Burkholderia pseudomallei multi-centre study to establish EUCAST MIC and zone diameter distributions and epidemiological cut-off values.

OBJECTIVES: Melioidosis, caused by Burkholderia pseudomallei, requires intensive antimicrobial treatment. However, standardized antimicrobial susceptibility testing (AST) methodology based on modern principles for determining breakpoints and ascertaining performance of methods are lacking for B. pseudomallei. This study aimed to establish MIC and zone diameter distributions on which to set epidemiological cut-off (ECOFF) values for B. pseudomallei using standard EUCAST methodology for non-fastidious organisms. METHODS: Non-consecutive, non-duplicate clinical B. pseudomallei isolates (9-70 per centre) were tested at eight study centres against eight antimicrobials by broth microdilution (BMD) and the EUCAST disc diffusion method. Isolates without and with suspected resistance mechanisms were deliberately selected. The EUCAST Development Laboratory ensured the quality of study materials, and provided guidance on performance of the tests and interpretation of results. Aggregated results were analysed according to EUCAST recommendations to determine ECOFFs. RESULTS: MIC and zone diameter distributions were generated using BMD and disc diffusion results obtained for 361 B. pseudomallei isolates. MIC and zone diameter ECOFFs (mg/L; mm) were determined for amoxicillin-clavulanic acid (8; 22), ceftazidime (8; 22), imipenem (2; 29), meropenem (2; 26), doxycycline (2; none), tetracycline (8; 23), chloramphenicol (8; 22) and trimethoprim-sulfamethoxazole (4; 28). CONCLUSIONS: We have validated the use of standard BMD and disc diffusion methodology for AST of B. pseudomallei. The MIC and zone diameter distributions generated in this study allowed us to establish MIC and zone diameter ECOFFs for the antimicrobials studied. These ECOFFs served as background data for EUCAST to set clinical MIC and zone diameter breakpoints for B. pseudomallei.

Development of a novel selective agar for the isolation and detection of Bacillus anthracis.

AIMS: The aim of this study was to develop a novel selective agar for the specific isolation and detection of Bacillus anthracis. METHODS AND RESULTS: Based on published data on antibiotic resistance and susceptibility of B. anthracis and other closely related species of the Bacillus cereus sensu lato group, a new selective agar formulation termed CEFOMA (Bacillus CEreus sensu lato group-specific antibiotics, FOsfomycin, MAcrolides) was developed and evaluated. All tested strains of B. anthracis were able to grow on CEFOMA with the same colony number as on non-selective media, whereas CEFOMA inhibited the growth of the other species within the B. cereus sensu lato group. In comparison to other selective agars, CEFOMA had a superior performance and considerably reduced the total amount of accompanying flora in soil. Furthermore, B. anthracis was successfully isolated from deliberately spiked soil samples. CONCLUSIONS: CEFOMA is a highly promising selective agar for the efficient isolation of B. anthracis from environmental samples with a large bacterial background flora. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolation of B. anthracis from environmental samples is severely impaired by the lack of adequate selective agars which suppress the growth of other bacteria. CEFOMA agar represents an important improvement and suitable alternative to currently used selective agars.

Analysis of a newly discovered antigen of Bacillus cereus biovar anthracis for its suitability in specific serological antibody testing.

AIMS: The aim of this work was to identify a protein which can be used for specific detection of antibodies against Bacillus cereus biovar anthracis (Bcbva), an anthrax-causing pathogen that so far has been described in African rainforest areas. METHODS AND RESULTS: Culture supernatants of Bcbva and classic Bacillus anthracis (Ba) were analysed by gel electrophoresis, and a 35-kDa protein secreted only by Bcbva and not Ba was detected. The protein was identified as pXO2-60 by mass spectrometry. Sequence analysis showed that Ba is unable to secrete this protein due to a premature stop codon in the sequence for the signal peptide. Immunization of five outbred mice with sterile bacterial culture supernatants of Bcbva revealed an immune response in ELISA against pXO2-60 (three mice positive, one borderline) and the protective antigen (PA; four mice). When supernatants of classic Ba were injected into mice or human sera from anthrax patients were analysed, only antibodies against PA were detected. CONCLUSIONS: In combination with PA, the pXO2-60 protein can be used for the detection of antibodies specific against Bcbva and discriminating from Ba. SIGNIFICANCE AND IMPACT OF THE STUDY: After further validation, serological assays based on pXO2-60 can be used to perform seroprevalence studies to determine the epidemiology of B. cereus bv anthracis in affected countries and assess its impact on the human population.

High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei.

Burkholderia (B.) mallei is the causative agent of glanders. A previous work conducted on single-nucleotide polymorphisms (SNP) extracted from the whole genome sequences of 45 B. mallei isolates identified 3 lineages for this species. In this study, we designed a high-resolution melting (HRM) method for the screening of 15 phylogenetically informative SNPs within the genome of B. mallei that subtype the species into 3 lineages and 12 branches/sub-branches/groups. The present results demonstrate that SNP-based genotyping represent an interesting approach for the molecular epidemiology analysis of B. mallei.

Test methods for estimating the efficacy of the fast-acting disinfectant peracetic acid on surfaces of personal protective equipment.

AIMS: The work aimed at developing and evaluating practically relevant methods for testing of disinfectants on contaminated personal protective equipment (PPE). METHODS AND RESULTS: Carriers were prepared from PPE fabrics and contaminated with Bacillus subtilis spores. Peracetic acid (PAA) was applied as a suitable disinfectant. In method 1, the contaminated carrier was submerged in PAA solution; in method 2, the contaminated area was covered with PAA; and in method 3, PAA, preferentially combined with a surfactant, was dispersed as a thin layer. In each method, 0·5-1% PAA reduced the viability of spores by a factor of ≥6 log10 within 3 min. The technique of the most realistic method 3 proved to be effective at low temperatures and also with a high organic load. Vaccinia virus and Adenovirus were inactivated with 0·05-0·1% PAA by up to ≥6 log10 within 1 min. The cytotoxicity of ricin was considerably reduced by 2% PAA within 15 min of exposure. CONCLUSIONS: PAA/detergent mixture enabled to cover hydrophobic PPE surfaces with a thin and yet effective disinfectant layer. SIGNIFICANCE AND IMPACT OF THE STUDY: The test methods are objective tools for estimating the biocidal efficacy of disinfectants on hydrophobic flexible surfaces.

High and novel genetic diversity of Francisella tularensis in Germany and indication of environmental persistence.

In Germany tularemia is a re-emerging zoonotic disease. Therefore, we investigated wild animals and environmental water samples for the presence and phylogenetic diversity of Francisella tularensis in the poorly studied Berlin/Brandenburg region. The phylogenomic analysis of three isolates from wild animals revealed three new subclades within the phylogenetic tree of F. tularensis [B.71 from a raccoon dog (Nyctereutes procyonoides); B.74 from a red fox (Vulpes vulpes), and B.75 from a Eurasian beaver (Castor fiber albicus)]. The results from histological, PCR, and genomic investigations on the dead beaver showed that the animal suffered from a systemic infection. Indications were found that the bacteria were released from the beaver carcass into the surrounding environment. We demonstrated unexpectedly high and novel phylogenetic diversity of F. tularensis in Germany and the fact that the bacteria persist in the environment for at least one climatic season. These findings support a broader host species diversity than previously known regarding Germany. Our data further support the assumption derived from previous serological studies of an underestimated frequency of occurrence of the pathogen in the environment and in wild animals. F. tularensis was isolated from animal species not previously reported as natural hosts in Germany.

Comparison of eleven commercially available rapid tests for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

UNLABELLED: Yersinia pestis, Bacillus anthracis and Francisella tularensis cause serious zoonotic diseases and have the potential to cause high morbidity and mortality in humans. In case of natural outbreaks and deliberate or accidental release of these pathogens rapid detection of the bacteria is crucial for limitation of negative effects of the release. In the present study, we evaluated 11 commercially available rapid test kits for the detection of Y. pestis, B. anthracis and F. tularensis in terms of sensitivity, specificity and simplicity of the procedure. The results revealed that rapid and easy-to-perform lateral flow assays for detection of highly pathogenic bacteria have very limited sensitivity. In contrast, the immunofiltration assays showed high sensitivity but limited specificity and required a too complicated procedure to be applied in the field by nonlaboratory workers (e.g. First Responders like fire, police and emergency medical personnel). Each sample - whether tested negative or positive by the rapid tests - should be retested in a reference laboratory using validated methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid detection of highly pathogenic bacteria causing anthrax, plague and tularemia is crucial for the limitation of negative effects of a potential release (natural, accidental or deliberate). In the study, commercially available rapid tests for detection of Bacillus anthracis, Yersinia pestis and Francisella tularensis were investigated in terms of sensitivity, specificity and ease-to-perform. The study showed problems which could be faced during testing and results interpretation. Conclusions from this study should be helpful not only in selection of the most appropriate test for particular group of First Responders but also in undertaking decisions in situation of a contamination suspicion which have high social and economical impacts.

Decontamination of a BSL3 laboratory by hydrogen peroxide fumigation using three different surrogates for Bacillus anthracis spores.

AIMS: Two independent trials investigated the decontamination of a BSL3 laboratory using vaporous hydrogen peroxide and compared the effect on spores of Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis as surrogates for Bacillus anthracis spores, while spores of Geobacillus stearothermophilus served as control. METHODS AND RESULTS: Carriers containing 1·0 × 10(6) spores were placed at various locations within the laboratory before fumigation with hydrogen peroxide following a previously validated protocol. Afterwards, carriers were monitored by plating out samples on agar and observing enrichment in nutrient medium for up to 14 days. Three months later, the experiment was repeated and results were compared. On 98 of 102 carriers, no viable spores could be detected after decontamination, while the remaining four carriers exhibited growth of CFU only after enrichment for several days. Reduction factors between 4·0 and 6·0 log levels could be reached. CONCLUSIONS: A validated decontamination of a laboratory with hydrogen peroxide represents an effective alternative to fumigation with formaldehyde. Spores of B. cereus seem to be more resistant than those of G. stearothermophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide important results in the field of hydrogen peroxide decontamination when analysing the effect on spores other than those of G. stearothermophilus.

Establishing a nurse-based psychiatric CL service in the accident and emergency department of a general hospital in Germany.

INTRODUCTION: Patients with mental health problems in accident and emergency departments (A&E) are frequent users and often difficult to handle. Failure in managing these patients can cause adversities to both patients and A&E staff. It has been shown that nurse-based psychiatric consultation-liaison (CL) services work successfully and cost effectively in English-speaking countries, but they are hardly found in European countries. The aim of this study was to determine whether such a liaison service can be established in the A&E of a German general hospital. We describe structural and procedural elements of this service and present data of A&E patients who were referred to the newly established service during the first year of its existence, as well as an evaluation of this nurse-led service by non-psychiatric staff in the A&E and psychiatrists of the hospital's department of psychiatry. SUBJECTS AND METHODS: In 2008 a nurse-based psychiatric CL-service was introduced to the A&E of the Königin Elisabeth Herzberge (KEH) general hospital in the city of Berlin. Pathways for the nurse's tasks were developed and patient-data collected from May 2008 till May 2009. An evaluation by questionnaire of attitudes towards the service of A&E staff and psychiatrists of the hospital's psychiatric department was performed at the end of this period. RESULTS: Although limited by German law that many clinical decisions to be performed by physicians only, psychiatric CL-nurses can work successfully in an A&E if prepared by special training and supervised by a CL-psychiatrist. The evaluation of the service showed benefits with respect to satisfaction and skills of staff with regard to the management of psychiatrically ill patients. CONCLUSION: Nurse-based psychiatric CL-services in A&E departments of general hospitals, originally developed in English-speaking countries, can be adapted for and implemented in a European country like Germany. Open access: This article is published with open access at link.springer.com.

Anthrax among heroin users in Europe possibly caused by same Bacillus anthracis strain since 2000.

Injection anthrax was described first in 2000 in a heroin-injecting drug user in Norway. New anthrax cases among heroin consumers were detected in the United Kingdom (52 cases) and Germany (3 cases) in 2009-10. In June 2012, a fatal case occurred in Regensburg, Bavaria. As of December 2012, 13 cases had been reported in this new outbreak from Germany, Denmark, France and the United Kingdom. We analysed isolates from 2009-10 and 2012 as well as from the first injection anthrax case in Norway in 2000 by comparative molecular typing using a high resolution 31 marker multilocus variable-number tandem repeat analysis (MLVA) and a broad single nucleotide polymorphism (SNP) analysis. Our results show that all cases may be traced back to the same outbreak strain. They also indicate the probability of a single source contaminating heroin and that the outbreak could have lasted for at least a decade. However, an additional serological pilot study in two German regions conducted in 2011 failed to discover additional anthrax cases among 288 heroin users.

Tularaemia seroprevalence of captured and wild animals in Germany: the fox (Vulpes vulpes) as a biological indicator.

A total of 2475 animals from Germany, both captive and wild, were tested for antibodies against Francisella tularensis to obtain more knowledge about the presence of this pathogen in Germany. An indirect and a competitive ELISA served as screening methods, positive and inconclusive samples were confirmed by Western blot. Of the zoo animals sampled between 1992 and 2007 (n = 1122), three (0·3%) were seropositive. The seroconversion of a hippopotamus in Berlin Zoo was documented. From 1353 serum samples of wild foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and wild boars (Sus scrofa), collected between 2005 and 2009 in the federal state of Brandenburg (surrounding Berlin), a total of 101 (7·5%) tested positive for antibodies to F. tularensis lipopolysaccharide. Our results indicate a higher seroprevalence of F. tularensis in wildlife in eastern Germany than commonly assumed. Furthermore, we found foxes and raccoon dogs to be biological indicators for tularaemia.

Surveillance of tularaemia in Kosovo, 2001 to 2010.

Tularaemia, caused by Francisella tularensis, had not been registered in Kosovo before an outbreak in 1999 and 2000. A national surveillance system has been implemented in Kosovo since 2000 to monitor a number of diseases, including tularaemia. Antibody detection in human sera was used for laboratory diagnosis of tularaemia and F. tularensis lipopolysaccharide antigen was used as a marker of infection. The purpose of this study is to describe the incidence of tularaemia in Kosovo after the 1999-00 outbreak. In 2001 and 2002, a second outbreak occurred, with 327 serologically confirmed cases. From 2001 to 2010, 25-327 cases were registered per year, giving a mean annual incidence of 5.2 per 100,000 population. The most likely sources of infection were contaminated drinking water and food. The dominant clinical manifestations were the glandular (79%) and ulcero-glandular (21%) forms. By 2010, the disease had spread throughout Kosovo. Presumably as a result of war and subsequent environmental disruption, mass population displacement and breakdown of sanitation and hygiene, the two major outbreaks of tularaemia resulted in the establishment of an active endemic area of tularaemia in Kosovo.

Fatal anthrax infection in a heroin user from southern Germany, June 2012.

Blood cultures from a heroin user who died in June 2012, a few hours after hospital admission, due to acute septic disease, revealed the presence of Bacillus anthracis. This report describes the extended diagnosis by MALDI-TOF and real-time PCR and rapid confirmation of the anthrax infection through reference laboratories. Physicians and diagnostic laboratories were informed and alerted efficiently through the reporting channels of German public health institutions, which is essential for the prevention of further cases.

Immunodetection of inactivated Francisella tularensis bacteria by using a quartz crystal microbalance with dissipation monitoring.

Francisella tularensis are very small, gram-negative bacteria which are capable of infecting a number of mammals. As a highly pathogenic species, it is a potential bioterrorism agent. In this work we demonstrate a fast immunological detection system for whole F. tularensis bacteria. The technique is based on a quartz crystal microbalance with dissipation monitoring (QCMD), which uses sensor chips modified by a specific antibody. This antibody is useful as a capture molecule to capture the lipopolysaccharide structure on the surface of the bacterial cell wall. The QCMD technique is combined with a microfluidic system and allows the label-free online detection of the binding of whole bacteria to the sensor surface in a wide dynamic concentration range. A detection limit of about 4 × 10(3) colony-forming units per milliliter can be obtained. Furthermore, a rather short analysis time and a clear discrimination against other bacteria can be achieved. Additionally, we demonstrate two possibilities for specific and significant signal enhancement by using antibody-functionalized gold nanoparticles or an enzymatic precipitation reaction. These additional steps can be seen as further proof of the specificity and validity.

Identification and subtyping of Francisella by pyrosequencing and signature matching of 16S rDNA fragments.

AIMS: To analyse the V1 region of the 16S rDNA gene by a universal pyrosequencing protocol to identify and subtype Francisella in 31 strains from a repository collection and 96 patient isolates. METHODS AND RESULTS: Pyrosequencing was used to determine the nucleotide sequence of PCR amplification products of the variable region (V1) of the 16S rDNA from 31 repository strains and 96 isolates from Swedish patients with ulceroglandular tularaemia. Pyrosequencing resulted in a 37 nucleotide sequence, specific for Francisella sp., for all repository strains and patient samples analysed. In addition, the isolates could be divided into two groups based on the analysis of a single nucleotide polymorphism in the sequence: one group included Francisella tularensis ssp. tularensis, ssp. holarctica and ssp. mediasiatica, whereas the other group included Francisella tularensis ssp. novicida and other species of Francisella. The analysis of samples taken from patients suffering from ulceroglandular tularaemia revealed that all isolates belonged to the first group comprising subspecies of F. tularensis virulent for humans. CONCLUSIONS: The pyrosequencing analysis of the 16S rDNA V1 is a useful molecular tool for the rapid identification of suspected isolates of Francisella sp. in clinical or environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulent F. tularensis ssp. causing ulceroglandular tularaemia, or those with a potential to be used in a bioterrorism event, could rapidly be discriminated from subspecies less virulent for humans.

Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens.

The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization.

Preliminary case report of fatal anthrax in an injecting drug user in North-Rhine-Westphalia, Germany, December 2009.

A fatal case of anthrax occurred in an injecting drug user in Germany, in December 2009. A potential link to similar cases in Scotland in the same time period is currently under investigation.

[Investigation of a family cluster of influenza A/H1N1 infections in Germany, 2009]. / Untersuchungen zu einem familiären Cluster von Infektionen durch Influenza A/H1N1 in Deutschland, 2009.

INTRODUCTION: On May 3, 2009, a first case of influenza A/H1N1 infection occurred in the federal state of Saxony-Anhalt, Germany. In order to stop the possible spread of the virus and to study the epidemiological and clinical characteristics of the infection, an investigation was launched by the local health authorities and the RKI. METHODS: Standardised questionnaires were used to assess demographic and clinical data. Specimens were collected from case patients and close contacts and were analysed for influenza A/H1N1 using real-time PCR. RESULTS: The index patient showed fever and coughing 3.5 days after returning from a holiday in Mexico. The local health authorities were informed on May 3, and measures were rapidly implemented. These measures included a trace-back of possible contact persons, isolation of the case and close contacts, prophylactic treatment with Oseltamivir. Virological investigations showed that the case shedded viral genome up until the last day of antiviral therapy. Viral genome was also detected in the spouse and the son of the patient. Both showed no symptoms under a prophylactic treatment with antiviral medication. No viral genome was detected in three other family members, and in six other contact persons outside of the family. DISCUSSION: The spread of the virus was contained due to the fast response of the local health authorities. Two secondary cases occurred in the family. These cases remained asymptomatic, possibly due to antiviral prophylaxis. Epidemiological and virological results suggest that the influenza A/H1N1 virus has a longer incubation period and that viral shedding may probably be prolonged when compared with seasonal influenza.