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1.
J Med Microbiol ; 71(4)2022 Apr.
Article in English | MEDLINE | ID: covidwho-1788579

ABSTRACT

Introduction. Carbapenem-resistant Acinetobacter baumannii (CRAB) is the primary pathogen causing hospital-acquired infections. The spread of CRAB is mainly driven by the dissemination of resistant clones, and in Latin America, International Clones IC-1 (also known as clonal complex CC1), IC-4 (CC15) and IC-5 (CC79) are the most prevalent.Gap Statement. There are no documented outbreaks of CRAB International Clone 2 (IC-2) reported in Brazil.Aim. To describe a large outbreak of CRAB caused by the uncommon IC-2 in a Brazilian COVID-19 hospital.Methodology. From May 2020 to May 2021, 224 patients infected or colonized with CRAB were identified in a single hospital; 92 % of them were also infected with SARS-CoV-2. From these patients, 137 isolates were recovered and subjected to antimicrobial susceptibility testing, PCR analysis and molecular typing. Whole-genome sequencing and downstream analysis were carried out on a representative isolate (the first available isolate).Results. In 76 % of the patients, a single OXA-23-producing CRAB IC-2 was identified. All the isolates were susceptible to polymyxin B, but highly resistant (>95 %) to aminoglycosides, fluoroquinolones and beta-lactams. Genomic analysis revealed that the representative isolate also carried the 16S rRNA Methylase ArmA, which was detected for the first time in this species in Brazil.Conclusion. We report the rapid spread of an emerging CRAB clone responsible for causing a large outbreak in a hospital in Brazil, a country with predominance of other CRAB clones. Continuous and prospective surveillance is warranted to evaluate the impact of this clone in Brazilian hospital settings.


Subject(s)
Acinetobacter Infections , Acinetobacter baumannii , COVID-19 , Acinetobacter Infections/epidemiology , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Brazil/epidemiology , COVID-19/epidemiology , Clone Cells , Humans , Microbial Sensitivity Tests , Multilocus Sequence Typing , Pandemics , Prospective Studies , RNA, Ribosomal, 16S , SARS-CoV-2/genetics , beta-Lactamases/genetics
2.
Nat Commun ; 13(1): 988, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1713165

ABSTRACT

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Despite the ribosome helicase, certain mRNA stem-loops stimulate programmed ribosomal frameshift by inhibiting translation elongation. Here, using mutagenesis, biochemical and single-molecule experiments, we examine whether high stability of three basepairs, which are unwound by the translating ribosome, is critical for inducing ribosome pauses. We find that encountering frameshift-inducing mRNA stem-loops from the E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) hinders A-site tRNA binding and slows down ribosome translocation by 15-20 folds. By contrast, unwinding of first three basepairs adjacent to the mRNA entry channel slows down the translating ribosome by only 2-3 folds. Rather than high thermodynamic stability, specific length and structure enable regulatory mRNA stem-loops to stall translation by forming inhibitory interactions with the ribosome. Our data provide the basis for rationalizing transcriptome-wide studies of translation and searching for novel regulatory mRNA stem-loops.


Subject(s)
Frameshifting, Ribosomal , RNA, Messenger/chemistry , Bacterial Proteins/genetics , DNA Polymerase III/genetics , Escherichia coli/genetics , Fluorescence Resonance Energy Transfer , HIV/genetics , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , Single Molecule Imaging , Thermodynamics
3.
Cell Rep ; 38(8): 110414, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1700507

ABSTRACT

Inflammasome activation exacerbates infectious disease caused by pathogens such as Listeria monocytogenes, Staphylococcus aureus, and severe acute respiratory syndrome coronavirus 2. Although these pathogens activate host inflammasomes to regulate pathogen expansion, the mechanisms by which pathogen toxins contribute to inflammasome activation remain poorly understood. Here we show that activation of inflammasomes by Listeria infection is promoted by amino acid residue T223 of listeriolysin O (LLO) independently of its pore-forming activity. LLO T223 is critical for phosphorylation of the inflammasome adaptor ASC at amino acid residue Y144 through Lyn-Syk signaling, which is essential for ASC oligomerization. Notably, a Listeria mutant expressing LLO T223A is impaired in inducing ASC phosphorylation and inflammasome activation. Furthermore, the virulence of LLO T223A mutant is markedly attenuated in vivo due to impaired ability to activate the inflammasome. Our results reveal a function of a pathogen toxin that exacerbates infection by promoting phosphorylation of ASC.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , CARD Signaling Adaptor Proteins/metabolism , Heat-Shock Proteins/metabolism , Hemolysin Proteins/metabolism , Inflammasomes/metabolism , Listeria monocytogenes/pathogenicity , Signal Transduction , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , CARD Signaling Adaptor Proteins/chemistry , CARD Signaling Adaptor Proteins/deficiency , CARD Signaling Adaptor Proteins/genetics , Gene Editing , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Hemolysin Proteins/chemistry , Hemolysin Proteins/genetics , Interleukin-18/metabolism , Listeria monocytogenes/metabolism , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/metabolism , Macrophages, Peritoneal/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutagenesis, Site-Directed , Phosphorylation , Syk Kinase/genetics , Syk Kinase/metabolism , Virulence , src-Family Kinases/genetics , src-Family Kinases/metabolism
4.
Cells ; 11(3)2022 01 27.
Article in English | MEDLINE | ID: covidwho-1662647

ABSTRACT

In this contribution, we report on the possibility that cryptococcal protease(s) could activate the SARS-CoV-2 spike (S) protein. The S protein is documented to have a unique four-amino-acid sequence (underlined, SPRRAR↓S) at the interface between the S1 and S2 sites, that serves as a cleavage site for the human protease, furin. We compared the biochemical efficiency of cryptococcal protease(s) and furin to mediate the proteolytic cleavage of the S1/S2 site in a fluorogenic peptide. We show that cryptococcal protease(s) processes this site in a manner comparable to the efficiency of furin (p > 0.581). We conclude the paper by discussing the impact of these findings in the context of a SARS-CoV-2 disease manifesting while there is an underlying cryptococcal infection.


Subject(s)
Aspartic Acid Proteases/metabolism , Bacterial Proteins/metabolism , Cryptococcus neoformans/enzymology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Aspartic Acid Proteases/genetics , Bacterial Proteins/genetics , Binding Sites , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , Cryptococcus neoformans/genetics , Fluorescent Dyes/chemistry , Furin/genetics , Furin/metabolism , Humans , Pandemics , Peptides/chemistry , Peptides/metabolism , Proteolysis , SARS-CoV-2/physiology
5.
Eur J Clin Microbiol Infect Dis ; 41(4): 573-580, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1640882

ABSTRACT

PURPOSE: To evaluate the prevalence of multi-carbapenemase-producing Enterobacterales (EB) and the activity of cefiderocol (CFDC), meropenem-vaborbactam (MEV), ceftazidime-avibactam (CZA), and combinations of CZA plus aztreonam (ATM), MEV plus ATM and CFDC plus CZA against them. METHODS: A collection of carbapenemase-producing EB clinical isolates (n = 1242) was investigated by lateral flow immunoassay NG-Test CARBA-5 and molecular testing. Cefiderocol MICs were determined using broth microdilution SensititreTM panel. MICs of CZA and MEV were determined by the gradient diffusion method. Antimicrobial synergy testing was performed using gradient diffusion strip crossing. RESULTS: KPC were the most frequent carbapenemases (83.2%), followed by VIM (9.2 %), OXA-48-like (4.3 %) and NDM enzymes (4.1%). Multi-carbapenemase producers were found in 10 (0.8%) isolates. Three combinations of two different carbapenemases were observed: KPC+VIM (n = 4), NDM+OXA-48-like (n = 4), and VIM+OXA-48-like (n = 2). CFDC showed potent activity against eight out of ten dual-carbapenemases producers, while resistance or reduced susceptibility was shown towards CZA and MEV. CFDC in combination with CZA showed no synergistic effects and only two additive effects on seven (87.5%) of the CFDC-susceptible strains. Conversely, CZA plus ATM and MEV plus ATM combinations were synergistic against all ATM-resistant strains regardless of dual-carbapenemases phenotype. CONCLUSIONS: The occurrence of multi-carbapenemase producers is not uncommon in Northern Italy area. MEV in combination with ATM might be considered as a potential therapeutic option, alternative to CZA plus ATM. CFDC susceptibility testing and synergy evaluation of ATM-based combinations should be performed in the lab routine to evaluate the most in vitro active antimicrobial regimen.


Subject(s)
Aztreonam , COVID-19 , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Azabicyclo Compounds , Aztreonam/pharmacology , Bacterial Proteins/genetics , Boronic Acids , Ceftazidime/pharmacology , Cephalosporins , Drug Combinations , Humans , Meropenem/pharmacology , Microbial Sensitivity Tests , beta-Lactamases/genetics
6.
J Antimicrob Chemother ; 77(4): 1140-1145, 2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1632142

ABSTRACT

OBJECTIVES: To report an outbreak of hypervirulent Klebsiella pneumoniae (hvKp) in COVID-19 patients. METHODS: Prospective, observational study including consecutive COVID-19 patients with hvKp infections admitted to the University Hospital of Pisa (Italy). Clinical data and outcome of patients were collected. All patients were followed-up to 30 days from the diagnosis of infection. Mortality within 30 days of the diagnosis of hvKp infection was reported. The hypermucoviscous phenotype was determined by the 'string test'. Molecular typing was performed on three strains collected during different periods of the outbreak. The strains underwent whole genome sequencing using the Illumina MiSeq instrument. The complete circular assemblies were also obtained for the chromosome and a large plasmid using the Unicycler tool. RESULTS: From November 2020 to March 2021, hvKp has been isolated from 36 COVID-19 patients: 29/36 (80.6%) had infections (15 bloodstream infections, 8 ventilator-associated pneumonias and 6 complicated urinary tract infections), while 7/36 (19.4%) had colonization (3 urine, 2 rectal and 2 skin). The isolates belonged to ST147 and their plasmid carried three replicons of the IncFIB (Mar), IncR and IncHI1B types and several resistance genes, including the rmpADC genes encoding enhancers of capsular synthesis. The hvKp isolates displayed an ESBL phenotype, with resistance to piperacillin/tazobactam and ceftolozane/tazobactam and susceptibility only to meropenem and ceftazidime/avibactam. The majority of patients were treated with meropenem alone or in combination with fosfomycin. Thirty-day mortality was 48.3% (14/29). CONCLUSIONS: ST147 ESBL-producing hvKp is associated with high mortality in COVID-19 patients. Strict microbiological surveillance and infection control measures are needed in this population.


Subject(s)
COVID-19 , Klebsiella Infections , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/genetics , Humans , Klebsiella Infections/epidemiology , Klebsiella Infections/microbiology , Klebsiella pneumoniae , Prospective Studies
7.
Eur J Clin Microbiol Infect Dis ; 41(3): 495-500, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1603573

ABSTRACT

The objective was to study ceftazidime-avibactam resistant and susceptible Klebsiella pneumoniae isolated from a patient admitted to the Policlinico Umberto I of Rome for SARS-CoV2. Data on the evolution of patient's conditions, antimicrobial therapies, and microbiological data were collected. Whole-genome sequencing performed by Illumina and Nanopore sequencing methods were used to type the strains. During the hospitalization, a SARS-CoV2-infected patient was colonized by a KPC-producing K. pneumoniae strain and empirically treated with ceftazidime-avibactam (CZA) when presenting spiking fever symptoms. Successively, ST2502 CZA-resistant strain producing the KPC-31 variant gave a pulmonary infection to the patient. The infection was treated with high doses of meropenem. The KPC-31-producing strain disappeared but the patient remained colonized by a KPC-3-producing K. pneumoniae strain. An interplay between highly conserved KPC-31- and KPC-3-producing ST2502 strains occurred in the SARS-CoV2 patient during the hospitalization, selected by CZA and carbapenem treatments, respectively.


Subject(s)
Anti-Bacterial Agents , COVID-19 , Klebsiella Infections , Meropenem , Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/genetics , COVID-19/complications , Ceftazidime/therapeutic use , Drug Combinations , Humans , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/genetics , Meropenem/therapeutic use , Microbial Sensitivity Tests , beta-Lactamases/genetics
8.
PLoS One ; 16(12): e0261442, 2021.
Article in English | MEDLINE | ID: covidwho-1593549

ABSTRACT

A laboratory validation study was conducted to assess the equivalence of Xpert MTB/RIF Ultra testing on the GeneXpert System and the GeneXpert Omni System ('Omni') for tuberculosis and rifampicin resistance. High concordance of the two devices was demonstrated for well-characterized clinical samples as well as control materials, with controls tested on Omni at normal and challenging environmental conditions (i.e. 35°C, 90% relative humidity). Equivalence of the Cts for all probes was also shown. Equivalence was demonstrated for the Omni and GeneXpert devices for tuberculosis and rifampicin resistance detection for a diverse range of clinical specimens and environmental conditions.


Subject(s)
Antibiotics, Antitubercular/pharmacology , Mycobacterium tuberculosis/drug effects , Point-of-Care Testing , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Pulmonary/diagnosis , Bacterial Proteins/genetics , DNA-Directed RNA Polymerases/genetics , Drug Resistance, Multiple, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Rifampin/pharmacology , Sputum/microbiology , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Pulmonary/drug therapy
9.
Infect Genet Evol ; 97: 105188, 2022 01.
Article in English | MEDLINE | ID: covidwho-1568934

ABSTRACT

The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.


Subject(s)
COVID-19 Vaccines/genetics , COVID-19/prevention & control , CRISPR-Cas Systems , Gene Editing/methods , Mutation Rate , SARS-CoV-2/genetics , Viral Proteins/genetics , APOBEC Deaminases/genetics , APOBEC Deaminases/immunology , Adenosine Deaminase/genetics , Adenosine Deaminase/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , COVID-19/immunology , COVID-19 Vaccines/biosynthesis , Endonucleases/genetics , Endonucleases/immunology , Gene Expression , Genome, Viral , Humans , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/immunology , Selection, Genetic , Vaccines, Attenuated , Viral Proteins/immunology
10.
RNA ; 28(2): 227-238, 2022 02.
Article in English | MEDLINE | ID: covidwho-1533393

ABSTRACT

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.


Subject(s)
Bacillus subtilis/genetics , Endoribonucleases/genetics , Endoribonucleases/metabolism , Bacillus subtilis/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Endoribonucleases/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Microorganisms, Genetically-Modified , Mutation , RNA Stability , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , Ribonucleases/genetics , Ribonucleases/metabolism , Substrate Specificity , Viral Nonstructural Proteins/metabolism
11.
Microbiol Spectr ; 9(3): e0112221, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1511426

ABSTRACT

Whole-genome sequencing was used to characterize carbapenemase-producing Enterobacterales (CPE) strains recovered from rectal screening swab samples obtained from children at a tertiary-care pediatric hospital in Qatar during a 3-year period. A total of 72 CPE isolates recovered from 61 fecal carriers were characterized. Escherichia coli (47 isolates [65.3%]) and Klebsiella pneumoniae (22 isolates [30.6%]) were the most common species identified. High levels of genetic diversity were observed for both species. These 72 isolates produced 78 carbapenemases, characterized as either NDM-type (41 enzymes [52.6%]) or OXA-48-type (37 enzymes [47.4%]). NDM-5 (24 enzymes [30.8%]), NDM-1 (15 enzymes [19.2%]), and OXA-181 (15 enzymes [19.2%]) were the most common variants detected within each type. Twenty-three NDM producers exhibited difficult-to-treat resistance, compared with only 2 of the OXA-48 producers. Multiple comorbidities were identified in 88.5% of the patients, whereas recent travel history to countries in which CPE are endemic was documented for 57.4% of the patients. All 9 blaOXA-48-type-gene-containing E. coli sequence type 38 (ST38) strains were isolated from patients without international travel history. The mean quarterly incidence of fecal carriage decreased more than 6-fold after the implementation of coronavirus disease 2019 (COVID-19)-related international travel restrictions in Qatar in mid-March 2020. Our data suggest that NDM-type and OXA-48-type carbapenemases expressed by a large diversity of E. coli and K. pneumoniae genotypes are largely dominant in the pediatric population of Qatar. Although our data indicate successful local expansion of E. coli ST38 strains harboring blaOXA-244 genes, at least within health care settings, blaOXA-48-type and blaNDM-type genes appear to have been mainly introduced sporadically by asymptomatic carriers who visited or received health care in some nearby countries in which the genes are endemic. IMPORTANCE To the best of our knowledge, this is the first study addressing the molecular characteristics of CPE in a pediatric population in Qatar using whole-genome sequencing. Since several countries in the Arabian Peninsula share relatively similar demographic patterns and international links, it is plausible that the molecular characteristics of CPE in children, at least in the middle and eastern parts of the region, are similar to those observed in our study.


Subject(s)
Bacterial Proteins/chemistry , Enterobacteriaceae/enzymology , Feces/chemistry , beta-Lactamases/chemistry , Adolescent , Anti-Bacterial Agents , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , COVID-19 , Child , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Escherichia coli/enzymology , Escherichia coli/genetics , Genotype , Humans , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests , Mutation , Qatar , Retrospective Studies , SARS-CoV-2 , Whole Genome Sequencing , beta-Lactamases/genetics , beta-Lactamases/isolation & purification
12.
Sci Rep ; 11(1): 21075, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1493212

ABSTRACT

Bats are potential natural reservoirs for emerging viruses, causing deadly human diseases, such as COVID-19, MERS, SARS, Nipah, Hendra, and Ebola infections. The fundamental mechanisms by which bats are considered "living bioreactors" for emerging viruses are not fully understood. Some studies suggest that tolerance to viruses is linked to suppressing antiviral immune and inflammatory responses due to DNA damage by energy generated to fly. Our study reveals that bats' gut bacteria could also be involved in the host and its microbiota's DNA damage. We performed screening of lactic acid bacteria and bacilli isolated from bats' feces for mutagenic and oxidative activity by lux-biosensors. The pro-mutagenic activity was determined when expression of recA increased with the appearance of double-strand breaks in the cell DNA, while an increase of katG expression in the presence of hydroxyl radicals indicated antioxidant activity. We identified that most of the isolated bacteria have pro-mutagenic and antioxidant properties at the same time. This study reveals new insights into bat gut microbiota's potential involvement in antiviral response and opens new frontiers in preventing emerging diseases originating from bats.


Subject(s)
Chiroptera/virology , Gastrointestinal Microbiome , Mutagens , Animals , Antioxidants/metabolism , Antiviral Agents , Bacillus , Bacterial Proteins/genetics , Biosensing Techniques , COVID-19 , DNA , DNA Damage , Disease Reservoirs/virology , Escherichia coli/metabolism , Feces , Immune System , Inflammation , Lactic Acid/metabolism , Mass Spectrometry , Mutagenesis , Oxidative Stress , Rec A Recombinases/metabolism , SARS-CoV-2 , Viruses/isolation & purification , Zoonoses/virology
13.
Eur J Clin Microbiol Infect Dis ; 40(11): 2295-2303, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1479485

ABSTRACT

The aim of this study is to present the first nationwide microbiological and epidemiological study of invasive group A Streptococcus (iGAS) disease in Spain. One thousand eight hundred ninety-three iGAS isolates were analyzed over 2007-2019. emm typing was performed by sequencing the gene's variable 5' end, exotoxin genes were identified by PCR, and antimicrobial susceptibility explored via the E test and disk diffusion. Five hundred twenty-three isolates were associated with sepsis, 292 with cellulitis, 232 with scarlet fever, 153 with pneumonia, 141 with streptococcal toxic shock syndrome, and 94 with necrotizing fasciitis. The most prevalent emm types were emm1 (449/1893 isolates), emm89 (210/1893), emm3 (208/1893), emm4 (150/1893), emm12 (112/1893) emm6 (107/1893), emm87 (89/1893), emm28 (88/1893), emm75 (78/1893), emm77 (78/1893), emm11 (58/1893), and emm22 (35/1893). emm1, emm3, emm4, and emm6 were the predominant types affecting children (mostly respiratory infections), while emm11, emm77, and emm89 prevailed in the elderly (mostly skin infections). Each emm type was associated with one or more exotoxin gene (spe, sme, and ssa) profiles. speA was detected in 660 isolates, speB in 1829, speC in 1014, speF in 1826, speG in 1651, speJ in 716, speH in 331, smeZ in 720, and ssa in 512. Isolates with speA were associated with the most severe infections. Penicillin susceptibility was universal. Two hundred twenty-four isolates were resistant to tetracycline, 169 to erythromycin, and 81 to clindamycin. Tetracycline, erythromycin, and clindamycin resistance rates declined over the study period. The above information could serve as the basis for continued surveillance efforts designed to control disease cause by this bacterium.


Subject(s)
Streptococcal Infections/microbiology , Streptococcus pyogenes/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Child , Child, Preschool , Erythromycin/pharmacology , Exotoxins/genetics , Exotoxins/metabolism , Female , Humans , Infant , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microbial Sensitivity Tests , Middle Aged , Penicillins/pharmacology , Spain/epidemiology , Streptococcal Infections/epidemiology , Streptococcus pyogenes/classification , Streptococcus pyogenes/drug effects , Streptococcus pyogenes/genetics , Young Adult
14.
Molecules ; 26(19)2021 Oct 02.
Article in English | MEDLINE | ID: covidwho-1463766

ABSTRACT

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N'-diacetyl-ß-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s-1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains-2x Fn3/Big3 and a carbohydrate binding module-that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.


Subject(s)
Bacterial Proteins/metabolism , Chitin/metabolism , Chitinases/metabolism , Clostridium/metabolism , Bacterial Proteins/genetics , Catalytic Domain , Chitinases/chemistry , Chitinases/genetics , Clostridium/growth & development , Clostridium/isolation & purification , Gastrointestinal Microbiome , Humans , Hydrogen-Ion Concentration , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
15.
BMC Infect Dis ; 21(1): 927, 2021 Sep 08.
Article in English | MEDLINE | ID: covidwho-1403227

ABSTRACT

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is among the most concerning cause of healthcare-associated infections (HAI) due to its high level of antibiotic resistance and high mortality. In the era of the COVID-19 pandemic, the key priority of infection control committees is to contain the dissemination of antibiotic resistant Gram-negative bacteria. Here, we aimed to timely recognize the emergence of CRAB in COVID-19 cases admitted to the wards of a tertiary referral hospital and to identify the genetic relatedness of the isolates. METHODS: From 30 March to 30 May 2020, a total of 242 clinical samples from COVID-19 cases were screened for CRAB isolates using standard microbiologic and antibiotic susceptibility tests. The PCRs targeting oxa23, oxa24, oxa58, blaTEM and blaNDM-1 genes were performed. Two multiplex PCRs for identifying the global clones (GC) of A. baumannii were also performed. The sequence type of CRABs was determined using Institut Pasteur (IP) multilocus sequence typing (MLST) scheme. RESULTS: Eighteen CRAB isolates were recovered from COVID-19 patients with the mean age of 63.94 ± 13.8 years. All but 4 COVID-19 patients co-infected with CRAB were suffering from an underlying disease. Death was recorded as the outcome in ICUs for 9 (50%) COVID-19 patients co-infected with CRAB. The CRAB isolates belong to GC2 and ST2IP and carried the oxa23 carbapenem resistance gene. CONCLUSION: This study demonstrated the co-infection of CRAB isolates and SARS-CoV-2 in the patients admitted to different ICUs at a referral hospital in Tehran. The CRAB isolates were found to belong to ST2IP, share the oxa23 gene and to have caused several outbreaks in the wards admitting COVID-19 patients.


Subject(s)
Acinetobacter Infections , COVID-19 , Coinfection , Acinetobacter Infections/epidemiology , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/genetics , Aged , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , COVID-19/epidemiology , COVID-19/microbiology , Carbapenems/pharmacology , Coinfection/epidemiology , Humans , Iran/epidemiology , Microbial Sensitivity Tests , Middle Aged , Multilocus Sequence Typing , Pandemics , Tertiary Care Centers , beta-Lactamases/genetics
16.
Sci Rep ; 10(1): 18149, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-1387454

ABSTRACT

Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer-stabilized in the prefusion conformation and fused with SpyCatcher-could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with 0.08 µg of SARS-CoV-2 spike-LuS nanoparticle elicited similar neutralizing responses as 2.0 µg of spike, which was ~ 25-fold higher on a weight-per-weight basis. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.


Subject(s)
Antigens/immunology , Betacoronavirus/metabolism , Nanoparticles/chemistry , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antigens/genetics , Antigens/metabolism , Aquifex , Bacteria/enzymology , Bacterial Proteins/genetics , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections , Ferritins/genetics , Helicobacter pylori/metabolism , Humans , Mice , Multienzyme Complexes/genetics , Neutralization Tests , Pandemics , Pneumonia, Viral , Protein Multimerization , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Surface Properties
17.
Int J Mol Sci ; 22(17)2021 Aug 26.
Article in English | MEDLINE | ID: covidwho-1374425

ABSTRACT

Bifidobacteria are some of the major agents that shaped the immune system of many members of the animal kingdom during their evolution. Over recent years, the question of concrete mechanisms underlying the immunomodulatory properties of bifidobacteria has been addressed in both animal and human studies. A possible candidate for this role has been discovered recently. The PFNA cluster, consisting of five core genes, pkb2, fn3, aaa-atp, duf58, tgm, has been found in all gut-dwelling autochthonous bifidobacterial species of humans. The sensory region of the species-specific serine-threonine protein kinase (PKB2), the transmembrane region of the microbial transglutaminase (TGM), and the type-III fibronectin domain-containing protein (FN3) encoded by the I gene imply that the PFNA cluster might be implicated in the interaction between bacteria and the host immune system. Moreover, the FN3 protein encoded by one of the genes making up the PFNA cluster, contains domains and motifs of cytokine receptors capable of selectively binding TNF-α. The PFNA cluster could play an important role for sensing signals of the immune system. Among the practical implications of this finding is the creation of anti-inflammatory drugs aimed at alleviating cytokine storms, one of the dire consequences resulting from SARS-CoV-2 infection.


Subject(s)
Bacterial Proteins/genetics , Bifidobacterium/physiology , COVID-19/therapy , /genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , COVID-19/immunology , COVID-19/virology , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/prevention & control , Cytokines/chemistry , Cytokines/metabolism , Humans , Immune System , Operon/genetics , /metabolism , SARS-CoV-2/isolation & purification
18.
PLoS One ; 16(7): e0254815, 2021.
Article in English | MEDLINE | ID: covidwho-1318322

ABSTRACT

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.


Subject(s)
African Swine Fever Virus/isolation & purification , African Swine Fever/diagnosis , Bacterial Proteins/genetics , CRISPR-Associated Proteins/genetics , Endodeoxyribonucleases/genetics , Swine Diseases/diagnosis , African Swine Fever/genetics , African Swine Fever/virology , African Swine Fever Virus/genetics , Animals , Bacterial Proteins/chemistry , CRISPR-Associated Proteins/chemistry , CRISPR-Cas Systems , DNA-Directed DNA Polymerase/chemistry , Endodeoxyribonucleases/chemistry , Molecular Diagnostic Techniques , Point-of-Care Systems , Recombinases/chemistry , Swine , Swine Diseases/genetics , Swine Diseases/pathology , Swine Diseases/virology
19.
Am J Infect Control ; 49(10): 1324-1326, 2021 10.
Article in English | MEDLINE | ID: covidwho-1309130

ABSTRACT

An outbreak of Klebsiella pneumoniae producing the carbapenemase NDM-1 occurred in our ICU during the last COVID-19 wave. Twelve patients were tested positive, seven remained asymptomatic whereas 5 developed an infection. Resistome and in silico multilocus sequence typing confirmed the clonal origin of the strains. The identification of a possible environmental reservoir suggested that difficulties in observing optimal bio-cleaning procedures due to workload and exhaustion contributed to the outbreak besides the inappropriate excessive glove use.


Subject(s)
COVID-19 , Klebsiella Infections , Anti-Bacterial Agents , Bacterial Proteins/genetics , Disease Outbreaks , Dreams , Humans , Intensive Care Units , Klebsiella Infections/epidemiology , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests , Multilocus Sequence Typing , Pandemics , SARS-CoV-2 , beta-Lactamases/genetics
20.
Microbiology (Reading) ; 167(3)2021 03.
Article in English | MEDLINE | ID: covidwho-1288221

ABSTRACT

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and spermine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c-di-GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane-bound c-di-GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.


Subject(s)
Bacterial Proteins/genetics , Biofilms , Vibrio cholerae/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Mutagenesis , Periplasm/genetics , Periplasm/metabolism , Protein Domains , Sequence Alignment , Signal Transduction , Vibrio cholerae/chemistry , Vibrio cholerae/physiology
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