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1.
Appl Environ Microbiol ; 86(22)2020 10 28.
Article in English | MEDLINE | ID: mdl-32887718

ABSTRACT

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage PIZ SAE-01E2 against S Abortusequi in a mouse model of abortion were investigated. Phage PIZ SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of PIZ SAE-01E2. All of these characteristics indicate that PIZ SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 103 CFU/mouse of S Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of PIZ SAE-01E2 (108 PFU/mouse, multiplicity of infection = 105) 1 h before or after S Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage PIZ SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased (<102 CFU/g) compared to those in the placenta and the uterus of the mice in the control group (>106 CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P < 0.05) compared to those in the placenta and blood of the mice in the control group. Altogether, these findings indicate that PIZ SAE-01E2 shows the potential to block abortions induced by S Abortusequi in vivoIMPORTANCES Abortusequi is an important pathogen that can induce abortions in mares. Although S Abortusequi has been well controlled in Europe and the United States due to strict breeding and health policies, it is still widespread in African and Asian countries and has proven difficult to control. In China, abortions caused by S Abortusequi have also been reported in donkeys. So far, there is no commercial vaccine. Thus, exploiting alternative efficient and safe strategies to control S Abortusequi infection is essential. In this study, a new lytic phage, PIZ SAE-01E2, infecting S Abortusequi was isolated, and the characteristics of PIZ SAE-01E2 indicated that it has the potential for use in phage therapy. A single intraperitoneal inoculation of PIZ SAE-01E2 before or after S Abortusequi challenge provided effective protection to all pregnant mice. Thus, PIZ SAE-01E2 showed the potential to block abortions induced by S Abortusequi in vivo.


Subject(s)
Abortion, Veterinary/prevention & control , Bacteriophages/physiology , Horse Diseases/prevention & control , Salmonella Infections, Animal/prevention & control , Salmonella/physiology , Abortion, Veterinary/microbiology , Abortion, Veterinary/virology , Animals , Female , Horse Diseases/microbiology , Horse Diseases/virology , Horses , Mice , Mice, Inbred ICR , Pregnancy , Salmonella Infections, Animal/microbiology , Salmonella Infections, Animal/virology
2.
Front Microbiol ; 11: 1461, 2020.
Article in English | MEDLINE | ID: mdl-32733409

ABSTRACT

Inflammation is the hallmark of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli-induced bovine mastitis. Organic selenium can activate pivotal proteins in immune responses and regulate the immune system. The present study aimed to investigate whether selenomethionine (SeMet) attenuates ESBL E. coli-induced inflammation in bovine mammary epithelial cells (bMECs) and macrophages. Cells were treated with 0, 5/10, 10/20, 20/40, or 40/60 µM SeMet for 12 h and/or inoculated with ESBL-E. coli [multiplicity of infection (MOI) = 5] for 4/6 h, respectively. We assessed inflammatory responses, including selenoprotein S (SeS), Toll-like receptor 4 (TLR4), Ikappa-B (IκB), phospho-NF-κB p65 (Ser536), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) activities. Treatment with 40/60 µM SeMet promoted cell viability and inhibited LDH activities in both bMECs and macrophages. Inoculation with ESBL-E. coli reduced cell viability, which was attenuated by SeMet treatment in bMECs and macrophages. SeMet increased ESBL E. coli-induced downregulation of SeS and decreased LDH activities, TLR4, IκB, phospho-NF-κB p65 (Ser536), IL-1ß, and TNF-α protein expressions in bMECs and macrophages. In addition, knockdown of SeS promoted protein expression of TLR4-mediated nuclear factor-kappa (NF-κB) pathway and BAY 11-708 inhibited TNF-α and IL-1ß protein levels in bMECs and macrophages after ESBL-E. coli treatment. Moreover, ESBL-E. coli inoculation increased monocyte chemoattractant protein 1 (MCP-1), C-C motif ligand 3 (CCL-3), and CCL-5 mRNA expressions in bMECs. In conclusion, ESBL-E. coli induced expression of MCP-1, CCL-3, and CCL-5 in bMECs and then recruited and activated macrophages, whereas SeMet attenuated ESBL E. coli-induced inflammation through activated SeS-mediated TLR4/NF-κB signaling pathway in bMECs and macrophages.

3.
Microb Drug Resist ; 26(6): 685-696, 2020 Jun.
Article in English | MEDLINE | ID: mdl-31755810

ABSTRACT

Both mcr-1 phosphoethanolamine transferase enzymes and extended-spectrum ß-lactamases (ESBLs) are the main plasmid-mediated mechanisms of resistance to colistin and third-generation cephalosporins, respectively, and currently considered a major concern to humans and food animals. Prevalence of mcr-1 gene in Escherichia coli from dairy cattle has rarely been reported. Our objective was to determine prevalence and characteristics of mcr-1 carrying E. coli isolated from clinical mastitis cases in large dairy farms (>500 cows) in 16 provinces of China. A total of 249 E. coli was isolated from 2,038 mastitic milk samples. Among these isolates, 2.0% (n = 5) and 19.7% (n = 49) were colistin resistant mcr-1-positive and ESBL-producing isolates, respectively. All mcr-1-positive isolates that produced ESBLs also carried the blaCTX-M-15 gene and belonged to phylogroup-A. Most mcr-1 and blaCTX-M-15 genes were located on conjugative plasmids (IncP and IncF, respectively) that were successfully transferred to transconjugants in conjugation experiments. All mcr-1-positive E. coli isolates were multidrug resistant, exhibiting resistance to common antimicrobials. Multilocus sequence typing of these mcr-1-carrying E. coli isolates revealed four sequence types, reflecting substantial diversity. Multilocus sequence analysis detected evolutionary connection of mcr-1 carrying isolates with our recently reported ESBL-producing E. coli isolates, raising concerns regarding fast dissemination between bacteria. To our knowledge, this was the first nation-wide report describing isolates of E. coli from mastitic milk samples collected on large dairy farms in China, carrying mcr-1 and blaCTX-M-15 genes on conjugative plasmids. We concluded that dairy cattle are a potential source of mcr-1-carrying and ESBL-producing E. coli.


Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli Proteins/genetics , Escherichia coli/drug effects , Escherichia coli/genetics , Milk/microbiology , beta-Lactamases/genetics , Animals , Cattle , China/epidemiology , Colistin , Female , Genes, Bacterial , Integrons/genetics , Microbial Sensitivity Tests , Multilocus Sequence Typing , Plasmids/genetics
4.
Infect Drug Resist ; 12: 2135-2149, 2019.
Article in English | MEDLINE | ID: mdl-31410033

ABSTRACT

PURPOSE: The coexistence of mobile colistin (COL)-resistant gene mcr-1 with extended-spectrum beta-lactamase (ESBL) gene in Escherichia coli has become a serious threat globally. The aim of this study was to investigate the increasing resistance to COL and in particular its coexistence with ESBL-producing E. coli recovered from pig farms in China. MATERIALS AND METHODS: E. coli were isolated from 14 pig farms in Jiangsu China. Susceptibility testing was identified by micro-dilution method. PCR assay and nucleotide sequencing were used to detect COL-resistant genes, mcr-1 to -5, as well as ESBL genes, bla CTX-M, bla SHV and bla TEM. Conjugation experiment, plasmid replicon typing of the multidrug resistance (MDR), S1-PFGE and DNA southern hybridization were performed to study the transferability of these genes. RESULTS: Overall, 275 E. coli isolates were recovered from a total of 432 cloacal and nasal swabs. More than 90% of the isolates were MDR, of which 70.18% were resistant to COL. Of these 275 isolates, mcr-1 was identified as the most predominant gene carried by 71.63% (197/275) of isolates, 39.59% (78/197) of the isolates were harboring both mcr-1 and ESBL genes (bla CTX-M, bla SHV and bla TEM). ESBL genotyping showed that bla CTX-M was the most predominant ESBL (68.49%) followed by bla SHV (16.4%) and bla TEM (15%). Sequencing revealed that the most common variants of bla CTX-M identified were, bla CTX-M-15 (69%), bla CTX-M-55 (29%) and bla CTX-M-1 (1.8%). IncHI2, IncFIB, IncFIC, IncN and IncX4 were found to be the most common Inc-types found both in donors and in transconjugants and were associated with the transfer of the mcr-1 and ESBL encoding genes. Six strains carried a total of five different plasmids: approximately 97-, 130-, 160-, 227- and 242-kb plasmids. CONCLUSION: The coexistence of the mcr-1- and bla CTX-M-15-carrying isolates displaying high MDR, recovered from E. coli of pig origin, is a major concern for both humans and veterinary medicine.

5.
Virus Genes ; 55(5): 696-706, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31254238

ABSTRACT

Bacteriophages have been recently revisited as an alternative biocontrol tool due to the limitations of antibiotic treatment. In this study, we reported on the biological characteristics and genomic information of vB_KpnS_GH-K3 (abbreviated as GH-K3), a Klebsiella phage of the Siphoviridae family, which was previously isolated from a hospital sewage system. One-step growth curve analysis indicated that the burst size of GH-K3 was 291 PFU/cell. GH-K3 maintained a stable titer in a broad range of pH values (6-10) and temperature (up to 50 °C). Based on bioinformatics analysis, GH-K3 comprises of 49,427 bp containing a total of 77 open reading frames (ORFs), which share high degree of nucleotide similarity and close evolutionary relationships with at least 12 other Klebsiella phages. Of note, GH-K3 gp32 was identified as a unique ORF. The major segment of gp32 sequence at the C-terminus (residues 351-907) was found highly variable as determined by its mismatch with the nucleotide and protein sequences available at NCBI database. Furthermore, HHpred analysis indicated that GH-K3 gp32 contains three domains (PDB ID: 5W6S_A, 3GQ8_A and 1BHE_A) similar to depolymerase (depoKP36) of Klebsiella phage KP36 suggestive of a potential depolymerase activity during host receptor-binding in the processes of phage infection. Altogether, the current data revealed a novel putative depolymerase-like protein which is most likely to play an important role in phage-host interaction.


Subject(s)
Bacteriophages/growth & development , Klebsiella/virology , Bacteriophages/drug effects , Bacteriophages/genetics , Bacteriophages/radiation effects , Genome, Viral , Hydrogen-Ion Concentration , Microbial Viability/drug effects , Microbial Viability/radiation effects , Open Reading Frames , Sequence Homology , Synteny , Temperature , Viral Load , Viral Proteins/genetics
6.
Front Microbiol ; 10: 1189, 2019.
Article in English | MEDLINE | ID: mdl-31191500

ABSTRACT

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage.

7.
Vet Microbiol ; 229: 72-80, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30642601

ABSTRACT

Staphylococcus aureus is one of the most important pathogens causing rabbit necrotizing pneumonia and brings huge economic losses to rabbit production. This study investigated the preventive effect of a phage on rabbit necrotizing pneumonia caused by S. aureus. S. aureus S6 was isolated from the lungs of rabbits suffering necrotizing pneumonia and identified. A novel phage named VB-SavM-JYL01 was isolated by using S. aureus S6 as a host and showed a broader host range than the phages GH15 and K. The genome of VB-SavM-JYL01 lacked bacterial virulence-, antibiotic resistance- and lysogenesis-related genes. A single intranasal administration of VB-SavM-JYL01 (3 × 109 PFU) could effectively improve the survival rate at 48 h to 90% (9/10) compared with the survival rate of 10% and 80% observed with the PBS or linezolid treatment, respectively. The bacterial count in the lungs of rabbits treated with the phage VB-SavM-JYL01 was 4.18 × 104 CFU/g at 24 h, which was significantly decreased compared to that of rabbits treated with PBS (7.38 × 107 CFU/g) or linezolid (3.12 × 105 CFU/g). The phage treatment significantly alleviated lung tissue damage. The levels of total proteins, Panton-Valentine leukocidin (PVL), alpha-toxin (Hla) and cytokines in the lungs of the rabbits treated with the phage were significantly lower than those of the rabbits treated with PBS and similar to those of the rabbits treated with linezolid. These data demonstrate the potential utility of phage as an alternative for preventing rabbit necrotizing pneumonia caused by S. aureus.


Subject(s)
Pneumonia, Necrotizing/veterinary , Pneumonia, Staphylococcal/veterinary , Rabbits/microbiology , Staphylococcus Phages , Staphylococcus aureus/virology , Animals , Female , Pneumonia, Necrotizing/microbiology , Pneumonia, Necrotizing/prevention & control , Pneumonia, Staphylococcal/prevention & control
8.
Folia Microbiol (Praha) ; 64(1): 101-111, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30090964

ABSTRACT

Biofilm is involved in a variety of infections, playing a critical role in the chronicity of infections. Enterobacter cloacae is a biofilm-forming and multi-drug-resistant (MDR) nosocomial pathogen leading to significant morbidity and mortality. This study aimed at isolation of a bacteriophage against MDR clinical strain of E. cloacae and its efficacy against bacterial planktonic cells and biofilm. A bacteriophage MJ2 was successfully isolated from wastewater and was characterized. The phage exhibited a wide range of thermal and pH stability and demonstrated considerable adsorption to host bacteria in the presence of CaCl2 or MgCl2. Transmission electron microscopy (TEM) showed MJ2 head as approximately 62 and 54 nm width and length, respectively. It had a short non-contractile tail and was characterized as a member of the family Podoviridae [order Caudovirales]. The phage MJ2 was found to possess 11 structural proteins (12-150 kDa) and a double-stranded DNA genome with an approximate size of 40 kb. The log-phase growth of E. cloacae both in biofilm and suspension was significantly reduced by the phage. The E. cloacae biofilm was formed under different conditions to evaluate the efficacy of MJ2 phage. Variable reduction pattern of E. cloacae biofilm was observed while treating it for 4 h with MJ2, i.e., biofilm under static conditions. The renewed media with intervals of 24, 72, and 120 h showed biomass decline of 2.8-, 3-, and 3.5-log, respectively. Whereas, the bacterial biofilm formed with dynamic conditions with refreshing media after 24, 72, and 120 h demonstrated decline in growth at 2.5-, 2.6-, and 3.3-log, respectively. It was, therefore, concluded that phage MJ2 possessed considerable inhibitory effects on MDR E. cloacae both in planktonic and biofilm forms.


Subject(s)
Biofilms/growth & development , Drug Resistance, Multiple, Bacterial , Enterobacter cloacae/virology , Podoviridae/isolation & purification , Podoviridae/physiology , Calcium Chloride/pharmacology , DNA, Viral , Genome Size , Genome, Viral/genetics , Host Specificity , Hydrogen-Ion Concentration , Magnesium Chloride/pharmacology , Microbial Viability , Molecular Weight , Podoviridae/ultrastructure , Temperature , Viral Proteins/chemistry , Virus Attachment/drug effects , Wastewater/virology
9.
Biomed Res Int ; 2018: 9519718, 2018.
Article in English | MEDLINE | ID: mdl-29780833

ABSTRACT

The ß-lactams-a large class of diverse compounds-due to their excellent safety profile and broad antimicrobial spectrum are considered to be the most widely used therapeutic class of antibacterials prescribed in human and veterinary clinical practices. This, unfortunately, has also given rise to a continuous increased resistance globally in health care settings as well as in the community due to their permanent selective force driving diversification of the resistance mechanism. Resistance against ß-lactams is increasing rapidly as novel ß-lactamases, enzymes that degrade ß-lactams, are being discovered each day such as recent emergence of extended spectrum ß-lactamases (ESBL) that have the ability to inactivate most of the cephalosporins. The complexity and diversity of ESBL are increasing so rapidly that more than 170 variants have thus far been described for only a single genotype, the blaCTX-M -encoding ESBL. This review is to organize all the current updated literature describing genomic features, organization, and mechanism of resistance and mode of dissemination of all known ESBLs.


Subject(s)
Bacteria , Bacterial Proteins , Cephalosporins/metabolism , beta-Lactam Resistance/genetics , beta-Lactamases , Bacteria/enzymology , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolism
10.
Adv Exp Med Biol ; 1057: 111-126, 2018.
Article in English | MEDLINE | ID: mdl-29453670

ABSTRACT

Prevalence of zoonotic Mycobacterium bovis (bTB) disease in human population is underreported from the North of Pakistan. Here, we report on the proportion of human bTB disease among the overall TB patients, drug resistance pattern of bTB isolates, and knowledge, attitude, and practices (KAP)-based analysis of bTB disease. For this purpose, sputum samples from a total of 300 clinically diagnosed TB patients and 100 randomly selected school children suspected of pulmonary TB were processed by culture as well as polymerase chain reaction (PCR) for isolation, identification, and confirmation of Mycobacterium tuberculosis (mTB) and bTB species. Isolates of bTB were processed for drug susceptibility tests. Data on KAP regarding TB were obtained on a pretested questionnaire. Sputum-based PCR results indicated that 288/300 (96%) were confirmed as mTB, while 12/300 (4%) were found as bTB diseases. Interestingly, none of the school child was declared positive for either mTB or bTB. Notably, 274/300 (91.3%) positively cultured samples were identified as mTB, 13/300 (4.3%) as bTB, while 5/300 (1.7%) as mixed containing both. Importantly, except one, all of the bTB isolates were found resistant to pyrazinamide. Surprisingly, most of the bTB isolates (~70%) were found resistant to a broad range of first- and second-line anti-TB drugs. SplitsTree and recombination analysis indicated no evidence of intergenic recombination. Finally, residence, occupation, presence of animals at home, and sleeping alongside animals were found significantly associated with occurrence of bTB disease. To the best of our knowledge, we report for the first time on the high (4%) burden of bTB disease in human TB patients in Peshawar, Pakistan.


Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial , Mycobacterium bovis/drug effects , Tuberculosis, Multidrug-Resistant/diagnosis , Animals , Child , Humans , Incidence , Mycobacterium tuberculosis , Pakistan
11.
Protein Expr Purif ; 144: 12-18, 2018 04.
Article in English | MEDLINE | ID: mdl-29180019

ABSTRACT

A thorough characterisation of the genetics, physiology and metabolism of Escherichia coli has led to the availability of a large number of strains and vectors suitable for recombinant protein expression. Despite the relative ease in using E. coli for achieving amplified expression of many recombinant proteins, for some proteins this can be a frustrating and time-consuming process leading to very low expression or no expression at all. This is especially true for membrane proteins, which introduce additional challenges. A number of factors can be considered and optimised for achieving required levels of amplified expression of recombinant proteins in E. coli that are broadly classified as host strain, expression vector and growth conditions. In this paper we summarise these factors and consolidate the common challenges encountered and approaches to overcome them, focusing in particular on cases where there is low amplified expression or no expression at all of the desired recombinant protein, due to various reasons.


Subject(s)
Cloning, Molecular/methods , Escherichia coli/genetics , Membrane Proteins/genetics , Recombinant Proteins/genetics
12.
Front Microbiol ; 8: 1797, 2017.
Article in English | MEDLINE | ID: mdl-29018414

ABSTRACT

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is widely used as a model organism for the analysis of infection biology. In this context, there is a current need to develop improved reporters for enhanced bioluminescence imaging (BLI) of the pathogen in infection models. We have developed a click beetle red luciferase (CBR-luc) based vector (pPL2CBRopt) expressing codon optimized CBR-luc under the control of a highly expressed Listerial promoter (PHELP) for L. monocytogenes and have compared this to a lux-based system expressing bacterial luciferase for BLI of the pathogen using in vitro growth experiments and in vivo models. The CBR-luc plasmid stably integrates into the L. monocytogenes chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR-luc labeled L. monocytogenes emits a bright signal in exponential phase that is maintained during stationary phase. In contrast, lux-labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR-luc labeled bacteria was more efficient than the signal from lux-labeled bacteria in penetrating an artificial tissue depth assay system. A cell invasion assay using C2Bbe1 cells and a systemic murine infection model revealed that CBR-luc is suited to BLI approaches and demonstrated enhanced sensitivity relative to lux in the context of Listeria infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to lux and may have significant applications in the analysis of L. monocytogenes pathogenesis.

13.
Biomed Res Int ; 2017: 3248483, 2017.
Article in English | MEDLINE | ID: mdl-29333439

ABSTRACT

There is a need to identify and select new promising immunodominant antigens that have the ability to provide protective immunity against E. coli causing bovine mastitis. Recently we showed that f17a was found to be the most prevalent and crucial virulent factor among the pathogenic E. coli isolated from bovine mastitis. Here, in this report, the recombinant F17A based subunit vaccine adjuvant with MF59 was tested for immunogenicity against E. coli in a murine model. The vaccinated mice did not show any abnormal behavioral changes and histopathological lesions after vaccination. The specific antibody level against F17A was significantly higher in MF59-adjuvant-group, and also lasted for longer duration with a significant (P < 0.01) production level of IgG1 and IgG2a. Moreover, we noted higher survival rate in mice injected with F17A-MF59-adjuvant group after challenging with the clinical E. coli strain. Our findings of bacterial clearance test revealed that elimination rate from liver, spleen, and kidney in MF59-adjuvant-group was significantly higher than the control group. Finally, the proportion of CD4+T cells was increased, while CD8+ was decreased in MF59-adjuvant group. In conclusion, the current study reveals the capability of F17A-MF59 as a potential vaccine candidate against pathogenic E. coli causing mastitis in dairy animals.


Subject(s)
Adhesins, Bacterial/genetics , Escherichia coli Proteins/genetics , Escherichia coli/drug effects , Mastitis, Bovine/drug therapy , Vaccines, Subunit/administration & dosage , Adhesins, Bacterial/administration & dosage , Adhesins, Bacterial/immunology , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cattle , Escherichia coli/immunology , Escherichia coli/pathogenicity , Escherichia coli Proteins/administration & dosage , Escherichia coli Proteins/immunology , Female , Immunoglobulin G/immunology , Mastitis, Bovine/immunology , Mastitis, Bovine/microbiology , Mice , Polysorbates/administration & dosage , Squalene/administration & dosage , Vaccines, Subunit/immunology
14.
Front Microbiol ; 7: 1931, 2016.
Article in English | MEDLINE | ID: mdl-27965653

ABSTRACT

The prevalence of pathogenic multi-drug resistant (MDR) extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli is rapidly increasing, becoming a global concern. In a veterinary context, ESBL-producing E. coli are mostly reported in poultry and pigs. Here, we report on the prevalence and characterize ESBL-producing E. coli isolated from diverse dairy farms in China. Overall, 36 (23.53%) out of 153 E. coli isolates from mastitic milk samples (n = 1252) were confirmed as ESBL-producers by double-disc synergy testing and PCR. Nucleotide analysis of PCR amplicons revealed that blaCTX-M was the predominant ESBL gene detected in 28 (77.78%) isolates, with blaCTX-M-15 being the major (78.57%) allele encoding for ESBLs. Also, 20 (55.56%) and 6 (16.67%) of the ESBL isolates were carrying blaTEM and blaSHV genes, respectively, in singlet or in combination. The majority of these isolates belonged to phylo-group A (69.44%) and D (16.67%). Strikingly, all these isolates were found to be MDR showing high resistance to cephalosporins including the fourth generation cefepime and common non ß-lactams. Additionally, class 1 integrons (intI1) were found in 30 (83.33%) isolates. Analysis of the class 1 integrons variable regions indicated that they were carrying up to five different gene cassettes conferring resistance to various drugs with a predominant combination of dfrA17-aadA5 genes in tandem, conferring resistance to aminoglycosides and trimethoprim. However, no ESBL encoding genes were found in the cassettes. Interestingly, 22 (66.11%) of the ESBL isolates were also carrying insertion sequence common region 1 (ISCR1) which was found to be associated with most of the CTX-M genes. Altogether, the current study reports on the high prevalence of ESBL-positive E. coli, particularly CTX-M-15, carrying clinical class 1 integrons and ISCR1 elements are likely indicative of their rapid and wider dissemination, posing threats to veterinary and public health. To the best of our knowledge, this is the first comprehensive study to report on the alarming high occurrence of ESBL-producing E. coli from mastitic cows in China.

15.
Infect Immun ; 84(9): 2534-40, 2016 09.
Article in English | MEDLINE | ID: mdl-27324486

ABSTRACT

Two-partner secretion (TPS) systems export large TpsA proteins to the surface and extracellular milieu. In meningococci, three different TPS systems exist, and of these, TPS system 2 (TPS2) and TPS3 can be detected by the host's immune system. We evaluated the distribution of TPS systems among clinical isolates from two prospective cohort studies comprising 373 patients with meningococcal meningitis. TPS system 1 was present in 91% of isolates, and system 2 and/or 3 was present in 67%. The TPS system distribution was related to clonal complexes. Infection with strains with TPS2 and/or TPS3 resulted in less severe disease and better outcomes than infection with strains without these systems. Using whole-blood stimulation experiments, we found no differences in the host cytokine response between patients infected with TPS system 2 and 3 knockout strains and patients infected with a wild-type strain. In conclusion, meningococcal TPS system 2 and/or 3 is associated with disease severity and outcome in patients with meningitis.


Subject(s)
Bacterial Secretion Systems/metabolism , Meningitis, Meningococcal/microbiology , Neisseria meningitidis/metabolism , Bacterial Proteins/metabolism , Humans , Meningitis, Meningococcal/metabolism , Prospective Studies
16.
J Biol Chem ; 289(28): 19799-809, 2014 Jul 11.
Article in English | MEDLINE | ID: mdl-24872418

ABSTRACT

The two-partner secretion (TPS) systems of Gram-negative bacteria secrete large TpsA exoproteins by a dedicated TpsB transporter in the outer membrane. TpsBs contain an N-terminal module located in the periplasm that includes two polypeptide transport-associated (POTRA) domains. These are thought to initiate secretion of a TpsA by binding its N-terminal secretion signal, called the TPS domain. Neisseria meningitidis encodes up to five TpsA proteins that are secreted via only two TpsB transporters: TpsB1 and TpsB2. Of these two, the TpsB2 recognizes the TPS domains of all TpsAs, despite their sequence diversity. By contrast, the TpsB1 shows a limited recognition of a TPS domain that is shared by two TpsAs. The difference in substrate specificity of the TpsBs enabled us to investigate the role of the POTRA domains in the selection of TPS domains. We tested secretion of TPS domains or full-length TpsAs by TpsB mutants with deleted, duplicated, and exchanged POTRA domains. Exchanging the two POTRA domains of a TpsB resulted in a switch in specificity. Furthermore, exchanging a single POTRA domain showed that each of the two domains contributed to the cargo selection. Remarkably, the order of the POTRA domains could be reversed without affecting substrate selection, but this aberrant order did result in an alternatively processed secretion product. Our results suggest that secretion of a TpsA is initiated by engaging both POTRA domains of a TpsB transporter and that these select the cognate TpsAs for secretion.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Secretion Systems/physiology , Carrier Proteins/metabolism , Neisseria meningitidis/metabolism , Bacterial Proteins/genetics , Carrier Proteins/genetics , Neisseria meningitidis/genetics , Protein Structure, Tertiary , Protein Transport/physiology
17.
J Bacteriol ; 195(4): 788-97, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23222722

ABSTRACT

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a large secreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ∼30-kDa TPS domain located at its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidis and the related species Neisseria lactamica but not domains of Haemophilus influenzae and Pseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcal systems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related to the meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Carrier Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Neisseria meningitidis/metabolism , Bacterial Outer Membrane Proteins/genetics , Carrier Proteins/genetics , Cloning, Molecular , Computer Simulation , Gene Deletion , Models, Molecular , Molecular Sequence Data , Mutation , Neisseria meningitidis/genetics , Protein Conformation , Protein Structure, Tertiary , Species Specificity
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