In this study, we discovered a novel mobilized colistin resistance (mcr-1) gene variant, named mcr-1.9, which was identified in a colistin-resistant enterotoxigenic Escherichia coli (ETEC) strain from a clinical diarrhea case. The mcr-1.9 gene differs from mcr-1 at position 1036 due to a single nucleotide polymorphism (G→A), which results in an aspartic acid residue being replaced by an asparagine residue (Asp346→Asn) in the MCR-1 protein sequence. Antimicrobial susceptibility testing showed that the mcr-1.9-harboring ETEC strain is resistant to colistin at a minimum inhibitory concentration of 4 μg/ml. Plasmid profiling and conjugation experiments also suggest that the mcr-1.9 variant can be successfully transferred into the E. coli strain J53, indicating that the gene is located on a transferable plasmid. Bioinformatics analysis of data obtained from genome sequencing indicates that the mcr-1.9 gene is located on a 64,005 bp plasmid which has been named pEC26. This plasmid was found to have high similarity to the mcr-1-bearing IncI2-type plasmids pWF-5-19C (99% identity and 99% coverage) and pmcr1-IncI2 (99% identity and 98% coverage). The mcr-1.9-harboring ETEC also shows multidrug resistance to nine classes of antibiotics, and contains several virulence and antimicrobial-resistance genes suggested by the genome sequence analysis. Our report is the first to identify a new mcr-1 variant in an ETEC isolated from a human fecal sample, raising concerns about the existence of more such variants in human intestinal flora. Therefore, we believe that an undertaking to identify new mcr-1 variants in the bacterial communities of human intestines is of utmost importance, and that measures need to be taken to control the spread of mcr-1 and its variants in human intestinal microflora.

We aimed to analyze the antimicrobial resistance phenotypes and to elucidate the molecular mechanisms underlying resistance to cephalosporins, ciprofloxacin, and azithromycin in Salmonella enterica serovar Typhimurium isolates identified from patients with diarrhea in Shanghai. The isolates showed high rates of resistance to traditional antimicrobials, and 20.6, 12.7, and 5.5% of them exhibited decreased susceptibility to cephalosporins, ciprofloxacin, and azithromycin, respectively. Notably, 473 (84.6%) isolates exhibited multidrug resistance (MDR), including 161 (28.8%) isolates that showed an ACSSuT profile. Twenty-two MDR isolates concurrently exhibited decreased susceptibility to cephalosporins and ciprofloxacin, and six of them were co-resistant to azithromycin. Of all the 71 isolates with decreased susceptibility to ciprofloxacin, 65 showed at least one mutation (D87Y, D87N, or D87G) in gyrA, among which seven isolates simultaneously had mutations of parC (S80R) (n = 6) or parC (T57S/S80R) (n = 1), while 49 isolates with either zero or one mutation in gyrA contained plasmid-mediated quinolone resistance (PMQR) genes including qnrB, qnrS, and aac(6')-Ib-cr. Among the 115 cephalosporin-resistant isolates, the most common ESBL gene was blaCTX-M, followed by blaTEM-1, blaOXA-1, and blaSHV -12. Eight subtypes of blaCTX-M were identified and blaCTX-M-14 (n = 22) and blaCTX-M-55 (n = 31) were found to be dominant. To the best of our knowledge, this is the first report of the presence of blaCTX-M-123 and blaCTX-M-125 in S. Typhimurium. Besides, mphA gene was identified in 15 of the 31 azithromycin-resistant isolates. Among the 22 isolates with reduced susceptibility to cephalosporins and ciprofloxacin, 15 contained ESBL and PMQR genes. Coexistence of these genes lead to the emergence of MDR and the transmission of them will pose great difficulties in S. Typhimurium treatments. Therefore, surveillance for these MDR isolates should be enhanced.

Worldwide, Shewanella putrefaciens is the predominant seafood spoilage microorganism during cold storage. This bacterium can attach to biotic/abiotic surfaces to form biofilms which contribute to seafood quality degradation and shelf-life reduction. The mechanism of S. putrefaciens biofilm formation is not yet described. Crystal violet staining in combination with confocal laser scanning microscopy (CLSM) was used to study the sequence of events leading to the establishment of a mature biofilm at 4, 15, and 30°C. In addition, the main chemical constituents of the mature biofilm were determined by Raman spectroscopy (RM), whereas, comparative proteomic analysis was used to quantify changes in metabolic pathways and to find out underlying protein determinants. The physical dimensions of the mature biofilm, i.e., biomass, biovolume, and mean thickness, were higher at 4°C when compared to 15 and 30°C. The variations of proteins measured by RM confirmed the importance of proteins during the formation of a mature biofilm. Comparative proteomic analysis showed that siderophore and iron chelate transport proteins were down-regulated during mature biofilm formation. The down-regulated aforementioned proteins are involved in promoting iron storage in response to a higher demand for metabolic energy, whereas, the upregulated proteins of the sulfur relay system, pyrimidine metabolism, and purine metabolism are related to bacterial adaptability. Synthesis of proteins related to cold stress was increased and proteins involved in aminoacyl-tRNA biosynthesis were up-regulated, whereas, proteins involved in aminopeptidase activity were down-regulated. Proteolysis to scavenge energy was reduced as proteins involved in pyrophosphatase activity were up-regulated. Also extracellular eDNA was found which may play an important role in maintaining the stability of mature S. putrefaciens biofilm structures under cold stress. This work provides a better understanding of the role of proteins in mature biofilms. In addition, the biofilm formation mechanism of a psychrotrophic spoilage bacterial species at low temperature is explored, which may contribute to generating biofilm controlling strategies during seafood preservation and processing.

Glutathione (GSH) plays a key role in regulating the cellular Redox Homeostasis, and appears to be essential for initiation and development of root nodules. Glutathione peroxidase (Gpx) catalyzes the reduction of H2O2 and organic hydroperoxides by oxidation of GSH to oxidized GSH (GSSG), which in turn is reduced by glutathione reductase (GR). However, it has not been determined whether the Rhizobium leguminosarum Gpx or GR is required during symbiotic interactions with pea. To characterize the role of glutathione-dependent enzymes in the symbiotic process, single and double mutants were made in gpxA (encoding glutathione peroxidase) and gshR (encoding glutathione reductase) genes. All the mutations did not affect the rhizobial growth, but they increased the sensitivity of R. leguminosarum strains to H2O2. Mutant in GpxA had no effect on intracellular GSH levels, but can increase the expression of the catalase genes. The gshR mutant can induce the formation of normal nodules, while the gpxA single and double mutants exhibited a nodulation phenotype coupled to more than 50% reduction in the nitrogen fixation capacity, these defects in nodulation were characterized by the formation of ineffective nodules. In addition, the gpxA and gshR double mutant was severely impaired in rhizosphere colonization and competition. Quantitative proteomics using the TMT labeling method was applied to study the differential expression of proteins in bacteroids isolated from pea root nodules. A total of 27 differentially expressed proteins were identified in these root bacteroids including twenty down-regulated and seven up-regulated proteins. By sorting the down-regulated proteins, eight are transporter proteins, seven are dehydrogenase, deoxygenase, oxidase, and hydrolase. Moreover, three down-regulating proteins are directly involved in nodule process.

Composition of the gut microbiota has been linked with human immunedeficiency virus (HIV)-infected patients on antiretroviral therapy (ART). Evidence suggests that ART-treated patients with poor CD4+ T-cell recovery have higher levels of microbial translocation and immune activation. However, the association of the gut microbiota and immune recovery remains unclear. We performed a cross-sectional study on 30 healthy controls (HC) and 61 HIV-infected individuals, including 15 immunological ART responders (IRs), 20 immunological ART non-responders (INRs) and 26 untreated individuals (VU). IR and INR groups were classified by CD4+ T-cell counts of ≥350 cells/mm3 and <350 cells/mm3 after 2 years of ART, respectively. Each subject's gut microbiota composition was analyzed by metagenomics sequencing. Levels of CD4+ T cells, CD8+HLA-DR+ T cells and CD8+CD38+ T cells were measured by flow cytometry. We identified more Prevotella and fewer Bacteroides in HIV-infected individuals than in HC. Patients in INR group were enriched with Faecalibacterium prausnitzii, unclassified Subdoligranulum sp. and Coprococcus comes when compared with those in IR group. F. prausnitzii and unclassified Subdoligranulum sp. were overrepresented in individuals in VU group with CD4+ T-cell counts <350 cells/mm3. Moreover, we found that the relative abundance of unclassified Subdoligranulum sp. and C. comes were positively correlated with CD8+HLA-DR+ T-cell count and CD8+HLA-DR+/CD8+ percentage. Our study has shown that gut microbiota changes were associated with CD4+ T-cell counts and immune activation in HIV-infected subjects. Interventions to reverse gut dysbiosis and inhibit immune activation could be a new strategy for improving immune reconstitution of HIV-1-infected individuals.

Shigellosis, caused by Shigella, is a major global health concern, with nearly 164.7 million cases and over a million deaths occurring annually worldwide. Shigella flexneri is one of the most common subgroups of Shigella with a high incidence of multidrug-resistance. The phage therapy approach is an effective method for controlling multidrug-resistant bacteria. However, only a few Shigella phages have been described to date. In this study, a novel lytic bacteriophage SFPH2 was isolated from a sewage sample obtained from a hospital in Beijing, China, using a multidrug-resistant S. flexneri 2a strain (SF2) isolated from the fecal sample of a dysentery patient. SFPH2 is a member of the Podoviridae virus family with an icosahedral capsid and a short, non-contractile tail. It was found to be stable over a wide range of temperatures (4-50°C) and pH values (pH 3-11). Moreover, SFPH2 could infect two other S. flexneri serotypes (serotypes 2 variant and Y). High-throughput sequencing revealed that SFPH2 has a linear double-stranded DNA genome of 40,387 bp with 50 open reading frames. No tRNA genes were identified in the genome. Comparative analysis of the genome revealed that the SFPH2 belongs to the subfamily Autographivirinae and genus T7virus. The genome shows high similarity with other enterobacterial T7virus bacteriophages such as Citrobacter phage SH4 (95% identity and 89% coverage) and Cronobacter phage Dev2 (94% identity and 92% coverage). A comparison of the fiber proteins showed that minor differences in the amino acid residues might specify different protein binding regions and determine host species. In conclusion, this is the first report of a T7virus that can infect Shigella; SFPH2 has a functional stability under a wide range of temperatures and pH values, showing the potential to be widely applied to control Shigella-associated clinical infections and reduce the transmission rates of S. flexneri serotype 2a and its variants in the environment.

The rapid emergence of multidrug resistant Salmonella is a global public-health concern as outbreaks in recent years have mostly been caused by multidrug resistant strains. Here, we evaluated an outbreak in China caused by multidrug resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) by employing an epidemiological and laboratory investigation using conventional methods and whole genome sequencing (WGS). Eleven of the 12 people who participated in a banquet showed gastrointestinal symptoms, and 8S. Typhimurium strains were recovered. Isolated outbreak strains showed multidrug resistance (MDR), and decreased susceptibility to ciprofloxacin, a first-line drug recommended by WHO for clinical treatment of intestinal infections. Antimicrobial resistance (AMR) gene analysis indicated that the MDR phenotype of these outbreak strains may be due to the presence of a number of AMR genes, including the blaOXA-1 and blaTEM-1 β-lactamase genes, which are often plasmid-borne and easily transferred. Further virulence gene analysis indicated that these outbreak strains also carried a large number of virulence genes, including 2 types of Salmonella pathogenicity islands (SPI-1 and SPI-2) and many adhesion-related virulence genes. Cluster analysis based on pulse-field gel electrophoresis data and phylogenetic analysis based on WGS revealed that the outbreak clone was closely related to and thus probably derived from local strains. This outbreak caused by multidrug resistant S. Typhimurium highlights the need for government improved strategies for the prevention and control of Salmonella infections.

Food spoilage by certain species of bacteria is reported to be regulated by quorum sensing (QS). Acinetobacter johnsonii and Pseudomonas fluorescens, the major specific spoilage organisms, are found to be limited in their QS and co-culture interactions. The aim of this study was to determine how QS-regulated proteins affect the spoilage potential of co-cultured A. johnsonii and P. fluorescens obtained from spoiled bigeye tuna (Thunnus obesus) using a proteomics approach. The A. johnsonii, P. fluorescens, and their co-culture tested the N-acyl-homoserine lactone (AHL) activities using reporter Chromobacterium violaceum CV026 and LC-MS/MS in qualitative and quantitative approaches, respectively. These latter showed that, of the 470 proteins and 444 proteins in A. johnsonii (A) and P. fluorescens (P), respectively, 80 were significantly up-regulated and 97 were significantly down-regulated in A vs. AP, whereas 90 were up-regulated and 65 were down-regulated in P vs. AP. The differentially expressed proteins included the AI-2E family transporter OS, 50S ribosomal protein L3, thioredoxin reductase OS, cysteine synthase CysM OS, DNA-binding response regulator, and amino acid ABC transporter ATPase OS. The cellular process (GO:0009987), metabolic process (GO:0008152), and single-organism process (GO:0044699) were classified into the gene ontology (GO) term. In addition, energy production and conversion, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, post-translational modification, protein turnover, and chaperones were distributed into the clusters of orthologous groups of proteins (COG) terms. The KEGG pathways revealed that 84 and 77 differentially expressed proteins were divided into 20 KEGG pathways in A vs. AP and P vs. AP, respectively, and amino acid metabolism, carbohydrate metabolism, energy metabolism, and translation were significantly enriched. Proteins that correlated with the spoilage-related metabolic pathways, including thioredoxin reductase OS, cysteine synthase OS, and pyridoxal phosphate-dependent enzyme family protein OS, were identified. AI-2E family transporter OS and LuxR family transcriptional regulator OS were identified that related to the QS system. These findings provide a differential proteomic profile of co-culture in A. johnsonii and P. fluorescens, and have potential applications in QS and the regulation of spoilage potential.