Molecular detection of ß-lactamase and integron genes in clinical strains of Klebsiella pneumoniae by multiplex polymerase chain reaction

Abstract INTRODUCTION: Infections caused by β-lactamase-producing gram-negative bacteria, such as Klebsiella pneumoniae, are increasing globally with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the prevalence of antibiotic resistance genes (β-lactamase and integron genes) using multiplex PCR. METHODS One-hundred K. pneumoniae isolates were collected from different clinical samples. Antibiotic susceptibility testing was performed with thirteen different antibiotics. Multiplex-PCR was used to detect β-lactamase (bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC) and integron genes (int I, int II, and int III). RESULTS: The highest and lowest rate of resistance was exhibited against amikacin (93%) and imipenem (8%), respectively. The frequency of β-lactamase-positive K. pneumoniae was 37%, and the prevalence of the bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC genes was 38%, 24%, 19%, 12%, 6%, 11%, 33%, 0%, 28%, and 23%, respectively. Of the 100 isolates, eight (8%) were positive for class I integrons; however, class II and III integrons were not detected in any of the strains. CONCLUSIONS: These results indicate co-carriage of a number of β-lactamase genes and antibiotic resistance integrons on the same plasmids harboring multi-drug resistance genes. It seems that these properties help to decrease treatment complications due to resistant bacterial infections by rapid detection, infection-control programs and prevention of transmission of drug resistance.

Characterization of Mycobacterium tuberculosis complex isolated from iranian and afghani patients by spoligotyping method / Caracterização do complexo Mycobacterium tuberculosis isolado de pacientes do Irã e Afeganistão pelo método de spoligotyping

Designing newer drugs, vaccines, and diagnostic techniques is dependent on better understanding of M. tuberculosis virulence mechanism. In this study the prevalence of pcaA gene was determined in M. tuberculosis strains typed by spoligotyping. The associated risk factors among patients with different nationalities residing in Iran were also determined. The isolated M. tuberculosis strains have been characterized by performing susceptibility tests against four first-line antituberculosis drugs and were then subjected to spoligotyping characterization. PCR was used for detection of pcaA gene and its nucleotide sequence was also determined. Spoligotyping of M. tuberculosis strains resulted in 140 different patterns. One hundred twenty two (87.1%) of these spoligotype isolates were unique and reported for the first time. The remaining18 (12.8%) spoligotype patterns were previously reported from other geographical regions of the world. Haarlem family was most prevalent than other genotype. Antibiotic resistances were higher in those isolated from the Iranian patients. The pcaA gene was detected in M. tuberculosis clinical isolates but not in saprophyte strains such as M. kansasi. The results showed that, spread of M. tuberculosis strains belonging to the Beijing family among Iranian patients has to be considered seriously. This study confirmed the widespread existenceof pcaA gene in almost all the clinical isolates. It is also important to undertake studiesto identify which factors are the most significant to considerin tuberculosis control program.

O desenvolvimento de novas drogas, vacinas e técnicas de diagnóstico depende de uma melhor compreensão dos mecanismos de virulência de Mycobacterium tuberculosis. Neste estudo, a prevalência do gene pcaA em cepas de M. tuberculosis foi avaliada através de da técnica de spoligotyping. Os fatores de risco associados nos pacientes de diferentes nacionalidades vivendo no Irã foram também determinados. As cepas de M. tuberculosis isoladas foram submetidas a testes de sensibilidade a quatro drogas anti-tuberculose de primeira linha e à caracterização por spoligotyping. Empregou-se PCR para detectar o gene pcaA, determinado-se também a sequencia de nucleotidios. A espoligotipagem resultou em 140 grupos diferentes, sendo 120 (87,1%) reportados pela primeira vez. Os demais espoligotipos (12,8%) já foram descritos em outras regiões geográficas no mundo. A família Haarlem foi mais comum que os demais genótipos. A resistencia a antibióticos foi maior nas cepas isoladas dos pacientes iranianos. O gene pcaA foi detectado em isolados clínicos de M. tuberculosis mas não em cepas saprófitas, como M. kansasi. Os resultados indicaram a existência de M. tuberculosis pertencente à família Beijing nos pacientes iranianos. Este estudo confirmou a presença do gene pcaA em quase todos os isolados clínicos. Estudos que identifiquem os fatores mais significantes nos programas de controle da tuberculose são necessários.