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Graphical abstract, Highlights • MRSA infection is a global threat to public health. • Vancomycin is one of the first-line drugs for the treatment of MRSA infections. • MRSA with complete resistance to vancomycin have emerged in recent years. • The total number of VRSA isolates is updated in this paper. • Resistance mechanisms, characteristics of VRSA infections, as well as clinical treatments are reviewed., The infection caused by methicillin-resistant Staphylococcus aureus (MRSA) is a global threat to public health. Vancomycin remains one of the first-line drugs for the treatment of MRSA infections. However, S. aureus isolates with complete resistance to vancomycin have emerged in recent years. Vancomycin-resistant S. aureus (VRSA) is mediated by a vanA gene cluster, which is transferred from vancomycin-resistant enterococcus. Since the first VRSA isolate was recovered from Michigan, USA in 2002, 52 VRSA strains have been isolated worldwide. In this paper, we review the latest progresses in VRSA, highlighting its resistance mechanism, characteristics of VRSA infections, as well as clinical treatments.

Chlamydia trachomatis Scc4 (formerly CT663) engages the transcription machinery and the pathogenic type III secretion system (T3SS). Both machines are required for Chlamydia infection. These requirements and the limited ability for genetic manipulation in Chlamydia have hampered dissection of Scc4’s contributions. Here, by developing bacterial systems that permit the controlled expression and stable maintenance of Scc4, we assess Scc4’s effects on chlamydial growth phenotype, secretion, and the patterns of T3SS gene expression. Expressing Scc4 in Escherichia coli lacking a T3SS injectisome causes a growth defect. This deficiency is rescued by overexpressing the β-subunit of RNA polymerase (RNAP) or by exploiting sigma 70 (σ(70)) (homologous to chlamydial σ(66)) mutants that strengthen the interaction between σ(70) region 4 and the β-flap, confirming Scc4’s distinction as a module of RNAP holoenzyme capable of modulating transcription. Yersinia pestis expressing Scc4 sustains a functional T3SS, through which CopN secretion is boosted by cooption of Scc4 and Scc1. Finally, conditional expression of Scc4 in C. trachomatis results in fast expansion of the Chlamydia-containing vacuole and accelerated chlamydial development, coupled to selective up- or downregulation of gene expression from different T3SS genes. This work reveals, for the first time, the context-dependent action of Scc4 linking it to diverse protein networks in bacteria. It establishes that Scc4, when overexpressed, exerts incredible effects on chlamydial development by reinforcing control of the T3SS. IMPORTANCE The T3SS is a key virulence factor required for C. trachomatis infection. The control of the T3SS has not been well studied in this obligate intracellular pathogen. Here, we show that Scc4 plays a major role for precise control of the pathogenic T3SS at the levels of gene expression and effector secretion through genetically separable protein networks, allowing a fast adaptive mode of C. trachomatis development during infection in human epithelial cells.

Vi capsular polysaccharide, a linear homopolymer of α-1,4-linked N-acetylgalactosaminuronate, is characteristically produced by Salmonella enterica serovar Typhi. The Vi capsule covers the surface of the producing bacteria and serves as an virulence factor via inhibition of complement-mediated killing and promoting resistance against phagocytosis. Furthermore, Vi also represents a predominant protective antigen and plays a key role in the development of vaccines against typhoid fever. Herein, we reviewed the latest advances associated with the Vi polysaccharide, from its synthesis and transport within bacterial cells, mechanisms involved in virulence, immunological characteristics, and applications in vaccine, as well as its purification and detection methods.

We recently found lytic action of the truncated yncE gene. When the truncated yncE gene of Salmonella enterica serovar Paratyphi A was expressed in Escherichia coli DH5α under the control of the Ara promoter, bacterial growth was markedly inhibited. In the present study, we characterized this lytic action. The N-terminal 103 aa of YncE, containing a signal peptide, was demonstrated to be essential for inhibition. Microscopic observation showed that the bacterial envelope of E. coli was damaged by the expression of truncated yncE, resulting in the release of cytoplasmic content and the formation of bacterial ghosts. The addition of MgSO4 or spermine, which is the stabilizer of bacterial membrane structure, dramatically reversed the cell lysis induced by the toxic truncated YncE. In contrast, the lytic action was significantly enhanced by the addition of SDS or EDTA. Our data indicated that the toxic truncated YncE could cause cell lysis by the disruption of the bacterial membrane.

Enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased in recent years and became a global health issue. Currently licensed typhoid vaccines do not confer adequate cross-immunoprotection against S. Paratyphi A infection. Therefore, vaccines specifically against enteric fever caused by S. Paratyphi A are urgently needed. In the present study, an attenuated vaccine strain was constructed from S. Paratyphi A CMCC50093 by the deletions of aroC and yncD. The obtained strain SPADD01 showed reduced survival within THP-1 cells and less bacterial burden in spleens and livers of infected mice compared with the wild-type strain. The 50% lethal doses of SPADD01 and the wild-type strain were assessed using a murine infection model. The virulence of SPADD01 is approximately 40,000-fold less than that of the wild-type strain. In addition, SPADD01 showed an excellent immunogenicity in mouse model. Single intranasal inoculation elicited striking humoral and mucosal immune responses in mice and yielded effective protection against lethal challenge of the wild-type strain. A high level of cross-reactive humoral immune response against LPS of Salmonella enterica serovar Typhi was also detected in immunized mice. However, SPADD01 vaccination only conferred a low level of cross-protection against S. Typhi. Our data suggest that SPADD01 is a promising vaccine candidate against S. Paratyphi A infection and deserves further evaluation in clinical trial. To date, no study has demonstrated a good cross-protection between serovars of S. Typhi and S. Paratyphi A, suggesting that the dominant protective antigens of both serovars are likely different and need to be defined in future study.

Orally administered vaccine bacteria usually persist for a period of time in the intestinal tracts of immunized individuals, and are excreted in feces to the environment resulting in a potential biosafety issue. The releasing risk can be minimized by immediate elimination of the persistent vaccine bacteria once adequate protective immune responses have been elicited by the vaccine bacteria. In a previous study, inducible expression of truncated yncE gene (yncE*) was found lethal to host bacteria. This feature has an application potential in biosafety control. Here, we assessed the efficacy of YncE* in eliminating an attenuated strain of Salmonella enterica serovar Typhimurium in a mouse model. To this end, a pBAD-derived plasmid containing yncE* under the control of the Ara promoter was transformed into a ΔphoPQ mutant of S. Typhimurium. Our data show that the induced expression of yncE* in the presence of arabinose eliminated the vaccine bacteria both in vitro and in vivo. BALB/c mice with or without streptomycin-pretreatment were used to assess the efficacy of YncE* in vivo. Oral administration of 500 μl of 20% arabinose at 24 h postvaccination removed the vaccine bacteria from the guts of the tested mice without streptomycin-pretreatment. For streptomycin-pretreated mice, which were colonized with higher levels of Salmonella, an additional gavage of arabinose was required to completely eliminate the vaccine bacteria in the guts of the tested mice. The orally administered arabinose did not affect the persistence of bacteria that had penetrated the intestinal mucosa of the immunized mice. Furthermore, there was no significant difference in the protection rate between the routine immunization and the immunization with the arabinose treatment. The results indicate that the yncE* element improves the biosafety of the bacterial vaccine, and can be taken in consideration in future design of live bacterial vaccines.

Methicillin-susceptible Staphylococcus aureus (MSSA) accounts for ∼40% of staphylococcal infections in China. However, the molecular characterization of MSSA is not well described. In this study, 124 MSSA strains collected in 2013 from a comprehensive teaching hospital in Chongqing, Southwestern China, were subjected to antibiotics susceptibility testing and molecular typing, including multilocus sequence typing, staphylococcal protein A (spa) gene typing, accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) typing, Panton-Valentine leukocidin (pvl) gene detection, and antibiotic-resistant gene detection. MSSA strains exhibited high genetic heterogeneity. A total of 10 PFGE groups, 26 sequence types, and 47 spa types were identified. Type I (62.9%) was the most frequent agr type, followed by type II (15.3%), type IV (11.3%), and type III (10.5%). The prevalence of pvl genes was 27.4% (34/124). Notably, 44.4% (55/124) of MSSA strains were multidrug resistance (MDR), and MDR isolates were mostly resistant to penicillin, erythromycin, and clindamycin. The resistance gene blaZ was present in 84.7% of strains, ermC was present in 85.5% of strains, ermA was present in 28.2% of strains, tetK was present in 16.1% of strains, tetM was present in 6.5% of strains, and aacA-aphD was present in 2.6% of strains. These data demonstrated the high prevalence of MDR MSSA in Chongqing, thereby indicating the need to control MSSA infection.

Streptococcus suis (S. suis) serotype 2 usually cause infection in swine. Recently, two large-scale outbreaks in China with severe streptococcal toxic shock syndrome (STSS) and high mortality raised worldwide concern to human S. suis infection. To reveal the molecular pathogenesis of S. suis 2 during human infection, in-vivo induced antigen technology (IVIAT) was applied to identify the in-vivo induced genes (ivi genes) of S. suis 05ZYH33. The ivi genes are specifically expressed or up-regulated in-vivo and always associated with the in-vivo survival and pathogenicity of pathogens. In present study, convalescent sera from S. suis 05ZYH33 infected patients were pooled and fully adsorbed with in-vitro grown S. suis 05ZYH33 and Escherichia coli BL21 (DE3). Genomic expression library of 05ZYH33 was repeatedly screened with colony immunoblot assay using adsorbed sera. Finally, 19 genes were assessed as ivi genes of 05ZYH33. Fifteen of 19 genes encode proteins with biological functions in substance transport and metabolism, cell structure biogenesis, cell cycle control, replication, translation and other functions. The 4 remaining genes encode proteins with unknown functions. Of the 19 ivi genes, five (SSU05_0247, 0437, 1577, 1664 and 2144) encode proteins with no immunoreactivity to control sera from healthy individuals never exposed to 05ZYH33. The successful identification of ivi genes not only sheds light on understanding the pathogenesis of S. suis 05ZYH33 during its human infection, but also provides potential targets for the developments of new vaccines, therapeutic drugs and diagnostic reagents against human S. suis infection.

Chlamydia trachomatis is an important human pathogen that undergoes a characteristic development cycle correlating with stage-specific gene expression profiles. Taking advantage of recent developments in the genetic transformation in C. trachomatis, we constructed a versatile green fluorescent protein (GFP) reporter system to study the development-dependent function of C. trachomatis promoters in an attempt to elucidate the mechanism that controls C. trachomatis adaptability. We validated the use of the GFP reporter system by visualizing the activity of an early euo gene promoter. Additionally, we uncovered a new ompA promoter, which we named P3, utilizing the GFP reporter system combined with 5′ rapid amplification of cDNA ends (RACE), in vitro transcription assays, real-time quantitative RT-PCR (RT-qPCR) and flow cytometry. Mutagenesis of the P3 region verifies that P3 is a new class of C. trachomatis σ66-dependent promoter, which requires an extended −10 TGn motif for transcription. These results corroborate complex developmentally controlled ompA expression in C. trachomatis. The exploitation of genetically labeled C. trachomatis organisms with P3-driven GFP allows for the observation of changes in ompA expression in response to developmental signals. The results of this study could be used to complement previous findings and to advance understanding of C. trachomatis genetic expression.

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that are usually involved in the uptake of certain key nutrients, for example iron. In the genome of Salmonella enterica ssp. enterica serovar Typhi, the yncD gene encodes a putative TBDT and was identified recently as an in vivo-induced antigen. In the present study, a yncD-deleted mutant was constructed to evaluate the role of the yncD gene in virulence. Our results showed that the mutant is attenuated in a mouse model by intraperitoneal injection and its virulence is restored by the transformation of a complement plasmid. The competition experiments showed that the survival ability of the yncD-deleted mutant decreases significantly in vivo. To evaluate its vaccine potential, the yncD-deleted mutant was inoculated intranasally in the mouse model. The findings demonstrated a significant immunoprotection against the lethal wild-type challenge. The regulation analysis showed that yncD gene promoter is upregulated under acidic condition. The present study demonstrates that the yncD gene plays an important role in bacterial survival inside the host and is suitable for the construction of attenuated vaccine strains as a candidate target gene.

Secretion of effector proteins into the eukaryotic host cell is required for Chlamydia trachomatis virulence. In the infection process, Scc1 and Scc4, two chaperones of the type III secretion (T3S) system, facilitate secretion of the important effector and plug protein, CopN, but little is known about the details of this event. Here we use biochemistry, mass spectrometry, nuclear magnetic resonance spectroscopy, and genetic analyses to characterize this trimolecular event. We find that Scc4 complexes with Scc1 and CopN in situ at the late developmental cycle of C. trachomatis. We show that Scc4 and Scc1 undergo dynamic interactions as part of the unique bacterial developmental cycle. Using alanine substitutions, we identify several amino acid residues in Scc4 that are critical for the Scc4-Scc1 interaction, which is required for forming the Scc4·Scc1·CopN ternary complex. These results, combined with our previous findings that Scc4 plays a role in transcription (Rao, X., Deighan, P., Hua, Z., Hu, X., Wang, J., Luo, M., Wang, J., Liang, Y., Zhong, G., Hochschild, A., and Shen, L. (2009) Genes Dev. 23, 1818-1829), reveal that the T3S process is linked to bacterial transcriptional events, all of which are mediated by Scc4 and its interacting proteins. A model describing how the T3S process may affect gene expression is proposed.

Bacterial swarming motility is a flagella-dependent translocation on the surface environment. It has received extensive attention as a population behavior involving numerous genes. Here, we report that Citrobacter freundii, an opportunistic pathogen, exhibits swarming movement on a solid medium surface with appropriate agar concentration. The swarming behavior of C. freundii was described in detail. Insertional mutagenesis with transposon Mini-Tn5 was carried out to discover genetic determinants related to the swarming of C. freundii. A number of swarming genes were identified, among which flhD, motA, motB, wzx, rfaL, rfaJ, rfbX, rfaG, rcsD, rcsC, gshB, fabF, dam, pgi, and rssB have been characterized previously in other species. In mutants related to lipopolysaccharide synthesis and RcsCDB signal system, a propensity to form poorly motile bacterial aggregates on the agar surface was observed. The aggregates hampered bacterial surface migration. In several mutants, the insertion sites were identified to be in the ORF of yqhC, yeeZ, CKO_03941, glgC, and ttrA, which have never been shown to be involved in swarming. Our results revealed several novel characteristics of swarming motility in C. freundii which are worthy of further study.

As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-β, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-β products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9 Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of α-factor by the STE13 protease. Expressed hPAB-β was secreted into the culture medium at a level of 241.2 ± 29.5 mg/L. Purified hPAB-β with 95% homogeneity was obtained by 10 kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose™ XL column. The two major protein species separated through a SOURCE™ 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-β with minimal inhibitory concentrations of 8–64 μg/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-β, and that expressed hPAB-β in P. pastoris may be a potential antimicrobial agent against MRSA infection.

Objectives: To characterize morphological, physicochemical and genomic features of a novel virulent coliphage which was isolated from an engineered Escherichia coli culture and termed engineered E. coli phage (EEP). Methods and Results: Electron microscopy revealed that EEP has an icosahedral head (62 nm in diameter) and a long, flexible tail (138 nm in length). EEP was able to infect all 10 engineered E. coli strains kept in our laboratory, showing a strong ability to lyse engineered E. coli. Sequencing of the EEP genome revealed a double-stranded DNA (39.8 kb) with 54.72% GC content. Fifty-two open reading frames were predicted to be coding sequences, 18 of which were functionally defined and organized in a modular format, which includes modules for DNA replication, DNA packaging, structural proteins and host cell lysis. This phage could not be inactivated at 90° for 45 min and was resistant to ethanol and alkali treatment. EEP is assigned to the Siphoviridae family based on its morphological, genomic and physicochemical properties. Conclusions: A novel coliphage was isolated from engineered E. coli strains, and its morphological, genomic and physicochemical properties were characterized, which will improve our knowledge of bacteriophage diversity.

During infectious disease episodes, pathogens express distinct subsets of virulence factors which allow them to adapt to different environments. Hence, genes that are expressed or upregulated in vivo are implicated in pathogenesis. We used in vivo induced antigen technology (IVIAT) to identify antigens which are expressed during infection with Salmonella enterica serovar Typhi. We identified 7 in vivo induced (IVI) antigens, which included BcfD (a fimbrial structural subunit), GrxC (a glutaredoxin 3), SapB (an ABC-type transport system), T3663 (an ABC-type uncharacterized transport system), T3816 (a putative rhodanese-related sulfurtransferase), T1497 (a probable TonB-dependent receptor) and T3689 (unknown function). Of the 7 identified antigens, 5 antigens had no cross-immunoreactivity in adsorbed control sera from healthy subjects. These 5 included BcfD, GrxC, SapB, T3663 and T3689. Antigens identified in this study are potential targets for drug and vaccine development and may be utilized as diagnostic agents.

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that can cause severe disease and even death in both humans and swine. No effective vaccine is clinically available. In this study, a reverse vaccinology method was first applied to identify protective antigens against S. suis 2. As a consequence, 153 genes encoding vaccine candidates were selected from the whole genome sequence by means of bioinformatics analysis, from which 10 genes were selected based on experimental evidences arising from the study of related bacteria such as Streptococcus pneumoniae, group B streptococcus, S. suis and so on. Of 10 target genes, 8 were successfully expressed in Escherichia coli Rosetta, and expressed proteins were purified and used as the immunogens for evaluating vaccine efficacy in a mouse infection model. The results have confirmed that RTX family exoprotein A (RfeA), epidermal surface antigen (ESA), immunoglobulin G (IgG)-binding protein (IBP), and suilysin (SLY) can induce a protective response of the vaccinated animals against S. suis 2, whereas RfeA, ESA, and IBP mainly induce humoral-mediated immunity, and SLY elicits a combined pattern of both humoral- and cellular-mediated immunity. Although immunoprotection of SLY against S. suis 2 was reported previously, RfeA, ESA, and IBP were explored first in this study.

The global epidemic features of enteric fever have changed greatly in recent years. The incidence of enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased. In some areas of Asia, infections with S. Paratyphi A have exceeded those with S. Typhi, resulting in S. Paratyphi A becoming the main causative agent of enteric fever. However, two currently licensed typhoid vaccines do not confer adequate cross-protection against S. Paratyphi A infection. Therefore, development of specific vaccines against enteric fever caused by S. Paratyphi A is urgently needed. In the present study, an attenuated strain was constructed by double deletion of the htrA and yncD genes in a wild-type strain of S. Paratyphi A and its safety and immunogenicity assessed. In a mouse model, the 50% lethal dose of the double deletion mutant and the wild-type strain were 3.0 × 10(8) CFU and 1.9 × 10(3) CFU, respectively, suggesting that the double deletion resulted in remarkably decreased bacterial virulence. Bacterial colonization of the double deletion mutant in the livers and spleens of infected mice was strikingly less than that of the wild-type strain. A single nasal administration of the attenuated vaccine candidate elicited high concentrations of anti-LPS and anti-flagellin IgG in a mouse model and protected immunized mice against lethal challenge with the wild-type strain. Thus, our findings suggest that the attenuated vaccine strain is a promising candidate worthy of further evaluation both as a human enteric fever vaccine and as a vaccine delivery vector for heterologous antigens.

Heterodimeric beta-galactosidase of Lactobacillus acidophilus belongs to glycoside hydrolase family 2, encoded by two overlapping and translational coupling genes, lacL and lacM. The lacL and lacM genes of the sequenced strain L. acidophilus NCFM encode polypeptides with calculated molecular masses of 73,253 and 35,817 Da, respectively.To clone, overexpress and characterize the enzyme in Escherichia coli.We cloned the fragment (2834 bp) containing ribose-binding site (RBS) and coding regions of the lacLM genes from L. acidophilus ATCC 4356 into expression vector pQE31. RBS and HIS-Tag of pQE31 were substituted by inserted fragment. Recombinant plasmid was electrotransformed into E. coli JM109. Expression product was purified by ammonium sulphate fractionation, anion-exchange, affinity chromatography and gel permeation. Native molecular mass of homogenous enzyme was measured by gel permeation, and beta-galactosidase activity was determined by using o-nitrophenyl-beta-D-galactopyranoside (ONPG) as the substrate.Overexpression of the soluble enzyme in E. coli was achieved. Amino acid residue 512 of recombinant LacL was different from that of L. acidophilus NCFM. Homogenous enzyme was obtained by purification. The homogenous enzyme had a specific activity of 226 U/mg protein, a native molecular mass of 96.3+/-4.6 kDa, an optimum temperature at 49 degrees C and an optimum pH of 7. The Km and Vmax values of the enzyme were 2.18+/-0.12 mmol/L, 273+/-5 U/mg protein respectively.

Peptide antibiotics are small peptides encoded by organism genomic DNA. They are recognized to play important roles in the innate host defense of most living organisms. The growing resistance of bacteria to conventional antibiotics and the need for discovery of new antibiotics have stimulated great interest in the development of peptide antibiotics as human therapeutics. However, preparation of peptide antibiotics at a large scale is a great challenge in developing these commercial products. In this study, tandem repeat multimers of peptide antibiotic hPAB-beta were designed and the recombinant plasmids containing one to eight copies of hPAB-beta gene were generated. Eight genetic engineered bacteria harboring pQE-hPAB-beta1-8 recombinant were able to express the repetitive hPAB-beta multimers of interest in inclusion bodies, respectively. The expressed proteins could reach 2.6-28% of the total proteins. The hPAB-beta trimer construct was selected out for the subsequent study based on its higher expression level (27.8%), which yields in wet cell weights (3.15+/-0.45 g/l) and the fusion protein inclusion bodies was able to completely dissolve in 8 M urea. The tandem trimers could easily be captured by Ni-NTA affinity chromatography and cleaved into monomers by hydroxylamine. Then, the monomer hPAB-beta of interest was purified to 95% homogeneity by reverse phase chromatography and gel filtration. The final yield of purified recombinant monomer hPAB-beta was 680+/-12 mg/100 g wet cells. The minimum inhibitory concentrations (MICs) of the purified recombinant hPAB-beta against type or clinical strains of microorganisms were about 31-250 microg/ml and these results showed that the recombinant hPAB-beta could retain its bioactivity.