Quinolone resistance among Salmonella Kentucky and Typhimurium isolates in Tunisia: first report of Salmonella Typhimurium ST34 in Africa and qnrB19 in Tunisia.

AIMS: Characterization of quinolone-resistant Salmonella Kentucky and Typhimurium isolates in Tunisia from various sources, detection of some plasmid-mediated quinolone resistance genes and the genetic relatedness. METHODS: A total of 1404 isolates of S. Kentucky (n = 1059)/S. Typhimurium (n = 345) from various sources from all over Tunisia were tested for quinolone resistance by disk diffusion method. Minimum inhibitory concentrations of nalidixic acid, ciprofloxacin and ofloxacin were determined. Quinolone-resistant isolates were screened for plasmid-mediated quinolone-resistance genes (qnrA,qnrB,qnrS, aac(6')-Ib-cr and qepA) by polymerase chain reaction (PCR). Mutations in the quinolone-resistance-determining regions of the gyrA and parC genes were detected by PCR and DNA sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were accomplished for isolates harbouring plasmid-mediated quinolone-resistance genes. RESULTS: According to our selection criteria (NAL = resistance phenotype; CIP = resistant with diameter 0, or intermediate), only 63 S. Kentucky/41 S. Typhimurium isolates were investigated: 49% (5/104) were multidrug resistant. Two S. Typhimurium isolates harboured qnrB19 with different PFGE profiles. A mutation was detected in the gyrA gene for each of these two isolates. MLST revealed the presence of ST313 and ST34, an endemic sequence type. CONCLUSION: Our study highlights the presence of quinolone multidrug-resistant Salmonella in humans and animals in Tunisia. This is the first report of S. Typhimurium ST34 in Africa and qnrB19 in Tunisia. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes not only the current epidemiological situation of the quinolone resistance in S. Kentucky and Typhimurium isolated from various sources and regions in Tunisia, but also, the genetic resistance determinants associated with phenotypic antibiotic resistance and the molecular mechanisms of their quinolone-resistance. Also, we provide the first report of S. Typhimurium ST34 in Africa, and the first report of qnrB19 in Salmonella in Tunisia.

Serological evidence of Francisella tularensis in febrile patients seeking treatment at remote hospitals, northeastern Kenya, 2014-2015.

Tularaemia is a highly contagious infectious zoonosis caused by the bacterial agent Francisella tularensis. The aim of this study was to investigate the presence of antibodies to F. tularensis in febrile patients in northeastern Kenya. During 2014-2015, 730 patients were screened for anti-F. tularensis antibodies using a combination of ELISA and Western blot. Twenty-seven (3.7%) individuals were positive for F. tularensis. Tularaemia was not suspected by the treating clinicians in any of them. Our results suggest that tularaemia may be present in Kenya but remain unreported, and emphasizes the need for local clinicians to broaden their diagnostic repertoire when evaluating patients with undifferentiated febrile illness.

A cluster of tularaemia after contact with a dead hare in the Netherlands.

Tularemia is thought to be rare in the Netherlands. Here we describe a cluster of two patients who contracted tularaemia after field dressing of a hare found dead. Additionally, infection from the same source is suggested in three animals.

[Threat of transmission of infectious pathogens by Ixodes ricinus ticks in Germany]. / Gefahren der Übertragung von Krankheitserregern durch Schildzecken in Deutschland.

Tick-transmitted diseases are of great importance for the general health of the German population. Several viruses, such as tick-borne encephalitis virus (TBEV), Uukuniemi virus, Tribec virus, Eyach virus or bacteria, such as Borrelia, Rickettsiae, Francisella tularensis, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis (CNM) and Coxiella burnetii were detected in the most prominent tick in Germany, the hard tick Ixodes ricinus. While infections, such as TBE and Lyme disease are well known, other infections are hardly known even among experts. Although there have been a few descriptions of isolated cases in Germany, a systematic investigation regarding the distribution and the pathogenic potential of these pathogens is still lacking. In particular elderly people and people with underlying diseases seem to be mostly affected. The importance of new infectious disease agents, such as Candidatus Neoehrlichia mikurensis but also of long known pathogens, such as Rickettsiae still remains unclear, while some of them could be detected in 20 % of investigated ticks. Whether climate change contributes to the further distribution of these infectious agents remains unclear and requires further investigation. The increasing initiatives to create natural environments and the trend towards spending more time in nature for recreational activities will increase the danger of coming into contact with ticks and the respective infectious agents. Considering these circumstances an increase of diseases caused by these pathogens is to be expected.

Elucidation of colonization time and prevalence of thermophilic Campylobacter species during turkey rearing using multiplex polymerase chain reaction.

Two turkey flocks (male and female) and the environment of their house were investigated for the presence of thermophilic Campylobacter. Sample DNA was extracted directly from fecal material and environmental samples. Bacterial identification was done using a modified Campylobacter species specific multiplex PCR. The times needed for colonization and prevalence in male and female turkeys were determined independently. All environmental samples collected before restocking were negative in the PCR analysis, showing a good hygiene and biosecurity system. The first positive PCR results were obtained in drinking water samples at 6 d of age. Colonization occurred between the second and third week of age, starting in female birds and then followed by the males. Campylobacter jejuni was detected by multiplex PCR at first; later on, Campylobacter coli and mixtures of both were seen. After the 9 wk of age, the colonization of the flocks was completed. Great attention should be given to drinking water as a supposed source of Campylobacter contamination. Multiplex PCR proved to be a rapid, sensitive, and cheap tool for the diagnosis of Campylobacter contamination.

Characterization and comparison of invasive Corynebacterium diphtheriae isolates from France and Poland.

Corynebacterium diphtheriae, the agent of diphtheria, is rarely responsible for bacteremia. However, high numbers of bacteremia have been reported in countries with extensive immunization coverage. Here, we used molecular and phenotypic tools to characterize and compare 42 invasive isolates collected in France (including New Caledonia) and Poland over a 23-year period.

Rapid field detection assays for Bacillus anthracis, Brucella spp., Francisella tularensis and Yersinia pestis.

Rapid detection is essential for timely initiation of medical post-exposure prophylactic measures in the event of intentional release of biological threat agents. We compared real-time PCR assay performance between the Applied Biosystems 7300/7500 and the RAZOR instruments for specific detection of the causative agents of anthrax, brucellosis, tularemia and plague. Furthermore, an assay detecting Bacillus thuringiensis, a Bacillus anthracis surrogate, was developed for field-training purposes. Assay sensitivities for B. anthracis, Brucella spp., Francisella tularensis and Yersinia pestis were 10-100 fg of target DNA per reaction, and no significant difference in assay performance was observed between the instrument platforms. Specificity testing of the diagnostic panels with both instrument platforms did not reveal any cross-reactivity with other closely related bacteria. The duration of thermocycling with the RAZOR instrument was shorter, i.e. 40 min as compared with 100 min for the Applied Biosystems 7300/7500 instruments. These assays provide rapid tools for the specific detection of four biological threat agents. The detection assays, as well as the training assay for B. thuringiensis powder preparation analysis, may be utilized under field conditions and for field training, respectively.

'Imported' melioidosis in Germany: relapse after 10 years.

A 62-year-old German patient with insulin-dependent diabetes and diverticulitis was hospitalized for abdominal pain of the left lower quadrant. Further examination revealed an abdominal abscess, which was punctured. Presumptively a Pseudomonas species was identified, but further examination revealed Burkholderia pseudomallei as the causative agent. Most probably this infection was acquired in 1996 during a trip to Thailand, where the patient had been hospitalized. After combined chemotherapy and surgical revision of the abscess, the patient's condition improved. Clinicians and microbiologists have to keep in mind that in some tropical infections such as melioidosis relapse may occur after such a long time.

Comparison of hand-held test kits, immunofluorescence microscopy, enzyme-linked immunosorbent assay, and flow cytometric analysis for rapid presumptive identification of Yersinia pestis.

An in-house immunochromatographic test, Plague BioThreat Alert test strips, ABICAP columns, enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence microscopy were compared for the detection of the fraction 1 capsular antigen of Yersinia pestis, using spiked buffer and clinical specimens. Hand-held test kits proved to be excellent benchtop tools.

Development and clinical evaluation of a PCR assay targeting the metalloprotease gene (mprA) of B. pseudomallei.

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin-embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei.

Re-emergence of Francisella tularensis in Germany: fatal tularaemia in a colony of semi-free-living marmosets (Callithrix jacchus).

Francisella tularensis was identified as the cause of a die-off which occurred among a colony of semi-free-living common marmosets (Callithrix jacchus). During the outbreak 5 out of 62 animals died of tularaemia in a research facility located in the district of Goettingen, Germany. All animals had been born at the facility suggesting an endemic infection. A total of five culture isolates were recovered and characterized as F. tularensis holarctica, biovar I. These cultures represent the first isolates obtained in the Federal Republic of Germany for more than 45 years. The outbreak area shows several geographical and ecological characteristics known to favour long-term presence of F. tularensis. Persistence of the pathogen in the remote region along the former German-German border, continuous re-introduction from eastern European countries after destruction of the 'Iron curtain' or introduction through migrating birds are testable hypotheses which could explain the emergence of tularaemia in this particular region.

[Glanders--a comprehensive review]. / Ein Ubersichtsreferat zur Rotzerkrankung.

Since 1990 the number of glanders outbreaks in race, military and pleasure horses in Asia and South America is steadily increasing. Glanders, which is eradicated in Western Europe, Australia and Northern America, is currently considered a re-emerging disease. Consequently, the disease may be introduced into glanders-free regions by subclinical carriers at any time. The causative agent of glanders, Burkholderia (B.) mallei, is highly contagious and leads to chronic disease in horses whereas in donkeys and mules the disease is acute and often fatal. Occurrence of the disease leads to international trading restrictions and infected animals immediately have to be culled and safely disposed off. In humans B. mallei infection results in a severe clinical course, and is fatal without appropriate therapy. Its pathogenicity makes B. mallei a potential biological agent that may be used in bioterroristic attacks. Due to the eradication of glanders in the second half of the last century, veterinarians in western European countries are no longer familiar with its clinical presentation in solipeds. Having these facts in mind, this review describes the epidemiology, clinical signs, pathology and the current eradication strategy of this interesting zoonosis. Pictures of imported endurance horses infected with glanders taken during an eradication campaign in Dubai, United Arab Emirates, in 2004 illustrate most typical clinical findings.

Seroprevalence of anti-Yersinia antibodies in healthy Austrians.

Yersiniosis is caused by Y. enterocolitica and Y. pseudotuberculosis mostly presenting as intestinal infection. The infection is usually acquired from contaminated food. The aim of this study was to determine the seroprevalence of anti-Yersinia antibodies in Austrians. Sera of 750 healthy Austrians from all nine states were tested for anti-Yersinia IgG antibodies using the recomBlot Yersinia Westernblot kit. Overall seroprevalence was 29.7%. Seroprevalence increased significantly with age from 24.7% in the group of the 19 to 24 year olds to 38.5% in the group of persons older than 44 years. The seroprevalence of anti-Yersinia antibodies varied within the states between 18% and 43.5%. The high seroprevalence of anti-Yersinia antibodies in contrast to only approximately 100 reported yersiniosis cases per year points to the fact that the majority of infections is either subclinical or mild.

Seroprevalence of brucellosis, tularemia, and yersiniosis in wild boars (Sus scrofa) from north-eastern Germany.

Brucellosis and tularemia are classical zoonotic diseases transmitted from an animal reservoir to humans. Both, wildlife and domestic animals, contribute to the spreading of these zoonoses. The surveillance of the animal health status is strictly regulated for domestic animals, whereas systematic disease monitoring in wildlife does not exist. The aim of the present study was to provide data on the prevalence of anti-Brucella, anti-Francisella and anti-Yersinia antibodies in wild boars from North-Eastern Germany to assess public health risks. A total of 763 sera of wild boars from Mecklenburg-Western Pomerania hunted in 1995/1996 were tested using a commercially available Brucella suis ELISA, an in-house lipopolysaccharide (LPS)-based Francisella ELISA, and commercially available Western blot kits for the detection of anti-Francisella and anti-Yersinia antibodies. The Yersinia enterocolitica O:9 LPS is able to induce serological cross-reactions indistinguishable from brucellosis due to a similar immunodominant epitope in the Brucella O-polysaccharide. The Yersinia Western blot assay was, therefore, based on five recombinant Yersinia outer proteins which have been proved to be specific for the serodiagnosis of yersiniosis. Anti-Brucella, anti-Francisella and anti-Yersinia antibodies were detected in 22.0%, 3.1%, and 62.6% of the wild boars, respectively. The high seroprevalence of tularemia and brucellosis in wild boars indicates that natural foci of these zoonoses are present in wildlife in Germany. However, the impact of transmission of zoonotic pathogens from wildlife to livestock is unknown. Only careful and systematic monitoring will help to prevent the (re)emergence of these zoonotic diseases in domestic animals and consequently human infection.

Diagnostic procedures in tularaemia with special focus on molecular and immunological techniques.

Tularaemia is a severe bacterial zoonosis caused by the highly infectious agent Francisella tularensis. It is endemic in countries of the northern hemisphere ranging from North America to Europe, Asia and Japan. Very recently, Francisella-like strains causing disease in humans were described from tropical northern Australia. In the last decade, efforts have been made to develop sensitive and specific immunological and molecular techniques for the laboratory diagnosis of tularaemia and also for the definite identification of members of the species F. tularensis and its four subspecies. Screening for the keyword 'Francisella' a Medline search over the last decade was performed and articles describing diagnostic methods for tularaemia and its causative agent were selected. Besides classical microbiological techniques (cultivation, biochemical profiling, susceptibility testing) several new immunological and molecular approaches to identify F. tularensis have been introduced employing highly specific antibodies and various polymerase chain reaction (PCR)-based methods. Whereas direct antigen detection by enzyme-linked immunosorbent assay (ELISA) or immunofluorescence might allow early presumptive diagnosis of tularaemia, these methods--like all PCR techniques--still await further evaluation. Therefore, diagnosis of tularaemia still relies mainly on the demonstration of specific antibodies in the host. ELISA and immunoblot methods started to replace the standard tube or micro-agglutination assays. However, the diagnostic value of antibody detection in the very early clinical phase of tularaemia is limited. Francisella tularensis is regarded as a 'highest priority' biological agent (category 'A' according to the CDC, Atlanta, GA, USA), thus rapid and reliable diagnosis of tularaemia is required not only for a timely onset of therapy, the handling of outbreak investigations but also for the surveillance of endemic foci. Only very recently, evaluated test kits for serological diagnosis of human tularaemia became available, while the introduction of standardized molecular techniques for detection and typing is still missing.

Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives.

Burkholderia mallei causes glanders or farcy in solipeds, a disease that must be reported to the OIE (Office International des Epizooties, Paris, France). The number of reported outbreaks has increased steadily during the last decade. Serodiagnosis is hampered by the considerable number of false-positives and -negatives of the internationally prescribed tests. The major problem leading to low sensitivity and specificity of complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e. crude preparations of whole cells. Future perspectives for the development and evaluation of serological test kits using well-characterized single antigens are discussed in the light of recent molecular research on B. mallei and the closely related saprozoonotic agent B. pseudomallei.

Human brucellosis in a nonendemic country: a report from Germany, 2002 and 2003.

Human brucellosis has become a rare disease in Germany since the eradication of bovine and ovine/caprine brucellosis in this country. Therefore, most physicians are unfamiliar with the illnesses clinical presentation, diagnostic tools, and therapeutic strategies. This retrospective study was carried out to evaluate the epidemiological, clinical, and laboratory features of human brucellosis in Germany in the years 2002 and 2003. Thirty-one bacterial isolates from 30 patients sent to the German national reference laboratory were characterized using the genus-specific bcsp31 real-time PCR, the species-specific AMOS-PCR, and standard microbiological methods for the detection and identification of Brucella spp. The medical records of all patients with bacteriologically confirmed brucellosis were evaluated. All 31 isolates proved to be Brucella (30 Brucella melitensis and 1 Brucella suis). Most of the brucellosis patients were infected in endemic countries while visiting friends and relatives during their summer holidays. One case of laboratory-acquired infection was identified. Brucellosis was transmitted mainly by the consumption of contaminated unpasteurized milk or cheese from goats and sheep. The patients presented primarily with flu-like symptoms, i.e. fever, chills, sweating, headaches, arthralgia, and myalgia. In most cases, however, symptoms and signs of focal complications, e.g. spondylitis, endocarditis, and meningoencephalitis, predominated. The rate of complications was much higher than that in endemic countries, presumably as a result of diagnostic delay due to a low index of suspicion. In summary, physicians in nonendemic countries such as Germany must be aware of brucellosis being a possible cause of fever of unknown origin in immigrants and tourists travelling from endemic countries.

Prevalence of anti-Yersinia outer protein antibodies in goats in lower saxony.

Little is known about the prevalence of caprine yersiniosis in Germany. Only few cases are reported every year. The intention of the survey was to provide representative data on the seroprevalence of anti-Yersinia antibodies in goats in the German state of Lower Saxony. A commercially available Western blot kit was used to identify caprine and ovine anti-Yersinia antibodies against five proteins [YopM, H, D, E and V-antigen (V-Ag)]. Of the 681 investigated goat sera, 449 (66%) had anti-Yop/V-Ag antibodies. Only two of 28 animal holdings housed sero-negative goats. Boxplot analysis showed that the number of non-reactive animals is correlated to the size of a herd and the fact of milk production, respectively. A tendency was observed that various management factors may influence the anti-Yersinia antibody status. No statement was possible on the impact of keeping additional carrier animals such as pigs, cows or sheep on a farm or the type of husbandry on the seroprevalence of anti-Yersinia antibodies. This study provides trend-setting data for yersiniosis in goat-holdings. The impact on consumer health, i.e. especially for risk groups-like people allergic to cow milk and the impact on the profit of a farm will have to be elucidated in further studies.

Management in der Behandlung von Patienten nach Einsatz biologischer Agenzien.

The risk of terrorist attacks with weapons of mass destruction like biological agents is increasing. Biological agents can be disseminated as aerosols or by contaminating food and beverages. The multitude of agents and the different pathways of transmission cause very different clinical presentations. Natural infections with potential biological agents in Germany are rare and in most cases imported from endemic areas abroad. It is crucial to include these diseases in the spectrum of differential diagnosis. Local and state health departments have to be notified as early as possible in dubious cases. Public health management can be efficient only, if there is high reporting discipline and all epidemic measures are well coordinated.

Helminthic infestations in the Tyrol, Austria.

At the federal public health laboratory, Innsbruck, 142 426 samples were examined for intestinal helminthosis from 1990 until 2000. Enterobius vermicularis accounted for half (49.7%) of the cases diagnosed, followed by Taenia saginata (28.3%), Ascaris lumbricoides (12.8%), and Trichuris trichiura (3.9%). Of all specimens tested for helminths, 26% had been positive in 1945, and 0.98% in 1985. The proportion of positive findings with respect to the total number of specimens tested was 0.24% in the time span 1990-2000. It appears to us that these numbers fairly reflect the real prevalence of helminthosis in Austria.