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Objective To investigate the mechanism of drug resistance mediated by micF gene and outer membrane porin F ( OmpF) in Shigella strains. Methods Shigella strains were isolated from stool samples of patients who presented to the Second Hospital of Tianjin Medical University with acute diar-rhea in 2015. Antibiotic susceptibility test was performed to screen out the multidrug-resistant and non-multi-drug-resistant strains. The ompF gene was amplified by PCR. The micF and ompF genes at transcriptional levels in the two groups of strains were detected by quantitative real-time RT-PCR. Intracellular concentra-tions of ciprofloxacin in the two groups of Shigella strains were measured by automatic microplate reader. Re-sults According to the result of antibiotic susceptibility test, 13 strains that were resistant to 3 or more than 3 antibiotics were classified into the multidrug-resistant group, while the other 8 strains that were sensitive to all antibiotics used in this study or only resistant to 1 or 2 antibiotic were classified into the non-multidrug-re-sistant group. All of the 21 Shigella strains carried the ompF gene. Compared with the non-multidrug-resist-ant strains, the multidrug-resistant strains showed higher expression of micF gene, but lower expression of ompF gene. The differences in micF and ompF genes between the two groups were statistically significant. The result of correlation analysis suggested that there was a negative correlation between micF and ompF genes (r=-0. 244). The intracellular concentrations of ciprofloxacin in multidrug-resistant strains were low-er than those in the non-multidrug-resistant strains (P<0. 001). Conclusion The decreased expression of OmpF was one of the possible mechanisms of multidrug-resistance in Shigella strains. The micF gene was negatively related to the expression of OmpF. Moreover, the decreased intracellular concentrations of cipro-floxacin in multidrug-resistant strains might be related to the decreased expression of OmpF.
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Após o surgimento e disseminação das ß-lactamases de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenemeertapenem) têm sido considerados a terapia de escolha devido à estabilidade apresentada contra estas enzimas. A desvantagem destes antibióticos é a sua capacidade de induzir resistência aos ß-lactâmicos e a outros antibióticos quimicamente não relacionados. O imipenem tem favorecido a indução de cefalosporinases cromossômicas (AmpC) e também tem sido relacionado, in vivo, com a seleção de mecanismos intrínsecos de resistência, contribuindo com o perfil multi -droga resistente (MDR). Esse perfil é freqüentemente associado à diminuição da permeabilidade por alteração na síntese de porinas em conjunto com um aumento da atividade de bombas de efluxo, as quais não permitem o estabelecimento de uma concentração ativa do antibiótico no interior da célula bacteriana. O presente trabalho teve como objetivo avaliar o estabelecimento do perfil MDR em enterobactérias provenientes de isolados clínicos em função da exposição a diferentes concentrações de imipenemin vitro. A seleção do grupo das amostras estudadas foi feito por meio da determinação do perfil de sensibilidade dos isolados, tipagem molecular e ensaio de hidrólise de Imipenem. Nos isolados selecionados para a indução foi realizada numa etapa inicial (etapa basal) a análise de porinas de membrana externa por SDS-PAGE e o estudo de genes codificadores de ß-lactamases pela técnica de PCR. O estudo do estabelecimento do perfil MDR foi feito por meio de passagens sucessivas das amostras em meio contendo concentrações sub-inibitórias de imipenem seguido de análise fenotípica (CIM e acúmulo do antibiótico intracelular e SDS-PAGE), e a análise da expressão gênica de genes associados a permeabilidade de membrana (ompC, ompF eAcrA) e genes reguladores(marA e ompR). Após a indução com o imipenem, 77% dos isolados induzidos aumentaram a CIM para os carbapenêmicos, mudando assim o perfil de resistência observado na etapa basal Também foi afetado o perfil de resistência para outros antibióticos não relacionados a ß-lactámicos, porém numa percentagem menor. Com relação à alteração da permeabilidade, a perda de porina foi observada apenas para um isolado, no entanto a diminuição na expressão gênica de Omp36 foi significativa desde o começo da indução. A expressão da bomba de efluxoAcrAB foi afetada pela indução com imipenem, aumentando significativamente a expressão de AcrA, enquanto os reguladores estudados, MarA e OmpR tiveram a sua expressão induzida pelo imipenem. Foi possível observar também associação do nível de expressão gênica do regulador MarA com a expressão de AcrA,porém não foi possível observar uma associação estatisticamente significativa deste regulador com o perfil de expressão de OMPs. A indução de OmpR foi associado com um aumento da expressão de RNAm de Omp35, já para Omp36 foi possível observar apenas uma tendência na repressão deste gene. O estudo da resposta destes genes reguladores e determinantes de resistência, em resposta à exposição ao com o imipenem in vitro, permitiu reportar o comportamento molecular da bactéria numa resposta adaptativa no estagio inicial do estabelecimento do fenótipo MDR. A utilização de isolados clínicos com diversos determinantes de resistência permitiu observar a variabilidade nas respostas adaptativas das enterobacterias, o que é fundamental para a compreensão dos mecanismos de adaptação da bactéria e sua contribuição na falha terapêutica
After emergence and broad dissemination of extended spectrum ß-lactamases into the Enterobacteriaceae family, the carbapenemic antibiotics (imipenem, meropenem and ertapenem) have been considered the chosen therapy in the treatment of nosocomial infections by the stability that these antibiotics show to these enzymes. The disadvantage of carbapenems is theirs capacity to induce resistance against ß-lactamics and to other chemically unrelated antibiotics. The imipenem has been shown to induce chromosomal cephalosporinases (AmpC) and it was also related, in vivo, with the selection of intrinsic mechanism leading to multi-drug resistance profile (MDR). This profile is usually associated with membrane impermeability due to reduced outer membrane porin synthesis with an incremented activity of efflux pumps, which results in a reduced concentration of antibiotics inside the bacteria. This study aimed to evaluate the establishment of the MDR profile in Enterobacteriaceae from clinical isolates by exposure to different concentrations of imipenem in vitro. The selection of the study group was performed by determination of antibiotic susceptibility profile,molecular typing and hydrolysis assay of imipenem. In the selected isolates submitted to induction, in an initial step (baseline), was performed the outer membrane porin analysis by SDS-PAGE and the gene-specific amplification of B-lactamase enzymes by PCR. The study of the establishment of MDR was performed by progressive passages with subclinical concentrations of imipenem, followed each one by the evaluation of phenotypic profile (MIC, accumulation antibiotic in celland SDS-PAGE) and gene expression analysisof genes related to membrane permeability (ompC, ompF and acrA) and regulatory genes(MarA and ompR). After induction with imipenem, 77 % of the isolates increased the MIC for the carbapenems, changing the resistance profile at the baseline. In a lesser percentage, the resistance profile to other ß-lactams-unrelated antibiotics was also affected. Loss of porin was observed only for an isolated, however a significantly decreased Omp36 mRNA expression was observed from the start of induction. The expression of the efflux pump AcrAB ,was also affected by the imipenem induction, significantly increasing the AcrA gene expression, whereas the studied regulatory genes,MarA and OmpR,were induced by the imipenem. It was also possible to observe an association between the expression of the regulator MarA and the expression of AcrA, nevertheless no association was observed between this regulator and OMPs . OmpR induction was associated with an increased Omp35mRNA expression, however only a trend for the repression of Omp36was observed. The study of the response of these regulatory genes and genetic determinants of resistance, in response to the imipenem exposure in vitro, allowed to report the molecular behavior of the bacteria in an adaptive response in the initial stage of the establishment of a MDR phenotype. The use of clinical isolates with diverse resistance determinants allowed observing the variability in adaptive responses in enterobacteria, which is important to understand the adaptive mechanisms of bacteria to this antibiotic, the involvement in the emergence of the MDR profile and its contribution to the treatment failure
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Fenotipo , Técnicas In Vitro/instrumentación , Imipenem , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Enterobacteriaceae/efectos de los fármacos , MicrobiologíaRESUMEN
Objective To study the drug resistance mechanism and homologous relationship of four clinical strains of carbapen-em-resistant Klebsiella pneumoniae isolated from different sites of the same one patient.Methods Four carbapenem-resistant K .pneumoniae strains were isolated from blood,urine,sputum and pelvic drainage of a patient after cystectomy in clinical mi-crobiology lab of Xinhua Hospital in March 2012.① Modified Hodge test was used to detect the expression of carbapenemase.② PCR amplification assay and DNA sequencing were used to detect resistance-related genes.③ SDS-PAGE analysis was per-formed to analyze outer membrane porin components of the four carbapenem-resistant K .pneumoniae isolates.ERIC-PCR as-say was used to analyze the homology of these carbapenem-resistant isolates.Results Modified Hodge test demonstrated that all the four carbapenem-resistant K.pneumoniae expressed carbapenemase.KPC-2 gene was identified in all the four isolates by PCR test and DNA sequencing.SDS-PAGE analysis indicated an outer membrane porin alteration common to all the four iso-lates which is distinct from that of the strain sensitive to antibiotics.The identical DNA fingerprinting of the four strains was confirmed by ERIC-PCR analysis.Conclusions The antibiotic resistance mechanism of the four carbapenem resistant K.pneu-moniae associated with this case includes the expression of KPC-2 and altered outer membrane permeability induced by abnormal expression of outer membrane porin.These four strains belong to one common clonal group.
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Objective To investigate resistant mechanisms of a pandrug-resistant Acinetobacter baumannii (js01) to β-1actams.Methods Strain js01 isolated from sputum sample of an inpatient from Ningbo First Municipal Hospital in December 2011 was confirmed by PCR amplifying and sequencing of gyrA and parC,and aligning with BLASTn.Thirty-three kinds of β-lactamase genes ( 13 kinds of class A,10kinds of class B,2 kinds of class C,8 kinds of class D),linkage detection of insertion sequences and β-lactamase genes,as well as outer membrane porin gene carO were analyzed by PCR.Genes encoding PBPI A were divided into three fragments,PCR amplified and bidirectional sequenced,and ligated to the full-length gene.Results Four kinds of β-lactamase genes were positive in js01:TEM-I,ADC-30, OXA-23 and OXA-66.Linkage detection of insertion sequences and β-lactamase genes showed that ISabal-ADC-30 and ISabal-OXA-23 were positive. When compared with sensitive strain (SDF) of Acinetobacter baumannii,sense mutations were found in carO gene of js01,and identity of amino acid sequence of carO gene between js01 and SDF was 76.0％ (189/249),and differences owed to loss of 3 amino acids.Sense mutations were also found in genes encoding PBP1A of js01,and identity of amino acid sequence of genes encoding PBP1A between js01 and SDF was 99.6％ ( 848/851 ),and differences owed to variations of 3amino acids.However,compared with three-dimensional structure of PBP1 A of SDF,PBP1 A of js01 lost 2helixes.Conclusion In strain js01,mutations of housekeeping genes ( genes encoding PBP1A and CarO),and genes producing β-lactamase mediated by mobile genetic elements,may play a key role in resistance to β-lactams.