PrfA activation at the single cell level in Listeria monocytogenes

The human pathogen Listeria monocytogenes is able to transition from environmental saprophyte to facultative intracellular bacteria. In this process, virulence gene expression is controlled by the positive regulatory factor A (PrfA). Recent studies at the single cell level have shown that gene expression in response to stress exposure is stochastic in individual bacteria cells. Current studies applied those general findings to Listeria cells, revealing that PrfA as well is not regulated consistently, but that PrfA activity differs between individual cells. The aim of this study was to elucidate the mechanism by which Listeria regulates PrfA activation at the single cell level and whether this property is heritable or not. A reporter fusion, namely an eGFP sequence, integrated following the PrfA dependent promoter (Phly), was used to visualize the activation after heat stress exposure. Fluorescence activated cell sorting (FACS) was used to distinguish PrfA positive and negative cells. After passaging and sorting PrfA activating versus non-activating cells over several generations, two stable fluorescent phenotypes emerged. A comparison between the genome of the PrfA positive and its parent strain revealed a single-nucleotide polymorphism (SNP) in the CDS of LMRG_02823, as well as a mutation in the 5'UTR of LMRG_00195, both LPXTG family cell wall associated proteins regulated via small RNAs. The link between those mutations and PrfA activation is currently being investigated

Identification of Glaesserella parasuis and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays

Glaesserella parasuis is the etiological agent of Glässer’s disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg–1.25 pg of input DNA for GPS-HRM1 and 125 fg–12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools

Development of a novel high resolution melting assay for identification and differentiation of all known 19 serovars of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed polymerase chain reaction (PCR) amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25 fg-125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen

Mycobacterium microti: not just a coincidental pathogen for cats

Public interest in animal tuberculosis is mainly focused on prevention and eradication of bovine tuberculosis in cattle and wildlife. In cattle, immunodiagnostic tests such as the tuberculin skin test or the interferon gamma (IFN-γ) assay have been established and are commercially available. Feline tuberculosis is rather unknown, and the available diagnostic tools are limited. However, infections with Mycobacterium tuberculosis complex members need to be considered an aetiological differential diagnosis in cats with granulomatous lymphadenopathy or skin nodules and, due to the zoonotic potential, a time-efficient and accurate diagnostic approach is required. The present study describes 11 independent cases of Mycobacterium microti infection in domestic cats in Switzerland. For three cases, clinical presentation, diagnostic imaging, bacteriological results, immunodiagnostic testing, and pathological features are reported. An adapted feline IFN-γ release assay was successfully applied in two cases and appears to be a promising tool for the ante mortem diagnosis of tuberculosis in cats. Direct contact with M. microti reservoir hosts was suspected to be the origin of infection in all three cases. However, there was no evidence of M. microti infection in 346 trapped wild mice from a presumptive endemic region. Therefore, the source and modalities of infection in cats in Switzerland remain to be further elucidated

Brucella canis infection in a young dog with epididymitis and orchitis

The following case report describes the clinical and diagnostic procedure for suspected brucellosis infection in a dog. A 21 month old intact male Border Collie was presented with an enlarged right testicle and epididymis. The dog was imported to Switzerland from Germany at the age of three months, but was never abroad since then. Clinical and laboratory diagnostic investigation included bacteriology and histology. An initial serological evaluation by means of rapid slide agglutination test (RSAT) was negative. Repeated examination of the same serum by a chromatographic immunoassay (ICT) revealed a positive result. Brucella canis infection was confirmed by culture. The present case is intended to underline the importance of the suspected diagnosis of 'brucellosis' in the presence of reproductive tract problems in dogs. In addition, Brucella canis has zoonotic potential and it is imperative to comply with strict hygiene management

Identification of <i>Glaesserella parasuis</i> and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays

Glaesserella parasuis is the etiological agent of Glässer’s disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg–1.25 pg of input DNA for GPS-HRM1 and 125 fg–12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools

Highly resistant Mycobacterium abscessus subsp. abscessus infection in a Swiss cat

A 2-year-old European shorthair cat was presented with a nonhealing skin lesion on the right abdominal wall. Repeated surgical excision and antimicrobial treatment with cefovecin, clindamycin, and trimethoprim-sulfamethoxazole did not lead to clinical improvement. Histopathology of the skin lesion showed pyogranulomatous dermatitis and panniculitis. Definitive diagnosis was made by mycobacterial culture and rpoB sequencing, revealing Mycobacterium abscessus subsp. abscessus. To the authors’ knowledge, this is the first report of Mycobacterium abscessus subsp. abscessus infection in a cat in Switzerland and the second case in Europe. The isolated strain carried a functional erm(41) gene that confers inducible macrolide resistance. Further phenotypic resistances to doxycycline, minocycline, and imipenem were detected, resulting in little chance of successful antimicrobial treatment. Mycobacterium abscessus subsp. abscessus is an emerging pathogen in human medicine and poses a non-negligible zoonotic risk for the owners of the cat

Mycobacterium nebraskense infection in a dog in Switzerland with disseminated skin lesions

BACKGROUND: Cutaneous disseminated mycobacteriosis is rare in dogs. To the best of the authors' knowledge, the slowly growing mycobacterial species Mycobacterium nebraskense has not been described before in this species. OBJECTIVE: Description of clinical features, laboratory analyses and treatment regimen of this unusual case. ANIMAL: A 9-year-old female-spayed West Highland white terrier dog presented with progressive nodules and ulcerations on both sides of the thorax and the rostral aspect of the chest. METHODS AND MATERIALS: Investigations involved histopathological examination of skin biopsies (including special stains for fungi, bacteria and mycobacteria), standard and mycobacterial culture (including susceptibility testing), 16S/23S rRNA sequencing and BLAST similarity searching. RESULTS: Ziehl-Neelsen staining of decontaminated biopsy material revealed acid-fast bacteria morphologically consistent with mycobacteria. Treatment with clarithromycin and marbofloxacin achieved partial resolution. A change in the treatment regimen to pradofloxacin and azithromycin resulted in rapid deterioration of skin lesions. Final healing occurred with the addition of prednisolone at an anti-inflammatory dose. The results of mycobacterial culture and susceptibility testing were received 10 and 12 months, respectively, after the first presentation of the dog. Therapy was stopped after 16 months without recurrence of skin lesions. CONCLUSIONS AND CLINICAL IMPORTANCE: This case is noteworthy for the description of a new mycobacterial species contributing to disseminated panniculitis in a dog and for the difficulties experienced in the lengthy empirical treatment of slowly growing nontuberculous mycobacterial infections. The addition of prednisolone to induce complete healing raises the question of whether the mycobacterial infection was primary or whether it occurred secondarily to an ongoing sterile panniculitis