Proteus mirabilis

The HiSeq and MiSeq metagenomes were built using 20 sets of bacterial whole-genome shotgun reads. These reads were found either as part of the GAGE-B project [21 (link)] or in the NCBI Sequence Read Archive. Each metagenome contains sequences from ten genomes (Additional file 1: Table S1). For both the 10,000 and 10 million read samples of each of these metagenomes, 10% of their sequences were selected from each of the ten component genome data sets (i.e., each genome had equal sequence abundance). All sequences were trimmed to remove low quality bases and adapter sequences.
The composition of these two metagenomes poses certain challenges to our classifiers. For example, Pelosinus fermentans, found in our HiSeq metagenome, cannot be correctly identified at the genus level by Kraken (or any of the other previously described classifiers), because there are no Pelosinus genomes in the RefSeq complete genomes database; however, there are seven such genomes in Kraken-GB’s database, including six strains of P. fermentans. Similarly, in our MiSeq metagenome, Proteus vulgaris is often classified incorrectly at the genus level because the only Proteus genome in Kraken’s database is a single Proteus mirabilis genome. Five more Proteus genomes are present in Kraken-GB’s database, allowing Kraken-GB to classify reads better from that genus. In addition, the MiSeq metagenome contains five genomes from the Enterobacteriaceae family (Citrobacter, Enterobacter, Klebsiella, Proteus and Salmonella). The high sequence similarity between the genera in this family can make distinguishing between genera difficult for any classifier.
The simBA-5 metagenome was created by simulating reads from the set of complete bacterial and archaeal genomes in RefSeq. Replicons from those genomes were used if they were associated with a taxon that had an entry associated with the genus rank, resulting in a set of replicons from 607 genera. We then used the Mason read simulator [22 ] with its Illumina model to produce 10 million 100-bp reads from these genomes. First we created simulated genomes for each species, using a SNP rate of 0.1% and an indel rate of 0.1% (both default parameters), from which we generated the reads. For the simulated reads, we multiplied the default mismatch and indel rates by five, resulting in an average mismatch rate of 2% (ranging from 1% at the beginning of reads to 6% at the ends) and an indel rate of 1% (0.5% insertion probability and 0.5% deletion probability). For the simBA-5 metagenome, the 10,000 read set was generated from a random sample of the 10 million read set.

The following bacterial species were used: Bacillus subtilis (ATCC 10707), Enterobacter cloacae (human isolate), Escherichia coli (ATCC 0157:H7), Micrococcus flavus (ATCC 9341),Proteus mirabilis (human isolate), Pseudomonas aeruginosa (ATCC 27853), Salmonella enteritidis (ATCC 13076), S. epidermidis (ATCC 12228) S. typhimurium (ATCC 13311) Staphylococcus aureus (ATCC 25923). The antibacterial assays were carried out by the disc-diffusion [25 (link)] and microdilution method [26 ,27 (link),28 (link)] in order to determine the antibacterial activity of oils and their components against the human pathogenic bacteria. The bacterial suspensions were adjusted with sterile saline to a concentration of 1.0 × 10⁵ CFU/mL. The inocula were prepared daily and stored at +4 °C until use. Dilutions of the inocula were cultured on solid medium to verify the absence of contamination and to check the validity of the inoculum.

This was a retrospective cross-sectional study of antimicrobial susceptibility of all nonduplicate (first isolate in 30 days) Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp. isolates from ICU and non-ICU patients collected from January 1, 2017, to December 31, 2017. Reporting institutions comprised 358 US hospitals included in the BD Insights Research Database (Becton, Dickinson and Company, Franklin Lakes, NJ). The electronic surveillance system and clinical research database (formerly the CareFusion Clinical Research Database) have been previously described [8–10 (link)]. This database provides good geographical representation across the United States and includes both small and large hospitals in urban and rural areas.
The analyses reported here include all nonduplicate Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp. (A. baumannii and A. haemolyticus) isolates from blood, respiratory, urine, skin/wound, intraabdominal, and other sources. Isolates from each source were considered separately; for example, if the patient had a blood and respiratory isolate for P. aeruginosa within 30 days, then an isolate was counted for each source. Isolates from the same patient within 30 days were included if they had different drug susceptibilities (>1 susceptibility difference). Isolates were classified as Carb-NS based on facility reports of intermediate susceptibility or resistance to at least 1 of the following agents: (a) ertapenem, imipenem, meropenem, or doripenem for Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, and Citrobacter freundii; (b) ertapenem, meropenem, or doripenem for Proteus mirabilis and Morganella morganii; and (c) imipenem, meropenem, or doripenem for P. aeruginosa and Acinetobacter spp.
Care settings were classified using the Centers for Disease Control and Prevention (CDC) National Healthcare Safety Network classification and further classified as ICU (critical care) and non-ICU (inpatient adult wards, specialty care areas, and step-down wards). Hospital-onset isolates were defined as those occurring >3 days after inpatient admission or within 14 days of previous discharge, whereas admission isolates were defined as those occurring ≤3 days of inpatient admission with no previous admission within the past 14 days. Admission period isolates were classified as ICU-associated if the isolate was collected in the admission period and the patient was admitted to an ICU within 3 days of inpatient admission, and they were classified as non-ICU-associated if the isolate was collected in the admission period and the patient was admitted to a non-ICU location within 3 days of inpatient admission and did not have an ICU admission within 3 days of the inpatient admission. Hospital-onset isolates were classified as ICU-associated if the patient was admitted to an ICU on the specimen collection date and as non-ICU-associated if the patient was admitted to a non-ICU location on the specimen collection date with no ICU admission on that date. The study was approved by the New England Institutional Review Board (Wellesley, MA).

The nutrient-deficient medium with phenol red was used to perform the bacterial urease activity test. The overnight cultures of different isolates were centrifuged at 4,000 rpm and washed with PBS to remove the secreted urease. Bacteria were resuspended in the nutrient-deficient medium, with or without urea supplementation, and diluted to a final OD of 0.03–0.04. The urease activity was measured by continuously monitoring the OD at 560 and 415 nm, every 30 min, on the Micro-ELISA autoreader. Proteus mirabilis and E. coli were used as urease-positive and -negative controls, respectively.

We conducted a retrospective, laboratory-based, multi-center study referring oncological patients suffering from UTI from January 2015 to December 2019 in Beijing, China. A total of three cancer hospitals participated in the study, including Cancer Hospital, Chinese Academy of Medical Sciences (CAMS), Beijing Chaoyang Sanhuan (SH) Cancer Hospital, and Cancer Hospital of Huanxing (HX) Chaoyang District Beijing. Cancer Hospital, CAMS served as a major tertiary-level referral center and clinic hospital for cancer patients in China. All three hospitals focused on the treatment of solid tumors and lymphoma, and none of them set up a hematological malignancy department other than lymphoma. All urine specimens were transported to the central laboratory (Department of Clinical Microbiology Laboratory, Cancer Hospital, CAMS) within two hours of collection for microbial culture. There are regular shuttle buses between the three hospitals, which can be driven in less than 10 min. The complete medical record was inquired from 2015 to 2019. The prevalence of ESBL UTI was observed for 5 years. Cancer patients older than 18 years who had underlying E. coli, K. pneumoniae, K. oxytoca or P. mirabilis related UTI were included in this study. Benign tumor cases, multi-bacteria UTI cases, episodes without antimicrobial susceptibility results, or complete case information were excluded from this study.