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1.
Arq. gastroenterol ; 55(4): 390-396, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-983850

ABSTRACT

ABSTRACT BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is one of the main acute and chronic diarrhea causes both in children and adults, mainly in developing countries. OBJECTIVE: The aim of the present study is to characterize EAEC strains isolated from faecal samples and to identify genes potentially contributing to virulence, biofilm production and antimicrobial resistance in children admitted to a pediatric hospital in Porto Velho, Rondônia State. METHODS: The total of 1,625 E. coli specimens were isolated from 591 children in the age group 6 years or younger who were hospitalized in Cosme and Damião Children Hospital in Porto Velho, between February 2010 and February 2012, with acute gastroenteritis. Colonies suggestive of E. coli were subjected to polymerase chain reaction testing in order to identify the virulence factors. The in vitro adhesion assays using HEp-2 adherence were tests. Biofilm detection through spectrophotometry and antimicrobial susceptibility tests were conducted in the disk diffusion method. RESULTS: The mentioned study examined 591 stool samples from children with diarrhea. Diarrheogenic E. coli was found in 27.4% (162/591) of the children. EAEC was the diarreagenic E. coli most frequently associated with diarrhea 52.4% (85/162), which was followed by enteropathogenic E. coli 43.8% (71/162), enterotoxigenic E. coli 2.4% (4/162), and enterohemorrhagic E. coli 1.2% (2/162). The aggR gene was detected in 63.5% (54/85) of EAEC isolates; moreover, statistically significant correlation was observed among typical EAEC (aggR) and aatA (P<0.0001), irp2 (P=0.0357) and shf (P=0.0328). It was recorded that 69% (59/85) of the 85 analyzed EAEC strains were biofilm producers; 73% (43/59) of the biofilm producers carried the aggR gene versus 42.3% (11/26) of non-producers (P=0.0135). In addition, there was association between the aatA gene and biofilm production; 61% (36/59) of the samples presented producer strains, versus 19.2% (5/26) of non-producers (P<0.0004). Antibiotic sensitivity test evidenced that most EAEC were ampicillin 70.6% (60/85), sulfamethoxazole 60% (51/85), tetracycline 44.7% (38/85) and cefotaxime 22.4% (19/85) resistant. CONCLUSION: As far as it is known, the present study is pioneer in Northern Brazil to investigate EAEC virulence factors and to show the antimicrobial susceptibility of EAEC strains isolated from children with diarrhea.


RESUMO CONTEXTO: A Escherichia coli enteroagregativa (EAEC) é um dos principais agentes causadores de diarreia aguda e crônica em crianças e adultos, principalmente em países em desenvolvimento. OBJETIVO: Caracterizar cepas de EAEC isoladas de amostras fecais e identificar genes que potencialmente contribuem para a virulência, produção de biofilme e resistência antimicrobiana em crianças internadas em um hospital pediátrico em Porto Velho, Rondônia. MÉTODOS: Um total de 1.625 cepas de E. coli foram isolados de 591 crianças com gastroenterite aguda na faixa etária de 6 anos que foram internadas no Hospital Infantil Cosme e Damião na cidade de Porto Velho, entre fevereiro de 2010 e fevereiro de 2012. Colônias sugestivas de E. coli foram submetidas a reação em cadeia da polimerase para identificação de fatores de virulência. O ensaio de adesão in vitro foi desenvolvido com célula HEp-2. A detecção de biofilme foi realizada através do teste de espectrofotometria e os testes de susceptibilidade aos antimicrobiana foram realizados através do método de difusão em disco. RESULTADOS: A E. coli diarreiogênica foi encontrada em 27,4% (162/591) das crianças e a EAEC foi a E. coli diarreiogênica mais frequentemente associada à diarreia com 52,4% (85/162), seguida pela E. coli enteropatogênica 43,8% (71/162), E. coli enterotoxigênica 2,4% (4/162) e E. coli enterohemorrágica 1,2% (2/162). O gene aggR foi detectado em 63,5% (54/85) dos isolados de EAEC com correlação estatisticamente significante entre esse gene com os genes aatA (P<0,0001), irp2 (P=0,0357) e shf (P=0,0328). Neste estudo 69% (59/85) das cepas de EAEC eram produtoras de biofilme, destas 73% (43/59) possuíam o gene aggR, ao passo que entre as não produtoras 42,3% (11/26) possuíam o gene (P=0,0135). Essa associação também foi observada com o gene aatA, presente em 61% (36/59) das cepas produtoras e em 19,2% (5/26) das não produtoras (P<0,0004). O teste de sensibilidade aos antibimicrobianos evidenciou que a maioria das EAEC eram resistentes a ampicilina 70,6% (60/85), ao sulfametoxazol 60% (51/85), a tetraciclina 44,7% (38/85) e a cefotaxima 22,4% (19/85). CONCLUSÃO: Este é o primeiro estudo no Norte do Brasil sobre a investigação dos fatores de virulência de EAEC mostrando a susceptibilidade antimicrobiana de cepas de EAEC isoladas de crianças com diarreia.


Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Biofilms/growth & development , Diarrhea/microbiology , Escherichia coli/isolation & purification , Escherichia coli/physiology , Escherichia coli Infections/microbiology , Virulence/genetics , Brazil/epidemiology , Microbial Sensitivity Tests , Polymerase Chain Reaction , Prevalence , Diarrhea/epidemiology , Escherichia coli/drug effects , Escherichia coli Infections/epidemiology , Feces/microbiology , Genes, Bacterial/genetics
2.
Braz. j. biol ; 78(3): 421-428, Aug. 2018. tab, graf
Article in English | LILACS | ID: biblio-951563

ABSTRACT

Abstract Wolbachia (Hertig) endosymbionts are extensively studied in a wide range of organisms and are known to be transmitted through the egg cytoplasm to the offsping. Wolbachia may cause several types of reproductive modifications in arthropods. In Trichogramma species, parthenogenesis-inducing Wolbachia bacteria allow females wasps to produce daughters from unfertilized eggs and these bacteria are present in at least 9% of all Trichogramma species. Phylogenetic studies have led to the subdivision of the Wolbachia clade in five supergroups (A, B, C, D and E) and Wolbachia from Trichogramma belong to supergroup B. Here, using the wsp gene, four groups of Wolbachia that infect Trichogramma species were distinguished and the addition of a new group "Ato" was suggested due to the addition of Wolbachia from Trichogramma atopovirilia (Oatman and Platner). Specific primers were designed and tested for the "Ato" group. Seventy-five percent of all evaluated Wolbachia strains from Trichogramma fell within "Sib" group.


Resumo Endosimbiontes do gênero Wolbachia (Hertig) são extensivamente estudados em uma ampla gama de organismos e são conhecidos por serem transmitidos via citoplasma do ovo hospedeiro para seu descendente. Wolbachia pode causar vários tipos de alterações reprodutivas nos artrópodes. Nas espécies de Trichogramma, a reprodução partenogenética induzida por Wolbachia, possibilita as fêmeas dos parasitoides a produção de fêmeas a partir de ovos não fertilizados e estas bactérias estão presentes em pelo menos 9% de todas as espécies de Trichogramma. Estudos filogenéticos têm levado a subdivisão do clado Wolbachia em cinco supergrupos (A, B, C, D and E). Wolbachia em Trichogramma pertence ao supergrupo B. Com o gene wsp foi possível se distinguir quatro grupos de Wolbachia que infectam Trichogramma e adicionar um novo grupo (Ato) devido a inclusão de Wolbachia detectada em Trichogramma atopovirilia (Oatman and Platner, 1983). Primers específicos foram construídos e testados para o grupo "Ato". Setenta e cinco por cento de todas as linhagens de Wolbachia que infectam Trichogramma se enquadraram dentro do grupo "Sib".


Subject(s)
Animals , Female , Bacterial Outer Membrane Proteins/metabolism , Wasps/microbiology , DNA Primers/genetics , Alphaproteobacteria/metabolism , Wolbachia/genetics , Genes, Bacterial/genetics , Phylogeny , Reproduction , Species Specificity , Symbiosis , Wasps/genetics
3.
Rev. Soc. Bras. Med. Trop ; 51(5): 603-609, Sept.-Oct. 2018. tab, graf
Article in English | LILACS | ID: biblio-957466

ABSTRACT

Abstract INTRODUCTION: The behavior of methicillin-resistant Staphylococcus aureus (MRSA) isolated from central venous catheter-related infection was evaluated to determine its biofilm potential, antimicrobial resistance, and adhesion genes. METHODS: A total of 1,156 central venous catheters (CVC) were evaluated to screen for pathogens. Antimicrobial sensitivity, biofilm formation potential, and molecular analysis of MRSA were examined following standard guidelines. RESULTS: Of the 1,156 samples, 882 (76%) were colonized by bacteria or candida. Among the infected patients, 69% were male and 36% were female with median age of 32 years. Staphylococcus aureus infected 39% (344/882) of CVCs in patients. Of the 59% (208/344) of patients with MRSA, 57% had community acquired MRSA and 43% had hospital acquired MRSA. Linezolid and vancomycin killed 100% of MRSA; resistance levels to fusidic acid, doxycycline, clindamycin, azithromycin, amikacin, trimethoprim-sulfamethoxazole, gentamycin, tobramycin, and ofloxacin were 21%, 42%, 66%, 68%, 72%, 85%, 95%, 97%, and 98% respectively. Strong biofilm was produced by 23% of samples, moderate by 27%, and weak by 50% of MRSA. The presence of adhesion genes, sdrC and sdrD (90%), eno (87%), fnbA (80%), clfA and sdrE (67%), fnbB, sdrD (61%), and cna (51%), in most MRSA samples suggested that the adhesion genes are associated with biofilm synthesis. CONCLUSIONS: The superbug MRSA is a major cause of CVC-related infection. Antibiotic resistance to major classes of antibiotics and biofilm formation potential enhanced superbug MRSA virulence, leading to complicated infection. MRSA causes infection in hospitals, communities, and livestock.


Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Young Adult , Staphylococcal Infections/microbiology , Cross Infection/microbiology , Community-Acquired Infections/microbiology , Biofilms/growth & development , Methicillin-Resistant Staphylococcus aureus/physiology , Catheter-Related Infections/microbiology , Anti-Bacterial Agents/pharmacology , Bacterial Adhesion/genetics , Microbial Sensitivity Tests , Biofilms/drug effects , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Methicillin-Resistant Staphylococcus aureus/drug effects , Genes, Bacterial/genetics , Middle Aged
4.
Mem. Inst. Oswaldo Cruz ; 113(10): e180192, 2018. tab, graf
Article in English | LILACS | ID: biblio-1040581

ABSTRACT

Raoultella planticola is an emerging zoonotic pathogen that is associated with rare but life-threatening cases of bacteremia, biliary tract infections, and urinary tract infections. Moreover, increasing antimicrobial resistance in the organism poses a potential threat to public health. In spite of its importance as a human pathogen, the genome of R. planticola remains largely unexplored and little is known about its virulence factors. Although lipopolysaccharides has been detected in R. planticola and implicated in the virulence in earlier studies, the genetic background is unknown. Here, we report the complete genome and comparative analysis of the multidrug-resistant clinical isolate R. planticola GODA. The complete genome sequence of R. planticola GODA was sequenced using single-molecule real-time DNA sequencing. Comparative genomic analysis reveals distinct capsular polysaccharide synthesis gene clusters in R. planticola GODA. In addition, we found bla TEM-57 and multiple transporters related to multidrug resistance. The availability of genomic data in open databases of this emerging zoonotic pathogen, in tandem with our comparative study, provides better understanding of R. planticola and the basis for future work.


Subject(s)
Polysaccharides, Bacterial/biosynthesis , Genome, Bacterial/genetics , Enterobacteriaceae/genetics , Genes, Bacterial/genetics , Polysaccharides, Bacterial/genetics , Bacterial Capsules/genetics , Enterobacteriaceae/classification
5.
Mem. Inst. Oswaldo Cruz ; 112(7): 499-503, July 2017. graf
Article in English | LILACS | ID: biblio-1040573

ABSTRACT

ABSTRACT Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.


Subject(s)
Humans , Operon/genetics , Arginine/genetics , Staphylococcus aureus/genetics , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Arginine/metabolism , Staphylococcus aureus/metabolism , Gene Expression Regulation, Bacterial/genetics , Interspersed Repetitive Sequences/genetics , Genes, Bacterial/genetics
6.
Braz. j. infect. dis ; 21(3): 282-289, May-June 2017. tab, graf
Article in English | LILACS | ID: biblio-839231

ABSTRACT

ABSTRACT The herein presented assay provided a bacteriological and molecular characterization of 100 samples of L. monocytogenes isolated from human (43) and food (57) sources, from several regions of Brazil, and collected between 1975 and 2013. Antigenic characterization defined 49% of serotype 4b samples, followed by 28% of serotype 1/2b, 14% of serotype 1/2c, 8% of serotype 1/2a, and 1% of serotype 3b. Both type of samples from human and food origin express the same serotype distribution. Multiplex PCR analysis showed 13 strains of type 4b with the amplification profile 4b-VI (Variant I). Virulence genes hly, inlA, inlB, inlC, inlJ, actA, plcA, and prfA were detected in all samples, highlighting a deletion of 105pb on the actA gene in 23% of serotype 4b samples. Macrorestriction profile with ApaI at PFGE showed 55 pulsotypes, with the occurrence of the same pulsotype in hospitalized patients in São Paulo in 1992 and 1997, and two other highly related pulsotypes in patients hospitalized in Rio de Janeiro in 2008. Recognized pulsotypes in listeriosis cases have also been detected in food. Thus, the prevalence of a serotype and the persistence of certain pulsotypes herald future problems.


Subject(s)
Humans , Virulence Factors/genetics , Food Microbiology , Listeria monocytogenes/genetics , Brazil , Serotyping , Electrophoresis, Gel, Pulsed-Field , Molecular Typing , Multiplex Polymerase Chain Reaction , Genes, Bacterial/genetics , Listeria monocytogenes/isolation & purification , Listeria monocytogenes/pathogenicity
7.
Mem. Inst. Oswaldo Cruz ; 111(9): 597-598, Sept. 2016.
Article in English | LILACS | ID: lil-794727

ABSTRACT

Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and has been commonly associated with high morbimortality. In Brazil, this resistance is mainly attributed to the spread of OXA-23-producing clones and, to a lesser extent, to OXA-143-producing clones. Here, we describe, for the first time, two OXA-72-producing A. baumannii isolates in southern Brazil to a broad spectrum of antibiotics, except polymyxin B and tigecycline. Molecular typing by multilocus sequence typing (MLST) demonstrated that both OXA-72-producing isolates belong to a new sequence type (ST), ST730, which was recently identified in OXA-23-producing A. baumannii isolates in São Paulo, Brazil. We demonstrate that the two A. baumannii ST730 isolates carrying blaOXA-72share a common ancestral origin with the blaOXA-23producers in Brazil. This observation reinforces the importance of strain-typing methods in order to clarify the dynamics of the emergence of new clones in a geographic region.


Subject(s)
Humans , Male , Aged , Acinetobacter baumannii/genetics , beta-Lactamases/genetics , Genes, Bacterial/genetics , Acinetobacter baumannii/enzymology , Bacterial Proteins/genetics , Brazil , Drug Resistance, Bacterial/genetics , Multilocus Sequence Typing
8.
Rev. chil. infectol ; 33(4): 410-418, ago. 2016. ilus, tab
Article in Spanish | LILACS | ID: biblio-830111

ABSTRACT

Introduction: Bacterial resistance is a global concern for public health. Reports of antimicrobial resistance, including that against methicillin, have increased in strains of coagulase positive Staphylococcus (CPS) isolated from pets, however in Chile this information is limited. Objectives: To determine the antimicrobial susceptibility profiles and to detect the mecA gene in CPS strains isolated from cats in Chile. Materials and Methods : 134 samples were obtained from healthy cats and cats with skin lesions. These strains were characterized in their coagulase production and identified by BBL Crystal kit. The antimicrobial susceptibility was determined by Kirby Bauer method against 12 antimicrobials, including oxacillin. All strains were subjected to PCR to detect the mecA gene. Results: 72 CPS strains were isolated, including S. aureus and S. intermedius. Antimicrobial resistance against at least one drug was detected in strains from both healthy cats (75%) and from cats with skin lesions (87.5%). The mecA gene was detected in eight methicillin-resistant strains and also in three sensitive strains, being in general multi-resistant. Discussion: These results highlight the role of pets as reservoirs of bacterial resistance, and their potential impact on national public health.


Introducción: La resistencia bacteriana constituye un tema de preocupación para la salud pública mundial. Últimamente han aumentado los reportes de resistencia a antimicrobianos, incluida meticilina, en cepas de Staphylococcus coagulasa positiva (SCP) aisladas desde mascotas. Sin embargo, en Chile esta información es escasa. Objetivos: Determinar el perfil de susceptibilidad antimicrobiana y detectar el gen mecA en cepas de SCP aisladas desde gatos en Chile. Materiales y Métodos: Se obtuvieron 134 muestras desde gatos sanos y con lesiones dermatológicas. Las cepas fueron caracterizadas en su producción de coagulasa e identificadas mediante kit BBL Crystal. La susceptibilidad antimicrobiana se determinó mediante el método de Kirby Bauer ante 12 antimicrobianos, incluida oxacilina. Todas las cepas fueron sometidas a RPC para la detección del gen mecA. Resultados: 72 cepas de SCP fueron aisladas, incluyendo S. aureus y S. intermedius. Se detectó resistencia antimicrobiana a al menos un antimicrobiano en cepas de gatos sanos (75%) y de gatos con lesiones cutáneas (87,5%). El gen mecA fue detectado en ocho cepas resistentes a meticilina y en tres cepas sensibles, siendo en general multi-resistentes. Discusión: Estos resultados destacan el rol de las mascotas como reservorios de resistencia bacteriana y su potencial impacto en la salud pública.


Subject(s)
Animals , Bacterial Proteins/genetics , Cats/microbiology , Polymerase Chain Reaction/veterinary , Penicillin-Binding Proteins/genetics , Methicillin-Resistant Staphylococcus aureus/genetics , Microbial Sensitivity Tests , Chile , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Genes, Bacterial/drug effects , Genes, Bacterial/genetics , Anti-Bacterial Agents/pharmacology
9.
Braz. j. infect. dis ; 20(3): 276-281, May.-June 2016. tab, graf
Article in English | LILACS | ID: lil-789481

ABSTRACT

Abstract Introduction There is a mechanism of macrolide resistance in Staphylococcus spp. which also affects the lincosamides and type B streptogramins characterizing the so-called MLSB resistance, whose expression can be constitutive (cMLSB) or inducible (iMLSB) and is encoded mainly by ermA and ermC genes. The cMLSB resistance is easily detected by susceptibility testing used in the laboratory routine, but iMLSB resistance is not. Therapy with clindamycin in cases of infection with isolated iMLSB resistance may fail. Objective To characterize the phenotypic (occurrence of cMLSB and iMLSB phenotypes) and molecular (occurrence of ermA and ermC genes) profiles of MLSB resistance of clinical isolates of susceptible and methicillin-resistant Staphylococcus aureus and CNS (coagulase-negative Staphylococcus) from patients of a university hospital, in Pernambuco. Methods The antimicrobial susceptibility of 103 isolates was determined by the disk diffusion technique in Mueller–Hinton agar followed by oxacillin screening. The iMLSB phenotype was detected by D test. Isolates with cMLSB and iMLSB phenotypes were subjected to polymerase chain reaction (PCR) for the detection of ermA and ermC genes. Results The cMLSB and iMLSB phenotypes were respectively identified in 39 (37.9%) and five (4.9%) isolates. The iMLSB phenotype was found only in four (10.8%) methicillin-susceptible S. aureus and one (4.5%) methicillin-resistant S. aureus. In the 44 isolates subjected to PCR, four (9.1%) only ermA gene was detected, a lower frequency when compared to only ermC 17 (38.6%) gene and to one (2.3%) isolate presenting both genes. Conclusion In the Staphylococcus spp. analyzed, the ermC gene was found more often than the ermA, although the iMLSB phenotype had been less frequent than the cMLSB. It was important to perform the D test for its detection to guide therapeutic approaches.


Subject(s)
Humans , Staphylococcus/drug effects , Staphylococcus/genetics , Macrolides/pharmacology , Streptogramin B/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Lincosamides/pharmacology , Phenotype , Brazil , Drug Resistance, Multiple, Bacterial/drug effects , Disk Diffusion Antimicrobial Tests , Genes, Bacterial/genetics , Hospitals, University
11.
Braz. j. microbiol ; 46(3): 885-892, July-Sept. 2015. tab, ilus
Article in English | LILACS | ID: lil-755834

ABSTRACT

This study was conducted to determine the species distribution, antimicrobial resistance pheno- and genotypes and virulence traits of mannitol-positive methicillin-resistant staphylococci (MRS) isolated from pigs in Nsukka agricultural zone, Nigeria. Twenty mannitol-positive methicillin-resistant coagulase-negative staphylococcal (MRCoNS) strains harboring the mecA gene were detected among the 64 Staphylococcus isolates from 291 pigs. A total of 4 species were identified among the MRCoNS isolates, namely, Staphylococcus sciuri (10 strains), Staphylococcus lentus (6 strains), Staphylococcus cohnii (3 strains) and Staphylococcus haemolyticus (one strain). All MRCoNS isolates were multidrug-resistant. In addition to β-lactams, the strains were resistant to fusidic acid (85%), tetracycline (75%), streptomycin (65%), ciprofloxacin (65%), and trimethoprim/sulphamethoxazole (60%). In addition to the mecA and blaZ genes, other antimicrobial resistance genes detected were tet(K), tet(M), tet(L), erm(B), erm(C), aacA-aphD, aphA3, str, dfrK, dfrG, catpC221, and catpC223. Thirteen isolates were found to be ciprofloxacin-resistant, and all harbored a Ser84Leu mutation within the QRDR of the GyrA protein, with 3 isolates showing 2 extra substitutions, Ser98Ile and Arg100Lys (one strain) and Glu88Asp and Asp96Thr (2 strains). A phylogenetic tree of the QRDR nucleotide sequences in the gyrA gene revealed a high nucleotide diversity, with several major clusters not associated with the bacterial species. Our study highlights the possibility of transfer of mecA ...


Subject(s)
Animals , Drug Resistance, Multiple, Bacterial/genetics , Fermentation/physiology , Mannitol/metabolism , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Methicillin-Resistant Staphylococcus aureus/metabolism , Staphylococcus haemolyticus/isolation & purification , Staphylococcus haemolyticus/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , DNA Gyrase/genetics , DNA, Bacterial/genetics , Genes, Bacterial/genetics , Microbial Sensitivity Tests , Methicillin-Resistant Staphylococcus aureus/genetics , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Nigeria , Penicillin-Binding Proteins/genetics , Staphylococcal Infections/microbiology , Staphylococcus haemolyticus/genetics , Staphylococcus haemolyticus/pathogenicity , Swine/microbiology
12.
Braz. j. infect. dis ; 19(3): 278-284, May-Jun/2015. tab, graf
Article in English | LILACS | ID: lil-751888

ABSTRACT

Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS) Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins 1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.


Subject(s)
Adult , Aged , Child , Female , Humans , Male , Middle Aged , /chemistry , Shiga Toxin 1/isolation & purification , /isolation & purification , Shigella dysenteriae/chemistry , DNA, Bacterial/genetics , Enzyme-Linked Immunosorbent Assay , /genetics , Feces/microbiology , Genes, Bacterial/genetics , Polymerase Chain Reaction , Sensitivity and Specificity , Shiga Toxin 1/genetics , /genetics , Shigella dysenteriae/genetics
13.
Braz. j. microbiol ; 45(4): 1131-1138, Oct.-Dec. 2014.
Article in English | LILACS | ID: lil-741262

ABSTRACT

Shigellosis produces inflammatory reactions and ulceration on the intestinal epithelium followed by bloody or mucoid diarrhea. It is caused by enteroinvasive E. coli (EIEC) as well as any species of the genus Shigella, namely, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. This current species designation of Shigella does not specify genetic similarity. Shigella spp. could be easily differentiated from E. coli, but difficulties observed for the EIEC-Shigella differentiation as both show similar biochemical traits and can cause dysentery using the same mode of invasion. Sequencing of multiple housekeeping genes indicates that Shigella has derived on several different occasions via acquisition of the transferable forms of ancestral virulence plasmids within commensal E. coli and form a Shigella-EIEC pathovar. EIEC showed lower expression of virulence genes compared to Shigella, hence EIEC produce less severe disease than Shigella spp. Conventional microbiological techniques often lead to confusing results concerning the discrimination between EIEC and Shigella spp. The lactose permease gene (lacY) is present in all E. coli strains but absent in Shigella spp., whereas β-glucuronidase gene (uidA) is present in both E. coli and Shigella spp. Thus uidA gene and lacY gene based duplex real-time PCR assay could be used for easy identification and differentiation of Shigella spp. from E. coli and in particular EIEC.


Subject(s)
Dysentery, Bacillary/microbiology , Escherichia coli/genetics , Escherichia coli/pathogenicity , Shigella/genetics , Shigella/pathogenicity , Virulence Factors/genetics , Bacteriological Techniques , Diagnosis, Differential , Dysentery, Bacillary/diagnosis , Dysentery, Bacillary/pathology , Escherichia coli/classification , Genotype , Genes, Bacterial/genetics , Molecular Diagnostic Techniques , Multiplex Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction , Shigella/classification
14.
Mem. Inst. Oswaldo Cruz ; 109(6): 712-715, 09/09/2014. graf
Article in English | LILACS | ID: lil-723999

ABSTRACT

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.


Subject(s)
Humans , Bacterial Proteins/genetics , Enterococcus faecium/genetics , Genes, Bacterial/genetics , Vancomycin-Resistant Enterococci/genetics , Anti-Bacterial Agents/pharmacology , Blotting, Southern , Bacterial Proteins/blood , Electrophoresis, Gel, Pulsed-Field , Enterococcus faecalis/genetics , Enterococcus faecium/drug effects , Enterococcus/drug effects , Enterococcus/genetics , In Situ Hybridization/methods , Microbial Sensitivity Tests , Multilocus Sequence Typing , Multigene Family/physiology , Polymerase Chain Reaction , Teicoplanin/pharmacology , Vancomycin Resistance/genetics , Vancomycin/pharmacology
15.
Mem. Inst. Oswaldo Cruz ; 109(3): 265-278, 06/2014. tab, graf
Article in English | LILACS | ID: lil-711727

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important bacterial pathogens based on its incidence and the severity of its associated infections. In addition, severe MRSA infections can occur in hospitalised patients or healthy individuals from the community. Studies have shown the infiltration of MRSA isolates of community origin into hospitals and variants of hospital-associated MRSA have caused infections in the community. These rapid epidemiological changes represent a challenge for the molecular characterisation of such bacteria as a hospital or community-acquired pathogen. To efficiently control the spread of MRSA, it is important to promptly detect the mecA gene, which is the determinant of methicillin resistance, using a polymerase chain reaction-based test or other rapidly and accurate methods that detect the mecA product penicillin-binding protein (PBP)2a or PBP2’. The recent emergence of MRSA isolates that harbour a mecA allotype, i.e., the mecC gene, infecting animals and humans has raised an additional and significant issue regarding MRSA laboratory detection. Antimicrobial drugs for MRSA therapy are becoming depleted and vancomycin is still the main choice in many cases. In this review, we present an overview of MRSA infections in community and healthcare settings with focus on recent changes in the global epidemiology, with special reference to the MRSA picture in Brazil.


Subject(s)
Animals , Humans , Bacterial Proteins/genetics , Methicillin-Resistant Staphylococcus aureus/genetics , Penicillin-Binding Proteins/genetics , Staphylococcal Infections/microbiology , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/classification , DNA, Bacterial/genetics , Genes, Bacterial/genetics , Microbial Sensitivity Tests , Methicillin-Resistant Staphylococcus aureus/drug effects , Polymerase Chain Reaction , Penicillin-Binding Proteins/classification
16.
Article in English | WPRIM | ID: wpr-194861

ABSTRACT

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.


Subject(s)
Animals , Anti-Bacterial Agents/pharmacology , Cattle/microbiology , Drug Resistance, Multiple, Bacterial , Electrophoresis, Gel, Pulsed-Field/veterinary , Escherichia coli Infections/epidemiology , Female , Genes, Bacterial/genetics , Latex Fixation Tests/veterinary , Microbial Sensitivity Tests/veterinary , Multilocus Sequence Typing/veterinary , Prevalence , Republic of Korea/epidemiology , Shiga Toxin 1/genetics , Shiga Toxin 2/genetics , Shiga-Toxigenic Escherichia coli/drug effects
17.
Mem. Inst. Oswaldo Cruz ; 108(6): 718-723, set. 2013. tab
Article in English | LILACS | ID: lil-685491

ABSTRACT

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.


Subject(s)
Adult , Female , Humans , Male , Middle Aged , Bacterial Proteins/genetics , Genes, Bacterial/genetics , Genetic Loci/genetics , Mycobacterium tuberculosis/genetics , Polyketide Synthases/genetics , Base Sequence , Drug Resistance, Multiple, Bacterial/genetics , Epidemiological Monitoring , Genetic Markers/genetics , Mexico , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/isolation & purification , Polymerase Chain Reaction , Sequence Deletion , Sputum/microbiology
18.
Rev. chil. infectol ; 30(4): 381-387, ago. 2013. ilus, graf, tab
Article in Spanish | LILACS | ID: lil-690526

ABSTRACT

Background: Escherichia coliis able to produce different infections in humans. It pathogenicity in the female genital tract is unknown. Objective:To determine the presence of virulence genes (VG) in E. colistrains isolated from the female genital tract. Material and Methods:146 E. colistrains isolated as monomicrobial cultures from vaginal infections were genetically characterized by search of hly, iucC, afa, fimH, neuC, sfa/foc, cnF1, papC, usp,and ibeAVG. Studies were performed by means PFGE and PCR. Results:Genetic analysis of the strains showed two groups with a similarity of approximately 80%. The similarity genetic intragroup was approximately 95%. The results showed strains with a high number of VG and the most common were cnf1andfimH.The afagene was not detected. Were identified eight VG combinations and the most common was papC+ hly+ iucC+ afa- neuC- fimH+ sfa/foc+ cnf1+ usp+ ibeA-. Discussion:The studied strains are concentrated in two genetic groups. Most of the strains contain a great number of VG present in E. coliisolated from extraintestinal infections. Conclusion:It is important to develop new research strategies in this area, to deepen the phylogenetic knowledge of these strains and confirm their true role in vaginal infection.


Introducción: Escherichia colies capaz de producir diferentes cuadros infecciosos en el ser humano. Su patogenicidad en el tracto genital femenino es discutible. Objetivo:Determinar la presencia de genes de virulencia (GV) en cepas de E. colide procedencia vaginal. Material y Métodos:146 cepas de E. coliaisladas desde infecciones vaginales a partir de cultivos monomicrobianos fueron estudiadas mediante EGCP y RPC. Los genes investigados fueron: papC, hly, iucC, afa, fimH, neuC, sfa/foc, cnf1, usp,e ibeA. Resultados:El análisis genético de las cepas demostró dos grupos con una similitud aproximada a 80% según Dice. La similitud genética intra-grupo fue aproximadamente de 95%. Los resultados mostraron cepas con un alto número de GV, siendo más comunes cnf1 y fimH.El gen afano fue detectado. Se determinaron ocho combinaciones de GV siendo la más común papC+ hly+ iucC+ afa- neuC- fimH+ sfa/foc+ cnf1+ usp+ibeA-. Discusión:las cepas estudiadas se concentran en dos grupos genéticos característicos y la mayoría de las cepas analizadas concentra un importante número de GV presentes en E. coliaisladas de infecciones extra-intestinales. Conclusión:Es importante desarrollar nuevas estrategias de investigación en esta área, que permitan profundizar el conocimiento filogenético de estas cepas y confirmar su verdadero rol en la infección vaginal.


Subject(s)
Female , Humans , Escherichia coli Infections/microbiology , Escherichia coli/genetics , Escherichia coli/pathogenicity , Vaginosis, Bacterial/microbiology , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Escherichia coli/isolation & purification , Genes, Bacterial/genetics , Phylogeny , Polymerase Chain Reaction , Virulence/genetics
19.
Mem. Inst. Oswaldo Cruz ; 108(4): 453-456, jun. 2013. tab
Article in English | LILACS | ID: lil-678299

ABSTRACT

Here we report the presence and expression levels of the vanC 1 and vanC 2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC 1 and vanC 2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC 1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.


Subject(s)
Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Enterococcus faecalis/drug effects , Enterococcus faecalis/genetics , Vancomycin Resistance/genetics , Vancomycin/pharmacology , Chickens , Cloaca/microbiology , Disk Diffusion Antimicrobial Tests , DNA, Bacterial/analysis , Enterococcus faecalis/isolation & purification , Genes, Bacterial/genetics , Real-Time Polymerase Chain Reaction
20.
Indian J Exp Biol ; 2013 Jun; 51(6): 470-476
Article in English | IMSEAR | ID: sea-147616

ABSTRACT

Guggal is tapped for extraction of medicinally important oleo–gum–resin (guggul) by inoculating the stem bark with natural gum suspension containing pathogenic bacterium Xanthomonas axonopodis pv. commiphorae (Xac). The tree dies in the process. In absence of any specific medium for isolation of Xac, it is difficult to assess spread of the pathogen within the plant. A PCR based molecular detection technique using fyuA and rpoD gene specific primers is described here. The primers amplified products only from Xac and not from host tissues or common saprophytes. The method was sensitive enough to produce positive signals for up to 4.4 bacterial cells or 2 pg target DNA per reaction mixture. However, PCR inhibitors present in plant tissues drastically reduced the limit of detection. A simple overnight incubation of surface sterilised plant tissues in nutrient medium was introduced to increase pathogen titre and to overcome this problem. This technique was successfully used to measure spread of Xac in plant tissues away from the site of inoculation. The pathogen showed preference for acropetal movement and did not spread to 7–8 cm below the site of inoculation till 15 days after inoculation. This suggests possibility to manage the disease through plant surgery.


Subject(s)
DNA Primers/chemistry , DNA, Bacterial/genetics , Genes, Bacterial/genetics , Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/microbiology , Limit of Detection , Plant Diseases/genetics , Plant Diseases/microbiology , Polymerase Chain Reaction , Resins, Plant/chemistry , Resins, Plant/metabolism , Triterpenes/chemistry , Triterpenes/metabolism , Xanthomonas axonopodis/genetics , Xanthomonas axonopodis/pathogenicity
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