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1.
Nature ; 625(7994): 352-359, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37992756

RESUMO

It was recently shown that bacteria use, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems1-4, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a 'sponge' that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms.


Assuntos
Fagos Bacilares , Bactérias , Proteínas Virais , Fagos Bacilares/classificação , Fagos Bacilares/genética , Fagos Bacilares/imunologia , Fagos Bacilares/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Bactérias/virologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Nature ; 625(7994): 360-365, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37992757

RESUMO

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.


Assuntos
Bactérias , Proteínas de Bactérias , Bacteriófagos , Evasão da Resposta Imune , Multimerização Proteica , Bactérias/genética , Bactérias/imunologia , Bactérias/metabolismo , Bactérias/virologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Bacteriófagos/genética , Bacteriófagos/imunologia , Bacteriófagos/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Desoxirribonucleases/ultraestrutura , DNA Viral/química , DNA Viral/metabolismo , DNA Viral/ultraestrutura
3.
Cell ; 186(9): 1863-1876.e16, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37030292

RESUMO

Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.


Assuntos
Bactérias , Bacteriófagos , Bactérias/genética , Bactérias/virologia , Bacteriófagos/genética , Sistemas CRISPR-Cas , Proteínas Virais/metabolismo , Mutação , Fenômenos Fisiológicos Bacterianos
4.
Nat Microbiol ; 7(11): 1849-1856, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36192536

RESUMO

Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.


Assuntos
Bacteriófagos , NAD , NAD/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , NAD+ Nucleosidase
5.
Cell Host Microbe ; 30(11): 1556-1569.e5, 2022 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-36302390

RESUMO

Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection.


Assuntos
Bacteriófagos , Bacteriófagos/genética , Bactérias/genética , Genoma Microbiano , Genômica , Sistema Imunitário
6.
Nature ; 611(7935): 326-331, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36174646

RESUMO

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.


Assuntos
Bactérias , Bacteriófagos , Domínios Proteicos , Receptores de Interleucina-1 , Transdução de Sinais , Receptores Toll-Like , Proteínas Virais , Bactérias/imunologia , Bactérias/metabolismo , Bactérias/virologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Receptores de Interleucina-1/química , Transdução de Sinais/imunologia , Bacteriófagos/química , Bacteriófagos/imunologia , Bacteriófagos/metabolismo , Proteínas Virais/química , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Receptores Toll-Like/química , Cristalografia por Raios X
7.
Nat Microbiol ; 7(8): 1200-1209, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35817891

RESUMO

DNA viruses and retroviruses consume large quantities of deoxynucleotides (dNTPs) when replicating. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral lifecycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. Here, we report that bacteria use a similar strategy to defend against bacteriophage infection. We identify a family of defensive bacterial deoxycytidine triphosphate (dCTP) deaminase proteins that convert dCTP into deoxyuracil nucleotides in response to phage infection. We also identify a family of phage resistance genes that encode deoxyguanosine triphosphatase (dGTPase) enzymes, which degrade dGTP into phosphate-free deoxyguanosine and are distant homologues of human SAMHD1. Our results suggest that bacterial defensive proteins deplete specific deoxynucleotides (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Our study shows that manipulation of the dNTP pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.


Assuntos
Bacteriófagos , Antivirais , Bactérias , Bacteriófagos/genética , Desoxiguanosina , Humanos , Proteína 1 com Domínio SAM e Domínio HD
8.
Nature ; 605(7910): 522-526, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35395152

RESUMO

The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3-10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.


Assuntos
Bacteriófagos , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriófago T4/metabolismo , Bacteriófagos/fisiologia , Sistemas CRISPR-Cas/genética , Endonucleases/metabolismo , Escherichia coli/metabolismo , Nucleotídeos Cíclicos/metabolismo , Oligonucleotídeos , Pirimidinas/metabolismo
9.
Cell ; 183(6): 1551-1561.e12, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33157039

RESUMO

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed.


Assuntos
Bactérias/genética , Bactérias/imunologia , Bacteriófagos/fisiologia , RNA não Traduzido/genética , DNA Polimerase Dirigida por RNA/genética , Bactérias/virologia , Ilhas de CpG/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , Filogenia
10.
Science ; 359(6379)2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29371424

RESUMO

The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in "defense islands" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.


Assuntos
Bacillus subtilis/imunologia , Bacillus subtilis/virologia , Bacteriófagos/imunologia , Bacteriófagos/patogenicidade , Escherichia coli/imunologia , Escherichia coli/virologia , Genes Bacterianos/fisiologia , Bacillus subtilis/genética , Escherichia coli/genética , Genoma Bacteriano , Família Multigênica
11.
Nature ; 541(7638): 488-493, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28099413

RESUMO

Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis-lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six amino-acids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the 'arbitrium' system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to 'communicate' with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.


Assuntos
Bacteriólise , Bacteriófagos/fisiologia , Lisogenia , Sequência de Aminoácidos , Bacillus/citologia , Bacillus/virologia , Bacteriólise/efeitos dos fármacos , Bacteriófagos/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , DNA Viral/metabolismo , Lisogenia/efeitos dos fármacos , Modelos Biológicos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Multimerização Proteica , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia
12.
Diagn Microbiol Infect Dis ; 79(2): 255-60, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24743043

RESUMO

We performed comparative sequence analysis of 3 blaKPC-2 encoding plasmids to examine evolution of these plasmids and their dissemination. We found that all of them have an IncN replicon with a newly determined IncN plasmid sequence type (ST), ST15. The 2 Klebsiella pneumoniae (KPN) plasmids also harbor an IncF2A1-B1- replicon. The blaKPC-2 is located in the Tn4401c transposon with a newly discovered mutation in the P2 promoter. Screening of the 27 additional blaKPC-2 carrying plasmids from Enterobacter cloacae, Escherichia coli (EC), and K. pneumoniae showed that: all KPN and EC plasmids are IncN plasmids belonging to ST15; 4/7 KPN and 1/6 EC plasmids contain an additional IncF2A1-B1- replicon; all Enterobacter plasmids belong to neither IncN nor IncF2A1-B1- replicon plasmids; 6/7 KPN and 2/5 EC plasmids carry the mutated P2 promoter. Study of the blaKPC-2 environment, transposon, pMLST, and Inc group suggests transposon and plasmid inter- and intra-species dissemination and evolution.


Assuntos
Enterobacter cloacae/genética , Escherichia coli/genética , Genômica , Klebsiella pneumoniae/genética , Plasmídeos/classificação , beta-Lactamases/genética , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Enterobacter cloacae/enzimologia , Escherichia coli/enzimologia , Evolução Molecular , Humanos , Klebsiella pneumoniae/enzimologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Replicon , Análise de Sequência de DNA
13.
Mol Cell ; 50(1): 136-48, 2013 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-23478446

RESUMO

Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using more than 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an "antidefense" protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.


Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , Clonagem Molecular/métodos , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genoma Bacteriano , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Bacteriófago T7/patogenicidade , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Família Multigênica , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Fatores de Tempo , Virulência
14.
Genome Res ; 22(4): 802-9, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22300632

RESUMO

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes, we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes, our assays revealed novel toxins and restriction enzymes, and new classes of small, non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short, toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator DnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.


Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos/genética , RNA Bacteriano/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA Bacteriano/metabolismo , DNA Bacteriano/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Dados de Sequência Molecular , Ligação Proteica , RNA Bacteriano/metabolismo , RNA Bacteriano/farmacologia , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência/farmacologia , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
15.
Antimicrob Agents Chemother ; 54(10): 4493-6, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20696875

RESUMO

We have determined the entire DNA sequence of plasmid pKpQIL, the bla(KPC-3)-carrying plasmid harbored by the carbapenem-resistant Klebsiella pneumoniae clone sequence type 258 (ST 258) in Israel. pKpQIL is a 113,637-bp, self-transmissible plasmid that belongs to the incompatibility group IncFII. It consists of a large backbone of a pKPN4-like plasmid and carries the bla(KPC-3)-containing Tn4401a transposon of a pNYC-like plasmid.


Assuntos
Proteínas de Bactérias/genética , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Plasmídeos/genética , beta-Lactamases/genética , Modelos Genéticos , Dados de Sequência Molecular
16.
Antimicrob Agents Chemother ; 54(7): 3002-6, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20350950

RESUMO

Sporadic isolates of carbapenem-resistant KPC-2-producing Klebsiella pneumoniae were isolated in Tel Aviv Medical Center during 2005 and 2006, parallel to the emergence of the KPC-3-producing K. pneumoniae sequence type 258 (ST 258). We aimed to study the molecular epidemiology of these isolates and to characterize their bla(KPC)-carrying plasmids and their origin. Ten isolates (8 KPC-2 and 2 KPC-3 producing) were studied. All isolates were extremely drug resistant. They possessed the bla(KPC) gene and varied in their additional beta-lactamase contents. The KPC-2-producing strains belonged to three different sequence types: ST 340 (n = 2), ST 277 (n = 2), and a novel sequence type, ST 376 (n = 4). Among KPC-3-producing strains, a single isolate (ST 327) different from ST 258 was identified, but both strains carried the same plasmid (pKpQIL). The KPC-2-encoding plasmids varied in size (45 to 95 kb) and differed among each of the STs. Two of the Klebsiella bla(KPC-2)-carrying plasmids were identical to plasmids from Escherichia coli, suggesting a common origin of these plasmids. These data indicate that KPC evolution in K. pneumoniae is related to rare events of interspecies spread of bla(KPC-2)-carrying plasmids from E. coli followed by limited clonal spread, whereas KPC-3 carriage in this species is related almost strictly to clonal expansion of ST 258 carrying pKpQIL.


Assuntos
Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Epidemiologia Molecular/métodos , Plasmídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbapenêmicos/uso terapêutico , Farmacorresistência Bacteriana/genética , Humanos , Israel/epidemiologia , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Filogenia , Reação em Cadeia da Polimerase , beta-Lactamases/genética , beta-Lactamases/metabolismo
17.
J Antimicrob Chemother ; 65(2): 243-8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19939824

RESUMO

OBJECTIVES: An extremely drug-resistant (XDR) clone of KPC-3-producing Klebsiella pneumoniae emerged in Israel in 2006, causing a nationwide outbreak. We aimed to characterize the local KPC-3-encoding plasmid carried by these isolates and study its contribution to antibiotic resistance. METHODS: Mechanisms of carbapenem resistance were investigated in seven selected isolates (isolated between 2006 and 2008) belonging to the epidemic clone. Isolates underwent MIC testing, and were examined for the presence of KPC, Tn4401, class I integron elements and additional antibiotic resistance genes. Plasmids were analysed by transformation, transconjugation, restriction mapping, curing and complementation experiments. Outer membrane protein (OMP) analysis was performed. RESULTS: OMP analysis did not reveal loss of porins. KPC-3-producing K. pneumoniae isolates possessed various plasmids but all harboured a common self-transmissible 105 kb plasmid, termed pKpQIL, encoding bla(TEM-1) and bla(KPC-3). Curing of pKpQIL led to a complete loss of resistance to cephalosporins and carbapenems, proving its crucial role in carbapenem resistance. Transformation of plasmid pKpQIL into the cured Klebsiella strain resulted in full reconstitution of carbapenem resistance. The presence of all Tn4401 transposon elements located upstream of the KPC-3 gene was detected by PCR and sequencing. pKpQIL lacked additional antibiotic resistance genes. CONCLUSIONS: Our findings demonstrate the presence of pKpQIL, a 105 kb KPC-3- and TEM-1-encoding plasmid, in the XDR K. pneumoniae epidemic strain in Israel. pKpQIL is unique and appears consistently in all isolates of this clone over the years. The extensive beta-lactam resistance phenotype of this clone is primarily mediated by this single self-transmissible plasmid.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Klebsiella pneumoniae/efeitos dos fármacos , Plasmídeos/análise , beta-Lactamases/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Surtos de Doenças , Humanos , Israel/epidemiologia , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Reação em Cadeia da Polimerase , beta-Lactamases/genética
18.
Pediatr Infect Dis J ; 28(11): 960-5, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19738508

RESUMO

BACKGROUND: Staphylococcus aureus colonization typically precedes infection but risk factors for colonization in children are not well defined. Our previous study suggested that S. aureus carriage in children is associated with parental carriage. Here we wished to distinguish the different components that play a role in the risk to a child of a S. aureus-carrying parent. METHODS: Between 2002 and 2005, children (0-40 months) and their parents were screened for carriage of S. aureus and Streptococcus pneumoniae during 1 of 6 surveys. Data were collected from the parents and the medical files. Multivariate analysis of possible associated factors and effect modifiers was carried out. Pulse-field gel electrophoresis was performed to determine strain relatedness. RESULTS: A total of 4648 children were screened. S. aureus was isolated from 342 (7.6%) children and 992 (22%) parents. Pairs of parent-child carriers were found in 155 cases, over twice the rate expected by chance (1.66%, P<0.0001). The variable that was most significantly associated with carriage in children was having a parent carrier (OR: 3.35; 95% CI: 2.59-4.33), whereas close contact with peers (as assessed by day care centers attendance or having young siblings) was not associated with carriage. Children<3 months had the highest carriage rate and children aged 6 to 12 months had the lowest (25.4% and 4.3%, respectively, P<0.0001). Breast-feeding was not associated with higher or lower carriage. In 30 of 150 strains studied, >70% parent-child strains were genetically identical. CONCLUSIONS: Parental S. aureus colonization, but not DCC attendance or having young siblings, is an independent predictor for staphylococcal carriage in young children.


Assuntos
Portador Sadio/epidemiologia , Pais , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/isolamento & purificação , Adulto , Técnicas de Tipagem Bacteriana , Portador Sadio/microbiologia , Pré-Escolar , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/isolamento & purificação , Adulto Jovem
19.
J Clin Microbiol ; 47(4): 969-74, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19213695

RESUMO

Ertapenem resistance in Klebsiella pneumoniae is rare. We report on an ertapenem-nonsusceptible phenotype among 25 out of 663 (3.77%) extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates in a multicenter Israeli study. These isolates originated from six different hospitals and were multiclonal, belonging to 12 different genetic clones. Repeat testing using Etest and agar dilution confirmed ertapenem nonsusceptibility in only 15/663 (2.3%) of the isolates. The molecular mechanisms of ertapenem resistance in seven single-clone resistant isolates was due to the presence of ESBL genes (CTX-M-2 in four isolates, CTX-M-10 and OXA-4 in one isolate, SHV-12 in one isolate, and SHV-28 in one isolate) combined with the absence of OMPK36. Seven of 10 isolates initially reported as ertapenem nonsusceptible and subsequently classified as susceptible showed an inoculum effect with ertapenem but not with imipenem or meropenem. Population analysis detected the presence of an ertapenem-resistant subpopulation at a frequency of 10(-6). These rare resistant subpopulations carried multiple ESBL genes, including TEM-30, SHV-44, CTX-M-2, and CTX-M-10, and they lacked OMPK36. The clinical and diagnostic significance of the results should be further studied.


Assuntos
Antibacterianos/farmacologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/biossíntese , beta-Lactamas/farmacologia , Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Eletroforese em Gel de Poliacrilamida , Ertapenem , Genótipo , Hospitais , Humanos , Israel , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
20.
Antimicrob Agents Chemother ; 53(2): 818-20, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19029323

RESUMO

A highly epidemic carbapenem-resistant clone of KPC-3-producing Klebsiella pneumoniae emerged in Israel in 2006, causing a nationwide outbreak. This clone was genetically related to outbreak strains from the United States isolated in 2000 but differed in KPC-carrying plasmids. The threat of the global spread of hyperepidemic, extensively drug-resistant bacterial strains should be recognized and confronted.


Assuntos
Proteínas de Bactérias/genética , Surtos de Doenças , Farmacorresistência Bacteriana/genética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Israel/epidemiologia , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estados Unidos/epidemiologia
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