A patient with community-acquired pneumonia was admitted to a designated hospital in Changde, Hunan, China, on December 13, 2019, and an alert was triggered twice due to two waves of high fever on December 20 and December 25–30, 2019. One week later, while caring for his father, the patient’s 30-year-old son also had a high fever. After treatment with doxycycline for 11 days, both his high fever and breathing difficulty were under control ( Supplementary Figure S1 ). Finally, he was discharged from the local hospital before the COVID-19 outbreak in Wuhan, approximately 260 km away from his hometown. During his hospital admission, samples were collected with his family’s consent ( Supplementary Table S1 ). Moreover, swabs, sera, and an alveolar wash were collected from his relatives and close contacts ( Supplementary Table S2 ). Before collecting any clinical samples, signed informed consent was sought from possible participants, including family members, the attending physician, and the nurses. Also, the study was approved by the institutional review board (IRB) of China Agricultural University. The collected throat swabs were kept in sucrose-phosphate-glutamate (SPG) solution at 4°C, while the sera were stored at −20°C for further testing.

All serum samples were detected using a commercial antibody kit (Lizhu Biotech Co., Ltd., Shenzhen, China), and RNA samples were analyzed in accordance with the qPCR protocol recommended by the WHO (Corman et al., 2020) using COVID-19 primers ( Supplementary Table S3 ).

Genomic sequences were amplified for Chlamydia tests using specific primers ( Table 1 ). The Chlamydia genus was determined using the following: 2× super mix 12.5 µL, 10 µM Chlamydia forward primer 1.5 µL, 10 µM reverse primer 1.5 µL, and 0.5–1 µg DNA. The PCR conditions were conducted as follows: 95°C, 5 min, denaturing at 95°C for 30 s, annealing at 56°C for 30 s, and extending at 72°C for 30 s. The cycle was repeated 30 times, and the final reaction was incubated at 72°C for 10 min. Whole-genome sequencing was performed by a commercial institute (Beijing Genomics Institution, BGI, Beijing, China). For C. psittaci detection, the ompA gene was determined using qPCR (Applied Biosystems™ 7500, Thermo Fisher, Beijing, China) as described previously (Fang et al., 2021). The qPCR reagents included 5 µL SYBR Reaction Mix (2×) (TransGen Biotech, Beijing, China), 1 µL forward primer (2.5 μM), 1 µL reverse primer (2.5 μM), 1 µL template DNA, and 2 µL ddH₂O. qPCR was performed as follows: 50°C for 10 min, 95°C for 5 min, 95°C for 10 s, 60°C for 30 s, 45 cycles of 95°C for 1 min, 55°C for 1 min, and 55°C–95°C (0.5°C increment) for 10 s. The ompA gene of C. psittaci, approximately 1,209 bp, was amplified as described previously (Fang et al., 2021). It was subjected to electrophoresis in a 1% (w/v) agarose gel. The PCR product was sequenced by a commercial company (Tsingke Biotechnology Co., Ltd., Beijing, China).

The throat swabs collected from the patient and his relatives were mixed in 900 μL phosphate buffered saline (PBS) containing 2,000 IU/mL gentamicin and 2,000 IU/mL streptomycin (Solarbio Science & Technology Co., Ltd., Beijing, China). After incubation for 30 min at 37°C and centrifugation at 500 × g for 5 min at 4°C, the supernatant (0.4 mL) was collected and injected into 7-day-old specific pathogen free (SPF) embryonated chicken eggs at 0.2 mL per egg. Afterward, the embryonated chicken eggs were incubated at 37°C for 1 week, and the embryonated chicken eggs were monitored twice per day. The second passage was carried out to observe the pathogenicity using the yolk membranes (Zhang et al., 2009). The typical inclusion bodies were monitored using the IMAGEN™ Chlamydia kit (Thermo Scientific, Beijing, China), and specific species of C. psittaci was verified using the aforementioned qPCR. For antibody detection, C. psittaci-specific human antibodies were detected using SeroFIA™ C. psittaci immunofluorescence detection assay (Savyon Diagnostics Ltd., Ashdod, Israel) following the manufacturer’s instructions (Ling et al., 2015).

DNA samples were extracted from the positive E. faecalis isolates using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The following specific primers were used in the study. The forward primer was 5′-GTACAGTTGCTTCAGGACGTATC-3′, and the reverse primer was 5′-ACGTTCGATTTCATCACGTTG-3′. A 197-bp fragment of the tuf gene was amplified and subjected to electrophoresis in a 1% (w/v) agarose gel. The PCR procedure comprised an initial incubation for 4 min at 95°C, 35 cycles for 60 s each at 95°C, annealing for 60 s at 59°C, and extension for 60 s at 72°C, with a final extension for 5 min at 72°C (Pan et al., 2012). The PCR product was sequenced, and then NCBI blast was used.

All throat swabs were grown on standard I nutrient agar (Merck, Darmstadt, Germany) with 5% sheep blood and incubated at 37°C for 24 h. The positive colonies were transferred into the Baird–Parker medium (Qingdao Hope Bio-Technology Co., Ltd., Shandong, China). Afterward, the typical colonies were identified using Gram staining and biochemical assays (Sigma-Aldrich, Beijing, China). The biochemical tests assayed for glucose, fructose, sucrose, maltose, lactose, galactose, mannose, mannitol, sorbitol, arabinose, inulin, urease, lysine decarboxylase, nitrate, gelatinase, motility, indole, 0.1% methylene blue milk, and growth on MacConkey agar in bouillon medium at pH 9.6, sodium hippurate medium, and 6.5% NaCl bouillon medium. Bacterial colony forming unit (CFU) was quantified on nutrient agar containing 5% sheep blood (Pan et al., 2012).

The animal test was designed in accordance with guidelines issued by the Institutional Animal Care and Use Committee (IACUC) and followed humane protocols to minimize animal pain. It was approved by the Ethics Review Committee of China Agricultural University (approval code: IACUC20191222). Forty C57BL/6 mice weighing 20–22 g were randomly assigned to five groups with eight animals per group ( Supplementary Table S4 ) and maintained in negative isolators in biosafety level 2 facilities. In the current study, Group 1 mice were infected intranasally with 0.1 mL of 1.0 × 10⁵ inclusion forming unit (IFU) of C. psittaci isolate alone. Group 2 mice were inoculated intranasally with 0.1 mL of 1.0 × 10⁷ CFU of E. faecalis isolate alone. Group 3 mice received intranasally 1.0 × 10⁷ CFU of E. faecalis and 0.1 mL of 1.0 × 10⁵ IFU of C. psittaci at the same time. Group 4 mice were first inoculated intranasally with 1.0 × 10⁵ IFU of C. psittaci isolate and then inoculated with 0.1 mL of 1.0 × 10⁷ CFU of E. faecalis through the same route after 3 days. This infection model was used to identify whether primary C. psittaci infection would aggravate E. faecalis-mediated respiratory distress. Group 5 mice received sterile 0.1 mL of PBS via the same route, serving as the healthy control. Mouse body weights were monitored weekly until the end of the experiment. After 14 days, the mice were anesthetized by intraperitoneal injection into the right lower quadrant of the abdomen with 2 mg ketamine and 0.2 mg xylazine (Jianglei Biotech Co., Ltd., Shanghai, China).

Lungs were harvested from each group of euthanized mice, examined for pathological lesions, and scored. Lung lesions were determined according to a previous description (Li et al., 2022). Briefly, for lungs, the grades were as follows: grade 0, lines normal; grade 1, 30% of the lung surface area showed hemorrhage, degeneration, or necrosis; grade 2, 60% of the lung surface area showed hemorrhage, degeneration, or necrosis; and grade 3, the entire lung surface showed hemorrhage, degeneration, or necrosis. For lung sections, the grades were as follows: grade 0, none; grade 1, slight edema of the alveolar walls; grade 2, moderate edematous thickening of alveolar walls with occasional alveoli containing coagulated edema fluid; and grade 3, extensive occurrence of alveolar and interstitial edema.

The mouse lungs were aseptically removed on day 14. Briefly, sterile lung tissues of eight mice from each group were taken and then minced, and 50 mg of lung tissues from each mouse and supernatant lung homogenates were obtained. The homogenates were stored at 4°C for 40 min and then centrifuged at 2,000 rpm/min for 5 min. The supernatant was used to determine the concentration of C. psittaci by qPCR (Fang et al., 2021). For E. faecalis determination, the samples from the lungs were inoculated on standard I nutrient agar (Merck, Germany) with 5% sheep blood and incubated at 37°C for 24–48 h. Finally, the number of bacterial colonies was measured.

Statistical significance was analyzed using one-way ANOVA with the least significant difference (LSD) post-hoc test. Data were expressed as the mean ± standard deviation. All data were calculated and analyzed using SPSS v26.0 (SPSS Inc., Chicago, IL, USA). Graphs were generated using the GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). Statistically significant differences were judged as p < 0.05.

After the patient’s hospital admission, his chest X-ray revealed bilateral pneumonia and ground-glass opacity, and he was prescribed oxygen therapy and doxycycline. Afterward, lesions with ground-glass opacity were reduced, but pulmonary consolidation was still observed in the lungs ( Supplementary Figure S2 ). Based on 23S rDNA amplification, a positive 172-bp band of C. psittaci was detected in the patient’s alveolar wash, while his tracheal mucosa and throat swabs were negative ( Figure 1A ). Other family members, the nurses, the attending physician, and the house chickens were negative for C. psittaci ( Figure 1B ). Afterward, qPCR results showed that the patient’s alveolar wash was positive for C. psittaci. Subsequently, positive C. psittaci was identified in the patient’s alveolar wash and throat swab, and his sons’ throat swab. However, a negative reaction was found in his tracheal mucosa and other close contacts’ samples ( Figure 2 ). The ompA gene sequence of the C. psittaci strain was determined and submitted to GenBank (accession number: OR616243). After inoculation into SPF embryonated eggs, typical intracellular inclusions were observed using immunofluorescence staining ( Figure 3 ).

For C. psittaci-specific antibodies, IgG antibody titers were arranged from 1:64 dilution on December 25 and increased to 1:128 dilution on December 27, 2019, in a time-course manner ( Figure 4 ). Compared to the patient’s sera, IgG titers were from 1:32 to 1:64 to 1:128, and the serum samples from other close contacts were negative in the test. Meanwhile, antibodies against COVID-19 were negative ( Table 2 ). Serum samples from the patient were also tested for antibodies against common respiratory pathogens, including influenza A/B virus, respiratory syncytial virus, C. pneumoniae, and M. pneumoniae. All results were negative.

E. faecalis was successfully isolated in the alveolar lavage and throat swab using single-colony purification. Subsequently, the isolate was classified using biochemical assays and conventional PCR, and E. faecalis isolates were found to be Gram-positive, chain-forming, coccus-shaped organisms upon microscopic inspection ( Figure 5A ). The isolated strains were determined as E. faecalis using PCR. The DNA extracted from the typical colony produced the expected 197-bp PCR product of the target gene ( Figure 5B ); the sequence of the tuf gene was submitted to GenBank (accession number: SUB15618628), and NCBI blast was used. A sequence analysis of the tuf segment showed that the sequence from this isolate was 98%–100% homologous to the E. faecalis reference strains (GenBank # EU156939 and AY266992).

Clinically, mice infected primarily with C. psittaci, followed by E. faecalis (C. psittaci/E. faecalis) inoculation or C. psittaci infection alone, showed ruffled feathers, inactivity, poor appetite, and low weight; especially severe signs were found in female mice compared to male mice. For body weights, those of the C. psittaci/E. faecalis group, C. psittaci alone group, and C. psittaci+E. faecalis group were lower 1 week later. More importantly, mouse body weights declined during the observation in the C. psittaci/E. faecalis group (p < 0.01). However, no decrease was found in the E. faecalis alone group and the control group ( Figure 6 ).

Postmortem, diffuse hemorrhagic lungs were observed in the mice with C. psittaci infection alone, while enlarged lung size was evident in the mice with E. faecalis infection ( Figure 7 ). For C. psittaci/E. faecalis group and the C. psittaci+E. faecalis group, atrophy, viscous exudation, and diffuse hemorrhage characterized severe lesions, while fibrinous consolidation developed in the C. psittaci+E. faecalis group ( Figure 7D ).

Under microscopic observation, severe consolidation lesions were observed in the C. psittaci+E. faecalis group and C. psittaci/E. faecalis group, diffuse hemorrhagic inflammation was evident in the lungs of the C. psittaci alone group, and no obvious lesions were found in the E. faecalis alone group ( Figure 8 ).

Regarding bacterial loads, high chlamydial loads were determined in the C. psittaci/E. faecalis group compared to the C. psittaci+E. faecalis group or C. psittaci alone group (p < 0.05) ( Figure 9A ). On the contrary, no significant difference in E. faecalis clearance was found between the C. psittaci/E. faecalis group and the C. psittaci+E. faecalis group ( Figure 9b ).