RESUMO
BACKGROUND/PURPOSE: The aim of this study was to characterize the Staphylococcus aureus strains isolated from periodontal lesions of patients, to determine the expression of genes involved in cell adhesion upon their infection of human epithelial cells using an in vitro model, its biofilm formation, and its resistance to antibiotics. METHODS: S. aureus was analysed by PCR, Kirby-Bauer, and pulsed-field gel electrophoresis (PFGE), measuring gene expression by real-time PCR after infection of human cells in vitro. RESULTS: S. aureus was identified in 18.6% (50/268) of the samples. All strains (n = 50) possessed the virulence genes spa (Staphylococcal protein A), coa (coagulase), and icaAB (intercellular adhesin); 96% (n = 48) possessed clfB (clumping factor B), and 88% (n = 44) possessed ebps (elastin-binding protein) and sdrD (serine aspartate repeat protein D). All strains were resistant to methicillin, ampicillin, dicloxacillin, cefotaxime, and penicillin, and were multidrug resistant to 6-12 antibiotics. PFGE analysis showed 37 different pulsed-field types and most strains (60.4%) had a unique pulsed-field type. Twenty-four distinct combinations of virulence genes and antibiotic-resistant phenotypes were identified. CONCLUSION: Although S. aureus has been considered a transient member of the oral microbiota, our results indicate a high-level expression of virulence genes and multidrug resistance in the strains isolated from periodontal lesions. These strains might complicate the successful treatment of the disease.
Assuntos
Adesinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Adesinas Bacterianas/efeitos dos fármacos , Antibacterianos/farmacologia , Antígenos de Bactérias/genética , Biofilmes/efeitos dos fármacos , Linhagem Celular , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana Múltipla/genética , Eletroforese em Gel de Campo Pulsado , Células Epiteliais , Feminino , Regulação Bacteriana da Expressão Gênica , Genótipo , Humanos , Masculino , México , Testes de Sensibilidade Microbiana , Microbiota , Boca/microbiologia , Fenótipo , Reação em Cadeia da Polimerase , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Virulência/genéticaRESUMO
Multi-drug resistant cervicovaginal Escherichia coli (CVEC) infections are a serious health problem. The aim of this study is to determine the patterns of virulence genes, antibiotic resistance and O-serogroups of CVEC isolated in Mexico. Two hundred strains of CVEC were isolated from women attending two Clinics at the Instituto Mexicano del Seguro Social. E. coli O-serogroups and virulence markers were identified by PCR. Antibiotic susceptibility was determined using the Kirby-Bauer disc-diffusion method. Serogroups O25 (50%), O75 (9%) and O15 (7.5%) were the most frequent among the CVEC strains isolated. The frequencies for antibiotic resistance were ampicillin 97%, (n = 194); carbenicillin 93.5%, (n = 187); cefalotin 77%, (n = 154); and nitrofurantoin 71%, (n = 142). The frequency of multiresistant isolates (3-12 drugs) was 197 (98.5%). The most frequent virulence genes found were feoB (91.5%), fimH (89.5%), kpsMT11 (75%), iutA (66%), and iroN (59%). One hundred and four distinct patterns of virulence markers with antibiotic-resistance genes associated with O-serogroups were identified amongst CVEC isolates. In conclusion: most CVEC strains isolated were multiresistant to antibiotics, belonged to three O-serogroups, and possessed a battery of virulence factors. This knowledge may lead to improved guidelines and standards for treating cervicovaginal infections.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli Uropatogênica/genética , Vaginose Bacteriana/microbiologia , Adolescente , Adulto , Idoso , Antibacterianos/uso terapêutico , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Infecções por Escherichia coli/tratamento farmacológico , Proteínas de Escherichia coli/genética , Feminino , Genótipo , Humanos , México , Pessoa de Meia-Idade , Sorogrupo , Escherichia coli Uropatogênica/isolamento & purificação , Escherichia coli Uropatogênica/patogenicidade , Vagina/microbiologia , Vaginose Bacteriana/tratamento farmacológico , Fatores de Virulência/genética , Adulto JovemRESUMO
Virulence and antibiotic resistance properties related to different Escherichia coli phylogenetic groups have not been studied in detail in Mexico. We aimed to identify patterns of virulence genes and multidrug resistance in phylogenetic groups of uropathogenic strains (UPEC). Strains of E. coli were isolated from outpatients with urinary tract infections (UTIs), who went to unit of the public health sector in the State of Mexico. E. coli virulence markers and phylogenetic groups were identified by PCR. Susceptibility to 12 antimicrobials was determined by Kirby-Bauer. E. coli was identified in 60.4% (n = 194) of the patients with UTIs. Phylogroups B2 51% (n = 99), A 13.4% (n = 26) and B1 10.3% (n = 20) were the most frequent. Resistance to three or up to eleven antibiotics was detected in most phylogroups (n = 188). The genes fimH (n = 146), feoB (n = 179), iutA (n = 178), sitA (n = 121), fyuA (n = 99), and traT (n = 142) were mainly detected in strains of phylogroups B2, A, B1, C, and D. Seventy-two patterns of virulence markers were distributed across eight E. coli phylogenetic groups. A high frequency of virulence markers and the multiple antibiotic resistance phenotypes was observed in the phylogroups. The genes of extended-spectrum ß-lactamases (ESBLs) found with higher frequency among UPEC strains were blaTEM, blaSHV y blaCTX-M group 1, CIT (plasmid-mediated AmpC ß-lactamase), and blaOXA-like. In conclusion, our findings show the importance of surveillance, permanent monitoring, and particularly controlled prescription of antibiotics by physicians in the social security health system to reduce the spread of highly virulent UPEC strains that are resistant to multiple antimicrobial agents.
Assuntos
Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/patogenicidade , Fatores de Virulência/análise , Adulto , Idoso , Antibacterianos/farmacologia , Infecções por Escherichia coli/epidemiologia , Feminino , Variação Genética , Genótipo , Humanos , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Pacientes Ambulatoriais , Filogenia , Reação em Cadeia da Polimerase , Infecções Urinárias/epidemiologia , Escherichia coli Uropatogênica/classificação , Escherichia coli Uropatogênica/isolamento & purificação , Virulência , Fatores de Virulência/genética , Adulto JovemRESUMO
Mannheimia haemolytica causes respiratory disease in cattle. Amyloid proteins are a major component of biofilms; they aid in adhesion and confer resistance against several environmental insults. The amyloid protein curli is highly resistant to protease digestion and physical and chemical denaturation and binds Congo red (CR) dye. The purpose of this study was to characterize an approximately 50-kDa CR-binding amyloid-like protein (ALP) expressed by M. haemolytica. This protein resisted boiling and formic acid digestion and was recognized by a polyclonal anti-Escherichia coli curli serum, suggesting its relationship with amyloid proteins. Immunolabeling and transmission electron microscopy showed that antibodies bound long, thin fibers attached to the bacterial surface. Mass spectrometry analysis indicated that these fibers are M. haemolytica OmpP2-like proteins. The purified protein formed filaments in vitro, and antiserum against it reacted positively with biofilms. An in silico analysis of its amino acid sequence indicated it has auto-aggregation properties and eight amyloid peptides. Rabbit polyclonal antibodies generated against this ALP diminished the adhesion of ATCC 31612 and BA1 M. haemolytica strains to A549 human epithelial cells, indicating its participation in cell adhesion. ALP expressed by M. haemolytica may be important in its pathogenicity and ability to form biofilms.
Assuntos
Proteínas Amiloidogênicas/química , Proteínas da Membrana Bacteriana Externa/química , Biofilmes/crescimento & desenvolvimento , Mannheimia haemolytica/química , Células A549 , Sequência de Aminoácidos , Proteínas Amiloidogênicas/biossíntese , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/isolamento & purificação , Animais , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Bovinos , Vermelho Congo/química , Expressão Gênica , Humanos , Soros Imunes/química , Soros Imunes/isolamento & purificação , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Mannheimia haemolytica/metabolismo , Modelos Moleculares , Peso Molecular , Pasteurelose Pneumônica , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Alinhamento de Sequência , OvinosRESUMO
In this study, we investigated distinct expression patterns of genes encoding iron-acquisition systems, adhesins, protectins, and toxins in human uroepithelial cells infected with 194 uropathogenic Escherichia coli (UPEC) strains in vitro. We assessed the association of these genes with antibiotic resistance genes in this group of UPEC strains, previously characterised by polymerase chain reaction (PCR). Strains were isolated from patients with urinary tract infections (UTIs) from Unidad Médica Familiar de Salud Pública, located in Estado de México, México. Antibiotic resistance genes were identified by PCR, and the expression of virulence genes was detected by reverse-transcriptase-PCR after in vitro infection of cultured A431 human keratinocytes derived from a vulvar epidermoid carcinoma. The most frequently expressed virulence genotypes among the investigated UPEC strains included usp (68%), iha (64.9%), kpsMT (61.3%), fim (58.2%), irp2 (48.4), papC (33.5%), set (31.4%) and astA (30.9%), whereas the most frequently detected antibiotic resistance genes were tet(A) (34%), sul1 (31.4%) and TEM (26.3%). Furthermore, the most abundant pattern of gene expression (irp2/fim/iha/kpsMT/usp), associated with 8 different combinations of antibiotic resistance genotypes, was exhibited by 28 strains (14.4%). Taken together, these results indicate collective participation of distinct virulence UPEC genotypes during in vitro infection of cultured human epithelial cells, suggesting their potential involvement in UTI pathogenesis.
Assuntos
Infecções por Escherichia coli/microbiologia , Regulação Bacteriana da Expressão Gênica , Escherichia coli Uropatogênica/genética , Antibacterianos/farmacologia , Linhagem Celular , Células Cultivadas , Farmacorresistência Bacteriana , Genes Bacterianos , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/patogenicidade , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Antifungal resistance and virulence properties of Candida albicans are a growing health problem worldwide. To study the expression of virulence and azole resistance genes in 39 clinical strains of C. albicans, we used a model of infection of human vaginal epithelial cells with C. albicans strains isolated from Mexican women with vulvovaginal candidiasis (VVC). The strains were identified by PCR amplification of the ITS1 and ITS2 regions of rRNA. The detection and expression of virulence genes and azole resistance genes MDR1 and CDR1 were performed using PCR and RT-PCR, respectively. All strains were sensitive to nystatin and 38 (97.4%) and 37 (94.9%) were resistant to ketoconazole and fluconazole, respectively. ALS1, SAP4-SAP6, LIP1, LIP2, LIP4, LIP6, LIP7, LIP9, LIP10, and PLB1-PLB2 were present in all strains; SAP1 was identified in 37 (94.8%) isolates, HWP1 in 35 (89.7%), ALS3 in 14 (35.8%), and CDR1 in 26 (66.6%). In nearly all of the strains, ALS1, HWP1, SAP4-SAP6, LIP1-LIP10, PLB1, and PLB2 were expressed, whereas CDR1 was expressed in 20 (51.3%) and ALS3 in 14 (35.8%). In our in vitro model of infection with C. albicans, the clinical strains showed different expression profiles of virulence genes in association with the azole resistance gene CDR1. The results indicate that the strains that infect Mexican patients suffering from VVC are highly virulent and virtually all are insensitive to azoles.