and antimicrobial susceptibility, serotyping, phylogeny, and follow-up traceback analysis. Mountain pastures turned out to be the most likely locations for transmission between cattle of different herd origins, as indicated by epidemiological data and genomic traceback analyses. In this context, S. Dublin shedding was also detected in asymptomatic herding dogs. Due to the high prevalence of S. Dublin in Upper Bavaria over the years, we suggest referring to this administrative region as "endemic". Consequently, cattle should be screened for salmonellosis before and after mountain pasturing.

Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years.

The zoonotic disease anthrax, caused by the endospore-forming bacterium Bacillus anthracis, is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009, resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture, and the presence of both virulence plasmids (pXO1 and pXO2) was confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA sequenced and phylogenetically grouped within the B.Br.CNEVA clade, which is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate from 2009, separated only by three single nucleotide polymorphisms (SNPs) on the chromosome, one on plasmid pXO2 and three indel regions. Further, B. anthracis DNA was detected by PCR from soil samples taken from spots in the pasture where the cow had fallen. New tools based on phage receptor-binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil samples. These environmental isolates were genotyped and found to be identical to BF-5 in terms of SNPs. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The area contaminated by the cadaver was subsequently disinfected with formaldehyde.

Epidemiological investigation of a tularaemia outbreak after a hare hunt in Bavaria, Germany, 2018.

In November 2018, a tularaemia outbreak occurred in Bavaria, Germany, among participants of a hare hunt and butchery employees handling the hares. We conducted an epidemiological outbreak investigation, including a retrospective cohort study among hunting participants, to identify likely transmission routes and activities associated with infection. Twelve of 41 participants were antibody-positive for Francisella (F.) tularensis (attack rate: 29%). Cases reported influenza-like symptoms (n = 11), lymphadenopathy (n = 1) and conjunctivitis (n = 1). Infection only occurred in those hunting participants present while hares were processed, while risk of infection was highest when directly involved (RR = 10.0; 95%CI: 2.6-392). F. tularensis was isolated from 1/4 hares. Only two individuals reported using some of the recommended personal protective equipment (PPE). Occurrence of mainly non-specific symptoms, likely due to early treatment, was not indicative of a specific transmission route. Transmissions via direct (skin/mucosa) contact and by inhalation of contaminated aerosols seem plausible. Promoting and increasing appropriate use of PPE among people processing hares is crucial to prevent future outbreaks.

Antimicrobial Resistance in Isolates from Cattle with Bovine Respiratory Disease in Bavaria, Germany.

Patterns of antimicrobial resistance (AMR) regarding Pasteurella multocida (n = 345), Mannheimia haemolytica (n = 273), Truperella pyogenes (n = 119), and Bibersteinia trehalosi (n = 17) isolated from calves, cattle and dairy cows with putative bovine respiratory disease syndrome were determined. The aim of this study was to investigate temporal trends in AMR and the influence of epidemiological parameters for the geographic origin in Bavaria, Germany, between July 2015 and June 2020. Spectinomycin was the only antimicrobial agent with a significant decrease regarding not susceptible isolates within the study period (P. multocida 88.89% to 67.82%, M. haemolytica 90.24% to 68.00%). Regarding P. multocida, significant increasing rates of not susceptible isolates were found for the antimicrobials tulathromycin (5.56% to 26.44%) and tetracycline (18.52% to 57.47%). The proportions of multidrug-resistant (MDR) P. multocida isolates (n = 48) increased significantly from 3.70% to 22.90%. The proportions of MDR M. haemolytica and P. multocida isolates (n = 62) were significantly higher in fattening farms (14.92%) compared to dairy farms (3.29%) and also significantly higher on farms with more than 300 animals (19.49%) compared to farms with 100 animals or less (6.92%). The data underline the importance of the epidemiological farm characteristics, here farm type and herd size regarding the investigation of AMR.

Mycobacterium microti Infections in Free-Ranging Red Deer (Cervus elaphus).

Infections with Mycobacterium microti, a member of the M. tuberculosis complex, have been increasingly reported in humans and in domestic and free-ranging wild animals. At postmortem examination, infected animals may display histopathologic lesions indistinguishable from those caused by M. bovis or M. caprae, potentially leading to misidentification of bovine tuberculosis. We report 3 cases of M. microti infections in free-ranging red deer (Cervus elaphus) from western Austria and southern Germany. One diseased animal displayed severe pyogranulomatous pleuropneumonia and multifocal granulomas on the surface of the pericardium. Two other animals showed alterations of the lungs and associated lymph nodes compatible with parasitic infestation. Results of the phylogenetic analysis including multiple animal strains from the study area showed independent infection events, but no host-adapted genotype. Personnel involved in bovine tuberculosis-monitoring programs should be aware of the fastidious nature of M. microti, its pathogenicity in wildlife, and zoonotic potential.

Antimicrobial Resistance, Serologic and Molecular Characterization of E. coli Isolated from Calves with Severe or Fatal Enteritis in Bavaria, Germany.

Worldwide, enterotoxigenic Escherichia coli (ETEC) cause neonatal diarrhea and high mortality rates in newborn calves, leading to great economic losses. In Bavaria, Germany, no recent facts are available regarding the prevalence of virulence factors or antimicrobial resistance of ETEC in calves. Antimicrobial susceptibility of 8713 E. coli isolates obtained from 7358 samples of diseased or deceased diarrheic calves were investigated between 2015 to 2019. Considerably high rates of 84.2% multidrug-resistant and 15.8% extensively drug-resistant isolates were detected. The resistance situation of the first, second and third line antimicrobials for the treatment, here amoxicillin-clavulanate, enrofloxacin and trimethoprim-sulfamethoxazole, is currently acceptable with mean non-susceptibility rates of 28.1%, 37.9% and 50.0% over the investigated 5-year period. Furthermore, the ETEC serotypes O101:K28, O9:K35, O101:K30, O101:K32, O78:K80, O139:K82, O8:K87, O141:K85 and O147:K89, as well as the virulence factors F17, F41, F5, ST-I and stx1 were identified in a subset of samples collected in 2019 and 2020. The substantially high rates of multi- and extensively drug-resistant isolates underline the necessity of continuous monitoring regarding antimicrobial resistance to provide reliable prognoses and adjust recommendations for the treatment of bacterial infections in animals.

Genotyping of Francisella tularensis subsp. holarctica from Hares in Germany.

Francisella tularensis is the causative agent of the zoonotic disease tularemia. In Germany, most human infections are caused by contact with infected hares. The aim of this study was to characterize Francisella tularensis subsp. holarctica strains isolated from hares in Germany and to develop bioinformatics tools to analyze their genetic relatedness. In total, 257 German isolates-obtained mainly from hares (n = 233), other vertebrate animals, and ticks, but also from humans (n = 3)-were analyzed within this study. Publically available sequence data from 49 isolates were used to put our isolates into an epidemiological context and to compare isolates from natural foci and humans. Whole-genome sequences were analyzed using core-genome Multi-Locus-Sequence-Typing, canonical Single Nucleotide Polymorphism (SNP) typing and whole-genome SNP typing. An overall conformity of genotype clustering between the typing methods was found, albeit with a lower resolution for canonical single SNP typing. The subclade distribution, both on local and national levels, among strains from humans and hares was similar, suggesting circulation of the same genotypes both in animals and humans. Whilst close to identical isolates of the same subclade were found distributed over large areas, small geographical foci often harbored members of different subclades. In conclusion, although genomic high-resolution typing was shown to be robust, reproducible and allowed the identification of highly closely related strains, genetic profiling alone is not always conclusive for epidemiological linkage of F. tularensis strains.

Outbreak of Tularemia in a Group of Hunters in Germany in 2018-Kinetics of Antibody and Cytokine Responses.

In November 2018, an outbreak of tularemia occurred among hare hunters in Bavaria, Germany. At least one infected hare was confirmed as the source of infection. A number of hunting dogs showed elevated antibody titers to Francisella tularensis, but the absence of titer increases in subsequent samples did not point to acute infections in dogs. Altogether, 12 persons associated with this hare hunt could be diagnosed with acute tularemia by detection of specific antibodies. In nine patients, the antibody and cytokine responses could be monitored over time. Eight out of these nine patients had developed detectable antibodies three weeks after exposure; in one individual the antibody response was delayed. All patients showed an increase in various cytokines and chemokines with a peak for most mediators in the first week after exposure. Cytokine levels showed individual variations, with high and low responders. The kinetics of seroconversion has implications on serological diagnoses of tularemia.

Phenotypic characterization and whole genome analysis of extended-spectrum beta-lactamase-producing bacteria isolated from dogs in Germany.

Asymptomatic colonization with extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae has been described for humans, various mammal species, and birds. Here, antimicrobial resistant bacteria were recovered from dog feces originating in Germany, Kosovo, Afghanistan, Croatia, and Ukraine, with a subset of mostly E. coli isolates obtained from a longitudinal collection over twelve months. In vitro antimicrobial resistance testing revealed various patterns of resistance against single or all investigated beta-lactam antibiotics, with none of the 101 isolates resistant against two tested carbapenem antibiotics. Whole genome sequence analysis revealed bacteria species-specific patterns for 23 antimicrobial resistance coding DNA sequences (CDS) that were unapparent from the in vitro analysis alone. Phylogenetic analysis of single nucleotide polymorphisms (SNP) revealed clonal bacterial isolates originating from different dogs, suggesting transmission between dogs in the same community. However, individual resistant E. coli clones were not detected over a period longer than seven days. Multi locus sequence typing (MLST) of 85 E. coli isolates revealed 31 different sequence types (ST) with an accumulation of ST744 (n = 9), ST10 (n = 8), and ST648 (n = 6), although the world-wide hospital-associated CTX-M beta-lactamase producing ST131 was not detected. Neither the antimicrobial resistance CDSs patterns nor the phylogenetic analysis revealed an epidemiological correlation among the longitudinal isolates collected from a period longer than seven days. No genetic linkage could be associated with the geographic origin of isolates. In conclusion, healthy dogs frequently carry ESBL-producing bacteria, independent to prior treatment, which may be transmitted between individual dogs of the same community. Otherwise, these antimicrobial resistant bacteria share few commonalities, making their presence eerily unpredictable.

Genotyping Yersinia pestis in Historical Plague: Evidence for Long-Term Persistence of Y. pestis in Europe from the 14th to the 17th Century.

Ancient DNA (aDNA) recovered from plague victims of the second plague pandemic (14th to 17th century), excavated from two different burial sites in Germany, and spanning a time period of more than 300 years, was characterized using single nucleotide polymorphism (SNP) analysis. Of 30 tested skeletons 8 were positive for Yersinia pestis-specific nucleic acid, as determined by qPCR targeting the pla gene. In one individual (MP-19-II), the pla copy number in DNA extracted from tooth pulp was as high as 700 gene copies/µl, indicating severe generalized infection. All positive individuals were identical in all 16 SNP positions, separating phylogenetic branches within nodes N07_N10 (14 SNPs), N07_N08 (SNP s19) and N06_N07 (s545), and were highly similar to previously investigated plague victims from other European countries. Thus, beside the assumed continuous reintroduction of Y. pestis from central Asia in multiple waves during the second pandemic, long-term persistence of Y. pestis in Europe in a yet unknown reservoir host has also to be considered.

Diverse Genotypes of Yersinia pestis Caused Plague in Madagascar in 2007.

BACKGROUND: Yersinia pestis is the causative agent of human plague and is endemic in various African, Asian and American countries. In Madagascar, the disease represents a significant public health problem with hundreds of human cases a year. Unfortunately, poor infrastructure makes outbreak investigations challenging. METHODOLOGY/PRINCIPAL FINDINGS: DNA was extracted directly from 93 clinical samples from patients with a clinical diagnosis of plague in Madagascar in 2007. The extracted DNAs were then genotyped using three molecular genotyping methods, including, single nucleotide polymorphism (SNP) typing, multi-locus variable-number tandem repeat analysis (MLVA), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) analysis. These methods provided increasing resolution, respectively. The results of these analyses revealed that, in 2007, ten molecular groups, two newly described here and eight previously identified, were responsible for causing human plague in geographically distinct areas of Madagascar. CONCLUSIONS/SIGNIFICANCE: Plague in Madagascar is caused by numerous distinct types of Y. pestis. Genotyping method choice should be based upon the discriminatory power needed, expense, and available data for any desired comparisons. We conclude that genotyping should be a standard tool used in epidemiological investigations of plague outbreaks.

[Human plague and pneumonic plague : pathogenicity, epidemiology, clinical presentations and therapy]. / Pest und Lungenpest : Pathogenität, Epidemiologie, Klinik und Therapie.

Yersinia pestis is a highly pathogenic gram-negative bacterium and the causative agent of human plague. In the last 1500 years and during three dreaded pandemics, millions of people became victims of Justinian's plague, the Black Death, or modern plague. Today, Y. pestis is endemic in natural foci of Asian, African and American countries. Due to its broad dissemination in mammal species and fleas, eradication of the pathogen will not be possible in the near future. In fact, plague is currently classified as a "re-emerging disease". Infection may occur after the bite of an infected flea, but also after oral ingestion or inhalation of the pathogen. The clinical presentations comprise the bubonic and pneumonic form, septicemia, rarely pharyngitis, and meningitis. Most human cases can successfully be treated with antibiotics. However, the high transmission rate and lethality of pneumonic plague require international and mandatory case notification and quarantine of patients. Rapid diagnosis, therapy and barrier nursing are not only crucial for the individual patient but also for the prevention of further spread of the pathogen or of epidemics. Therefore, WHO emergency schedules demand the isolation of cases, identification and surveillance of contacts as well as control of zoonotic reservoir animals and vectors. These sanctions and effective antibiotic treatment usually allow a rapid containment of outbreaks. However, multiple antibiotic resistant strains of Y. pestis have been isolated from patients in the past. So far, no outbreaks with such strains have been reported.

Pneumonic plague outbreak, Northern Madagascar, 2011.

Yersinia pestis, the causative agent of plague, is endemic to Madagascar, particularly to the central highlands. Although plague has not been previously reported in northern Madagascar, an outbreak of pneumonic plague occurred in this remote area in 2011. Over a 27-day period, 17 suspected, 2 presumptive, and 3 confirmed human cases were identified, and all 15 untreated 20 patients died. Molecular typing of Y. pestis isolated from 2 survivors and 5 Rattus rattus rat samples identified the Madagascar-specific 1.ORI3-k single-nucleotide polymorphism genotype and 4 clustered regularly interspaced short palindromic repeat patterns. This outbreak had a case-fatality rate of 100% for nontreated patients. The Y. pestis 1.ORI3-k single-nucleotide polymorphism genotype might cause larger epidemics. Multidrug-resistant strains and persistence of the pathogen in natural foci near human settlements pose severe risks to populations in plague-endemic regions and require outbreak response strategies.

Genotyping of Burkholderia mallei from an outbreak of glanders in Bahrain suggests multiple introduction events.

BACKGROUND: Glanders, caused by the gram-negative bacterium Burkholderia mallei, is a highly infectious zoonotic disease of solipeds causing severe disease in animals and men. Although eradicated from many Western countries, it recently emerged in Asia, the Middle-East, Africa, and South America. Due to its rareness, little is known about outbreak dynamics of the disease and its epidemiology. METHODOLOGY/PRINCIPAL FINDINGS: We investigated a recent outbreak of glanders in Bahrain by applying high resolution genotyping (multiple locus variable number of tandem repeats, MLVA) and comparative whole genome sequencing to B. mallei isolated from infected horses and a camel. These results were compared to samples obtained from an outbreak in the United Arab Emirates in 2004, and further placed into a broader phylogeographic context based on previously published B. mallei data. The samples from the outbreak in Bahrain separated into two distinct clusters, suggesting a complex epidemiological background and evidence for the involvement of multiple B. mallei strains. Additionally, the samples from Bahrain were more closely related to B. mallei isolated from horses in the United Arab Emirates in 2004 than other B. mallei which is suggestive of repeated importation to the region from similar geographic sources. CONCLUSION/SIGNIFICANCE: High-resolution genotyping and comparative whole genome analysis revealed the same phylogenetic patterns among our samples. The close relationship of the Dubai/UAE B. mallei populations to each other may be indicative of a similar geographic origin that has yet to be identified for the infecting strains. The recent emergence of glanders in combination with worldwide horse trading might pose a new risk for human infections.

Parallel independent evolution of pathogenicity within the genus Yersinia.

The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.

Yersinia pestis and the plague of Justinian 541-543 AD: a genomic analysis.

BACKGROUND: Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic. METHODS: Teeth were removed from two individuals (known as A120 and A76) from the early medieval Aschheim-Bajuwarenring cemetery (Aschheim, Bavaria, Germany). We isolated DNA from the teeth using a modified phenol-chloroform method. We screened DNA extracts for the presence of the Y pestis-specific pla gene on the pPCP1 plasmid using primers and standards from an established assay, enriched the DNA, and then sequenced it. We reconstructed draft genomes of the infectious Y pestis strains, compared them with a database of genomes from 131 Y pestis strains from the second and third pandemics, and constructed a maximum likelihood phylogenetic tree. FINDINGS: Radiocarbon dating of both individuals (A120 to 533 AD [plus or minus 98 years]; A76 to 504 AD [plus or minus 61 years]) places them in the timeframe of the first pandemic. Our phylogeny contains a novel branch (100% bootstrap at all relevant nodes) leading to the two Justinian samples. This branch has no known contemporary representatives, and thus is either extinct or unsampled in wild rodent reservoirs. The Justinian branch is interleaved between two extant groups, 0.ANT1 and 0.ANT2, and is distant from strains associated with the second and third pandemics. INTERPRETATION: We conclude that the Y pestis lineages that caused the Plague of Justinian and the Black Death 800 years later were independent emergences from rodents into human beings. These results show that rodent species worldwide represent important reservoirs for the repeated emergence of diverse lineages of Y pestis into human populations. FUNDING: McMaster University, Northern Arizona University, Social Sciences and Humanities Research Council of Canada, Canada Research Chairs Program, US Department of Homeland Security, US National Institutes of Health, Australian National Health and Medical Research Council.

Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

Yersinia pestis DNA from skeletal remains from the 6(th) century AD reveals insights into Justinianic Plague.

Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19(th) and 20(th) centuries, during which plague was spread around the world, and the second pandemic of the 14(th)-17(th) centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6(th)-8(th) centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics.