Energy conservation in acetogenic bacteria : caffeate reduction in Acetobacterium woodii

Aus Fruktose-gezogenen Zellen von A. woodii, die in Gegenwart von Caffeat als Elektronenakzeptor gewachsen waren, wurde durch Behandlung mit Lysozym und anschliessendem French-Press-Aufschluß bei niedrigem Druck ein zellfreier Rohextrakt unter strikt anaeroben Bedingungen hergestellt. Dieser katalysierte eine H2-abhängige Caffeatreduktion mit Raten von 8,7 – 18,7 nmol/min x mg Protein. Die Aktivität war strikt ATP-abhängig. Nach einer Auftrennung des Rohextraktes in Cytoplasma- und Membranfraktion war die H2-abhängige Caffeatreduktion ausschliesslich in der cytoplasmatischen Fraktion lokalisiert. Die Zugabe von Membranen führte zu keiner Stimulierung der Aktivität. Die Membranfraktion selbst wies keine Caffeatreduktionsaktivität auf. Verschiedene Verbindungen wurden auf ihre Fähigkeit getestet, als artifizielle Elektronendonatoren für die Caffeatreduktion zu fungieren. TMPD, 1,5-Diphenylcarbazid und reduziertes Methylviologen konnten Caffeat nicht reduzieren. In Gegenwart von Phenylendiamin wurde in zellfreiem Rohextrakt und in der Cytoplasmafraktion Caffeatreduktionsaktivität beobachtet. In der Membranfraktion wurde dagegen keine Reduktion von Caffeat mit Phenylendiamin als Elektronendonor nachgewiesen. NADH + H+ konnte als physiologischer Elektronendonor für die Reduktion von Caffeat fungieren. Die NADH-abhängige Caffeatreduktion war ausschliesslich in der cytoplasmatischen Fraktion lokalisiert und strikt abhängig von ATP. Die Beobachtung, dass NADH + H+ als Elektronendonor für die Caffeareduktion fungieren kann, führte zu der Frage, wie im Zuge H2-abhängiger Caffeatreduktion NADH + H+ gebildet wird und welche Enzyme daran beteiligt sein könnten. NAD+-abhängige Hydrogenaseaktivität wurde an Membranen und im Cytoplasma nachgewiesen. Rund 70% der Aktivität waren in der löslichen Fraktion lokalisiert. Die Gegenwart einer Elektronendonor:NAD+-Oxidoreduktase in A. woodii wurde untersucht. Gewaschene Membranen vermittelten die Oxidation von NADH + H+ mit Kaliumhexacyanoferrat oder Benzylviologen als Elektronenakzeptor. Diese Beobachtung wurde als Hinweis auf eine NAD+-Reduktase gewertet, da solche Enzyme in der Regel reversibel sind. Eine Hydrogenase konnte hierfür ausgeschlossen werden, da die NADH-oxidierende Aktivität Sauerstoff-unempfindlich war. Die Aktivität des NADH-oxidierenden Enzyms konnte durch zugesetztes Na+ nicht stimuliert werden. In Analogie zu Na+-translozierenden NADH:Chinon-Reduktasen wurde die NADH-oxidierende Aktivität an gewaschenen Membranen aber durch Ag+ oder Cu2+ in mikromolaren Konzentrationen vollständig inhibiert. Membranen von A. woodii vermittelten die Reduktion von NAD+ mit reduzierten Ferredoxin als Elektronendonor. Ob diese Aktivität durch das gleiche membranständige Enzyme katalysiert wurde, das auch die NADH-Oxidation bewerkstelligte, konnte nicht geklärt werden. Die Ferredoxin-abhängige NAD+-Reduktion wurde durch micromolare Konzentrationen Ag+ vollständig inhibiert. Die Inihibition war aber wahrscheinlich unspezifischer Natur. Mittels vergleichender 2D-Gelelektrophorese wurden zwei Caffeat-induzierte Proteine identifiziert. Ein Vergleich der Peptidsequenzen, die durch ESI-MS/MS-Analyse der Proteine erhalten wurden, mit in Datenbanken hinterlegten Proteinsequenzen, ergab eine große Übereinstimmung zu der großen alpha- bzw. kleinen beta-Untereinheit von heterodimeren Elektronentransfer-Flavoproteinen (ETFP). Die Proteine wurden mit EtfA und EtfB bezeichnet. Anhand der Peptidsequenzen von EtfA und EtfB wurden degenerierte Oligonukleotide abgeleitet, die zur Amplifikation von Fragmenten der kodierenden Gene herangezogen wurden. Die Analyse der abgeleiteten Aminosäuresequenzen der erhaltenen PCR-Produkte untermauerte die Zuordnung von EtfA und EtfB als Untereinheiten eines ETFP. Die Fähigkeit zur Caffeatreduktion ist in A. woodii nicht konstitutiv vorhanden, sondern wird in erst durch Gegenwart des Phenylacrylats induziert. Die Spezifität dieser Induktion wurde untersucht. Suspensionen ruhender Zellen, die aus Caffeat-induzierten Zellen hergestellt worden waren, reduzierten neben Caffeat auch die Phenylacrylate Ferulat oder p-Cumarsäure mit H2 als Elektronendonor. Analoge Beoabachtungen wurden mit Ferulat-induzierten und p-Cumarsäure-induzierten Zellsuspensionen gemacht. Die Ergebnisse legen den Schluß nahe, dass in A. woodii die Reduktion von Phenylacrylaten durch ein induzierbares enzymatisches System bewerkstelligt wird. EtfA und EtfB wurden als MalE-Fusionsproteine dargestellt und gereinigt. Dagegen wurden Antiseren hergestellt. Immunologische Untersuchungen zeigten, dass die Produktion von EtfA und EtfB durch Gegenwart verschiedener Phenylacrylate induziert wird. Die Induktion war unabhängig vom Wachstumssubstrat. In Gegenwart von zu Phenylacrylaten ähnlichen Verbindungen erfolgte keine Induktion. Für EtfA und EtfB wurde eine Funktion als universeller Elektronenüberträger des Phenylacrylat-Reduktionssystems in A. woodii postuliert. Für die H2-abhängige Reduktion von Caffeat und anderer Phenylacrylate wurde die folgende Reaktionssequenz postuliert: die Oxidation des Elektronendonors H2 durch eine Hydrogenase geht einher mit der Bildung von reduziertem Ferredoxin. Dieses wird durch eine membranständige Ferredoxin-NAD+-Oxidoreduktase oxidiert, die den Transfer der Elektronen auf NAD+ mit der Translokation von Na+ koppelt. Ein aus EtfA und EtfB gebildetes ETFP fungiert dann als Elektronenüberträger zwischen NADH + H+ und einer löslichen Phenylacrylatreduktase.Acetobacterium woodii was grown with fructose in the presence of caffeate as electron acceptor. Cells were treated with lysozyme and broken up applying low preassure by passage through a „French Press“ cell yielding cell-free extract. All manipulations were performed under strictly anaerobic conditions. This cell-free extract mediated H2-dependent caffeate reduction with rates between 8.7 and 18.7 nmol/min x mg protein. The activity was strictly dependent on the presence of ATP. After separating cell-free extract into the cytoplasmic and the membraneous fraction H2-dependent caffeate reduction was exclusively located in the cytoplasm. The addition of membranes lead to no stimulation of the activity. No activity was found in the membraneous fraction. Various compounds were tested to act as artficial electron donors for caffeate reduction. Tetramethyl phenylendiamine, 1,5-diphenylcarbazide and reduced methyl viologen did not mediate caffeate reduction. In the presence of phenylenediamine caffeate reduction was observed in cell-free extract and the cytoplasmic fraction. In the membraneous fraction no reduction of caffeate was observed with phenylenediamine serving as electron donor. NADH acted as a physiological electron donor for the reduction of caffeate. NADH-dependent caffeate reduction was exclusively located in the cytoplasmic fraction and was strictly dependent on the presence of ATP. The observation that NADH acts as an electron donor for caffeate reduction raised the question how NADH is generated during H2-dependent caffeate reduction and which enzymes could be involved. NAD+-dependent hydrogenase activity was identified in the membraneous and the cytoplasmic fraction. About 70% of the activity was located in the soluble fraction. The presence of an electron donor:NAD+-oxidoreductase in A. woodii was investigated. Washed membranes mediated the oxidation of NADH with ferricyanide or benzyl viologen serving as electron acceptors. This observation was taken as a hint for the presence of a NAD+-reductase, since these enzymes are usually reversible. A hydrogenase could be excluded to mediate this activity since the NADH-oxidizing activity was insensitive towards oxygen. The activity of the NADH-oxidizing enzyme could not be stimulated by Na+. In analogy to Na+-translocating NADH:quinone-reductases the NADH-oxidizing activity located at washed membranes was completly inhibited by micro-molar concentrations of Ag+ or Cu2+. Washed membranes of A. woodii mediated the reduction of NAD+ with reduced ferredoxin serving as electron donor. It could not be clarified, whether this activity was mediated by the membrane-bound NADH-oxidizing enzyme. Ferredoxin-dependent NAD+-reduction was completely inhibited by micro-molar concentrations of Ag+. This inihibition was most likely unspecific. Using 2D gel electrophoresis two caffeate-induced proteins were identified that were subsequently subjected to ESI-MS/MS. A comparision of the obtained peptide sequences with protein sequences from data bases revealed great identities to the large alpha-subunit and the small beta-subunit respectively of heterodimeric electron transfer flavorproteins (ETFP). The caffeate-induced proteins were designated EtfA and EtfB respectively. Based on the peptide sequences degenerated oligonucleotides were deduced and gene fragements coding for EtfA and EtfB were amplified. Analysis of the amino acid sequences deduced from the PCR products confirmed that EtfA and EtfB are subunits of an ETFP. The ability to reduce caffeate is not a constitutive feature of A. woodii but is induced in the presence of the phenyl acrylate. The specificity of this induction was investigated. Suspensions of resting cells prepared from caffeate-induced cells reduced besides caffeate other phenyl acrylates like ferulate or p-coumarate with H2 serving as electron donor. This observation implies that the reduction of phenyl acrylates in A. woodii is accomplished by one single induceable enzymatic system. EtfA and EtfB were heterologously overproduced as MalE-fusion proteins and were purified. Antibodies were generated against MalE-EtfA and MalE-EtfB. Western blot analysis revealed that the production of EtfA and EtfB is induced by various phenyl acrylates. This induction was independent of the growth substrate. In the presence of compounds structurally similar to phenyl acrylates no induction was observed. Thus, it is postulated that EtfA/EtfB act as the universal electron mediator of the phenyl acrylate reducing system in A. woodii. The following reaction sequence was postulated for the reduction of caffeate and other phenyl acrylates: the oxidation of the electron donor H2 is accompanied by the generation of reduced ferredoxin. The latter is oxidized by a membrane-bound ferredoxin-NAD+-oxidoreductase that couples electron transfer to NAD+ with the translocation of Na+. An ETFP consisting of EtfA and EtfB mediates the electron transfer between NADH and a soluble phenyl acrylate reductase

Pupylation-dependent and -independent proteasomal degradation in mycobacteria

Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway

Pupylation-dependent and -independent proteasomal degradation in mycobacteria

Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway

Mycoplasma penetrans bacteremia in an immunocompromised patient detected by metagenomic sequencing: a case report.

Background Mycoplasma sp. are well recognized as etiological agents of respiratory and sexually transmitted disease. Mycoplasma penetrans, a species of Mycoplasma sp., has been frequently detected in HIV-positive patients and associated with the progression of HIV-associated disease. To date, there is only a single case report describing M. penetrans as the causative agent of a severe respiratory tract infection in a HIV-negative patient. Case presentation In this report, we describe the case of M. penetrans bacteremia in a HIV-negative, 38-year-old, female, immunocompromised, solid organ transplant patient (combined kidney and pancreas transplantation in 2016), who was admitted to our hospital with anemic uterine bleeding and fever of 38.3 °C. Several hours before her admission at our university hospital, a latex bladder catheter was inserted into her uterus and she complained about fatigue, dizziness and ongoing vaginal bleeding. Laboratory examination showed severe anemia, but microbiological examination was inconspicuous (culture negative vaginal and cervical smears, negative urine culture). Bacterial blood cultures showed a growth signal after 4 h, but microscopic examination with Gram staining and subcultures on different agar media did not identify bacterial pathogens. To identify the bacterial cause of malignancy in the patient, metagenomic sequencing of the blood culture was performed that identified M. penetrans. Conclusion Metagenomic sequencing identified M. penetrans in an immunosuppressed patient with culture-negative bacteremia. Clinicians should be aware of the opportunistic potential of M. penetrans that may cause severe infections in certain vulnerable patient populations and the limitations of culture and Gram staining for confirming the presence of fastidious bacterial pathogens like Mycoplasma spp

Antimicrobial susceptibility testing is crucial when treating Finegoldia magna infections

Finegoldia magna is an anaerobic gram-positive bacterium that can cause invasive human infections. Recently, a 52-year-old patient suffering from a periprosthetic joint infection (PJI) due to F. magna was treated with cefepime on hemodialysis; however, treatment failed due to relapse caused by antibiotic-resistant strains. Reports on the antimicrobial susceptibility of F. magna clinical isolates are rare. We collected 57 clinical F. magna isolates from Zurich, Switzerland, between September 2019 and July 2020 and tested their antimicrobial susceptibility to investigate the local resistance pattern. Antimicrobial susceptibility testing (AST) was evaluated for nine antibiotics (benzylpenicillin, amoxicillin/clavulanic acid, cefuroxime, cefepime, levofloxacin, rifampicin, metronidazole, doxycycline, and clindamycin) by E-test according to CLSI guidelines. All F. magna strains were susceptible to benzylpenicillin, amoxicillin/clavulanic acid, and metronidazole, while 75% to clindamycin. F. magna isolates showed MIC values lower than species-unrelated breakpoints for cefuroxime, levofloxacin, and cefepime in 93%, 56%, and 32% of the cases, respectively. MIC values for rifampicin and doxycycline were lower than locally determined ECOFFs in 98% and 72% of the cases, respectively. In summary, we recommend the use of benzylpenicillin, amoxicillin/clavulanic acid, or metronidazole without prior AST as first-line treatment option against F. magna PJI infections. If cefuroxime, cefepime, levofloxacin, rifampicin, doxycycline, or clindamycin are used, AST is mandatory. Keywords: Antimicrobial susceptibility; Cefepime; Finegoldia magna; Periprosthetic joint infectio

A retrospective analysis of blood culture-negative endocarditis at a tertiary care centre in Switzerland

AIMS OF THE STUDY Numerous studies from different countries have contributed to an improved understanding of blood culture-negative infective endocarditis. However, little is known about its epidemiology and microbiology in Switzerland. We aimed to assess the epidemiology and microbiology of blood culture-negative endocarditis at the University Hospital Zurich, Switzerland. METHODS We screened all patients hospitalised between 1997 and 2020 with possible or definite endocarditis at our institution. Thereof, we identified all cases with blood culture-negative endocarditis and retrospectively retrieved patient characteristics, microbiological, histopathological, radiographic and surgical data from medical records. RESULTS Among 861 patients screened, 66 (7.7%) cases of blood culture-negative endocarditis were identified. Thereof, 31 cases could be microbiologically documented or not documented (n = 30), and in five cases a non-infectious aetiology was confirmed. Endocarditis predominantly affected men (77%) and the left heart (79%); predisposing factors were prosthetic valves (42%), congenital heart disease (35%) and prior endocarditis (14%). The most common reasons for negative blood cultures were antibiotic treatment prior to blood culture sampling (35%), fastidious and slow growing microorganisms (30%) and definite non-infective endocarditis (8%). Coxiella burnetii and Bartonella spp. were the most common fastidious bacteria identified. In addition to serology, identification of causative microorganisms was possible by microbiological and/or histopathological analysis of tissue samples, of which polymerase chain reaction testing (PCR) of the 16S ribosomal RNA proved to be most successful. CONCLUSIONS The present study provides a detailed analysis of blood culture-negative endocarditis over a time span of more than 20 years in Zurich, Switzerland. Antibiotic treatment prior to blood collection, and fastidious and slow growing organisms were identified as main reasons for sterile blood cultures. Typical culture-negative bacteria were mainly found by PCR and/or culture of tissue samples

Broad Range Eubacterial Polymerase Chain Reaction of Cerebrospinal Fluid Reduces the Time to Exclusion of and Costs Associated with Ventriculostomy-Related Infection in Hemorrhagic Stroke

BACKGROUND: Patients with hemorrhagic stroke and an external ventricular drain in situ are at risk for ventriculostomy-related-infections (VRI). Because of the contamination of the cerebrospinal fluid (CSF) with blood and the high frequency of false negative CSF culture, the diagnosis of VRI remains challenging. This study investigated the introduction of CSF broad range eubacterial polymerase chain reaction (ePCR) and its effect on frequency and duration of antibiotic therapy for VRI, neurocritical care unit (NCCU) length of stay, related costs, and outcome. METHODS: Between 2020 and 2022, we prospectively included 193 patients admitted to the NCCU of the University Hospital of Zürich with hemorrhagic stroke and an external ventricular drain for more than 48 h. Patient characteristics, serum inflammatory markers, white blood cell count in CSF, use and duration of antibiotic treatment for VRI, microbiological findings (CSF cultures and ePCR tests), and NCCU length of stay were compared in patients with no infection, noncerebral infection, suspected VRI, and confirmed VRI. Data of patients with suspected VRI of this cohort were compared with a retrospective cohort of patients with suspected VRI treated at our NCCU before the introduction of CSF ePCR testing (2013-2019). RESULTS: Out of 193 patients, 12 (6%) were diagnosed with a confirmed VRI, 66 (34%) with suspected VRI, 90 (47%) with a noncerebral infection, and 25 (13%) had no infection at all. Compared with the retrospective cohort of patients, the use of CSF ePCR resulted in a reduction of patients treated for suspected VRI for the whole duration of 14 days (from 51 to 11%). Furthermore, compared with the retrospective group of patients with suspected VRI (n = 67), after the introduction of CSF ePCR, patients with suspected VRI had shorter antibiotic treatment duration of almost 10 days and, hence, lower related costs with comparable outcome at 3 months. CONCLUSIONS: The use of CSF ePCR to identify VRI resulted in shorter antibiotic treatment duration without changing the outcome, as compared with a retrospective cohort of patients with suspected VRI

Comparison of Disk Diffusion, E-Test, and Broth Microdilution Methods for Testing In Vitro Activity of Cefiderocol in Acinetobacter baumannii

The reference method for cefiderocol antimicrobial susceptibility testing is broth microdilution (BMD) with iron-depleted-Mueller-Hinton (ID-MH) medium, whereas breakpoints recommended for disk diffusion (DD) are based on MH-agar plates. We aimed to compare the performance of the commercial BMD tests ComASP (Liofilchem) and UMIC (Bruker), and DD and E-test using MH- and ID-MH-agar plates with the reference BMD method using 100 carbapenem-resistant-A. baumannii isolates. Standard BMD was performed according to the EUCAST guidelines; DD and E-test were carried out using two commercial MH-agar plates (BioMérieux and Liofilchem) and an in-house ID-MH-agar plate, while ComASP and UMIC were performed according to the manufacturer's guidelines. DD performed with the ID-MH-agar plates led to a higher categorical agreement (CA, 95.1%) with standard BMD and fewer categorization errors compared to the commercial MH-agar plates (CA BioMérieux 91.1%, Liofilchem 89.2%). E-test on ID-MH-agar plates exhibited a significantly higher essential agreement (EA, 75%) with standard BMD compared to the two MH-agar plates (EA BioMérieux 57%, Liofilchem 44%), and showed a higher performance in detecting high-level resistance than ComASP and UMIC (mean log2 difference with standard BMD for resistant isolates of 0.5, 2.83, and 2.08, respectively). In conclusion, DD and E-test on ID-MH-agar plates exhibit a higher diagnostic performance than on MH-agar plates and the commercial BMD methods. Therefore, we recommend using ID-MH-agar plates for cefiderocol susceptibility testing of A. baumannii

High Rates of Asymptomatic Mycoplasma genitalium Infections With High Proportion of Genotypic Resistance to First-Line Macrolide Treatment Among Men Who Have Sex With Men Enrolled in the Zurich Primary HIV Infection Study

Background Mycoplasma genitalium (Mg) is an emerging sexually transmitted pathogen among men who have sex with men (MSM). Resistance to recommended antimicrobial agents are of public health concern. Few data exist on Mg infections in MSM diagnosed with human immunodeficiency virus (HIV) during primary HIV infection. Methods Participants of the Zurich Primary HIV Study (ClinicalTrials.gov Identifier NCT00537966) were systematically offered screening for sexually transmitted infections (STIs) between April 2019 and September 2020. Screening was performed using an in-house polymerase chain reaction panel comprising Mg including genotypic resistance testing for macrolides and quinolones, Chlamydia trachomatis including serovars L1-L3, Neisseria gonorrhoeae, Treponema pallidum, and Hemophilus ducreyi. Results We screened 148 of 266 (55.6%) participants, with an overall total of 415 follow-up visits. Ninety-one percent were MSM. The incidence rate for all STIs was 47.0 (95% confidence interval [CI], 32.2-68.6) per 100 person-years. Mycoplasma genitalium was the most frequently detected pathogen: 30 participants (20%) presented with at least 1 Mg infection, corresponding to a period prevalence of 20.3% and incidence rate of 19.5 Mg infections (95% CI, 11.8-32.4). Most Mg infections (93%) were asymptomatic, and 9 (30%) participants showed spontaneous clearance. We detected high rates of antibiotic resistance: 73.3% to macrolides, 3.3% to quinolones, and 13.3% resistance to both antibiotics. Conclusions The high prevalence of mostly asymptomatic Mg infections and high rate of spontaneous clearance support cautious initiation for treatment. The high proportion of macrolide-resistant strains suggests that a genotypic determination of resistance should be standard of care. Moxifloxacin should be the preferred treatment option for symptomatic Mg infections among MSM if resistance testing is unavailable

Zoonotic Chlamydiae as rare causes of severe pneumonia

Zoonotic species of the Chlamydiaceae family should be considered as rare pathogenic agents of severe atypical pneumonia. A fatal case of a severe pneumonia due to Chlamydia psittaci was traced back to pet birds, and pneumonia in a pregnant woman was attributed to abortions in a sheep and goat flock, being the source of Chlamydia abortus. The two SARS‑CoV‑2-negative pneumonia cases presented here were investigated in an inter-disciplinary approach involving physicians and veterinarians. State-of-art molecular methods allowed the identification and genotyping of zoonotic Chlamydiae