Oliver Nolte

Genotypic Variation and Mixtures of Lyme Borrelia in Ixodes Ticks from North America and Europe

Background Lyme disease, caused by various species of Borrelia, is transmitted by Ixodes ticks in North America and Europe. Studies have shown the genotype of Borrelia burgdorferi sensu stricto (s.s.) or the species of B. burgdorferi sensu lato (s.l.) affects the ability of the bacteria to cause local or disseminated infection in humans. Methodology/Principal Findings We used a multilocus PCR electrospray mass spectrometry assay to determine the species and genotype Borrelia from ticks collected in New York, Connecticut, Indiana, Southern Germany, and California and characterized isolates from parts of the United States and Europe. These analyses identified 53 distinct genotypes of B. burgdorferi sensu stricto with higher resolution than ospC typing. Genotypes of other members of the B. burgdorferi sensu lato complex were also identified and genotyped including B. afzelii, B. garinii, B. lusitaniae, B. spielmanii, and B. valaisiana. While each site in North America had genotypes unique to that location, we found genotypes shared between individual regions and two genotypes found across the United States. Significant B. burgdorferi s.s. genotypic diversity was observed between North America and Europe: only 6.6% of US genotypes (3 of 45) were found in Europe and 27% of the European genotypes (3 of 11) were observed in the US. Interestingly, 39% of adult Ixodes scapularis ticks from North America were infected with more than one genotype of B. burgdorferi s.s. and 22.2% of Ixodes ricinus ticks from Germany were infected with more than one genotype of B. burgdorferi s.l. Conclusions/Significance The presence of multiple Borrelia genotypes in ticks increases the probability that a person will be infected with more than one genotype of B. burgdorferi, potentially increasing the risks of disseminated Lyme disease. Our study indicates that the genotypic diversity of Borrelia in ticks in both North America and Europe is higher then previously reported and can have potential clinical consequences.

Oncocin (VDKPPYLPRPRPPRRIYNR-NH2): a novel antibacterial peptide optimized against gram-negative human pathogens.

Small proline-rich antimicrobial peptides (AMP) have attracted considerable interest, as they target specific intracellular bacterial components and do not act by lytic mechanisms. Here, a novel peptide, termed oncocin (VDKPPYLPRPRPPRRIYNR-NH(2)), is reported that was optimized for the treatment of Gram-negative pathogens. Its minimal inhibitory concentrations in tryptic soy broth medium ranged from 0.125 to 8 microg/mL for 34 different strains and clinical isolates of Enterobacteriaceae and nonfermenters, such as Escherichia coli , Pseudomonas aeruginosa , and Acinetobacter baumannii . Substitutions of two arginine residues by ornithine increased the half-lives in full mouse serum from about 20 min to greater than 180 min and the activity. Both optimized oncocin derivatives were neither toxic to human cell lines nor hemolytic to human erythrocytes. They could freely penetrate lipid membranes and were washed out completely without any sign of lytic activity, as assessed by quartz crystal microbalance. Fluorescence labeled peptides entered the periplasmic space within 20 min at room temperature and homogeneously stained E. coli within 50 min. In conclusion, the optimized oncocin represents a very promising candidate for future in vivo work and may serve as a novel lead compound for an antibacterial drug class.

Api88 Is a Novel Antibacterial Designer Peptide To Treat Systemic Infections with Multidrug-Resistant Gram-Negative Pathogens

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a K(D) of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin.

Prevalence of Borrelia miyamotoi in Ixodes Ticks in Europe and the United States

Infection rates of Ixodes ticks with Borrelia miyamotoi in Europe and the United States vary greatly based upon location.

The designer proline-rich antibacterial peptide A3-APO is effective against systemic Escherichia coli infections in different mouse models.

Antimicrobial peptides are considered to be viable alternatives to conventional antibiotics. However, they rarely show systemic efficacy in animal models when added at non-toxic doses. The dimer A3-APO was designed to attack both the bacterial membrane and the Enterobacteriaceae-specific domain of the heat shock protein DnaK in order to reduce toxicity whilst maintaining activity. The peptide exhibited a minimal inhibitory concentration (MIC) range of 2-128 mg/L against 28 clinical Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium strains, with a median MIC of 30 mg/L. At this concentration, A3-APO was bactericidal to E. coli 5770, a fluoroquinolone-resistant extended-spectrum beta-lactamase-producing strain. The No Observed Adverse Effect Limit (NOAEL) at repeated intraperitoneal peptide administration was 20mg/kg. When administered at this dose three times starting immediately after E. coli Neumann infection, A3-APO cured 100% of mice in a standard bacteraemia model used by the pharmaceutical industry. In a more stringent assay, when treatment started after E. coli 5770 bacteraemia had already been established, three doses of 10mg/kg A3-APO prolonged early survival at a rate similar to that of imipenem and reduced the bacterial counts to base level. When the second assay was repeated in kidney clearance conditions resembling those in humans, 10mg/kg A3-APO was as efficacious as imipenem in the long-term. The increased in vivo efficacy compared with the in vitro bactericidal figures can potentially be explained by the generally observable immunostimulatory properties of antimicrobial peptides. Peptide A3-APO shows promising features as a member in our antibiotic arsenal against multidrug-resistant bacterial pathogens.

Molecular diagnosis of microbial aetiologies using SepsiTest™ in the daily routine of a diagnostic laboratory.

A universal PCR and sequencing test, SepsiTest™ (Molzym, Germany) was evaluated for its applicability during daily diagnostic routine in a privately operated laboratory. In total, 96 specimens originating from 66 patients under suspect of infectious endocarditis, infections of joints, encephalitis/meningitis, systemic infections and infections of unknown genesis were PCR analysed and compared to culture results. Samples comprised cultured and non-cultured blood, synovial fluid, synovial tissue, heart valves, pacemakers, spinal tissue, cerebrospinal fluid, and swabs. PCR and culture were concordant in 26 negative and 8 positive cases (51.5%). A group of 25 patients was culture-negative but PCR-positive (37.9%). In at least 14 of these, common and/or rare aetiologies were identified, while for 4 patients the results of 16S PCR could not be unequivocally linked with the underlying disease. Benefits and limitations of the molecular test are discussed with special emphasis on technical and economic issues. In conclusion, SepsiTest™ proved to be a valuable tool for the diagnosis of aetiologies, particularly in cases of culture-negative patients who are under strong suspicion for an infection.

Broad-Range Survey of Tick-Borne Pathogens in Southern Germany Reveals a High Prevalence of Babesia microti and a Diversity of Other Tick-Borne Pathogens

Abstract Ticks harbor numerous pathogens of significance to human and animal health. A better understanding of the pathogens carried by ticks in a given geographic area can alert health care providers of specific health risks leading to better diagnosis and treatments. In this study, we tested 226 Ixodes ricinis ticks from Southern Germany using a broad-range PCR and electrospray ionization mass spectrometry assay (PCR/ESI-MS) designed to identify tick-borne bacterial and protozoan pathogens in a single test. We found 21.2% of the ticks tested carried Borrelia burgdorferi sensu lato consisting of diverse genospecies; a surprisingly high percentage of ticks were infected with Babesia microti (3.5%). Other organisms found included Borrelia miyamotoi, Rickettsia helvetica, Rickettsia monacensis, and Anaplasma phagocytophilum. Of further significance was our finding that more than 7% of ticks were infected with more than one pathogen or putative pathogen.

Severe Wound Infection with Photobacterium damselae ssp. damselae and Vibrio harveyi, following a Laceration Injury in Marine Environment: A Case Report and Review of the Literature

Marine microorganisms are uncommon etiologies of skin and skin structure infections, that is, wound infections. We report a case of severe wound infection, caused by the marine Photobacterium damselae (Vibrionaceae), in a 64-year-old male patient, returning from Australia. The isolate tested positive for pPHDD1, a plasmid conferring high-level virulence. Furthermore, the wound was coinfected with Vibrio harveyi, a halophile bacterium, which has never been reported from human infections before. Identification was achieved by use of Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry (MALDI-TOF) and confirmed by 16S rDNA sequencing. Data retrieval from bibliography was complicated since P. damselae has been renamed often with a number of synonyms present in the literature: Photobacterium damsela, Vibrio damselae, Vibrio damsela, Pasteurella damselae, and Listonella damsela. With all synonyms used as query terms, a literature search provided less than 20 cases published worldwide. A majority of those cases presenting as severe wound infection are even fatal following progression into necrotizing fasciitis. Management with daily wound dressing and antibiotic therapy (ofloxacin empirically, followed by doxycycline after availability of microbiology) led in the reported case to a favorable outcome, which seems to be, however, the exception based on a review of the available literature.

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s.

Landouzy septicemia (sepsis tuberculosa acutissima) due to Mycobacterium microti in an immunocompetent man.

Even in developed countries, tuberculosis still contributes significantly to morbidity and mortality. The most frequent causative agent is Mycobacterium tuberculosis, while infections due to other mycobacterial species are usually associated with immunocompromised patients. In the following, we describe the case of a previously healthy man who underwent laparotomy for suspected adrenal carcinoma. Peritoneal “cancerous nodules” turned out to be tuberculous granulomas. After surgery the patient developed a protracted septic shock and died 6 days after surgery. Isolation and identification of the causative agent yielded Mycobacterium microti, an uncommon species of the M. tuberculosis complex. No other pathogen could be isolated during the clinical course, which finally led to the diagnosis of Landouzy septicemia (sepsis tuberculosa acutissima).