Influence of human motion patterns on epidemic spreading dynamics.

Extensive real-data indicate that human motion exhibits novel patterns and has a significant impact on the epidemic spreading process. The research on the influence of human motion patterns on epidemic spreading dynamics still lacks a systematic study in network science. Based on an agent-based model, this paper simulates the spread of the disease in the gathered population by combining the susceptible-infected-susceptible epidemic process with human motion patterns, described by moving speed and gathering preference. Our simulation results show that the emergence of a hysteresis loop is observed in the system when the moving speed is slow, particularly when humans prefer to gather; that is, the epidemic prevalence of the systems depends on the fraction of initial seeds. Regardless of the gathering preference, the hysteresis loop disappears when the population moves fast. In addition, our study demonstrates that there is an optimal moving speed for the gathered population, at which the epidemic prevalence reaches its maximum value.

Transposition mechanism of ISApl1-the determinant of colistin resistance dissemination.

Multidrug-resistant Enterobacteriaceae, a prominent family of gram-negative pathogenic bacteria, causes a wide range of severe diseases. Strains carrying the mobile colistin resistance (mcr-1) gene show resistance to polymyxin, the last line of defense against multidrug-resistant gram-negative bacteria. However, the transmission of mcr-1 is not well understood. In this study, genomes of mcr-1-positive strains were obtained from the NCBI database, revealing their widespread distribution in China. We also showed that ISApl1, a crucial factor in mcr-1 transmission, is capable of self-transposition. Moreover, the self-cyclization of ISApl1 is mediated by its own encoded transposase. The electrophoretic mobility shift assay experiment validated that the transposase can bind to the inverted repeats (IRs) on both ends, facilitating the cyclization of ISApl1. Through knockout or shortening of IRs at both ends of ISApl1, we demonstrated that the cyclization of ISApl1 is dependent on the sequences of the IRs at both ends. Simultaneously, altering the ATCG content of the bases at both ends of ISApl1 can impact the excision rate by modifying the binding ability between IRs and ISAPL1. Finally, we showed that heat-unstable nucleoid protein (HU) can inhibit ISApl1 transposition by binding to the IRs and preventing ISAPL1 binding and expression. In conclusion, the regulation of ISApl1-self-circling is predominantly controlled by the inverted repeat (IR) sequence and the HU protein. This molecular mechanism deepens our comprehension of mcr-1 dissemination.

Prevalence of multidrug-resistant hypervirulent Klebsiella pneumoniae without defined hypervirulent biomarkers in Anhui, China: a new dimension of hypervirulence.

Klebsiella pneumoniae is an opportunistic pathogen that mainly causes nosocomial infections and hospital-associated pneumonia in elderly and immunocompromised people. However, multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKp) has emerged recently as a serious threat to global health that can infect both immunocompromised and healthy individuals. It is scientifically established that plasmid-mediated regulator of mucoid phenotype genes (rmpA and rmpA2) and other virulence factors (aerobactin and salmochelin) are mainly responsible for this phenotype. In this study, we collected 23 MDR-hvKp isolates and performed molecular typing, whole genome sequencing, comparative genomic analysis, and phenotypic experiments, including the Galleria mellonella infection model, to reveal its genetic and phenotypic features. Meanwhile, we discovered two MDR-hvKp isolates (22122315 and 22091569) that showed a wide range of hypervirulence and hypermucoviscosity without rmpA and rmpA2 and any virulence factors. In phenotypic experiments, isolate 22122315 showed the highest hypervirulence (infection model) with significant mucoviscosity, and conversely, isolate 22091569 exhibited the highest mucoviscosity (string test) with higher virulence compared to control. These two isolates carried carbapenemase (blaKPC - 2), ß-lactamase (blaOXA - 1, blaTEM - 1B), extended-spectrum ß-lactamase (ESBL) genes (blaCTX - M - 15, blaSHV - 106), outer membrane protein-coding genes (ompA), fimbriae encoding genes (ecpABCDER), and enterobactin coding genes (entAB, fepC). In addition, single nucleotide polymorphism analysis indicated that both isolates, 22122315 and 22091569, were found to have novel mutations in loci FEBNDAKP_03184 (c. 2084A > C, p. Asn695Thr), and EOFMAFIB_02276 (c. 1930C > A, p. Pro644Thr), respectively. Finally, NCBI blast analysis suggested these mutations are located in the wzc of the capsule polysaccharide (cps) region and are responsible for putative tyrosine kinase. This study would be a strong reference for enhancing the current understanding of identifying the MDR-hvKp isolates that lacked both mucoid regulators and virulence factors.

Epidemiological characteristics of multidrug-resistant Acinetobacter baumannii ST369 in Anhui, China.

IMPORTANCE: Acinetobacter baumannii is a major health threat due to its antibiotic resistance and ability to cause nosocomial infections. Epidemiological studies indicated that the majority of globally prevalent ST369 clones originated from China, indicating a significant impact on public health in the country. In this study, we conducted whole-genome sequencing, comparative genomics, and Galleria mellonella infection model on eight A. baumannii ST369 isolates collected from a provincial hospital in China to comprehensively understand the organism. We identified two mutations (G540A and G667D) on the wzc gene that can affect bacterial virulence and viscosity. We confirmed their impact on resistance and virulence. We also investigated the potential involvement of AB46_0125 and AB152_03903 proteins in virulence. This finding provides a theoretical reference for further research on A. baumannii ST369 clinical isolates with similar mutations.

Corrigendum: LcpB is a pyrophosphatase responsible for wall teichoic acid synthesis and virulence in Staphylococcus aureus clinical isolate ST59.

[This corrects the article DOI: 10.3389/fmicb.2021.788500.].

Corrigendum: Epidemiological characteristics an outbreak of ST11 multidrug-resistant and hypervirulent Klebsiella pneumoniae in Anhui, China.

[This corrects the article DOI: 10.3389/fmicb.2022.996753.].

Nitrate Reductase NarGHJI Modulates Virulence via Regulation of agr Expression in Methicillin-Resistant Staphylococcus aureus Strain USA300 LAC.

Staphylococcus aureus is a pathogenic bacterium with a widespread distribution that can cause diverse severe diseases. The membrane-bound nitrate reductase NarGHJI serves respiratory function. However, little is known about its contribution to virulence. In this study, we demonstrated that narGHJI disruption results in the downregulation of virulence genes (e.g., RNAIII, agrBDCA, hla, psmα, and psmß) and reduces the hemolytic activity of the methicillin-resistant S. aureus (MRSA) strain USA300 LAC. Moreover, we provided evidence that NarGHJI participates in regulating host inflammatory response. A mouse model of subcutaneous abscess and Galleria mellonella survival assay demonstrated that the ΔnarG mutant was significantly less virulent than the wild type. Interestingly, NarGHJI contributes to virulence in an agr-dependent manner, and the role of NarGHJI differs between different S. aureus strains. Our study highlights the novel role of NarGHJI in regulating virulence, thereby providing a new theoretical reference for the prevention and control of S. aureus infection. IMPORTANCE Staphylococcus aureus is a notorious pathogen that poses a great threat to human health. The emergence of drug-resistant strains has significantly increased the difficulty of preventing and treating S. aureus infection and enhanced the pathogenic ability of the bacterium. This indicates the importance of identifying novel pathogenic factors and revealing the regulatory mechanisms through which they regulate virulence. The nitrate reductase NarGHJI is mainly involved in bacterial respiration and denitrification, which can enhance bacterial survival. We demonstrated that narGHJI disruption results in the downregulation of the agr system and agr-dependent virulence genes, suggesting that NarGHJI participates in the regulation of S. aureus virulence in an agr-dependent manner. Moreover, the regulatory approach is strain specific. This study provides a new theoretical reference for the prevention and control of S. aureus infection and reveals new targets for the development of therapeutic drugs.

Transcription tuned by S-nitrosylation underlies a mechanism for Staphylococcus aureus to circumvent vancomycin killing.

Treatment of Staphylococcus aureus infections is a constant challenge due to emerging resistance to vancomycin, a last-resort drug. S-nitrosylation, the covalent attachment of a nitric oxide (NO) group to a cysteine thiol, mediates redox-based signaling for eukaryotic cellular functions. However, its role in bacteria is largely unknown. Here, proteomic analysis revealed that S-nitrosylation is a prominent growth feature of vancomycin-intermediate S. aureus. Deletion of NO synthase (NOS) or removal of S-nitrosylation from the redox-sensitive regulator MgrA or WalR resulted in thinner cell walls and increased vancomycin susceptibility, which was due to attenuated promoter binding and released repression of genes involved in cell wall metabolism. These genes failed to respond to H2O2-induced oxidation, suggesting distinct transcriptional responses to alternative modifications of the cysteine residue. Furthermore, treatment with a NOS inhibitor significantly decreased vancomycin resistance in S. aureus. This study reveals that transcriptional regulation via S-nitrosylation underlies a mechanism for NO-mediated bacterial antibiotic resistance.

Genotypic and Phenotypic Characterization of Some psms Hypervirulent Clinical Isolates of Staphylococcus aureus in a Tertiary Hospital in Hefei, Anhui.

Background: Staphylococcus aureus is a highly successful pathogen that can cause various infectious diseases, from relatively mild skin infections to life-threatening severe systemic diseases. The widespread pathogenicity of S. aureus is mainly due to its ability to produce many virulence factors that help destroy various host cells, causing disease. Our primary goal in this study was to explore the genes of highly virulent strains, to identify genes closely associated with high virulence, and to provide ideas for the treatment of infection by highly virulent clinical strains. Results: This study collected 221 clinical strains from The First Affiliated Hospital Of The University of Science and Technology of China (USTC); their hemolytic abilities were tested. Eight isolates were selected based on their highly hemolytic ability and tested their hemolytic activity again; their phenotypes and gene sequences were also explored. Whole-genome sequencing (WGS) showed six plasmids (pN315, pNE131, pSJH901, pSJH101, SAP106B, and MSSA476), eight antibiotic resistance genes [blaR1, blaI, blaZ, mecA, erm(C), erm(T), tet(38), and fosB-Saur] and seventy-two virulence related genes. Three highly virulent strains, namely X21111206, 21092239, and 21112607, were found according the Galleria mellonella infection model. Therefore, we selected 10 representative virulence genes for qRT-PCR: psmα, psmß, hlgA, hlgB, hlgC, hla, clfA, clfB, spa, and sak. Among them, the expression levels of psmα and psmß, the three isolates, were significantly higher than the positive control NCTC8325. Conclusion: Significant differences appear in the expression of virulence genes in the highly virulent strains, particularly the psmα and psmß, It may be that the high expression of psm gene is the cause of the high virulence of Staphylococcus aureus. We can reduce the pathogenicity of Staphylococcus aureus by inhibiting the expression of psm gene, which may provide a strong basis for psm as a new target for clinical treatment of S. aureus infection.

Molecular Epidemiology of Staphylococcus aureus in a Tertiary Hospital in Anhui, China: ST59 Remains a Serious Threat.

Purpose: This study aimed to investigate the molecular characteristics, antimicrobial resistance and hemolytic phenotype of Staphylococcus aureus isolated from Anhui, China. Results: From August 2021 to January 2022, 214 S. aureus isolates were collected from the Anhui Provincial Hospital. This study identified 117 methicillin-resistant S. aureus and 97 methicillin-sensitive S. aureus isolates, and the detection rate of methicillin-resistant isolates was 1.8-fold higher than the average isolates reported in China (53.9% vs 30.5%). S. aureus isolates share identity at five or more of the seven MLST-based housekeeping loci, referred to as the clonal complex (CC). Forty ST types were found in 214 clinical S. aureus isolates, with the most extensive distribution of ST59 and ST6697 typing numbers and higher CC5 detection rates than any other clonal group. (The ST typing is the result of the MLST typing website query.) To detect the virulence of ST types of S. aureus, hemolysis experiments were performed on 214 clinical isolates, and it was concluded that ST59 had a relatively robust hemolytic capacity. Conclusion: Anhui S. aureus isolates have unique molecular and antibiotic resistance profiles. The antibiotic resistance profile may be related to the random use of antibiotics.

Phage transcription activator RinA regulates Staphylococcus aureus virulence by governing sarA expression.

BACKGROUND: Staphylococcus aureus is a major human pathogen, that can lead to various community- and hospital-acquired infections. RinA is a transcription activator of S. aureus phage φ 11 involved in phage packaging and virulence gene transfer. However, little is known about the molecular mechanism of RinA in the regulation of virulence. OBJECTIVE: We aimed to explore a novel contribution of RinA in the regulation of virulence and provide a new drug target in the treatment of S. aureus infections. METHODS: The specific functions of RinA in S. aureus were analyzed by the methods of growth curve, real-time quantitative PCR (RT-qPCR), subcellular localization, electrophoretic mobility shift assay (EMSA), infection model of Galleria mellonella larvae and the mouse subcutaneous abscess model. RESULTS: In this study, we demonstrated that RinA is a protein evenly distributed in the cytoplasm of S. aureus, and its deletion could cause the growth defects. RT-qPCR and EMSA determined that rinA could negatively regulate the expression of sarA by directly binding to its promoter, and vice versa. The Galleria mellonella larvae infection and mouse subcutaneous abscess models revealed that the rinA mutant strain exhibited obvious virulence defects. When sarA is knocked out, the virulence of S.aureus had no significantly changes whether rinA is knocked out or not. CONCLUSION: Our fndings demonstrated that phage transcription activator RinA regulates S. aureus virulence by governing sarA expression.

Epidemiology of carbapenem-resistant Klebsiella pneumoniae ST15 of producing KPC-2, SHV-106 and CTX-M-15 in Anhui, China.

BACKGROUND: It is well known that carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a more problematic public health issue due to its widespread spread worldwide. In China, ST11-type CRKP is the most prevalent CRKP, but ST15-type CRKP, a recently prevalent high-risk clone, has emerged widely throughout China, posing a serious public health risk. Therefore, we conducted an epidemiological of an outbreak of ST15 CRKP of producing CTX-M-15, KPC-2 and SHV-106 in a tertiary hospital in Anhui, China, to Understanding the potential risks of the current STT15 CRKP outbreak. RESULTS: From July 2021 to December 2021, 13 ST15 CRKP isolates were identified by collecting non-repeated clinical multidrug-resistant isolates, with all capsular typing of serotype KL19. All ST15 CRKP isolates were resistant to cephalosporins, carbapenems and quinolones, but were sensitive to amikacin, tigecycline and polymyxin B. In addition, isolates carried blaSHV-106 (100%), blaKPC-2 (69%), blaCTX-M-15 (69%), blaTEM-1B (69%), blaOXA-1 (62%) and blaLAP-2 (8%), as well as iron chelators (iutA, ybt, fyuA, ent, fepA, irp1, irp2, 100%) were detected. In phenotyping experiments, all ST15 CRKP exhibited lower growth rates than NTUH-K2044, and all ST15 CRKP did not exhibit mucoviscositty characteristics. However, in the Galleria mellonella infection model, isolates 21081212, 21081241 and 21091216 were more lethal than the hypervirulent isolates NTUH-K2044. Sequencing results showed that the genetic environment surrounding the genes blaSHV-106, blaKPC-2, blaCTX-M-15, blaOXA-1 and blaTEM-1B were all identical in the ST15 CRKP isolates. Phylogenetic analysis showed that 13 ST15 CRKP isolates were divided into three subgroups, and when placed in global analysis, 10 of them were highly homologous to isolates from Jiangsu, two were highly homologous to isolates from Zhejiang, and one was homologous to an isolate from an unlabelled region. CONCLUSION: Our research shows that ST15 CRKP, which carries multiple ß-lactamases genes and siderophores-encoding genes, may be evolving to hypervirulence and may have spread widely in localised areas. Therefore, environmental surveillance and clinical infection control in hospitals should be strengthened to prevent further spread of ST15 CRKP.

Epidemiological characteristics an outbreak of ST11 multidrug-resistant and hypervirulent Klebsiella pneumoniae in Anhui, China.

Klebsiella pneumoniae has become a primary threat to global health because of its virulence and resistance. In 2015, China reported multidrug-resistant (MDR) and hypervirulent K. pneumoniae (hvKp) isolates. The emergence of MDR-hvKp poses a significant threat to public health. We collected 76 MDR K. pneumoniae isolates from the same hospital, of which there were a total of six MDR-hvKp isolates. We performed multilocus sequence typing (MLST) and capsular typing, whole genome sequencing, comparative genome analysis, and phylogenetic analysis as well as phenotypic experiments, including growth curves, mucoviscosity assay, Galleria mellonella infection model, human whole blood survival, and human neutrophil bactericidal assay to further characterize the samples. We identified six large plasmids carrying extended spectrum ß-lactamase (ESBL) genes or carbapenemase genes (bla CTX-M-65, bla KPC-2, bla SHV-12, bla SHV-158), 9 plasmids containing other drug resistance genes, and 7 hypervirulence plasmids carrying rmpA and rmpA2 in ST11 MDR-hvKp isolates. Some of these plasmids were identical, whereas others differed only by insertion elements. In addition, we identified a plasmid, p21080534_1, that carries hypervirulence genes (iucABCD, iutA, rmpA2), a carbapenemase gene (bla KPC-2), and an ESBL gene (bla SHV-12), as well as MDR-hvKp 21072329, which did not carry rmpA or rmpA2, but exhibited hypervirulence and hypermucoviscosity. ST11 MDR-hvKp derived from hypervirulence and multidrug resistance plasmids not only causes significant treatment difficulties, but also represents an unprecedented challenge to public health. Therefore, urgent measures are needed to limit further spread.

Analysis of gastric microbiome reveals three distinctive microbial communities associated with the occurrence of gastric cancer.

BACKGROUND: Gastric microbial dysbiosis were reported to be associated with gastric cancer (GC). This study aimed to explore the variation, diversity, and composition patterns of gastric bacteria in stages of gastric carcinogenesis based on the published datasets. METHODS: We conducted a gastric microbial analysis using 10 public datasets based on 16S rRNA sequencing, including 1270 gastric biopsies of 109 health control, 183 superficial gastritis (SG), 135 atrophic gastritis (AG), 124 intestinal metaplasia (IM), 94 intraepithelial neoplasia (IN), 344 GC, and 281 adjacent normal tissues. And QIIME2-pipeline, DESeq2, NetMoss2, vegan, igraph, and RandomForest were used for the data processing and analysis. RESULTS: We identified three gastric microbial communities among all the gastric tissues. The first community (designate as GT-H) was featured by the high abundance of Helicobacter. The other two microbial communities, namely GT-F, and GT-P, were featured by the enrichment of phylum Firmicutes and Proteobacteria, respectively. The distribution of GC-associated bacteria, such as Fusobacterium, Peptostreptococcus, Streptococcus, and Veillonella were enriched in tumor tissues, and mainly distributed in GT-F type microbial communities. Compared with SG, AG, and IM, the bacterial diversity in GC was significantly reduced. And the strength of microbial interaction networks was initially increased in IM but gradually decreased from IN to GC. In addition, Randomforest models constructed in in GT-H and GT-F microbial communities showed excellent performance in distinguishing GC from SG and precancerous stages, with varied donated bacteria. CONCLUSIONS: This study identified three types of gastric microbiome with different patterns of composition which helps to clarify the potential key bacteria in the development of gastric carcinogenesis.

Fecal Microbiota Transplantation Ameliorates Active Ulcerative Colitis by Downregulating Pro-inflammatory Cytokines in Mucosa and Serum.

Background: Ulcerative colitis (UC) is a multi-factor disease characterized by alternating remission periods and repeated occurrence. It has been shown that fecal microbiota transplantation (FMT) is an emerging and effective approach for UC treatment. Since most existing studies chose adults as donors for fecal microbiota, we conducted this study to determine the long-term efficacy and safety of the microbiota from young UC patient donors and illustrate its specific physiological effects. Methods: Thirty active UC patients were enrolled and FMT were administered with the first colonoscopy and two subsequent enema/transendoscopic enteral tubing (TET) practical regimens in The First Affiliated Hospital of Anhui Medical University in China. Disease activity and inflammatory biomarkers were assessed 6 weeks/over 1 year after treatment. The occurrence of adverse events was also recorded. The samples from blood and mucosa were collected to detect the changes of inflammatory biomarkers and cytokines. The composition of gut and oral microbiota were also sampled and sequenced to confirm the alteration of microbial composition. Results: Twenty-seven patients completed the treatment, among which 16 (59.3%) achieved efficacious clinical response and 11 (40.7%) clinical remission. Full Mayo score and calprotectin dropped significantly and remained stable over 1 year. FMT also significantly reduced the levels of C-reactive protein (CRP), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6). The gut microbiota altered significantly with increased bacterial diversity and decreased metabolic diversity in responsive patients. The pro-inflammatory enterobacteria decreased after FMT and the abundance of Collinsella increased. Accordingly, the altered metabolic functions, including antigen synthesis, amino acids metabolism, short chain fatty acid production, and vitamin K synthesis of microbiota, were also corrected by FMT. Conclusion: Fecal microbiota transplantation seems to be safe and effective for active UC patients who are nonresponsive to mesalazine or prednisone in the long-term. FMT could efficiently downregulate pro-inflammatory cytokines to ameliorate the inflammation.

Comparative genomic analyses of Polymyxin-resistant Enterobacteriaceae strains from China.

BACKGROUND: Mobile colistin resistance like gene (mcr-like gene) is a new type of polymyxin resistance gene that can be horizontally transferred in the Enterobacteriaceae. This has brought great challenges to the treatment of multidrug-resistant Escherichia coli and K. pneumoniae. RESULTS: K. pneumoniae 16BU137 and E. coli 17MR471 were isolated from the bus and subway handrails in Guangzhou, China. K. pneumoniae 19PDR22 and KP20191015 were isolated from patients with urinary tract infection and severe pneumonia in Anhui, China. Sequence analysis indicated that the mcr-1.1 gene was present on the chromosome of E. coli 17MR471, and the gene was in the gene cassette containing pap2 and two copies of ISApl1.The mcr-1.1 was found in the putative IncX4 type plasmid p16BU137_mcr-1.1 of K. pneumoniae 16BU137, but ISApl1 was not found in its flanking sequence. Mcr-8 variants were found in the putative IncFIB/ IncFII plasmid pKP20191015_mcr-8 of K. pneumoniae KP20191015 and flanked by ISEcl1 and ISKpn26. CONCLUSION: This study provides timely information on Enterobacteriaceae bacteria carrying mcr-like genes, and provides a reference for studying the spread of mcr-1 in China and globally.

Gastrointestinal Microbiota Changes in Patients With Gastric Precancerous Lesions.

Background: Gastric microbiota may be involved in gastric cancer. The relationship between gastrointestinal microbes and the risk of gastric cancer is unclear. This study aimed to explore the gastric and intestinal bacteria associated with gastritis and gastric precancerous lesions. We conducted a case-control study by performing 16S rRNA gene analysis of gastric biopsies, juices, and stool samples from 148 cases with gastritis or gastric precancerous lesions from Anhui and neighboring provinces, China. And we validated our findings in public datasets. Results: Analysis of microbial sequences revealed decreased bacterial alpha diversity in gastric bacteria during the progression of gastritis. Helicobacter pylori was the main contributor to the decreased microbial composition and diversity in the gastric mucosa and had little influence on the microbiota of gastric juice and feces. The gastric mucosal genera Gemella, Veillonella, Streptococcus, Actinobacillus, and Hemophilus had the higher degree of centrality across the progression of gastric precancerous lesions. And Acinetobacter may contribute to the occurrence of intraepithelial neoplasia. In addition, the microbial model of H. pylori-positive gastric biopsies and feces showed value in the prediction of gastric precancerous lesions. Conclusions: This study identified associations between gastric precancerous lesions and gastric microbiota, as well as the changes in intestinal microbiota, and explored their values in the prediction of gastric precancerous lesions.

VraCP regulates cell wall metabolism and antibiotic resistance in vancomycin-intermediate Staphylococcus aureus strain Mu50.

OBJECTIVES: Vancomycin-intermediate Staphylococcus aureus (VISA) is increasingly being reported. Previous studies have shown that vraC and vraP may be involved in vancomycin resistance, although the molecular mechanism remains elusive. METHODS: The vraC (SAV0577), vraP (SAV0578) and vraCP mutants were constructed in Mu50 by allelic replacement. Some common VISA phenotypes were assessed in mutants, such as, susceptibility to the cell wall-associated antibiotics, cell wall thickness, autolysis activity and growth rate. RT-qPCR was performed to reveal the differential genes associated with these phenotypes. The binding abilities of VraC and VraCP to the promoters of target genes were determined by electrophoretic mobility shift assay (EMSA). RESULTS: VraP forms a stable complex with VraC to preserve their own stability. The vraC, vraP and vraCP mutants exhibited increased susceptibility to the cell wall-associated antibiotics and thinner cell walls compared with the WT strain. Consistent with these phenotypes, RT-qPCR revealed downregulated transcription of glyS, sgtB, ddl and alr2, which are involved in cell wall biosynthesis. Moreover, the transcription of cell wall hydrolysis genes, including sceD, lytM and isaA, was significantly downregulated, supporting the finding that mutants exhibited reduced autolysis rates. EMSA confirmed that both VraC and VraCP can directly bind to the sceD, lytM and isaA promoter regions containing the consensus sequence (5'-TTGTAAN2AN3TGTAA-3'), which is crucial for the binding of VraCP with target genes. GFP-reporter assays further revealed VraC and VraCP can enhance promoter activity of sceD to positively regulate its expression. CONCLUSIONS: vraCP plays a significant role in cell wall metabolism and antibiotic resistance in Mu50.

Evolution of Resistance to Phenazine Antibiotics in Staphylococcus aureus and Its Role During Coinfection with Pseudomonas aeruginosa.

In the niches that Staphylococcus aureus and Pseudomonas aeruginosa coinhabit, the later pathogen produces phenazine antibiotics to inhibit the growth of S. aureus. Recently, a group of halogenated phenazines (HPs) has been shown to have potent antimicrobial activities against Staphylococci; however, no HP-resistant mutant has been reported. Here, we demonstrate that S. aureus develops HP-resistance via single amino acid change (Arg116Cys) in a transcriptional repressor TetR21. RNA-seq analysis showed that the TetR21R116C variation caused drastic up-regulation of an adjacent gene hprS (halogenated phenazine resistance protein of S. aureus). Deletion of the hprS in the TetR21R116C background restored bacterial susceptibility to HP, while hprS overexpression in S. aureus conferred HP-resistance. The expression of HprS is under tight transcriptional control of the TetR21 via direct binding to the promoter region of hprS. The R116C mutation in TetR21 significantly reduced its DNA binding affinity. Moreover, natural phenazine antibiotics (phenazine-1-carboxylic acid and pyocyanin) and a HP analog (HP-22) are ligands for the TetR21, regulating its repressor activity. Combining homology analysis and LC-MS/MS assay we demonstrated that HprS is a phenazine efflux pump. To the best of our knowledge, we provide the first report of phenazine efflux pump in S. aureus. Interestingly, the TetR21R116C variation has been found in some clinical S. aureus isolates, and a laboratory strain of S. aureus with TetR21R116C variation showed enhanced growth competitiveness toward P. aeruginosa and promoted coinfection with P. aeruginosa in the host environment, demonstrating significance of the mutation in host infections.