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Background Infections by Streptococcus gallolyticus subsp. pasteurianus (SGSP) is often underestimated. Herein, the epidemiological features and resistant characteristics of SGSP in mainland China are characterized to enable a better understanding of its role in clinical infections. Methods In the present work, 45 SGSP isolates were collected from the samples of bloodstream, urine, aseptic body fluid, and fetal membrane/placenta from patients in 8 tertiary general hospitals of 6 cities/provinces in China from 2011 to 2017. The identification of all isolates was performed using traditional biochemical methods, 16S rRNA and gyrB sequencing, followed by the characterization of their antibiotic resistance profiling and involved genes. Results Among 34 non-pregnancy-related patients, 4 (4/34,11.8%) patients had gastrointestinal cancer, 10 (10/34, 29.4%) patients had diabetes, and one patient had infective endocarditis. Moreover, 11 cases of pregnant women were associated with intrauterine infection (9/11, 81.2%) and urinary tract infection (1/11, 9.1%), respectively. Except one, all other SGSP isolates were correctly identified by the BD Phoenix automated system. We found that all SGSP isolates were phenotypically susceptible to penicillin, ampicillin, cefotaxime, meropenem, and vancomycin. Forty strains (40/45, 88.9%) were both erythromycin and clindamycin-resistant, belonging to the cMLSB phenotype, and the majority of them carried erm(B) gene (39/40, 97.5%). Although the cMLSB/erm(B) constituted the most frequently identified phenotype/genotype combination (25/40, 62.5%) among all erythromycin-resistant cMLSB isolates, erm(B)/erm(A), erm(B)/mef(A/E), and erm(B)/erm(T) was detected in 7, 4, and 3 isolates, respectively. Furthermore, 43 strains (43/45, 95.6%) were tetracycline-resistant, and out of these, 39 strains (39/45, 86.7%) carried tet(L), 27(27/45, 60.0%) strains carried tet(O), and 7 (7/45, 15.6%) strains carried tet(M), alone or combined, respectively. All erythromycin-resistant isolates were also resistant to tetracycline. Conclusions It is important to study and draw attention on SGSP, an underreported opportunistic pathogen targeting immunodeficient populations, notably elderly subjects, pregnant women and neonates.

Purpose Community-associated Staphylococcus aureus (CA S. aureus) is the most common causative pathogen of the skin and soft tissue infections (SSTIs). This study aims to determine clonal distribution, virulence factors of CA S. aureus clinical isolates from purulent SSTIs in Beijing, China. Materials and methods CA-S. aureus isolates were collected from 115 outpatients with purulent SSTIs from the department of dermatology from April 2015 to April 2017. Multilocus sequence typing and Staphylococcus cassette chromosome mec typing were performed to explore molecular characteristics. Phylogenetic analysis of 16S rRNA of dominant S. aureus isolates was performed using MEGA-X software. Virulence genes were detected by PCR, while biofilm formation was evaluated by a microtiter plate method. The antimicrobial susceptibility was tested by an automatic VITEK system. Results Forty-four CA-S. aureus isolates identified from SSTIs contain 9 methicillin-resistant S. aureus (MRSA) isolates (20.4%) and 35 methicillin-susceptible S. aureus isolates (MSSA) (79.6%). The dominant sequence types (STs) were ST22 (40.9%) and clonal complex 59 (CC59; 77.8%) in Community-associated methicillin resistant methicillin-resistant S. aureus. 27.8% of ST22 isolates were homologous to the epidemic ST22 EMRSA-15 in Europe. The prevalence of virulence genes lukS/lukF, tst-1, etA, edinA, icaA, and icaD was 50%, 93.2%, 4.5%, 4.5%, 100%, and 100%, respectively. All CC59 isolates exhibited stronger biofilm-forming capability than ST22 clones. Among the MSSA subgroup, the poor biofilm producers had significantly higher sensitivity to sulfamethoxazole/Trimethoprim. Conclusion The dominant epidemic clone PVL+ ST22 MSSA containing tst-1 occurs in Beijing, indicating that a PVL+ ST398 clone which was previously predominant in this district had been replaced by a new clone.

Nocardia spp. is a pathogen responsible for a variety of clinical infections, ranging from skin and soft tissue infections, to the respiratory tract and central nervous system infections. Its epidemiological characteristics, including species distribution, clinical features, and antimicrobial susceptibility profiles, should be under surveillance for the prevention and treatment of nocardiosis. In the present study, over a 9-year period (from 2009 to 2017), 53 non-repetitive Nocardia isolates were collected from 8 tertiary general hospitals of 7 cities in China. These isolates were identified to species level by multilocus sequence analysis(MLSA). The clinical data were also reviewed. The susceptibilities to 10 commonly-used antibiotics for Nocardia were determined by E-test stripes, and the resistance rates, MIC50 and MIC90 to each antibiotic by different species were analyzed. Of 53 Nocardia isolates, N. farcinica was the most common species (24.5%, 13/53), followed by N. cyriacigeorgica (20.8%, 11/53), N. terpenica (15.1%, 8/53), N. abscessus (9.43%, 5/53), N. otitidiscaviarum (7.55%, 4/53), respectively. Furthermore, 31 Nocardia (58.5%) isolates were recovered from lower respiratory tract (sputum and BALF), 15 (28.3%) from superficial Infection, 3 (5.7%) from pleural effusion, 2 (3.8%) from CSF, and 1 from bone marrow and 1 from synovial fluid, respectively. The antibiotic resistance profiles varied between different Nocardia species. All Nocardia isolates were susceptible to linezolid, followed by imipenem and amikacin (both 92.5% susceptibility rate). N. terpenica, rarely documented elsewhere, showed a different antimicrobial susceptibility profile. In summary, herein, the clinical and antibiotic resistance features of Nocardia species reported would be helpful for understanding the diversity of Nocardia species circulating in China and for decision making in the context of empiric therapy.

Burkholderia pseudomallei is the causative agent of melioidosis, endemic mainly in tropical and subtropical areas. Its clinical manifestation is broad ranging from a localized skin lesion to a life-threatening systemic disease. Osteomyelitis and septic arthritis caused by B. pseudomallei are a rare, fatal illness, whose clinical features have not been illustrated in mainland China. Over 10 years (2010 to 2019), of 334 culture-confirmed melioidosis in Hainan province, China, 44 patients (13.2%) were confirmed to have osteomyelitis and septic arthritis through the combination of clinical features, imaging examination and microbiological culture. Herein, we summarized these 44 patients’ clinical manifestations, demographical features, antibiotic treatment, and outcomes. Of them, osteomyelitis and septic arthritis accounted for 25 (56.8%) and 15 (34.1%), respectively, and 4 patients (9.1%) had both. The gender ratio of male/female was approximately 13.7:1; diabetes mellitus was the most common risk factor (38/44, 86.4%); imipenem and trimethoprim/sulfamethoxazole were the most frequently used antibiotics. Most B. pseudomallei strains were isolated from blood samples (41/44, 93.2%). After surgical handling, antibiotic treatment, or both, 9 patients died, with a mortality rate of 20.5%. In summary, in melioidosis endemic areas, for patients with both localized manifestations of joint and bone and a positive B. pseudomallei blood culture, increased awareness is required for melioidotic osteomyelitis and septic arthritis.

Background: The diagnosis of bacterial pathogens in lower respiratory tract infections (LRI) using conventional culture methods remains challenging and time-consuming. Methods: We enrolled 292 consecutively hospitalized patients suspected to have LRI between November 2018 and June 2019 in a single-center, prospective cohort study. Rapid clinical metagenomics test was performed on-site, and the results were compared with those of routine microbiology tests and evaluated for potential clinical benefit. Results: 171 bronchoalveolar lavage fluid (BAL) and 121 sputum samples were collected from patients with six kinds of LRI. The turn around-time (from sample registration to result) for the rapid metagenomics test was 6.4 ± 1.4 hours, compared to 94.8 ± 34.9 hours for routine culture. Compared with culture and real-time PCR tests, rapid metagenomics achieved 96.6% sensitivity and 88.0% specificity and identified pathogens in 63 out of 161 (39.1%) culture-negative samples. Correlation of anaerobes enrichment with lung abscess was demonstrated as 38 anaerobic species failed in culture but identified by metagenomics sequencing. The hypothetical impact of metagenomics test proposed antibiotic de-escalation in 34 patients compared to 1 using routine culture. Conclusions: Rapid clinical metagenomics test increased pathogen detection yield in the diagnosis of LRI. Empirical antimicrobial therapy could be de-escalated if rapid metagenomics test results were hypothetically applied to clinical management. Funding: The funder of the study had no role in study design, data collection,data analysis, data interpretation, or writing of the report. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. Declaration of Interest: None to declare Ethical Approval: The study was carried out in China-Japan Friendship Hospital, Beijing, China. Ethical approval was obtained from the China-Japan Friendship Hospital Ethics Committee(2018-145-k102). All subjects provided written consents.

Rapid pathogen detection is critical for prompt treatment, interrupting transmission routes, and decreasing morbidity and mortality. The V-type CRISPR system had been used for rapid pathogen detection. However, whether single-stranded DNA in CRISPR system can cause false positives remains undetermined. Herein, we show that high molar concentration of Cas12a effector tolerated more mismatches on ssDNA and activated its trans-cleavage activity at six base matches. Reducing Cas12a and crRNA molar concentration increased the minimal base-match number required for Cas12a ssDNA activation to 11, which reducing nonspecific activation. We then established a Cas12a-based M tuberculosis detection system with a primer having an 8 bp overlap with crRNA. This system did not exhibit primer-induced false positives, and minimum detection copy reached 1 copy/uL (inputting 1-μL sample) in standard strains. The Cas12a-based M tuberculosis detection system showed 80.0% sensitivity and 100.0% specificity in verification using clinical specimens, compared with Xpert MTB/RIF, which showed 72.0% sensitivity and 90.9% specificity. All these results prove that appropriate concentration of cas12a effector can effectively perform nucleic acid detection.

Few studies have described the key features and prognostic roles of lung microbiota in patients with severe community-acquired pneumonia (SCAP). We prospectively enrolled consecutive SCAP patients admitted to ICU. Bronchoscopy was performed at bedside within 48 h of ICU admission, and 16S rRNA gene sequencing was applied to the collected bronchoalveolar lavage fluid. The primary outcome was clinical improvements defined as a decrease of 2 categories and above on a 7-category ordinal scale within 14 days following bronchoscopy. Sixty-seven patients were included. Multivariable permutational multivariate analysis of variance found that positive bacteria lab test results had the strongest independent association with lung microbiota (R2 = 0.033; P = 0.018), followed by acute kidney injury (AKI; R2 = 0.032; P = 0.011) and plasma MIP-1β level (R2 = 0.027; P = 0.044). Random forest identified that the families Prevotellaceae, Moraxellaceae, and Staphylococcaceae were the biomarkers related to the positive bacteria lab test results. Multivariable Cox regression showed that the increase in α-diversity and the abundance of the families Prevotellaceae and Actinomycetaceae were associated with clinical improvements. The positive bacteria lab test results, AKI, and plasma MIP-1β level were associated with patients’ lung microbiota composition on ICU admission. The families Prevotellaceae and Actinomycetaceae on admission predicted clinical improvements. Electronic Supplementary Material Supplementary material is available in the online version of this article at 10.1007/s11684-021-0856-3 and is accessible for authorized users.

Background: Azithromycin, widely used in recent years, can change the airway microbiota in patients with chronic lung diseases. Little data exists regarding the effects of azithromycin administration on airway microbiota among healthy adults. Therefore, we conducted a randomized, double-blind, placebo-controlled trial to assess the process of variation and re-establishment of the airway microbiota after azithromycin exposure in healthy adults. Methods: Forty-eight healthy volunteers were enrolled and randomly assigned into two groups. 500mg azithromycin or placebo was administered once daily for 3 days. We collected the induced sputum at the day before the drugs administration (D0), the day after the treatment course was completed (D4), 14 days, 30 days and 60 days post-dosing. 16S rRNA gene sequencing and quantification were applied to the induced sputum samples. We collected the environmental information including air quality data [particulate matter (PM 2.5 ) and PM 10 , air quality index (AQI) values] that might have an influence on the airway microbiota during the study. The subjects’ respiratory tract infection (RTI) events during sampling were recorded. Results: Azithromycin didn’t alter bacterial load but significantly reduced species richness and Shannon index. Azithromycin exposure resulted in decrease in the detection rate and relative abundance of different families belonging to Veillonellaceae , Pasteurellaceae , Leptotrichiaceae , Neisseriaceae and Fusobacteriaceae . By contrast, the relative abundance of taxa belonging to Streptococcus increased immediately after azithromycin intervention. The shifts in the microbial community composition require about 14 days to recover while alpha-diversity recovered later. The high concentration of PM 2.5 contributed to a novel variability in microbial community composition of azithromycin group at D30 (30 days after baseline). The network analysis found that azithromycin altered the microbial interactions within airway microbiota. The influence was still obvious at D14 when the relative abundance of most taxa had returned to the baseline level. Conclusions: Azithromycin has transient effect in airway microbiota of healthy adults and decreases the ability of the airway microbiota resilient from PM 2.5 stress. The influence of azithromycin on microbial interactions is noteworthy though the airway microbiota has returned to the near-baseline level.Registration: Chinese Clinical Trial Registry (ChiCTR1800018494), September, 2018. http://www.chictr.org.cn/edit.aspx?pid=31269&htm=4

Background: The presentation of psittacosis can vary from subclinical infection to fatal pneumonia with a high mortality rate. Chlamydia psittaci infection during pregnancy is rare and might result in placental involvement, premature delivery or miscarriage. Herein, we report a case of severe pneumonia in a pregnant woman caused by C. psittaci.Case presentation: A 27-year-old female with a pregnancy at 22 weeks was admitted with hyperpyrexia, dry cough and dyspnea. Laboratory tests showed increased white blood cell count and slightly-elevated procalcitonin. Chest computed tomography scan revealed consolidation in the lingual and inferior lobe of the left lung. She suffered rapidly progressing respiratory failure and required mechanical ventilation and extracorporeal membrane oxygenation (ECMO). Her sample of bronchoalveolar lavage fluid (BALF) was collected, from which C. psittaci was detected using next-generation sequencing (NGS). The pathogen was further confirmed by polymerase chain reaction (PCR) with the primers specific for the microorganism. Moreover, paired serum tests showed elevated C. psittaci group titer in both IgG and IgM. She was treated with azithromycin and tigecycline. After 24-day hospitalization, the patient was clinically well and discharged home.Conclusion: We report a case of severe pneumonia in a pregnant woman caused by C. psittaci, which diagnosed by NGS, and was cured without adverse effects on infant with tigecycline. The introduction of NGS and other effective assays might increase the detection rate. C. psittaci infection in pregnant women is very rare, but critical. Early diagnosis and correct treatment may save the mother and fetus. New tetracycline agent, tigecycline, may also be an effective alternative and should be further evaluated in psittacosis.

Background: The pharmacokinetics and appropriate dose regimens of favipiravir are unknown in hospitalized influenza patients; such data are also needed to determine dosage selection for favipiravir trials in COVID-19. Methods: In this dose-escalating study, favipiravir pharmacokinetics and tolerability were assessed in critically ill influenza patients. Participants received one of two dosing regimens; Japan licensed dose (1600 mg BID on day 1 and 600 mg BID on the following days) and the higher dose (1800 mg/800 mg BID) trialed in uncomplicated influenza. The primary pharmacokinetic endpoint was the proportion of patients with a minimum observed plasma trough concentration (Ctrough) ≥20 mg/L at all measured time points after the second dose. Results: Sixteen patients were enrolled into the low dose group and 19 patients into the high dose group of the study. Favipiravir Ctrough decreased significantly over time in both groups (p 80% of the duration of treatment of the two dose regimens evaluated (18.8% and 42.1% of patients for low and high dose regimen, respectively). Increasing the favipravir dosage predicted a higher proportion of patients reaching this threshold of 20 mg/L, suggesting that dosing regimens of ≥3600/2600 mg might be required for adequate concentrations. The two dosing regimens were well-tolerated in critical ill patients with influenza. Conclusion: The two dosing regimens proposed for uncomplicated influenza did not achieve our pre-defined treatment threshold.

Background: Few studies have described the key features of the lung microbiota in patients with severe community-acquired pneumonia (SCAP). We conducted the study to identify the association between the lung microbiota on admission and the clinical prognosis in SCAP patients. Methods: The consecutive SCAP patients admitted from intensive care unit (ICU) were enrolled prospectively. 16S rRNA gene sequencing was applied to bronchoalveolar lavage fluid (BALF) collected within 48 hours. The clinical information was recorded during the stay of hospitalization. The primary endpoint was the rate of clinical improvements defined as a decrease of 2 categories and above on a 7-category ordinal scale within 14 days following bronchoscopy.Results: Sixty-seven patients were included. Multivariable Permutational multivariate analysis of variance (PERMANOVA) found that bacterial detection had the strongest independent relationship with the lung microbiota (R2=0.033; p=0.018), followed by acute kidney injury (AKI R2=0.032; p=0.011) and plasma MIP-1beta level (R2=0.027; p=0.044). Random forest identified that families Prevotellaceae, Moraxellaceae, Staphylococcaceae were the biomarkers related to bacterial detection. In the patients with positive bacteriology results, the mean relative abundance of families Prevotellaceae and Actinomycetaceae decreased while families Moraxellaceae, Staphylococcaceae and Streptococcaceae increased. Multivariable Cox regression showed that the increase in alpha-diversity and the abundance of families Prevotellaceae and Actinomycetaceae were associated with clinical improvements.Conclusions: The bacterial detection and patients’ intrinsic factors were associated with the lung microbiota. The increased alpha diversity and the enrichment of families Prevotellaceae and Actinomycetaceae in the lung microbiota were associated with clinical improvements.

With the introduction of molecular diagnostic techniques over the past decades, different kinds of viral pathogens in the same sample are detected simultaneously more frequently. Nevertheless, influenza virus (Flu) and respiratory syncytial virus (RSV) coinfection in adults was reported only occasionally. Moreover, the clinical implications of Flu/RSV coinfection in the respiratory tract of adults remain unclear.This retrospective study analyzed adult patients with acute respiratory infection from January 2017 to June 2019 in China-Japan Friendship Hospital.A total of 574, 235 and 113 patients were positive for influenza A-only (FA-only), influenza B-only (FB-only) and RSV-only in influenza seasons (from Nov 2017 to Mar 2018 and from Nov 2018 to Mar 2019), respectively. Of these, 19 cases were coinfected by Flu and RSV and admitted to this hospital. Compared with 809 Flu-only infected patients and 113 RSV-only infected patients, both the rates of intensive care unit(ICU) admission and use of invasive mechanical ventilation in Flu/RSV coinfected patients were higher (ICU admission: 47.4% vs. 20.1%, P=0.004; 47.4% vs. 22.1%, P=0.020; invasive mechanical ventilation: 47.4% vs.13.2%, P0.001; 47.4% vs. 17.7%, P=0.004). Furthermore, 60-day all-cause mortality attributed to Flu/RSV coinfections was significantly greater than that for Flu and RSV mono-infected patients (36.8% vs. 8.0%,P0.001; 36.8% vs. 11.5%, P=0.004.The findings of this study suggest that coinfection of Flu/RSV in adults is associated with a high adverse outcome. Thus, Flu/RSV coinfections should be increasingly appreciated and given appropriate management.

Cryptococcal meningitis, mainly caused by Cryptococcus neoformans/gattii species complexes, is a lethal infection in both immunosuppressive and immunocompetent populations. We characterized 110 Cryptococcus strains collected from Xiangya Hospital of Central South University in China during the 6-year study period between 2013 and 2018, and performed their antifungal susceptibility testing. Furthermore, the clinical features, laboratory and imaging data, treatment strategies and outcomes of the subjects were retrospectively analyzed. Of 110 Cryptococcus strains, C. neoformans species complexes accounted for 96.4% (106/110), including C. neoformans sensu stricto (VNI molecular type, 95.5%, 105/110) and Cryptococcus deneoformans (VNIV molecular type, 0.9%, 1/110), and Cryptococcus deuterogattii (VGII molecular type) accounted for 3.6% (4/110). The strains were further classified into 17 individual sequence types (STs) by using multilocus sequence typing (MLST). 89.1% (98/110) were represented by ST5; seven C. deuterogattii strains and one Cryptococcus deneoformans strain were assigned as ST7 and ST260, respectively. Antifungal minimal inhibitory concentrations above the epidemiological cutoff values (ECVs) were found mainly in C. neoformans species complexes strains (nine for amphotericin B, nine for fluconazole and seven for 5-fluorocytosine). Furthermore, 60.9% (67/110) of the subjects were male, and 40.0% (44/110) did not have underlying diseases. Hepatic diseases (hepatitis/HBV carrier status and cirrhosis) were the most common underlying health conditions (11.8%, 13/110), followed by autoimmune disorders (10.9%, 12/110) and chronic kidney disease (6.36%, 7/110). Only 4.5% (5/110) of the patients were HIV/AIDS positives. For clinical presentation, headache (77.3%, 85/110), fever (47.3%, 52/110), and stiff neck (40.9%, 45/110) were commonly observed. The mortality rate was 35.0% (36/103). In conclusion, our data were characterized by a high prevalence of the Cryptococcal meningitis patients without HIV/AIDS and other underlying health conditions, a relatively high non-wild-type rate of fluconazole and amphotericin B resistance, and low genetic diversity in Cryptococcus strains. The present study will provide evidence for further improvement of the diagnosis and treatment of cryptococcosis in China.

Background: Upper respiratory tract specimens are widely applicable for the diagnosis of COVID-19. To date, no study has analyzed the actual viral loads in upper respiratory tract and its relationship with the severity of lung lesions, Ct value of RT-PCR and transmission capacity in COVID-19 patients.Methods: We retrospectively enrolled nine COVID-19 patients. Clinical data and close contacts of these patients were investigated. Respiratory samples were tested for SARS-CoV-2 with both normal RT-PCR and droplet digital PCR.Results: All the COVID-19 patients complicated with pneumonia. Viral loads in nasopharyngeal swabs were accurately quantified, and they had no direct correspondence with the severity of lung lesions. The Cycle Threshold (Ct) value of RT-PCR was approximately consistent with the absolute quantification of digital PCR. The spearman correlation coefficient between them was -0.952 with P value < 0.001. Close contacts of patients with very low viral load or no detected virus were not infected.Conclusions: Viral loads in nasopharyngeal swabs, could not predict the severity of lung lesions revealed by CT in COVID-19 patients. The infectious capacity of patients with low or absent viral load in upper respiratory tract was relatively weak, and wearing mask might be helpful for lower its spread.

Background Members of the genus Proteus are mostly opportunistic pathogens that cause a variety of infections in humans. The molecular evolutionary characteristics and genetic relationships among Proteus species have not been elucidated to date. In this study, we developed a multilocus sequence analysis (MLSA) approach based on five housekeeping genes (HKGs) to delineate phylogenetic relationships of species within the genus Proteus. Results Of all 223 Proteus strains collected in the current study, the phylogenetic tree of five concatenated HKGs (dnaJ, mdh, pyrC, recA and rpoD) divided 223 strains into eleven clusters, which were representative of 11 species of Proteus. Meanwhile, the phylogenetic trees of the five individual HKGs also corresponded to that of the concatenated tree, except for recA, which clustered four strains at an independent cluster. The evaluation of inter- and intraspecies distances of HKG concatenation indicated that all interspecies distances were significantly different from intraspecies distances, which revealed that these HKG concatenations can be used as gene markers to distinguish different Proteus species. Further web-based DNA-DNA hybridization estimated by genome of type strains confirmed the validity of the MLSA, and each of eleven clusters was congruent with the most abundant Proteus species. In addition, we used the established MLSA method to identify the randomly collected Proteus and found that P. mirabilis is the most abundant species. However, the second most abundant species is P. terrae but not P. vulgaris. Combined with the genetic, genomic and phenotypic characteristics, these findings indicate that three species, P. terrae, P. cibarius and Proteus genospecies 5, should be regarded as heterotypic synonyms, and the species should be renamed P. terrae, while Proteus genospecies 5 has not been named to date. Conclusions This study suggested that MLSA is a powerful method for the discrimination and classification of Proteus at the species level. The MLSA scheme provides a rapid and inexpensive means of identifying Proteus strains. The identification of Proteus species determined by the MLSA approach plays an important role in the clinical diagnosis and treatment of Proteus infection.

We report co-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus in a patient with pneumonia in China. The case highlights possible co-detection of known respiratory viruses. We noted low sensitivity of upper respiratory specimens for SARS-CoV-2, which could further complicate recognition of the full extent of disease.

Background: The members of the genus Proteus are commonly opportunistic pathogens that cause a variety of infections in humans. The molecular evolutionary characteristics and genetic relationships among Proteus species are remain unelucidated. In this study, we developed a multilocus sequence analysis (MLSA) approach based on five housekeeping genes (HKGs) to delineate phylogenetic relationships of species within genus of Proteus. Results: Of all 223 Proteus strains collected in current study, phylogenetic tree of concatenated five HKGs (dnaJ, mdh, pyrC, recA and rpoD) divided into eleven clusters, which representative of their counterpart species. Meanwhile, phylogenetic trees of the five individual HKGs were also corresponded to that of the concatenated tree, except for recA, which clustered four strains at an independent cluster. The evaluation of inter- and intra-species distance of HKGs concatenation, all inter-species indicated more significant different distances than those of intra-species, which revealed these HKGs concatenation can be used as gene marker to distinguish different Proteus species. Further web-based DNA-DNA hybridization estimated by genome of type strains confirmed the validity of the MLSA and each of eleven clusters were congruent with eleven different Proteus species. In addition, we used the established MLSA method to identify the randomly collected Proteus, and found that P. mirabilis is the most species of Proteus. However, the top second is P. terrae, but not P. vulgaris. Combined with the genetic, genomic and phenotypic characteristics, three species, P. terrae, P. cibarius and Proteus genospecies 5 should be regarded as the heterotypic synonyms.Conclusions: Our data suggested the MLSA was a powerful method for the discrimination and classification of the Proteus at species level. The MLSA scheme provides a rapid, economical and precise identification of Proteus strains. The identification of Proteus species determined by the MLSA approach plays an important role in clinical diagnosis and treatment of Proteus infection.

Background: Adenovirus pneumonia account for 5%–10% of respiratory tract infections in children. However, it is rarely observed in immunocompetent patients. We sought to describe clinical and CT characteristics of community-acquired Adenovirus pneumonia in immunocompetent adults.Methods: 20 patients with clinically diagnosed adenovirus pneumonia from two hospitals were retrospectively included from January 2018 to December 2019. Medical records and chest CT of all patients were reviewed and summarized.Results: A total of 20 patients who were consisted of 18 men and 2 women with a median age of 36 years-old (range, 18-48). The clinical findings of patients with adenovirus pneumonia is high fever (>39oC) with the normal white blood cell (WBC) count and decreased Lymphocyte and elevated C-reactive protein. 10 cases complicated with mycoplasma infection when admission. Staphylococcus epidermidis was found in blood culture of 2 patients. CT findings included consolidation in all patients, ground glass opacity (GGO) in 13 patients, and pleural effusion in 12 patients. 13 patients were mechanically ventilated and 2 patients died during hospitalization.Conclusions: The most frequent finding of adenovirus pneumonia on CT was consolidation in the early stage. In adult patients with high fever, normal WBC count and decreased Lymphocyte, consolidation with/without GGO in the early stage may be a clue for suspecting adenovirus pneumonia.

Objectives ST11 Klebsiella pneumoniae is among the most important clinical pathogens in China, and KL47 and KL64 are the dominant K types of these strains. Understanding the genomic characteristics of these strains would be critical to their anti-infection treatment. Methods There were 364 genome sequences of ST11 K. pneumoniae strains isolated and collected from 13 countries from 2003 to 2018. These genome sequences included 338 downloaded from the National Center for Biotechnology Information (NCBI) database and 26 newly sequenced. Phylogenetic analyses of pan-genome and unique genes, and resistance and virulence gene analyses, were carried out to elucidate the molecular characteristics of these strains. Results A total of 19 732 genes were identified from the 364 ST11 strains, and the pan-genome was open, indicating the genetic diversity of ST11 K. pneumoniae. These strains were clustered into three clades. Clade 1 contained the most various K types (14/15, 93.3%) and unique genes. KL47 and KL64 were the dominant K types of clades 2 and 3, accounting for 100% and 99.4% of strains in each clade, respectively. KL64 strains contained the most virulence genes, including iucA and rmpA, and the two genes tend to coexist. In addition, strains in clade 1 were isolated from all 13 countries; the strains in clades 2 and 3 were isolated mainly from China. Conclusions The ST11 K. pneumoniae strain of KL64 is a newly emerging superbug, with more resistance and virulence genes in China; this was significantly different from other countries, and we should be alert to the dissemination of this subclone.

Biopsy tissue is a difficult sample to gain for the identification of potential pathogen in the clinical laboratory. At present, there are no effective culture methods for small-sized biopsy tissue. In this study, we propose a novel tissue biopsy culture method based on Myco/F lytic liquid culture system, namely, tissue was grinded before injected into culture bottles. More types and numbers of fungi are cultivated using this culture system than sputum or BALF culture. A few of mycobacteria were also detected with ground tissues. The method may be a promising alternative to or supplement for the traditional plate culture in clinical practice.

Additional file 1: Figure S1. Intra- and inter-species distances of eleven species infer by five individual genes. In each boxplot, from bottom to top: minimum, median and maximum. Table S1. Intra- and inter-species genetic distance median values and ranges of concatenated 5-gene and five individual genes.

Purpose. Group B Streptococcus (GBS) is one of the major pathogens in severe materno-neonatal infections. We aimed to describe the clinical and molecular characteristics of GBS isolates causing infections in 45 maternal and 50 neonatal subjects, collected from eight healthcare centres in mainland China over the period 2010– 2017. Methodology. The phenotypic and genotypic features of the GBS isolates, including capsular polysaccharide (cps) serotypes, pilus island (PI) genes and antibiotic resistance profiles and genes, were characterized by both conventional and molecular methods. The clonal relationship between these strains was investigated using multilocus sequence typing (MLST). Results. Of the 95 isolates, the predominant serotypes were III (51, 53.7 %), V (13, 13.7 %) and Ib (13, 13.7 %). All GBS strains carried at least one pilus island, with 32.6 % carrying PI-2b and 67.4 % PI-2a, singly or in combination. The most frequently-detected pilus island pattern was the combination of PI-1 and PI-2a, accounting for 56.8 % (54 isolates), followed by PI-1 combined with PI-2b (28, 29.5 %), PI-2a (10, 10.5 %) and PI-2b (3, 3.2 %). The strains were classified into 17 individual sequence types, and further clustered into six clonal complexes (CCs). A high prevalence of CC17/PI-1 and PI-2b (17, 34.0 %) was detected in 50 GBS isolates causing neonatal infections. No strain was resistant to penicillin, ampicillin, ceftriaxone or vancomycin, whereas 78.9, 76.8 and 81.5 % were resistant to erythromycin, clindamycin and tetracycline, respectively. Conclusion. Our study highlights the high genotypic diversity of GBS strains causing materno-neonatal infections, and the CC17/PI-1 and PI-2b sublineages should be noted in neonatal infections.

Purpose. Candida pathogens are commonly found in women and can cause vulvovaginal candidiasis (VVC), whose infection rate is further increased during pregnancy. We aimed to study the Candida prevalence and strain distribution in pregnant Chinese women with a molecular beacon assay. Methodology. From March 2016 to February 2017, a total of 993 pregnant women attending routine antenatal visits at the Beijing Obstetrics and Gynecology Hospital were enrolled. For Candida detection and identification, a unique molecular beacon assay was presented and compared with a traditional phenotypic method. Antifungal susceptibility was tested with the following agents: 5-flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole. Results. The prevalence of Candida was found to be 21.8 % when using the molecular method and 15.0 % when using the phenotypic method. The distribution of the Candida spp. was listed in order of decreasing prevalence: Candida albicans (79.8 %), Candida glabrata (13.5 %), Candida parapsilosis (3.7 %), Candida krusei (2.2 %) and Candida tropicalis (1.1 %). We found that 90.7 % of the Candida detection results were consistent between the molecular and the phenotypic methods. In the cases where the sequencing analyses for the Candida isolates resulted in inconsistent identification, the molecular method showed higher sensitivity than the phenotypic method (96.0 vs 64.6 %). C. albicans, C. glabrata and C. parapsilosis were essentially susceptible to all five antifungal agents tested, whereas C. tropicalis and C. krusei were susceptible to voriconazole and amphotericin B. Conclusion. By exhibiting good sensitivity and specificity, the molecular assay may offer a fast and accurate Candida screening platform for pregnant women.

The H7N9 virus underwent dynamic evolution during infection within individual patients. Signature amino acids in PB2 and NA demonstrated high polymorphism, and signature sites related to antiviral resistance and mammalian adaption underwent dynamic variation during disease progression., Background Signature amino acids of H7N9 influenza A virus play critical roles in human adaption and pathogenesis, but their dynamic variation is unknown during disease development. Methods We sequentially collected respiratory samples from H7N9 patients at different timepoints and applied next-generation sequencing (NGS) to the whole genome of the H7N9 virus to investigate the variation at signature sites. Results A total of 11 patients were involved, from whom 29 samples were successfully sequenced, including samples from multiple timepoints in 9 patients. Neuraminidase (NA) R292K, basic polymerase 2 (PB2) E627K, and D701N were the 3 most dynamic mutations. The oseltamivir resistance-related NA R292K mutation was present in 9 samples from 5 patients, including 1 sample obtained before antiviral therapy. In all patients with the NA 292K mutation, the oseltamivir-sensitive 292R genotype persisted and was not eliminated by antiviral treatment. The PB2 E627K substitution was present in 18 samples from 8 patients, among which 12 samples demonstrated a mixture of E/K and the 627K frequency exhibited dynamic variation. Dual D701N and E627K mutations emerged but failed to achieve predominance in any of the samples. Conclusions Signature amino acids in PB2 and NA demonstrated high polymorphism and dynamic variation within individual patients during H7N9 virus infection.

Group B streptococcus (GBS) is an increasing pathogen threat to newborns and adults with immunodepressive diseases. Here, a total of 193 GBS, including 51 invasive and 142 noninvasive isolates, were collected from the patients with infections in 7 tertiary hospitals from 5 cities in China during the year 2008 to 2015. The strains of GBS were characterized by classical and molecular techniques for capsular polysaccharide serotyping, genes for pilus island (PI) and α-like protein (alp), and antibiotic resistance profiling. Of 193 isolates, the predominant serotypes were III (45.6%) and Ia (18.7%). All strains carried at least 1 PI gene. The combination of PI-2b and PI-1 was present in 46.1% isolates, followed by PI-2a alone (80, 41.5%) and PI-2b alone (23, 11.9%). The most prevalent alp gene was rib (87, 45.1%), followed by α-C (47, 24.4%), ε (33, 17.1%), alp2/3 (7, 3.6%) and alp4 (2, 1.0%), respectively. The clonal relationships between strains were investigated using multilocus sequence typing. The strains were distinguished into 26 individual sequence typing, and further clustered into 6 clonal complexes. A significant association was noted between the distributions of alp genes, serotyping and PI profiles, such as serotype III-rib-PI+PI-2a, Ib-α-C, and Ia-ε-PI-2a. No penicillin-resistant strains were detected, and 74.1%, 64.2%, and 68.9% were resistant to erythromycin, clindamycin, and tetracycline, respectively. The infective GBS isolates in China demonstrated epidemical features.

Burkholderia pseudomallei is the causative agent of melioidosis, endemic in Southeast Asia and Northern Australia, and increasingly recognized in southern China, especially in Hainan Province. Mycotic aneurysm caused by B. pseudomallei is a rare but potentially severe illness with a high mortality rate. The clinical features of the mycotic aneurysm secondary to melioidosis have not been illustrated in China. Over a seven-year period (2013 to 2019), 159 patients with bacteremic melioidosis were retrospectively analyzed in Hainan province, China, of whom eight patients were confirmed to have mycotic aneurysm through the combination of imaging examination, pathologic examination and aneurysm tissue culture. We summarized these eight patients’ clinical characteristics, demographical features, treatments and outcomes. The susceptibilities to five commonly-used antibiotics for these eight B. pseudomallei isolates were also determined by E-test strips. Furthermore, the mycotic aneurysm cases secondary to melioidosis retrieved from the literature were also reviewed. Of the eight cases, six had abdominal mycotic aneurysms, one had a left iliac aneurysm, and the other one had an infectious mesenteric aneurysm. They were aged from 48 to 69 years old, and had the underlying risk factors of diabetes mellitus (2 patients), long-term smoking (4 patients), hypertension (6 patients), and soil and water contact history (6 patients), respectively. The positive arterial aneurysm imaging was observed in all patients via computed tomography (CT) or angiography. Eight B. pseudomallei isolates collected from both blood and mycotic aneurysm tissues remained 100% susceptible to imipenem and ceftazidime. After surgery combined with antibiotic administration, six patients survived, with a mortality rate of 25%. In melioidosis endemic areas, the mycotic aneurysm secondary to melioidosis might be underdiagnosed, and increased awareness of predisposing risk factors and clinical features of the mycotic aneurysm is required. Following a positive B. pseudomallei blood culture, the diagnosis of mycotic aneurysm should be under consideration in those with abdominal pain and/or hypertension. Imaging by CT or angiography is indispensable for its timely diagnosis and management., Author summary Burkholderia pseudomallei has the potential to cause mycotic aneurysm, an infrequently-occurred complication of melioidosis, with a high mortality rate despite appropriate antibiotic therapy and vascular surgery. The comprehensive clinical features of mycotic aneurysm due to B. pseudomallaei have not been documented in the People’s Republic of China and might be underrecognized. Therefore, we described eight patients with mycotic aneurysm from 159 bacteremic melioidosis patients in Hainan, China, over a seven-year period (between 2013 to 2019). We summarized their clinical characteristics, demographical features, treatments and outcomes. To our knowledge, this is the first comprehensive report of mycotic aneurysm secondary to melioidosis in China. In summary, in melioidosis endemic areas, or for those returning from endemic areas, increased awareness of the risk factors is required for early diagnosis and management of mycotic aneurysm secondary to melioidosis.

Purpose Community-associated Staphylococcus aureus (CA S. aureus) is the most common causative pathogen of the skin and soft tissue infections (SSTIs). This study aims to determine clonal distribution, virulence factors of CA S. aureus clinical isolates from purulent SSTIs in Beijing, China. Materials and methods CA-S. aureus isolates were collected from 115 outpatients with purulent SSTIs from the department of dermatology from April 2015 to April 2017. Multilocus sequence typing and Staphylococcus cassette chromosome mec typing were performed to explore molecular characteristics. Phylogenetic analysis of 16S rRNA of dominant S. aureus isolates was performed using MEGA-X software. Virulence genes were detected by PCR, while biofilm formation was evaluated by a microtiter plate method. The antimicrobial susceptibility was tested by an automatic VITEK system. Results Forty-four CA-S. aureus isolates identified from SSTIs contain 9 methicillin-resistant S. aureus (MRSA) isolates (20.4%) and 35 methicillin-susceptible S. aureus isolates (MSSA) (79.6%). The dominant sequence types (STs) were ST22 (40.9%) and clonal complex 59 (CC59; 77.8%) in Community-associated methicillin resistant methicillin-resistant S. aureus. 27.8% of ST22 isolates were homologous to the epidemic ST22 EMRSA-15 in Europe. The prevalence of virulence genes lukS/lukF, tst-1, etA, edinA, icaA, and icaD was 50%, 93.2%, 4.5%, 4.5%, 100%, and 100%, respectively. All CC59 isolates exhibited stronger biofilm-forming capability than ST22 clones. Among the MSSA subgroup, the poor biofilm producers had significantly higher sensitivity to sulfamethoxazole/Trimethoprim. Conclusion The dominant epidemic clone PVL+ ST22 MSSA containing tst-1 occurs in Beijing, indicating that a PVL+ ST398 clone which was previously predominant in this district had been replaced by a new clone.

The current knowledge of invasive Scopulariopsis/Microascus infection in lung transplantation has been derived from only four case reports. Although these fungi are uncommon compared with Aspergillus, they are highly resistant to the current antifungal agents, and the mortality is extremely high. To explore the risk factors, clinical manifestations, notable diagnostic characteristics and outcomes of positive Scopulariopsis/Microascus isolation in lung transplantation patients. We included all cases with positive Scopulariopsis/Microascus isolation from lower respiratory tracts or bronchial mucosa biopsies in our lung transplantation centre. Proven cases from the literature were added. Positive isolation occurred in 2% (3/157) in our centre. Four cases from the literature were added. The mortality could be considered as high as 80%, once the two cases of colonisation were excluded. The average interval between transplantation and positive isolation was 106 (19-131) days. A total of 57.1% of patients had experienced a combination of infection with Aspergillus or other fungi as well as long-term azole antifungal agent treatment before the positive isolation, which may be possible risk factors. The combination of micafungin, posaconazole and terbinafine may be an effective treatment. The peak time of positive isolation was consistent with that of some opportunistic pathogens, and the possible risk factors were the infection of other fungi as well as prior long-term azole antifungal administration. In addition to its high mortality, Scopulariopsis/Microascus was also highly resistant to common antifungal agents and the combination of two or three drugs for therapy was recommended.

Eight swarming motile bacteria were isolated from food and clinical samples in China. Cells were Gram-stain-negative, facultatively anaerobic and rod-shaped (0.5-0.8×1.0-3.0 μm) with hairlike pili and flagella. The 16S rRNA and partial rpoB housekeeping gene sequence analyses indicated that the strains belong to the genus Proteusin the family Enterobacteriaceae. Of the eight strains studied, seven and a single isolate formed two separate clades in the phylogeny of Proteusspecies, indicating two separate species. Both the in silico DNA-DNA hybridization and the average nucleotide identity values between these two groups and to the type strains of the genus Proteuswere below the recommended threshold for signifying their candidature as two separate species. The DNA G+C contents of strains TJ1636T and FJ2001126-3T were 37.8 and 38.1 mol%, respectively. The major cellular fatty acids of the two novel type strains were C16:0, cyclo C17:0, summed feature 3 and summed feature 8. The results supported that the strains belong to different taxonomic positions in the genus Proteus. The isolates were named Proteus faecis sp. nov., with type strain TJ1636T (=DSM 106180T=GDMCC 1.1245T), and Proteuscibi sp. nov., with type strain FJ2001126-3T (=DSM 106178T =GDMCC 1.1244T).

Background: Many unnecessary antibiotics are prescribed to patients with viral lower respiratory tract infections (LRTIs). The FilmArray® Respiratory Panel is a new molecular point-of-care testing (POCT) procedure, which provides faster and comprehensive identification of viruses and atypical pathogens. But whether POCT could potentially help optimize antibiotics use for LRTIs remains a controversial topic. Methods: In this single-center, open-label, randomized controlled study, hospitalized adult patients diagnosed with community-acquired pneumonia, acute exacerbation of chronic obstructive pulmonary disease, or acute exacerbation of bronchiectasis were enrolled in the study. Patients were randomly assigned to the POCT group or the routine real-time PCR assays (control) group. The primary outcome was the duration of intravenous antibiotics used during hospitalization. The secondary outcomes included the length of hospital stay and the total cost of hospitalization. We also evaluated the effects of a combination of viral testing with serum procalcitonin (PCT) levels on the duration of antibiotic use. Findings: Between October 2017 and July 2018, 761 eligible patients were enrolled (383 in the POCT group and 378 in the control group). The positive rates of virus and atypical pathogens in the POCT group was 31·7% (121/383) compared with 17·2% (32/378) in the control group (difference 14·5%, 95% CI 7·3% to 21·6%; P = 0·0003). The median duration of intravenous antibiotics in the POCT group was significantly shorter than that in the control group (7·0 days [IQR, 5·0, 9·0] in the POCT group vs 8·0 [IQR, 6·0, 11·0] days in the control group; mean difference -1·6 days, 95% CI -2·2 to -1·0 days; P < 0·0001). The median length of hospital stay of patients in the POCT group was significantly shorter than that in the control group (8·0 days [IQR, 6·0, 10·0] in the POCT group vs 9·0 days [IQR, 7·0, 12·0] in the control group; mean difference -1·1 days, 95% CI -1·7 to -0·5 days; P < 0·0001). The median cost of hospitalization was significantly lower in the POCT group ($1,761·1 [IQR, 1,261·1, 2581·3] in the POCT group vs $2,017·3 [IQR, 1,442·9, 2,894·3] in the control group). Furthermore, the median duration of intravenous antibiotic treatment was significantly shorter and the median cost of hospitalization was significantly lower in patients with a positive FilmArray Respiratory Panel test result and PCT < 0·25 ng/mL compared with a negative FilmArray test result and PCT ≥ 0·25 ng/mL (6·0 days [IQR, 4·5, 7·5] vs 7·0 days [IQR, 6·0, 10·5]; mean difference 2·3 days, 95% CI 0·6 to 4·0 days; P = 0·0079 and $1,374·6 [IQR, 1,034·2, 1991·7] vs $1,939·6 [IQR, 1,307·6, 2,955·7]). Interpretation: The use of POCT testing for respiratory viruses and atypical pathogens could help to optimize antibiotic use and improve clinical outcomes in hospitalized LRTI patients. Clinical Trial Number: clinicaltrials.gov Identifier: NCT03391076. Funding Statement: National Science Grant for Distinguished Young Scholars (81425001/H0104) Declaration of Interests: We declare that we have no competing interests. Ethics Approval Statement: The study was approved by the Ethics Committee of CJFH. Written informed consent was obtained from each participant before data collection.

S. dysgalactiae subsp. equisimilis (SDSE) is infrequently associated with maternal infections during delivery in pregnant women. A rare case is presented of a woman with intrauterine infection and surgical-site infection due to SDSE after cesarean section, which had colonized her genital tract and, via the ascending pathway, reached her intact fetal membrane. All isolates were identified as Streptococcus Lancefield group G, and their emm genes that coded M protein belonged to stG6.1. The isolates tested negative for a series of streptococcal superantigen virulence genes but positive for nonsuperantigenic virulence genes. In particular, molecular typing using pulsed-field gel electrophoresis analysis disclosed that the three isolates from the different infection sites had identical profiles. Furthermore, multilocus sequence typing indicated that the three isolates belonged to a new sequence typing. Our results indicated that SDSE is potentially pathogenic for pregnant women and newborns if colonized.

BACKGROUND: Infections produced by extensively drug-resistant (XDR) gram-negative bacilli (GNB) in solid organ transplant (SOT) are an important cause of morbidity and mortality. Ceftazidime/avibactam (CAZ-AVI) is a novel β-lactam/β-lactamase combination antibiotic with anti-GNB activity, but experience in real clinical practice with CAZ-AVI in lung transplant (LT) recipients is limited. METHODS: We conducted a retrospective study of patients with XDR-GNB infection who received at least 3 days of CAZ-AVI in the Department of Lung Transplantation Between December 2017 and December 2018 at China-Japan friendship hospital (CJFH). The general information, clinical manifestations, laboratory examinations, treatment course, and outcomes were summarized. RESULTS: A total of 10 patients who underwent LT at our center were included. They were all males with a mean age 51 years. Infections after LT included pneumonia and/or tracheobronchitis [n=9; 90% (9/10)], cholecystitis and blood stream infection (BSI) (n=1, patient 8). In these 10 LT recipients, the incidence of various airway complications was 70% (7/10). Carbapenem-resistant Klebsialla pneumoniae (CRKP) was the predominant pathogen, being detected in 9 patients. Multilocus sequence typing (MLST) analysis showed that all 9 CRKP isolates belonged to ST11. Six patients (6/10, 60%) started CAZ-AVI as salvage therapy after a first-line treatment with other antimicrobials. CAZ-AVI was administered as monotherapy or in combination regimens in 20% (2/10) and 80% (8/10) of patients respectively. There were no difference in temperature before and after CAZ-AVI treatment (P>0.05). White blood cell (WBC) at 7 days, and procalcitonin (PCT) at 7 days and 14 days significantly dropped (P

Background: The Xpert Xpress Flu/RSV assay is released by FDA for rapid detection of influenza A (FluA), influenza B (FluB), and respiratory syncytial virus (RSV). This study aimed to evaluate its clinical performance in comparison to that of the RT-PCR assay cleared by China FDA (CFDA-PCR). Methods: Nasopharyngeal specimens were collected from patients and tested by the two assays side by side. Discordant results were tested with a laboratory-developed real-time PCR for resolution. Viral load in the sample was quantified with a droplet digital PCR. Results: A total of 658 specimens were involved and gave 94.7%–99.1% agreement between the two assays. The Xpert assay showed higher sensitivity for FluA (100% vs. 89.8%) and FluB detection (100% vs. 95.3%), and also higher accuracy (98.9% vs. 95.7%) for FluA than the CDFA-PCR. The positive and negative predictive values (NPV) for the three viruses ranged from 90.5% to 100% in the two assays, with higher NPV for FluA and FluB in Xpert assay. Moreover, the Xpert Ct values showed a linear correlation with virus titer in specimens tested. Conclusion: Overall, the Xpert assay is a reliable and sensitive tool for the detection of FluA, FluB and RSV in our clinical settings. Keywords: Xpert Xpress Flu/RSV, RT-PCR, Digital PCR, Sensitivity

The emergence of multidrug-resistant Salmonella genomic island 1 (SGI1) and Proteus genomic island (PGI) bearing P. mirabilis present a serious threat to public health. In this study, we screened 288 Proteus isolates recovered from seven provinces in China. Fourteen strains (4.9%) all belonged to P. mirabilis were positive for SGI1/PGI2, including twelve from clinical samples (5.3%) and two from food (3.3%). A Blastn search against GenBank and phylogenetic analyses identified eight different SGI1 variants and one PGI2 variant from the fourteen SGI1/PGI2 variants. All SGI1 variants shared a common backbone and harbored different resistance gene(s), except the sul1 gene at its multidrug-resistant (MDR) region. Among the variants, three novel SGI1 variants, designated as SGI1-PmCA11, SGI1-PmCA14 and SGI1-PmCA46, contained different gene cassettes, which were similar to sequences in plasmids or class 1 integrons of Klebsiella pneumoniae, P. mirabilis, Escherichia coli and Salmonella. Moreover, one novel PGI2, designated as PGI2-PmCA72, had an identical gene cassette to the first class 1 integron from PGI2 (GenBank accession no. MG201402.1) in P. mirabilis, but varied due to missing, replaced, inserted and inverted gene clusters. The four novel SGI1/PGI2 variants contained the cmlA5, dfrA14, blaOXA-10, aadA15, blaOXA-1, catB3 and dfrA16 resistance genes, which have never been reported in SGI1/PGI2 variants. Phenotypically, all fourteen SGI1/PGI2-containing strains showed multidrug resistance. All except four strains were resistant to the first, or the second and/or-third generation cephalosporins. Considering the increasing number and the emergence of new SGI1/PGI2 variants, further surveillance is needed to prevent the spreading of the MDR genomic islands among Proteus isolates from human and food.

This study reports the simplified carbapenem inactivation method (sCIM) to detect carbapenemase-producing gram-negative bacilli in a simple and accurate manner. This method is based on the modified carbapenem inactivation method (mCIM) with the improvement of experimental procedures. Instead of incubating the antibiotic disk in the organism culture media, the organism to be tested was smeared directly onto the antibiotic disk in the sCIM. For evaluating the sensitivity and specificity of the method, a total of 196 Enterobacteriaceae, 73 Acinetobacter baumannii, and 158 Pseudomonas aeruginosa isolates were collected. Polymerase chain reaction (PCR) was used to detect the carbapenemase genes. Phenotypic evaluations were performed using both the sCIM and the mCIM. PCR results showed that, of the 196 Enterobacteriaceae strains, 147 expressed the carbapenemase genes blaKPC-2 (58.5%), blaIMP-4 (21.8%), blaIMP-2 (2.0%), blaVIM-1 (6.1%), blaNDM-1 (10.2%), and blaOXA-48 (1.4%). sCIM results had high concordance with PCR results (99.5%) and mCIM results (100%) with the exception of one Klebsiella pneumoniae strain, which had an minimal inhibitory concentration (MIC) for imipenem of 0.25 mg/L. PCR demonstrated that 53 of the 73 A. baumannii isolates expressed the carbapenemase genes blaOXA-23 (98.1%) and blaVIM-2 (1.8%). sCIM and PCR results corresponded but all A. baumannii isolates were carbapenemase negative by the mCIM. PCR demonstrated that 25 of the 158 P. aeruginosa isolates expressed carbapenemase genes blaVIM-1 (52%) , blaVIM-2 (8%) , blaVIM-4 (36%), and blaIMP-4 (4%). sCIM results had high concordance with PCR results (100%) and the mCIM results (99.4%) with the exception of one P. aeruginosa isolate that expressed the blaVIM-4 gene. The sCIM offers specificity and sensitivity comparable to PCR but has the advantage of being more user-friendly. This method is suitable for routine use in most clinical microbiology laboratories for the detection of carbapenemase-producing gram-negative bacilli.

BACKGROUND PCR-ESI/MS is a commercial molecular method that can identify bloodstream infection (BSI) pathogens directly from blood. Previous studies showed its sensitivity varied greatly. Its diagnostic accuracy has not been systematically evaluated yet, thus we aimed to assess its accuracy by systematic review and meta-analysis. METHODS Studies were searched on PubMed and Embase up to November 2017, for studies using PCR/ESI-MS to diagnose BSI directly from blood and providing sufficient data to construct two-by-two tables. Subgroup analysis and meta-regression were used to assess heterogeneity. RESULTS A total of nine studies including 3,392 patients met the inclusion criteria. Their pooled sensitivity and specificity was 0.66 (95% CI: 0.57 - 0.74) and 0.84 (95% CI: 0.78 - 0.89), respectively. The positive and negative likelihood ratios were 4.2 (95% CI: 3.0 - 5.9) and 0.40 (95% CI: 0.31 - 0.50), respectively. The diagnostic odds ratio (DOR) was 10.61 (95% CI: 6.67 - 16.88). The area under the SROC was 0.82 (95% CI: 0.78 - 0.85). High heterogeneity was found. Subgroup analysis and meta-regression showed that regions, BSI prevalence, blood volume, and settings may cause heterogeneity. CONCLUSIONS PCR-ESI/MS using blood specimens directly has the potential for early diagnosis of BSI, compared with blood culture. Its rule-in value is higher than rule-out value. Due to the heterogeneity of currently available studies, further high-quality studies are still needed.

Purpose Candida pathogens are commonly found in women and can cause vulvovaginal candidiasis (VVC), whose infection rate is further increased during pregnancy. We aimed to study the Candida prevalence and strain distribution in pregnant Chinese women with a molecular beacon assay. Methodology From March 2016 to February 2017, a total of 993 pregnant women attending routine antenatal visits at the Beijing Obstetrics and Gynecology Hospital were enrolled. For Candida detection and identification, a unique molecular beacon assay was presented and compared with a traditional phenotypic method. Antifungal susceptibility was tested with the following agents: 5-flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole. Results The prevalence of Candida was found to be 21.8 % when using the molecular method and 15.0 % when using the phenotypic method. The distribution of the Candida spp. was listed in order of decreasing prevalence: Candida albicans (79.8 %), Candida glabrata (13.5 %), Candida parapsilosis (3.7 %), Candida krusei (2.2 %) and Candida tropicalis (1.1 %). We found that 90.7 % of the Candida detection results were consistent between the molecular and the phenotypic methods. In the cases where the sequencing analyses for the Candida isolates resulted in inconsistent identification, the molecular method showed higher sensitivity than the phenotypic method (96.0 vs 64.6 %). C. albicans, C. glabrata and C. parapsilosis were essentially susceptible to all five antifungal agents tested, whereas C. tropicalis and C. krusei were susceptible to voriconazole and amphotericin B. Conclusion By exhibiting good sensitivity and specificity, the molecular assay may offer a fast and accurate Candida screening platform for pregnant women.

Two strains, 08MAS2251T and LZ2016-166, were isolated from diverse samples in China collected from the surface of crucian carp and the faeces of a diarrhoea patient, respectively. Both strains were pink-orange coloured, Gram-negative, oxidase- and catalase-positive, facultative anaerobic and motile bacteria, produced H2S and reduced nitrates to nitrites. Growth occurred in the presence of 0–9 % (w/v) NaCl and at 10–42 °C. The optimum conditions were with 1 % (w/v) NaCl and at 35 °C. The phylogenetic tree of 16S rRNA gene demonstrated that strains 08MAS2251T and LZ2016-166 clustered in a distinctive clade next to the species Shewanella chilikensis JC5T within the genus Shewanella . Meanwhile, gyrB gene sequence analysis indicated that the two strains formed an independent branch that was clearly separate from all the other Shewanella species with sequence similarities from 68.49 to 95.74 %. The DNA G+C content of strain 08MAS2251T was 52.68 mol%. Genomic relatedness of in silico DNA–DNA hybridization between strain 08MAS2251T and phylogenetic neighbours ranged from 50.5–51.8 %, below the cutoff of 70 %. In addition, corresponding average nucleotide identity values were between 93.01 to 93.49%, which were lower than 95 % threshold. The major fatty acids of strain 08MAS2251T were C17 : 1ω8c (27.2 %), iso-C15 : 0 (22.5 %), summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 8.7 %), C16 : 0 (6.2 %), iso-C13 : 0 (5.6 %) and C17 : 0 (4.5 %). Based on phenotypic and genetic analysis, strains 08MAS2251T and LZ2016-166 are identified as a novel species of the genus Shewanella , for which the name Shewanella carassii sp. nov. is proposed. The type strain is 08MAS2251T (=DSM 104682T=CGMCC 1.16033T).

In vitro fertilization (IVF) has become a very common procedure in the infertility practice due to its accessibility. The study is aiming at presenting two twin pregnancy-related infections caused by Listeria monocytogenes and reviewing the reported cases of listeriosis in multiple pregnancies.Two listeriosis cases with twin pregnancy after IVF were described and the literature on pregnancy-associated listeriosis was reviewed.The risk of listeriosis should be underscored in pregnant women after IVF, and timely diagnosis and rational treatment might result in a better outcome.The current study highlights that the infections due to L. monocytogenes should be noted in multiple pregnancies after IVF.

Background Bacterial species belonging to the genus Exiguobacterium are facultative anaerobic, non-spore-forming, Gram-positive bacilli, and rarely associated with human infections. Herein, we reported the first case of community-acquired pneumonia (CAP) and bacteremia due to Exiguobacterium spp. in China. Case presentation An adult male with severe CAP was hospitalized. The pathogen was isolated from his bloodstream and broncho-alveolar lavage fluid. The correct identification of the micro-organism was achieved using 16S rRNA sequencing, and its antibiotic susceptibility test was performed by microdilution method. The Whole Genome Sequencing (WGS) was used to characterize its genetic features and to elucidate its potential pathogenic mechanisms. Furthermore, its genome sequence was also compared with those of 3 publicly-available Exiguobacterium strains. A PubMed search was performed for further understanding the features of Exiguobacterium infections. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain GX59 was most closely related to Exiguobacterium AT1b (99.7%). The genome of GX59 was 2,727,929 bp in size, harbouring 2855 putative protein-coding genes, 5 rRNA operons, 37 tRNA genes and 1 tmRNA. The multiple genome comparison of 4 Exiguobacterium strains demonstrated that Exiguobacterium contained 37 genes of secretion systems, including sec, tat, FEA, Type IV Pili and competence-related DNA transformation transporter (Com). Virulence factors of the micro-organism included tlyC, NprR, MCP, Dam, which might play a critical role in causing lethal infection. Conclusions The study highlighted the potential pathogenicity of the genus Exiguobacterium for its unique genes encoding various virulence factors and those associated with antibiotic resistance, therefore, its clinical significance should be valued.

Objective: To develop a novel clinical diagnostic method to distinguish bacteremia from blood contamination due to coagulase-negative staphylococci (CNS) by using the ratio of the time to positivity (TTP) of a blood culture with positive results to its serum procalcitonin (PCT) level. Methods: We retrospectively reviewed the clinical and microbiological records of 102 patients who had 1 or more blood cultures that tested positive for CNS from August 2007 through February 2012. Results: Receiver operating characteristic (ROC) curve analysis indicated that, at a cutoff of 1.24 ng per mL, 21.5 hours, and 22.5 (hours × mL)/ng,the TTP to PCT ratio, PCT level, and TTP yielded the most favorable discrimination for bacteremia caused by CNS, with sensitivities of 85.7%, 78.6%, and 61.9%,respectively, and specificities of 80.0%, 78.3%, and 61.7%,respectively. The TTP to PCT ratio was the most accurate of the studied variables in predicting CNS-triggered bacteremia levels. Conclusion: The TTP to PCT ratio is a useful predictor to differentiate the culture samples that test positive, revealing the presence of CNS bacteremia, from those that are merely contaminated.

Brevundimonas diminuta is rarely associated with invasive infections. We report the case of a previously healthy young man with pleural effusion, in which B. diminuta was recovered but incorrectly identified as Kingella kingae when it was freshly isolated. Consequently, the misidentification resulted in initial treatment failure. The correct identification was achieved through further incubation, sequencing of the 16S rRNA gene and MS.

SummaryBacteraemia due to non-O1/non-O139 Vibrio cholerae is rarely documented in mainland China. We report such a case in a 70-year-old male with type 2 diabetes mellitus. The clinical features, phenotypic analyses, and presence of a panel of known virulence genes in the isolated strain are described. To the best of our knowledge, this is the first reported case of bacteraemia due to this strain in a T2DM patient without other coexisting underlying diseases in mainland China.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is presently considered as a human pathogen associated with clinical infection. We characterized 56 SDSE isolates collected from two tertiary hospitals in Beijing, China. Sixteen distinct emm types/subtypes were detected, dominated by stG245.0 (32.1%), stG652.0 (10.7%), stG6.1 (10.7%) and stG485.0 (10.7%), and a novel stG840.0 variant type was identified. All isolates possessed virulence genes of sagA and scpA, and most carried slo (98.2%), ska (98.2%) and speG(dys) (35.7%). By multilocus sequence typing (MLST) analysis, 17 individual sequence types (STs) were distinguished, including 7 newly-identified STs (26.8% of isolates), of which ST127 (30.4%), ST7 (12.5%) and ST44 (10.7%) dominated. Meanwhile, pulsed-field gel electrophoresis (PFGE) analysis revealed 33 pattern types (PTs), which were further combined into 16 pattern clusters (PCs), and 59.3% of isolates were distributed into 2 dominant PCs. Notably, emm types had both close relationship and consistency with STs and PFGE PCs. Furthermore, of 56 SDSE isolates, the predominant antibiotic resistances were erythromycin (71.4%), clindamycin (71.4%) and tetracycline (60.7%). Correspondingly, the prevalent resistance genes of macrolide and tetracycline were erm(B) (78.6%) and tet(M) (73.2%). In addition, multiple point mutations of parC, one of fluoroquinolone resistance genes, were observed (accounting for 75%), and were divided into 12 types, with parC 07 as the predominant type. Our data suggested the wide molecular diversity and distinctive regional features of SDSE from clinical infection in Beijing, China.