Pseudomonas aeruginosa: patogenicidad y resistencia antimicrobiana en la infección urinaria / Pseudomonas aeruginosa: pathogenicity and antimicrobial resistance in urinary tract infection

Resumen Dentro de las infecciones nosocomiales más frecuentes asociadas a bacterias multi-resistentes y de peor pronóstico, se encuentran las producidas por Pseudomonas aeruginosa. Esta bacteria posee una alta capacidad de adaptación a condiciones adversas como por ejemplo el pH y la osmolaridad de la orina. Pseudomonas aeruginosa es uno de los principales patógenos implicados en infecciones nosocomiales y de pacientes inmunosuprimidos. Esta bacteria se considera un agente infeccioso oportunista que posee diversos mecanismos de patogenicidad, así como de resistencia a antimicrobianos, lo que contribuye a la dificultad en el tratamiento de estas infecciones. En la presente revisión bibliográfica se analizan la taxonomía, los mecanismos de patogenicidad y genes de resistencia de P. aeruginosa. Así también, se abordan los factores microambientales de la infección urinaria producida por esta bacteria, haciendo un acercamiento al entendimiento de las bases fisiopatológicas de esta infección.

Among the most frequent nosocomial infections associated with polyresistant bacteria and with a worse prognosis, are those produced by Pseudomonas aeruginosa. This bacterium has a high capacity to adapt to adverse conditions such as pH and osmolarity of urine. Pseudomonas aeruginosa is one of the main pathogens involved in nosocomial infections and immunosuppressed patients. This bacterium is considered an opportunistic infectious agent that has diverse mechanisms of pathogenicity, as well as resistance to antimicrobials, which contributes to the difficulty in the treatment of these infections. In the present bibliographic review, the taxonomy, pathogenicity mechanisms and resistance genes of P. aeruginosa are analyzed. Likewise, the micro-environmental factors of the urinary infection produced by this bacterium are approached, making an approach to the understanding of the pathophysiological bases of this infection.

Clonal relation and antimicrobial resistance pattern of extended-spectrum ß-lactamase- and AmpC ß-lactamase-producing Enterobacter spp. isolated from different clinical samples in Tehran, Iran

Abstract INTRODUCTION: Here, we determined the genes encoding antibiotic resistance enzymes and virulence factors and evaluated the genetic relationship between Enterobacter spp. isolated from different clinical samples. METHODS: A total of 57 clinical isolates of Enterobacter spp. were tested for the production of extended-spectrum β-lactamases (ESBLs), carbapenemase, and AmpC using phenotypic and genotypic methods. RESULTS: The most common ESBLs and AmpC β-lactamases were bla TEM (63.3%) and bla EBC (57.7%), respectively. The most prevalent virulence gene was rpos (87.7%). The random amplified polymorphic DNA (RAPD) patterns of strains were genetically unrelated. CONCLUSIONS: RAPD polymerase chain reaction analysis revealed high genetic diversity among isolates.

Isolation of blaNDM-producing Enterobacteriaceae in a public hospital in Salvador, Bahia, Brazil

ABSTRACT Carbapenemases have great importance in the global epidemiological scenario since infections with carbapenemase-producing bacteria are associated with high mortality, especially in hospitalized patients in intensive care units. This study describes two microorganisms producers of the New Delhi Metallo-b-lactamase, Klebsiella pneumoniae and Citrobacter freundii, from two patients admitted to a public hospital in Salvador, Bahia. These are the first clinical cases of New Delhi Metallo-b-lactamase described in microorganisms in the north and northeast Brazil. The isolates were characterized by antimicrobial susceptibility test, with resistance to all β-lactams including carbapenems, negative Modified Hodge Test and the synergy test with Ethylenediaminetetraacetic acid, Phenylboronic Acid and Cloxacillin was positive only with Ethylenediaminetetraacetic acid (difference of >5 mm in the inhibition zone between the disk without and with the inhibitor). Analysis by multiplex PCR for blaIMP, blaVIM, blaNDM, blaKPC and blaOXA-48 enzymes confirmed the presence of blaNDM gene. This report of two different New Delhi Metallo-b-lactamase-producing microorganisms in a different region of Brazil confirms the risk of spreading resistance genes between different species and emphasizes the need for prevention and control of infections caused by these pathogens, which have limited treatment options and have been linked to high mortality rates.

Bases moleculares de la resistencia a meticilina en Staphylococcus aureus / Molecular basis of methicillin-resistance in Staphylococcus aureus

Resumen Desde el inicio de la era antimicrobiana se han ido seleccionando gradualmente cepas de Staphylococcus aureus resistentes a antimicrobianos de amplio uso clínico. Es así como en 1960 se describen en Inglaterra las primeras cepas resistentes a meticilina, y algunos años después son informadas en hospitales de Chile. Actualmente, S. aureus resistente a penicilinas antiestafilocóccicas es endémico en los hospitales de nuestro país y del mundo, siendo responsable de una alta morbimortalidad. La resistencia es mediada habitualmente por la síntesis de una nueva transpeptidasa, denominada PBP2a o PBP2' que posee menos afinidad por el β-lactámico, y es la que mantiene la síntesis de peptidoglicano en presencia del antimicrobiano. Esta nueva enzima se encuentra codificada en el gen mecA, a su vez inserto en un cassette cromosomal con estructura de isla genómica, de los cuales existen varios tipos y subtipos. La resistencia a meticilina se encuentra regulada, principalmente, por un mecanismo de inducción de la expresión del gen en presencia del β-lactámico, a través de un receptor de membrana y un represor de la expresión. Si bien se han descrito mecanismos generadores de resistencia a meticilina mec independientes, son categóricamente menos frecuentes.

Staphylococcus aureus isolates resistant to several antimicrobials have been gradually emerged since the beginning of the antibiotic era. Consequently, the first isolation of methicillin-resistant S. aureus occurred in 1960, which was described a few years later in Chile. Currently, S. aureus resistant to antistaphylococcal penicillins is endemic in Chilean hospitals and worldwide, being responsible for a high burden of morbidity and mortality. This resistance is mediated by the expression of a new transpeptidase, named PBP2a or PBP2', which possesses lower affinity for the β-lactam antibiotics, allowing the synthesis of peptidoglycan even in presence of these antimicrobial agents. This new enzyme is encoded by the mecA gene, itself embedded in a chromosomal cassette displaying a genomic island structure, of which there are several types and subtypes. Methicillin resistance is mainly regulated by an induction mechanism activated in the presence of β-lactams, through a membrane receptor and a repressor of the gene expression. Although mec-independent methicillin resistance mechanisms have been described, they are clearly infrequent.

Viability and Effects on Bacterial Proteins by Oral Rinses with Hypochlorous Acid as Active Ingredient

Abstract: This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients.

Resumo: Este estudo investigou o efeito de enxaguantes à base de ácido hipocloroso (HOCl) e clorexidina (CHX) sobre a viabilidade bacteriana de S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae e E. cloacae. O percentual de bactérias sobreviventes foi testado pelo método de fluorescência utilizando Live/Dead kit(r) e BacLight (Molecular Probes(r)), fazendo comparação entre os grupos com os testes de Kruskal-Wallis e U Mann-Whitney e correção de Bonferroni (p<0,012). O efeito de HOCl e CHX sobre P. gingivalis e S. mutans foi determinado por SDS-PAGE. O CHX mostrou eficácia superior ao HOCl contra S. mutans, A. israelii, E. corrodens e E. cloacae (p<0,001), ao passo que P. gingivalis, A. actinomycetemcomitans, C. rectus e K. oxytoca foram melhores que o CHX para o HOCl (p=0,001). O K. pneumoniae teve efeito similar para o CHX e para o HOCl. As proteínas de P. gingivalis e S. mutans foram afetadas de modo semelhante por CHX e HOCl. O HOCl reduziu a viabilidade bacterial, especialmente nas bactérias periodontopáticas, o que pode recomendar o uso no controle do biofilme subgingival em pacientes periodentais.

Docking studies on novel alkaloid tryptanthrin and its analogues against enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis.

Isoniazid resistance is a serious threat in the battle against the treatment of multi-drug resistant tuberculosis (MDR-TB) and extremely drug-resistant tuberculosis (XDR-TB). Isoniazid is an inhibitor of enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis, which is an important and functional enzyme of the type II fatty acid synthesis system and important therapeutic target. Natural alkaloid tryptanthrin and its analogues have shown anti-tubercular activity against MDR-TB, but their cellular target is unknown. In this work, in silico molecular docking was performed using docking server in order to see the interaction of tryptanthrin and its 15 analogues with InhA of M. tuberculosis. Results showed that among tryptanthrin and its 15 analogues, tryptanthrin and its two analogues exhibited good affinity to the binding site of InhA with free binding energy of -7.94 kcal/mol and inhibition constant (Ki) of 1.50 µm. Active site residues of InhA interacting with tryptanthrin were Ser13, Thr39, Phe41, Leu63, Asp64, Val65, Ile95, Phe97 and Ile122. In binding mode, polar bond were found between O1 (1) with Asp64 of bond length (3.34 Å) and hydrophobic bonds were found between Leu63 with C15 and C12, Val65 with C7, Val65 with C12 and C4, Ile95 with C6 and C7, Ile95 with C10, C12 and C14. Important pi-pi bonds were found between Phe41 with C2, C5, C7, C12, C4, C6, C8, C9, C13 and Phe97 with C9. These interactions indicated stability of tryptanthrin in active residue and suggested it as a potential drug candidate. Thus, good affinity of tryptanthrin to binding site of InhA may lead to synthesis of anti-tubercular drug capable of combating MDR strains of M. tuberculosis

Multidrug-resistant tuberculosis in Port-au-Prince, Haiti / Tuberculosis multirresistente en Puerto Príncipe, Haití

OBJECTIVE: To determine the prevalence of multidrug-resistant tuberculosis (MDR-TB) among patients with new smear-positive pulmonary TB in Port-au-Prince, Haiti. METHODS: Sputum samples were cultured from 1 006 patients newly diagnosed with TB in 2008. The core region of the rpoB gene that is associated with resistance to rifampin was sequenced. All isolates with rpoB mutations were sent to the New York State reference laboratory for conventional drug susceptibility testing (DST). All isolates were also tested with the GenoType MTBDRplus line-probe assay. RESULTS: Mycobacterium tuberculosis was isolated from 906 patients. Twenty-six (2.9 percent) of the isolates had missense mutations or deletions in rpoB and were resistant to rifampin by DST. All 26 were also resistant to isoniazid and classified as MDR-TB. Forty-six control isolates without rpoB mutations were found to be rifampin sensitive by DST. The GenoType MTBDRplus line-probe assay correctly identified 26 MDR-TB strains. It misclassified one pansusceptible isolate as rifampin resistant. CONCLUSIONS: This study shows an MDR-TB prevalence of 2.9 percent in newly diagnosed TB patients in Haiti and suggests that rpoB sequencing and hybridization assays are good screening tools for early detection of MDR-TB.

OBJETIVO: Determinar la prevalencia de tuberculosis (TB) multirresistente en pacientes con TB pulmonar nueva con baciloscopia positiva en Puerto Príncipe, Haití. MÉTODOS: Se cultivaron muestras de esputo de 1 006 pacientes con diagnóstico reciente de tuberculosis efectuado durante el 2008. Se secuenció la región nuclear del gen rpoB, que se asocia con la resistencia a la rifampicina. Todos los aislados con mutaciones de rpoB se enviaron al laboratorio de referencia del estado de Nueva York para llevar a cabo un antibiograma convencional. Todos los aislados se estudiaron también con el ensayo de sonda lineal GenoType MTBDRplus. RESULTADOS: Se aisló Mycobacterium tuberculosis de 906 pacientes. Veintiséis (2,9 por ciento) de los aislados presentaban mutaciones de sentido erróneo o deleciones en rpoB y fueron resistentes a la rifampicina en el antibiograma. Los 26 aislados fueron resistentes también a la isoniacida y se clasificaron como TB multirresistente. Cuarenta y seis aislados de control sin mutaciones de rpoB resultaron sensibles a la rifampicina en el antibiograma. El ensayo de sonda lineal GenoType MTBDRplus identificó correctamente a las 26 cepas de TB multirresistente y clasificó de manera errónea un aislado sensible a múltiples fármacos como resistente a la rifampicina. CONCLUSIONES: Este estudio revela una prevalencia de TB multirresistente de 2,9 por ciento en los pacientes con TB recién diagnosticada en Haití e indica que los ensayos de secuenciación e hibridación de rpoB son estudios de detección sistemática adecuados para la detección temprana de la TB multirresistente.

Functional characterization of MexXY and OpmG in aminoglycoside efflux in Pseudomonas aeruginosa.

MexXY is an active efflux system that contributes to intrinsic resistance to aminoglycosides in Pseudomonas aeruginosa. MexXY can function in combination with OprM in aminoglycoside efflux but may also functionally associate with another as yet unidentified outer membrane channel. The possible role of OpmG as a third component of MexXY in aminoglycoside efflux was investigated by construction of unmarked opmG mutants. Loss of OpmG did not have any impact on minimum inhibitory concentrations for aminoglycosides regardless of the presence of oprM, indicating that MexXY does not interact with OpmG in aminoglycoside efflux. In a clinical isolate PAJ010, (mexXY) enhanced streptomycin susceptibility but neither oprM nor opmG could, suggesting that MexXY functionally associates with an unidentified outer membrane protein for aminoglycoside efflux. Expression of an opmG-lacZ transcriptional fusion revealed that OpmG expression was neither constitutive nor inducible by gentamicin. Growth rates of wildtype P. aeruginosa and opmG mutant derivatives were not different, indicating that expression of opmG is not essential for P. aeruginosa growth.

Utility of the ceftazidime-imipenem antagonism test (CIAT) to detect and confirm the presence of inducible AmpC beta-lactamases among enterobacteriaceae

Detection of AmpC beta-lactamase production by enterobacteria has been problematic. Contrary to ESBLs, no specific guidelines are available for detection and confirmation of AmpC production by clinical relevant microorganisms. Moreover, some bacterial species may produce inducible AmpC beta-lactamases that can be easily overlooked by routine susceptibility tests. We reported here a new test based on the strong inducible effect of imipenem on AmpC genes and the consequent antagonism with ceftazidime. This test is very simple and proved to be helpful in detecting AmpC-inducible enzymes among several species of clinical isolates.

SCCmec typing and detection of VISA-related genes in methicillin-resistant Staphylococcus aureus clinical strains from Kobe University Hospital, Japan.

A total of 50 clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) were collected from Kobe University Hospital in 2003. Molecular typing of SCCmec was performed by multiplex polymerase chain reaction (PCR) and the presence of six genes (vraR, vraG, vraA, vraF, fruA, and fruB) associated with vancomycin (VCM) resistance was examined by simple PCR analysis. Out of 50 MRSA strains isolated 47 strains contained Type II SCCmec and the remaining contained Type IV SCCmec. Thirty seven strains contained pUB110 plasmid. VraA was present in 69% of the strains, vraF in 10%, vraG in 53%, and vraR in 16%. Noteworthy, strains without pUB110 contained vraR in relatively higher frequency (31%) compared with strains with pUB110 (11%).

Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.

Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil

In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > ou = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.