Xiangna Zhao

A Novel New Delhi Metallo-β-Lactamase Variant, NDM-14, Isolated in a Chinese Hospital Possesses Increased Enzymatic Activity against Carbapenems

ABSTRACT A novel New Delhi metallo-β-lactamase (NDM) variant, NDM-14, was identified in clinical isolate Acinetobacter lwoffii JN49-1, which was recovered from an intensive care unit patient at a local hospital in China. NDM-14, which differs from other existing enzymes by an amino acid substitution at position 130 (Asp130Gly), possesses enzymatic activity toward carbapenems that is greater than that of NDM-1. Kinetic data indicate that NDM-14 has a higher affinity for imipenem and meropenem.

Proteomic analysis of the interaction of Bifidobacterium longum NCC2705 with the intestine cells Caco-2 and identification of plasminogen receptors.

To identify proteins with a potential role in the interaction of Bifidobacterium longum with intestinal epithelial cells, we profiled the protein response of B. longum NCC2705 following interaction with Caco-2 cells. Thirty-one protein spots, belonging to a total of 23 proteins, which exhibited a change in abundance of at least 3-fold were identified in B. longum NCC2705 following co-culture with Caco-2 cells, and were subsequently identified. Changes in expression were confirmed at the transcriptional level for a selection of these proteins. Enolase (Eno) and elongation factor Tu (EF-Tu) were amongst the proteins that showed the most prominent increase in abundance. Interaction of these proteins with plasminogen (Plg) was analyzed by Plg overlay assays, glutathione S-transferase (GST)-pull down, and western blot analysis. The results suggested that EF-Tu and Eno serve as surface receptors for B. longum NCC2705 binding to human plasminogen. Purified GST-EF-Tu and GST-Eno inhibited adhesion of B. longum NCC2705 to Caco-2 cells. Collectively, our data suggest that Eno and EF-Tu moonlight as adhesions, and are possibly involved in the protective role played by B. longum NCC2705 in defense against enteric pathogens. Biological significance The interaction of bifidobacteria with the human host plasminogen/plasmin system confirms the existence of a new component in the molecular cross-talk between bacteria and the host. Our study analyzed proteins EF-Tu and Eno with Plg binding activity, and they can inhibit adhesion of B. longum NCC2705 to Caco-2 cells, suggesting their role in the bacterial adherent to the enterocyte surface.

Characterization of a novel Achromobacter xylosoxidans specific siphoviruse: phiAxp-1

Bacteriophages have recently been considered as an alternative biocontrol tool because of the widespread occurrence of antimicrobial-resistant Achromobacter xylosoxidans. Herein, we isolated a virulent bacteriophage (phiAxp-1) from a water sample of the Bohai sea of China that specifically infects A. xylosoxidans. Transmission electron microscopy revealed that phage phiAxp-1 belongs to the Siphoviridae. We sequenced the genome of phiAxp-1, which comprises 45,045u2009bp with 64 open reading frames. Most of the proteins encoded by phiAxp-1 have no similarity to sequences in the public databases. Twenty-one proteins with assigned functions share weak homology with those of other dsDNA bacteriophages infecting diverse hosts, such as Burkholderia phage KL1, Pseudomonas phage 73, Pseudomonas phage vB_Pae-Kakheti25, Pseudomonas phage vB_PaeS_SCH_Ab26, Acinetobacter phage IME_AB3 and Achromobacter phage JWX. The genome can be divided into different clusters for the head and tail structure, DNA replication and mazG. The sequence and genomic organization of bacteriophage phiAxp-1 are clearly distinct from other known Siphoviridae phages; therefore, we propose that it is a member of a novel genus of the Siphoviridae family. Furthermore, one-step growth curve and stability studies of the phage were performed, and the specific receptor of phiAxp-1 was identified as the lipopolysaccharide of A. xylosoxidans.

Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage

Achromobacter xylosoxidans, an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans, from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales). With a burst size of 9000 plaque-forming units and a latent period of 80u2009min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825u2009bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed.

Proteomic Profiling of Bifidobacterium bifidum S17 Cultivated Under In Vitro Conditions.

Bifidobacteria are frequently used in probiotic food and dairy products. Bifidobacterium bifidum S17 is a promising probiotic candidate strain that displays strong adhesion to intestinal epithelial cells and elicits potent anti-inflammatory capacity both in vitro and in murine models of colitis. The recently sequenced genome of B. bifidum S17 has a size of about 2.2 Mb and encodes 1,782 predicted protein-coding genes. In the present study, a comprehensive proteomic profiling was carried out to identify and characterize proteins expressed by B. bifidum S17. A total of 1148 proteins entries were identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), representing 64.4% of the predicted proteome. 719 proteins could be assigned to functional categories according to cluster of orthologous groups of proteins (COGs). The COG distribution of the detected proteins highly correlates with that of the complete predicted proteome suggesting a good coverage and representation of the genomic content of B. bifidum S17 by the proteome. COGs that were highly present in the proteome of B. bifidum S17 were Translation, Amino Acid Transport and Metabolism, and Carbohydrate Transport and Metabolism. Complete sets of enzymes for both the bifidus shunt and the Embden-Meyerh of pathway were identified. Further bioinformatic analysis yielded 28 proteins with a predicted extracellular localization including 14 proteins with an LPxTG-motif for cell wall anchoring and two proteins (elongation factor Tu and enolase) with a potential moonlighting function in adhesion. Amongst the predicted extracellular proteins were five of six pilin proteins encoded in the B. bifidum S17 genome as well as several other proteins with a potential role in interaction with host structures. The presented results are the first compilation of a proteomic reference profile for a B. bifidum strain and will facilitate analysis of the molecular mechanisms of physiology, host-interactions and beneficial effects of a potential probiotic strain.

Rapid detection of Acinetobacter baumannii and molecular epidemiology of carbapenem-resistant A. baumannii in two comprehensive hospitals of Beijing, China.

Acinetobacter baumannii is an important opportunistic pathogen associated with a variety of nosocomial infections. A rapid and sensitive molecular detection in clinical isolates is quite needed for the appropriate therapy and outbreak control of A. baumannii. Group 2 carbapenems have been considered the agents of choice for the treatment of multiple drug-resistant A. baumannii. But the prevalence of carbapenem-resistant A. baumannii (CRAB) has been steadily increasing in recent years. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of A. baumannii in clinical samples by using high-specificity primers of the blaOXA-51 gene. Then we investigated the OXA-carbapenemases molecular epidemiology of A. baumannii isolates in two comprehensive hospitals in Beijing. The results showed that the LAMP assay could detect target DNA within 60 min at 65°C. The detection limit was 50 pg/μl, which was about 10-fold greater than that of PCR. Furthermore, this method could distinguish A. baumannii from the homologous A. nosocomialis and A. pittii. A total of 228 positive isolates were identified by this LAMP-based method for A. baumannii from 335 intensive care unit patients with clinically suspected multi-resistant infections in two hospitals in Beijing. The rates of CRAB are on the rise and are slowly becoming a routine phenotype for A. baumannii. Among the CRABs, 92.3% harbored both the blaOXA-23 and blaOXA-51 genes. Thirty-three pulsotypes were identified by pulsed-field gel electrophoresis, and the majority belonged to clone C. In conclusion, the LAMP method developed for detecting A. baumannii was faster and simpler than conventional PCR and has great potential for both point-of-care testing and basic research. We further demonstrated a high distribution of class D carbapenemase-encoding genes, mainly OXA-23, which presents an emerging threat in hospitals in China.

Characterization of phiCFP-1, a virulent bacteriophage specific for Citrobacter freundii

Citrobacter freundii, a Gram‐negative bacterium, causes many opportunistic infections. Bacteriophage phiCFP‐1 was isolated and characterized by its ability to lyse the multidrug‐resistant clinical C. freundii strain P10159. Transmission electron microscopy showed that the phage has an icosahedral head and a short tail, making it a Podoviridae family member. In a single‐step growth experiment, phiCFP‐1 exhibited an eclipse period of 20u2009min and a burst size of 100 particles per cell. Its genome assembled as a circular molecule when genomic sequencing was completed. However, based on genome content and organization, it was categorized as a classic T7‐related phage, and such phages are known to have linear genomes with direct terminal repeats. With the quick and simple method established herein, the 38,625‐bp linear double‐stranded DNA with 229‐bp direct terminal repeats was accurately identified. The genome contained 43 putative open reading frames and no tRNA genes. Using a proteomics‐based approach, seven viral and two host proteins from purified phiCFP‐1 particles were identified. Comparative genomics and recombination analyzes revealed close genetic relatedness among phiCFP‐1, phiYeO3‐12/vB_YenP_AP5 (from Yersinia enterocolitica O3), and phiSG‐JL2 (from Salmonella enterica). J. Med. Virol. 88:895–905, 2016.

Survey and Visual Detection of Zaire ebolavirus in Clinical Samples Targeting the Nucleoprotein Gene in Sierra Leone.

Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect Zaire ebolavirus using the nucleoprotein gene (NP) as a target sequence. Two different techniques were used, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.

Cirrhosis related functionality characteristic of the fecal microbiota as revealed by a metaproteomic approach.

BackgroundIntestinal microbiota operated as a whole and was closely related with human health. Previous studies had suggested close relationship between liver cirrhosis (LC) and gut microbiota.MethodsTo determine the functional characteristic of the intestinal microbiota specific for liver cirrhosis, the fecal metaproteome of three LC patients with Child-Turcotte-Pugh (CTP) score of A, B, and C, and their spouse were first compared using high-throughput approach based on denaturing polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry in our study.ResultsA total of 5,020 proteins (88xa0% from bacteria, 12xa0% form human) were identified and annotated based on the GO and KEGG classification. Our results indicated that the LC patients possessed a core metaproteome including 119 proteins, among which 14 proteins were enhanced expressed and 7 proteins were unique for LC patients compared with the normal, which were dominant at the function of carbohydrate metabolism. In addition, LC patients have unique biosynthesis of branched chain amino acid (BCAA), pantothenate, and CoA, enhanced as CTP scores increased. Those three substances were all important in a wide array of key and essential biological roles of life.ConclusionsWe observed a highly comparable cirrhosis-specific metaproteome clustering of fecal microbiota and provided the first supportive evidence for the presence of a LC-related substantial functional core mainly involved in carbohydrate, BCAA, pantothenate, and CoA metabolism, suggesting the compensation of intestinal microbiota for the fragile and innutritious body of cirrhotic patients.

Isolation and characterization of a bacteriophage phiEap-2 infecting multidrug resistant Enterobacter aerogenes

Enterobacter aerogenes (Enterobacteriaceae) is an important opportunistic pathogen that causes hospital-acquired pneumonia, bacteremia, and urinary tract infections. Recently, multidrug-resistant E. aerogenes have been a public health problem. To develop an effective antimicrobial agent, bacteriophage phiEap-2 was isolated from sewage and its genome was sequenced because of its ability to lyse the multidrug-resistant clinical E. aerogenes strain 3-SP. Morphological observations suggested that the phage belongs to the Siphoviridae family. Comparative genome analysis revealed that phage phiEap-2 is related to the Salmonella phage FSL SP-031 (KC139518). All of the structural gene products (except capsid protein) encoded by phiEap-2 had orthologous gene products in FSL SP-031 and Serratia phage Eta (KC460990). Here, we report the complete genome sequence of phiEap-2 and major findings from the genomic analysis. Knowledge of this phage might be helpful for developing therapeutic strategies against E. aerogenes.