key: cord-0898171-0cb3iykb
authors: Zhou, Hong; Li, Cixiu; Hu, Tao; Liu, Ti; Ni, Nan; Chen, Weijun; Zhao, Huailong; Ruan, Shiman; Li, Juan; Wu, Honglong; Holmes, Edward C.; Kang, Dianmin; Hou, Peiqiang; Shi, Weifeng
title: Total Infectomes of 162 SARS-CoV-2 Cases Using Meta-Transcriptomic Sequencing
date: 2020-12-08
journal: J Infect
DOI: 10.1016/j.jinf.2020.12.004
sha: 5a3a56eca1a1163712a9f1a7a0685d13ad59e418
doc_id: 898171
cord_uid: 0cb3iykb

nan

Hong Zhou 1, 9 , Cixiu Li 1,9 , Tao Hu 1,9 , Ti Liu 2,9 , Nan Ni 3,9 , Weijun Chen 4,9 , Huailong Zhao 5 , Shiman Ruan 5 , Juan Li 1 , Honglong Wu 4 , Edward C. Holmes 6 sequencing reads after mapping to the human genome were compared against the non-redundant nucleotide (nt) database using blastn to identify potential viruses. For a broader microbe discovery, we utilized MetaPhlan2 7 , which covers ~1 million unique clade-specific marker genes from bacterial, archaeal and eukaryotic reference genomes.

Reads mapping to SARS-CoV-2 were found in all samples, with the absolute read numbers from 14 to 115,299,054, and the number of Reads Per Million (RPM) ranging from 0.07 to 717538.52. Overall, 82 out of the 162 SARS-CoV-2 cases (50.62%) were co-infected by at least one additional potentially pathogenic microbe (Table 1) . Among these, 42 cases (25.93%) were co-infected with one pathogen, including viruses (n=9), bacteria (n=33). The remaining 40 cases (24.69%) were co-infected with two or more pathogens, including multiple virus co-infection (n=1), virus and bacteria co-infections (n=4), virus, bacteria and 3 fungi co-infections (n=1), and multiple bacterial co-infections (n=34). Meanwhile, the abundance of SARS-CoV-2 was lower in samples co-infected with at least one microbe (P<0.05) compared to that of the samples without co-infections (Fig S1c) . Further analysis also showed a significantly lower abundance of SARS-CoV-2 in samples with Streptococcus pneumoniae, Haemophilus parainfluenzae and Neisseria meningitidis (P<0.05) (Fig S2a, 2d,   2f ). However, we did not find a positive correlation between associated factors (age or sex of the patients, variety or abundance of the co-infected microbes) and the abundance of SARS-CoV-2 (Fig S1a-b and Fig S2g-l) .

We identified 7 viruses with potential pathogenicity in 15 of the 162 (9.26%) COVID-19 cases. Human alphaherpesvirus 2 was the most frequently detected virus (n=6), followed by Human H3N2 influenza virus (n=3), Human coronavirus 229E (n=2), Human metapneumovirus (n=2), and Human coronavirus NL63 (n=1) (Fig 1a, Table S1 ). In addition, two rare respiratory viruses, Human rhinovirus C11 (n=1, RPM: 6.84) first reported in 2020 and Human enterovirus C105 (n=1, RPM: 6969.31) first reported in 2019 in China, were also identified (Fig 1a, Table S1 ), sharing 91% and 96% nucleotide identity to known reference viruses, respectively. Human enterovirus C105 has been associated with acute flaccid paralysis in children and respiratory tract infection in teenagers 8 . Our results revealed a relative low co-infection rate of other respiratory viruses with SARS-CoV-2: 9.26% (15/162) versus 20.7% (24/116) in a previous report 9 .

High rates of co-infection of common, but important pathogenic or opportunistic bacteria were also detected (Fig 1a) , including Streptococcus pneumoniae (n=37), Stenotrophomonas maltophilia (n=31), Pseudomonas putida (n=21), Haemophilus parainfluenzae (n=19), Haemophilus influenzae (n=14), Neisseria meningitidis (n=11), Moraxella catarrhalis (n=3), Streptococcus pyogenes (n=1), Streptococcus epidermidis (n=1), as well as two species of mycoplasma: Mycoplasma hyorhinis (n=3) and Mycoplasma pneumoniae (n=1) (Fig 1a) .

Importantly, all bacterial infections were confirmed by PCR using species-specific primers.

As a commensal microbe in the respiratory tract of pigs and rarely seen in humans, Mycoplasma hyorhinis was unexpected identified in three cases, although it was present at a relatively low abundance (39-79 RPM). Overall, 72 of the 162 SARS-CoV-2 patients 4 (44.44%) possessed at least one additional bacterium. One species of fungi, Candida albicans (n=1, RPM: 600) was identified (Fig 1a) . Our results highlighted more potential co-infections with bacteria (44.44%, 72/162) than with viruses (9.26%, 15/162) or fungi (0.62, 1/162), in contrast to a recent study 10 .

We further analyzed the expression of 52 unique antibiotic resistance genes (ARGs) associated with phenotypic resistance to seven classes of antibiotics: aminoglycoside, nitroimidazole, sulphonamide, phenicol, tetracycline, beta-lactam and macrolide (Figure 1b) . sulphonamide (24/82) (Fig 1b) . Unsurprisingly, a greater diversity of resistance genes was identified in samples with more bacteria (P<0.05) (Fig S1d) .

In sum, our mNGS analysis did not reveal high co-infection rates of other respiratory viruses (such as influenza viruses), mycoplasma and fungi with SARS-CoV-2. However, we did document high rates of co-infection between SARS-CoV-2 and multiple bacteria with numerous ARGs. Although the potential influence of co-infections of SARS-CoV-2 and other microbes in disease progress and severity remains poorly understood, the treatment of bacterial infection might provide clinical benefit for COVID-19 in cases where therapeutics are available. 

The authors declare no conflicts of interest. 

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This work was supported by Key research and development project of Shandong province